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A1 adenosine receptor–stimulated exocytosis in bladder umbrella cells requires phosphorylation of ADAM17 Ser-811 and EGF receptor transactivation

A1 adenosine receptor–stimulated exocytosis in bladder umbrella cells requires phosphorylation of... MB oC | ARTICLE A adenosine receptor–stimulated exocytosis in bladder umbrella cells requires phosphorylation of ADAM17 Ser-811 and EGF receptor transactivation H. Sandeep Prakasam, Luciana I. Gallo, Hui Li, Wily G. Ruiz, Kenneth R. Hallows, and Gerard Apodaca Departments of Medicine and Cell Biology, University of Pittsburgh, Pittsburgh, PA 15261 ABSTRACT Despite the importance of ADAM17-dependent cleavage in normal biology and Monitoring Editor Keith E. Mostov disease, the physiological cues that trigger its activity, the effector pathways that promote its University of California, function, and the mechanisms that control its activity, particularly the role of phosphorylation, San Francisco remain unresolved. Using native bladder epithelium, in some cases transduced with adenovi- ruses encoding small interfering RNA, we observe that stimulation of apically localized A 1 Received: Mar 20, 2014 adenosine receptors (A ARs) triggers a G -Gβγ-phospholipase C-protein kinase C (PKC) cas- Revised: Sep 10, 2014 1 i Accepted: Sep 11, 2014 cade that promotes ADAM17-dependent HB-EGF cleavage, EGFR transactivation, and apical exocytosis. We further show that the cytoplasmic tail of rat ADAM17 contains a conserved serine residue at position 811, which resides in a canonical PKC phosphorylation site, and is phosphorylated in response to A AR activation. Preventing this phosphorylation event by S811A expression of a nonphosphorylatable ADAM17 mutant or expression of a tail-minus con- struct inhibits A AR-stimulated, ADAM17-dependent HB-EGF cleavage. Furthermore, ex- S811A pression of ADAM17 in bladder tissues impairs A AR-induced apical exocytosis. We con- clude that adenosine-stimulated exocytosis requires PKC- and ADAM17-dependent EGFR transactivation and that the function of ADAM17 in this pathway depends on the phosphory- lation state of Ser-811 in its cytoplasmic domain. INTRODUCTION Protein ectodomain shedding, a process regulated by proteolysis, factors, and cell adhesion molecules (Reiss and Saftig, 2009) and is is a fundamental mechanism for the release of cytokines, growth altered in cancer, autoimmune and inflammatory diseases, cardio - vascular disease, and neurodegeneration (Murphy, 2008). The best- understood sheddases include the “a disintegrin and a metallopro- This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E14-03-0818) on September 17, 2014. teinase” (ADAM) family members ADAM10 and ADAM17 (also H.S.P., L.I.G., W.G.R., and H.L. designed and executed the experiments and as- known as TACE), both of which shed a variety of substrates, includ- sisted in the writing of the manuscript. K.R.H. and G.A. assisted in the design of ing the transmembrane ligands for the epidermal growth factor re- the experiments and wrote and prepared the manuscript. ceptor (EGFR). Whereas ADAM10 targets betacellulin, EGF, and The authors have no conflicts of interest to report. Address correspondence to: Gerard Apodaca (gla6@pitt.edu). neuregulin, ADAM17 is the principal sheddase for transforming Abbreviations used: A AR, A adenosine receptor; ADAM, a disintegrin and a growth factor (TGF) α, amphiregulin, epiregulin, epigen, and hepa- 1 1 metalloproteinase; CCPA, 2-chloro-N6-cyclopentyladenosine; DFV, discoidal rin-binding (HB) EGF (Jackson et al., 2003; Sahin et al., 2004; Sahin and/or fusiform-shaped vesicle; EGFR, epidermal growth factor receptor; ERK, and Blobel, 2007; Blobel, 2005; Horiuchi et al., 2005; Sternlicht extracellular signal-regulated kinase; GPCR, G protein–coupled receptor; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; PLC, phospholipase C; et al., 2005; Hassemer et al., 2010; Luo et al., 2011). The physiologi- PMA, phorbol-12-myristate-13-acetate; TIMP, tissue inhibitor of metalloprotei- cal cues that trigger shedding remain to be specified; however, nase. ADAM17-elicited shedding occurs in response to some G protein– © 2014 Prakasam et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is avail- coupled receptor (GPCR) ligands as well as treatment with ionomy- able to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported cin or phorbol-12-myristate-13-acetate (PMA), a diacylglycerol Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). ® ® (DAG) mimic and activator of classical protein kinase C (PKC) iso- “ASCB ,” “The American Society for Cell Biology ,” and “Molecular Biology of the Cell ” are registered trademarks of The American Society for Cell Biology. forms (Horiuchi et al., 2007; Le Gall et al., 2009; Dang et al., 2011). 3798 | H. S. Prakasam et al. Molecular Biology of the Cell 2+ In contrast, ADAM10-dependent shedding responds to Ca iono- (A AR). Finally, PKC likely promotes the phosphorylation of a previ- phores (e.g., ionomycin) but generally not PMA (Horiuchi et al., ously unreported canonical PKC site centered at Ser-811 in the cyto- 2007; Le Gall et al., 2009; Dang et al., 2011). In addition, our under- plasmic domain of rat ADAM17. This phosphorylation event is re- standing of the signaling and associated effector pathways that act quired for adenosine-induced and ADAM17-dependent HB-EGF downstream of these stimuli remains incomplete. The extracellular cleavage, EGFR transactivation, and exocytosis. These studies lend signal-regulated kinase (ERK), as well as the related p38 mitogen- support to the hypothesis that posttranslational modification of activated protein kinase (MAPK), PKCα, PKCδ, and the PKC-regu- ADAM17—phosphorylation in particular—can control its activity in lated protein phosphatase inhibitor 14D, has also been linked to a physiologically relevant setting. ADAM17-dependent shedding, and PKCδ is required for shedding of neuregulin (Bell and Gooz, 2010; Killock and Ivetic, 2010; Dang RESULTS et al., 2011, 2013; Hall and Blobel, 2012). A AR-stimulated apical exocytosis occurs through An additional unresolved question is how these effectors pro- transactivation of the EGFR mote ADAM-dependent shedding of EGFR ligands. Data indicate Late-phase exocytosis is triggered when uroepithelial tissue is maxi- that they work through multiple mechanisms and perhaps in a cell- mally stretched (Balestreire and Apodaca, 2007; Yu et al., 2009), but and stimulus-dependent manner (Fan et al., 2003; Mifune et al., it is unknown whether other stimuli promote a similar response. Be- 2005; Xu and Derynck, 2010). Potential regulatory steps include cause adenosine release is significantly increased as the bladder membrane trafficking of the ADAM or its ligands (Soond et al., reaches its capacity (Prakasam et al., 2012), and because extracel- 2005), effects on the ADAM17 dimer–monomer equilibrium, and lular adenosine also triggers exocytosis (Yu et al., 2006), we tested association with tissue inhibitor of metalloproteinases (TIMP) 3 (Xu the hypothesis that adenosine stimulates exocytosis by way of EGFR et al., 2012) or changes in the redox potential of the extracellular transactivation. Adenosine can stimulate exocytosis when added to protein disulfide isomerase (Willems et al., 2010), which is hypoth- either the serosal or mucosal surfaces of isolated uroepithelium; esized to alter ADAM17 activity by rearrangement of its disuld fi e however, in this study, we focused our attention on the mucosal bonds. An additional regulatory mechanism may be phosphoryla- events, as these are primarily mediated by the A AR, and EGFR tion of EGFR receptor substrates (Dang et al., 2013) or the ADAM transactivation occurs at this surface (Yu et al., 2006; Balestreire and metalloproteinase itself. For example, phosphorylation of ADAM17 Apodaca, 2007). Consistent with our previous reports (Yu et al., cytoplasmic residues Tyr-702, Ser-791, Ser-819, and Thr-735 have 2011; Prakasam et al., 2012), the A AR was localized to the apical been reported (Fan et al., 2003; Hall and Blobel, 2012; Niu et al., pole of rat umbrella cells (as well as in the underlying lamina propria; 2013). Tyr-702 is reportedly phosphorylated by the Src kinase when Figure 1A). When adenosine was added to the mucosal surface of myogenic precursor cells are stretched (Niu et al., 2013). In contrast, isolated rabbit tissue, it stimulated increased tissue capacitance (C ; Ser-791 is phosphorylated before stimulation, and mutations of Ser- 1 μF ≈ 1 cm ; Figure 1B), which in this tissue correlates well with 819 do not appear to affect shedding (Fan et al., 2003). In the case other measures of apical exocytosis (Truschel et al., 2002; Wang of Thr-735, very high doses of PMA (1 μM) are reported to promote et al., 2003a). Similar results were obtained when the highly selec- ERK-dependent phosphorylation of this residue (Soond et al., 2005). tive and high-affinity A AR agonist 2-chloro-N6-cyclopentyladenos- However, others report that phosphorylation of this residue is medi- ine (CCPA) was used. Unlike adenosine, CCPA is not rapidly con- ated by the p38 MAPK and is required for ADAM17-dependent verted to inosine or to AMP (Manjunath and Sakhare, 2009; Prakasam shedding in response to various forms of stress but not PMA (Xu and et al., 2012) and was thus used in our subsequent studies. We next Derynck, 2010). A significant argument against the role for phos - determined whether A AR-stimulated exocytosis, like that observed phorylation relies on reports that show that stimulus-evoked shed- in response to excess stretch, is dependent on protein synthesis or ding of ADAM17 occurs in cells expressing truncated versions of secretion. Indeed, treatment with cycloheximide or brefeldin A sig- this metalloproteinase that lack a C-terminus (Reddy et al., 2000; Le nificantly blocked CCPA-mediated exocytosis (Figure 1C). The ef - Gall et al., 2010; Hall and Blobel, 2012). fect of the latter was particularly pronounced. A potentially useful and physiologically relevant model system in We also assessed whether CCPA triggered apical exocytosis by which to study the signals, effector pathways, and mode of ADAM transactivating the EGFR. We r fi st tested the effect of treating the activation is the uroepithelium, a tissue that can be studied both ex mucosal surface of the tissue with CRM197, a mutant version of vivo and in vivo (Khandelwal et al., 2008, 2010, 2013). Bladder lfi ling diphtheria toxin that binds selectively and with high affinity to mem - triggers the exocytosis of an abundant subapical pool of discoidal- brane-bound HB-EGF and prevents its cleavage (Uchida et al., and/or fusiform-shaped vesicle (DFVs) in the outer umbrella cell 1973). Indeed, we observed that CRM197 almost completely layer (Wang et al., 2003a,b). Stretch-induced exocytosis progresses blocked CCPA-mediated apical exocytosis (Figure 2A). Next we in two phases. “Early-phase” exocytosis occurs during bladder fill - pretreated tissue with AG1478, a small-molecule inhibitor of EGFR, ing, as the epithelium is bowing outward, and is triggered by apical which also significantly blocked CCPA-mediated apical exocytosis 2+ Ca entry, likely conducted by a nonselective cation channel (Yu (Figure 2B). As further evidence that the EGFR was transactivated, et al., 2009). In contrast, “late-phase” exocytosis is initiated once we generated lysates from CCPA-treated epithelium and then the tissue is maximally bowed outward (i.e., in response to a full probed Western blots with an antibody that detects phosphoryla- bladder) and requires metalloproteinase-dependent cleavage of tion of the EGFR at Tyr-1173. This residue promotes assembly of a HB-EGF, leading to “transactivation” of apical EGFRs (Balestreire MAPK signaling cascade downstream of other GPCRs (Balestreire and Apodaca, 2007). The latter initiates a downstream ERK pathway and Apodaca, 2007). Phosphorylation of Tyr-1173 was maximally that culminates in protein synthesis and exocytosis (Balestreire and stimulated 10 min after continuous treatment with CCPA, decreased Apodaca, 2007). We now report that adenosine, which is released at 30 min posttreatment, and returned to control levels by 60 min from the epithelium in response to stress (Prakasam et al., 2012), (Figure 2, C and D). The Tyr-1173 phosphorylation was prevented spurs a late phase–like response in umbrella cells. We also find that when the tissue was pretreated with the EGFR inhibitor AG1478 a G -, Gβγ-, phospholipase C (PLC)-, and PKC-dependent effector before CCPA treatment (Figure 2, C and D), confirming that phos - cascade is initiated downstream of the adenosine A receptor phorylation of Tyr-1173 likely results from autophosphorylation. Volume 25 November 15, 2014 ADAM17-dependent exocytosis | 3799 DFVs and the apical surface of umbrella cells (Figure 3C; Apodaca, 2004). Consistent with a role for ADAM17 in DFV trafc fi king, we observed that the broad-spectrum metallo- proteinase inhibitor GM6001 and the ADAM17-selective inhibitor Tapi-2 (Bal- estreire and Apodaca, 2007; Kveiborg et al., 2011) both significantly inhibited the CCPA- mediated increases in C (Figure 3D). More- over, Tapi-2 blocked CCPA-mediated phos- phorylation of EGFR Tyr-1173 (Figure 2, C and D). To provide further evidence that ADAM17 is required for EGFR transactiva- tion, we exploited our previously de- scribed in situ viral transduction approach (Khandelwal et al., 2008, 2010), in this case to express ADAM17-specific short hairpin RNAs (shRNAs) or scrambled shR- NAs in the rat bladder uroepithelium. We used rat bladders in these studies because the volume capacity of the bladder, and therefore the number of virus particles FIGURE 1: A AR-stimulated exocytosis is dependent on protein synthesis and secretion. needed, was relatively small (∼500 μl) (A) Cryosection of rat bladder epithelium labeled with an A AR-specic fi antibody (green), compared with the volumes required to fill rhodamine–phalloidin to label the actin cytoskeleton (red), and TOPRO-3 (blue) to label the the rabbit bladder (∼60–100 ml). This tech- nucleus. Right, merge. An umbrella cell is marked with an asterisk and the white arrows mark the nique targets the umbrella cell layer and apicolateral borders of the cell. Scale bar, 12 m μ . (B) Rabbit uroepithelium was mounted in Ussing chambers and after equilibration left untreated (no drug), or 1 μM adenosine or 500 nM achieves transduction efficiencies of 70– CCPA was added to the mucosal hemichamber. Percentage changes in C were monitored. 95% (Khandelwal et al., 2008, 2010). In- (C) Rabbit tissue was left untreated, pretreated with 5 g μ /ml brefeldin A (BFA) for 30 min, or deed, we were able to achieve >90% pretreated with 100 ng/ml cyclohexamide for 60 min. CCPA (500 nM) was then added to the knockdown of ADAM17 expression (Figure mucosal hemichamber and C recorded. Data for CCPA alone are reproduced from B. 4, A and B). On examining the expression (B, C) Mean changes in C ± SEM (n ≥ 4). Significant differences ( p < 0.05), relative to no drug in and distribution of ADAM17 in cross sec- A or CCPA in B, are indicated with an asterisk. tions of uroepithelium, we observed that knockdown of ADAM17 was largely con- Finally, we observed that U0126, an inhibitor of MEK activity, also fined to the umbrella cells, and expression of ADAM17 in the caused a significant decrease in CCPA-stimulated changes in C underlying cell layers was largely undisturbed (Figure 4C). (Figure 2A). Next we used ADAM17-specific shRNAs to test whether ADAM17 Taken together, these data indicate that, like the previously de- was required for CCPA-stimulated EGFR transactivation and exocy- scribed late-phase response (Balestreire and Apodaca, 2007), A AR tosis in rat epithelium. Treatment with ADAM17-shRNA, but not activation stimulates apical exocytosis by promoting transactivation scrambled shRNA, decreased EGFR phosphorylation (Figure 4, D of the EGFR downstream of HB-EGF cleavage, leading to MEK/ERK and E). We show later in Figure 6, F and G, that the effects of the activation and protein synthesis. ADAM17-shRNA are specific, and ADAM17 function is restored when the shRNA is coexpressed with shRNA-resistant variant of wild- ADAM17 is localized to the apical surface of umbrella cells, type ADAM17. We also attempted to measure changes in C in the where it stimulates CCPA-induced EGFR transactivation rat bladders treated with apical CCPA; however, we did not observe and exocytosis a response. Because C is dependent on the rates of membrane ad- In vivo studies implicate ADAM17 as the physiologically relevant dition and removal, we reasoned that one possible explanation was sheddase in HB-EGF–mediated transactivation (Jackson et al., 2003; that CCPA stimulated both exocytosis and endocytosis in rat epithe- Sahin et al., 2004). Consistent with these observations, we observed lium, thus obviating any change in apparent C . Indeed, we observed that ADAM17 was localized to small vesicular elements under the that wheat germ agglutinin–fluorescein isothiocyanate (WGA-FITC), apical surface of the rat umbrella cells (Figure 3A), the likely site of added to the apical hemichamber of mounted rat bladders, was en- HB-EGF cleavage and EGFR transactivation during the late-phase docytosed in CCPA-treated tissue but much less so in control, un- response (Balestreire and Apodaca, 2007). Rat tissues were used in treated tissue (Figure 4F). To circumvent the effects of endocytosis, these experiments because our antibody was produced in rabbits. we instead measured release of exogenously expressed human ADAM17 was also detected in the intermediate and basal cell layers growth hormone (hGH), which we and others previously showed is of the uroepithelium, as well as in cells in the underlying lamina packaged into DFVs and released from the luminal surface of the propria (Figure 3, A and C). The signal for ADAM17 was diminished bladder into the urinary space (Kerr et al., 1998; Khandelwal et al., when the anti-ADAM17 antibody was preincubated with immuniz- 2008). Because of rapid dilution, there is little secreted hGH that is ing peptide, confirming the specificity of the antibody (Figure 3B). endocytosed. Compared to control bladders, CCPA stimulated a ADAM17 showed a high degree of colocalization with uroplakin 3a large increase in the mucosal release of hGH from the tissue (Figure (Manders coefc fi ient of colocalization, 0.92); this is associated with 4, G and H). Moreover, expression of ADAM-17-specific shRNA, but 3800 | H. S. Prakasam et al. Molecular Biology of the Cell compared with tissues transduced with scrambled shRNA (Figure 4, G and H). Taken together, our results provide strong evi- dence that in rat tissues, ADAM17 is critical for CCPA- and stretch-induced EGFR trans- activation and exocytosis. G , PLC, and PKC act upstream of ADAM17 and HB-EGF to promote A AR-mediated EGFR transactivation We next addressed how ADAM17 activity is coupled to A AR activation. Previous stud- ies showed that A AR signals through Gα 1 i to inhibit the activity of adenylyl cyclase, whereas the βγ subunits of G increase the activity of phospholipase C-β (PLCβ), which hydrolyzes phosphatidylinositol 4,5-bispho- sphate to generate inositol trisphosphate (IP3) and diacylgycerol (Freund et al., 1994; Bucheimer and Linden, 2004; Chang et al., 2008). The latter stimulates the activity of PKC, a well-known regulatory kinase that was previously implicated in ADAM17 acti- vation (Dang et al., 2011; Kveiborg et al., 2011; Lemjabbar-Alaoui et al., 2011). We found that pertussis toxin–mediated inhibi- tion of G or inhibition of G subunit activity i βγ by the inhibitor M119K (Kirui et al., 2010) significantly impaired CCPA-mediated api - cal exocytosis in rabbit bladder umbrella cells (Figure 5A). Furthermore, the PLC-se- lective antagonist U73122 caused marked inhibition of CCPA-induced changes in C (Figure 5A). Thus ADAM17 activation down- stream of A AR likely occurs by way of a classical G -stimulated signaling cascade in- volving G and PLCβ. βγ Next we examined whether PKC is im- portant for A AR-dependent EGFR transac- tivation and exocytosis. Strikingly, the PKC FIGURE 2: The A AR transactivates the EGF receptor. (A) The mucosal surface of rabbit uroepithelium was pretreated with 25 ng/ml CRM197 for 25 min or with 10 M μ U0126 for inhibitor calphostin C caused a marked de- 60 min. CCPA (500 nM) was then added to the mucosal hemichamber, and C was recorded. crease in CCPA-stimulated changes in C (B) Rabbit uroepithelium was pretreated with 1 μM AG1478 for 30 min, and then CCPA (500 nM) and EGFR transactivation (Figures 2, C and was added to the mucosal hemichamber and C was recorded. (A, B) CCPA control data are D, and 5B). In contrast, treatment with PMA, reproduced from Figure 1B. Mean changes in C ± SEM (n ≥ 3). Statistically significant an activator of classical PKCs (Nishizuka, differences (p < 0.05), relative to CCPA treatment alone, are marked with an asterisk. 1992), caused robust stimulation of exocyto- (C, D) Rabbit uroepithelium was either left untreated (left) or treated with AG1478 (1 μM) for sis in the absence of CCPA (Figure 5B). Of 25 min, Tapi-2 (15 M μ ) for 90 min, or calphostin C (500 nM) for 60 min (right). CCPA (500 nM) interest, the kinetics of PMA-mediated exo- was then added to the mucosal hemichamber. Left, cells were lysed at the indicated time points. cytosis were faster than those mediated by Right, cells were lysed at the 10-min time point. Equal amounts of proteins were resolved by CCPA, particularly during the first 30 min, SDS–PAGE and immunoblots probed with a rabbit anti–EGFR-phospho-Y antibody or rabbit anti-EGFR antibody. (D) Quantification of Y phosphorylation. Data (mean ± SEM, n ≥ 3) are and then appeared to increase at a similar reported as fold increase above untreated tissue samples at t = 0. Statistically signic fi ant values rate to CCPA-treated tissue. This may indi- (p < 0.05) above t = 0 are marked by an asterisk. cate that PKC stimulates not only late-phase- like responses, but perhaps early-phase not scrambled shRNA, caused a large inhibition (>90%) in mucosal ones as well. To conr fi m that PMA acted by way of ADAM17, we hGH release (Figure 4, G and H). treated the tissue with Tapi-2, which significantly inhibited PMA- Finally, because stretch-mediated, late-phase exocytosis is de- mediated apical exocytosis (Figure 5C). We also observed that pendent on EGFR transactivation and sensitive to metalloproteinase AG1478 impaired PMA-mediated exocytosis (Figure 5C). Our data inhibitors (Balestreire and Apodaca, 2007), we determined whether pointed to the possibility that PKC acted upstream of ADAM17, ADAM17 also played a role in stretch-mediated exocytosis. We which acted before HB-EGF cleavage. We reasoned, therefore, that observed that shRNA-mediated ADAM17 knockdown signic fi antly the calphostin C–or TAPI-2–mediated inhibition of C should be reduced the stretch-induced apical release of hGH by ∼80% relieved by addition of HB-EGF. To test this possibility, we pretreated Volume 25 November 15, 2014 ADAM17-dependent exocytosis | 3801 requires G , Gβγ, PLC, PKC, and ADAM17, and these effectors likely act upstream of HB-EGF release and EGFR transactivation. CCPA-stimulated HB-EGF shedding and exocytosis are dependent on phosphorylation of ADAM17 Ser-811 To explore how PKC might act to stimu- late ADAM17 activity, we compared the amino acid sequences from multiple ver- tebrate species using the proteomics tool Scansite and identified a conserved, canonical PKC phosphorylation motif (X-R/K-X-X-S/T-X-R/K-X; Pearson and Kemp, 1991; Nishikawa et al., 1997) in the cytoplas- mic tail of the protein centered at Ser-811 (equivalent to Ser-808 in the human protein; Figure 6A). To our knowledge, a functional role for this residue was not explored previ- ously. We r fi st conr fi med that ADAM17 was phosphorylated in response to CCPA by ex- pressing the A AR in combination with epitope-tagged ADAM17-HA (this variant of ADAM17 is resistant to shRNA; see Figure 6F) in P-orthophosphate–labeled HEK cells (Figure 6B). HEK cells were used in these assays because these experiments were difficult to perform in whole tissues. On addition of CCPA, we observed that phosphorylation of both the “immature” proform of ADAM17-HA (∼120 kDa) and the “mature” cleaved form of the enzyme (∼93 kDa) was stimulated approximately threefold (Figure 6, B and C). In contrast, CCPA had no significant effect when wild- type ADAM17-HA was substituted with a nonphosphorylatable mutant of ADAM17 in which Ser-811 was changed to an Ala resi- S811A r due (ADAM17 -HA ; Figure 6, B and C). These results indicate that in response to A AR activation, Ser-811 may be a major site of ADAM17 phosphorylation. Because the physiological role of ADAM17 phosphorylation is a matter of some controversy (Reddy et al., 2000; Horiuchi et al., 2007; Le Gall et al., 2010; Dang et al., 2013), we next determined FIGURE 3: ADAM17 expression in the uroepithelium. (A–C) Cryosections of rat bladder whether phosphorylation of Ser-811 was bi- uroepithelium were reacted with antibodies specic fi for ADAM17 (A), a mixture of antibody and ologically relevant. First, we measured shed- inhibitory peptide (B), or antibodies specific for ADAM17 and uroplakin 3a (C). After incubation ding of a chimeric protein in which alkaline with fluorophore-labeled secondary antibodies, the samples were analyzed using confocal phosphatase (AP) was fused to the extracel- microscopy. Where indicated, actin was labeled with phalloidin and nuclei were labeled with lular domain of HB-EGF (HB-EGF-AP; Sahin TOPRO-3. The umbrella cells are marked with the asterisk in the merged images and the et al., 2004; Uttarwar et al., 2011), an apicolateral junction is indicated by arrowheads; scale bar, 10 m μ . (D) Rabbit uroepithelium was ADAM17 substrate that was coexpressed in pretreated with Tapi-2 (15 μM) or GM6001 (15 μM) for 90 min, CCPA (500 nM) was added, and C was recorded. Control CCPA data are reproduced from Figure 1B. Data are mean ±SEM (n ≥ 3), HEK cells along with the A AR and ADAM17- and values significantly different ( p < 0.05) from CCPA alone are marked with an asterisk. HA (Figure 6D). In response to CCPA, we observed a significant ∼70% increase in HB- cells with calphostin C or TAPI-2, added CCPA at t = 0, and then EGF-AP release. In contrast, when we substituted ADAM17-HA S811A r added HB-EGF 2 h later (Figure 5, D and E). In both cases, we ob- with ADAM17 -HA , there was no significant increase in CCPA- served that HB-EGF signic fi antly stimulated exocytosis, even in the stimulated HB-EGF-AP release. We also tested a mutant in which S811D r presence of the PKC or ADAM17 inhibitor. Together our data are Ser-811 was mutated to an Asp residue (ADAM17 -HA ). In the consistent with a model in which A AR-mediated transactivation absence of CCPA, this variant did not significantly affect constitutive 3802 | H. S. Prakasam et al. Molecular Biology of the Cell HG-EGF-AP release. However, AD- S811D r AM17 -HA retained the ability to pro- mote HB-EGF-AP release in response to CCPA (Figure 6D), indicating that under these conditions it acted as a “phosphomi- metic.” In addition, we determined whether HB-EGF-AP release was stimulated in cells expressing a mutant of ADAM17 that lacked all but two of its cytoplasmic amino acids (ADAM17-∆CT), This construct was previ- ously used to show that activation of ADAM17-dependent shedding occurred in- dependent of its cytoplasmic domain (Le Gall et al., 2010; Hall and Blobel, 2012). However, this mutant was unable to stimu- late HB-EGF-AP release in CCPA-treated cells (Figure 6D). In addition, we assessed whether inhibition of PKC affected HB-EGF release. As predicted, calphostin C caused significant inhibition of CCPA-stimulated HB-EGF-AP release (Figure 6E). We also tested whether phosphory lation of Ser-811 might be critical for CCPA-induced exocytosis in umbrella cells. We silenced endogenous ADAM17 by transducing rat bladders in situ with adenovirus encoding shRNA and then expressed hGH in con- junction with shRNA-resistant ADAM17- r S811A r S811D HA , ADAM17 -HA , or ADAM17 - HA . The expression levels of the three ADAM constructs were approximately equal (e.g., see Figure 6F) and similar to the expression levels of the endogenous pro- tein (∼50–90% of endogenous levels). The exogenous expression of ADAM17-HA re- stored CCPA-induced hGH secretion in the shRNA background (Figure 6, F and G). In S811A r contrast, expression of ADAM17 -HA caused a significant, ∼80% reduction in hGH release, again indicating a critical role for this amino acid in A AR-mediated acti- vation of ADAM17 (Figure 6, F and G). S811D r Finally, ADAM17 -HA was able to res- FIGURE 4: A AR-stimulated exocytosis is dependent on ADAM17. (A) Uroepithelial lysates obtained from rat tissues transduced in situ with scrambled shRNA or ADAM17 shRNA were cue shRNA-mediated inhibition of hGH resolved by SDS–PAGE and Western blots probed with rabbit-anti ADAM17 antibody (top) or secretion (Figure 6, F and G). mouse anti–β-actin (bottom). (B) Quantic fi ation of ADAM17 expression in rat tissue treated with In sum, our data indicate that activation scrambled shRNA or ADAM17 shRNA. Data are mean ± SEM (n ≥ 4). The means of the two of the A AR stimulates phosphorylation of treatment groups are significantly different ( p < 0.05). (C) Immunolocalization of ADAM17 ADAM17 Ser-811, and this phosphorylation expression (red) in rat tissue treated with scrambled or ADAM17-specific shRNAs. Nuclei (blue) event is likely necessary for A AR-induced are labeled with TOPRO-3. Scale bar, 13 μm. The cell junctions are marked with arrows and the HB-EGF cleavage and exocytosis in um- umbrella cells with an asterisk. (D) CCPA (500 nM) was added to rat tissue treated with brella cells. scrambled or ADAM17 shRNA and total EGFR or receptor phosphorylated at Y was detected by Western blot. (E) Quantic fi ation of effects of scrambled or ADAM17-specic fi shRNA treatment on EGFR activation. Data are mean ± SEM (n = 4). The two treatment groups were significantly different ( p < 0.05). (F) WGA-FITC (50 μg/ml) was added to the mucosal mucosal hemichamber. The mucosal u fl id was hemichamber of untreated rat tissue (control) or that treated with 500 nM CCPA. After 120 min, collected and concentrated, the tissues were the tissue was incubated at 4°C with N-acetylglucosamine to remove surface lectin, fixed, and lysed, and hGH was detected in the secreted then processed for immunofluorescence. In these whole-mounted tissues, internalized WGA- fraction (1/20 of total) or tissue lysates FITC is shown in green, phalloidin-labeled actin in red, and TOPRO-3-labeled nuclei in blue. Bar, (1/13 of total) using Western blot. 15 μm. Quantification of the fluorescence intensity of WGA-FITC below the apical membrane. (H) Quantification of effects of ADAM17- Data are mean ± SEM (n ≥ 8). The two treatment groups were significantly different ( p < 0.05). specic fi shRNAs on hGH secretion. Data are (G) Rat tissues were transduced with hGH alone (control) or transduced with hGH and either mean ± SEM (n ≥ 3). Statistically signic fi ant scrambled or ADAM17 shRNAs. The excised bladder tissue was mounted in an Ussing stretch effects (p < 0.05) are indicated with an chamber and left untreated (control), treated with CCPA (500 nM), or stretched by lfi ling the asterisk. Volume 25 November 15, 2014 ADAM17-dependent exocytosis | 3803 promote ADAM17 activation, or the mecha- nisms that control ADAM17-dependent sheddase activity. Our studies show that 1) like stretch, the A AR stimulates umbrella cell exocytosis by way of EGFR transactiva- tion and ADAM17 is the physiologically rel- evant proteinase; 2) an A AR → G → G 1 αi βγ → PLC → PKC signaling cascade likely acts upstream of ADAM17 to promote HB-EGF cleavage; and 3) phosphorylation of Ser-811 in the cytoplasmic domain of ADAM17 ap- pears to be required for A AR-stimulated HB-EGF shedding and exocytosis. A AR-mediated exocytosis requires ADAM17 and EGFR transactivation Similar to the effects of maximal stretch (Bal- estreire and Apodaca, 2007), we found that activation of the A AR stimulated a slow and gradual increase in apical exocytosis, which required metalloproteinase-dependent HB- EGF cleavage, EGFR transactivation, protein synthesis, and secretion. Although these two stimuli appear dissimilar at first blush, adenosine is released from the uroepithe- lium in response to maximal stretch (Yu et al., 2006; Prakasam et al., 2012) and can func- tion as a stress hormone in some settings (Fredholm et al., 2001; Hasko et al., 2008; Wilson and Mustafa, 2009). Thus EGFR transactivation may be a common pathway the uroepithelium uses to cope with stressful stimuli. A critical step in transactivation is the cleavage of EGFR substrates by a metallo- proteinase, whose identity in umbrella cells was previously unknown. We focused our studies on ADAM17 in part because it is the FIGURE 5: Role for G , Gβγ, PLC, and PKC in A AR-stimulated exocytosis. (A) Rabbit tissue was major sheddase for several EGFR ligands, i 1 left untreated or pretreated with 100 ng/ml pertussis toxin (PTX) for 90 min, 10 μM M119K for including HB-EGF (Sahin et al., 2004; Blobel, 60 min, or 10 M μ U73122 for 60 min. CCPA (500 nM) was added to the mucosal hemichamber, 2005; Hassemer et al., 2010). In addition, we and C was recorded. (B) Rabbit tissue was left untreated or pretreated with calphostin C found that in umbrella cells, ADAM17 was (500 nM) for 90 min. Subsequently, tissue was treated with CCPA (500 nM) or PMA (10 nM). localized to uroplakin 3a–positive DFVs, the (C) Rabbit tissue was pretreated with 15 μM Tapi-2 for 90 min or 1 μM AG1478 for 30 min and major apically directed vesicle population in then treated with 10 nM PMA. The data for PMA treatment alone were reproduced from B. these cells. Of importance, these vesicles (A–C) Data for CCPA treatment alone were reproduced from Figure 1B. (D, E) Rabbit tissue was position ADAM17 at or near the apical sur- pretreated with calphostin C (D) or Tapi-2 (E) for 90 min, and then at t = 0, CCPA was added to face of the umbrella cells, which we previ- the mucosal hemichamber. After 120 min, HG-EGF (1 nM) was added to the mucosal hemichamber (indicated with an arrow), and the tissue was incubated for additional 180 min. In ously showed is a primary site for EGFR- and D, data for calphostin C + CCPA are reproduced from B. (A–E) Data are presented as mean ± HB-EGF-dependent receptor transactivation SEM (in A–C, n ≥ 3; in D and E, n ≥ 6). In A–C, values that are significantly different from CCPA (Balestreire and Apodaca, 2007). Further- alone (p < 0.05) are marked with an asterisk. In D and E, values that are significantly different more, our use of adenovirally mediated ex- from calphostin C + CCPA or Tapi-2 + CCPA (p < 0.05) are marked with an asterisk. pression of shRNAs allowed us to signifi - cantly decrease ADAM17 expression in the DISCUSSION uroepithelium, which caused >90% decrease in CCPA-induced hGH ADAM17 is expressed in a variety of tissues, including heart, lungs, release. Although we cannot rule out a role for other ADAMs in brain, kidney, skeletal muscles, and the bladder (Gooz, 2010), and A AR-mediated exocytosis, the knockdown of ADAM17 in rat um- there exists a large body of evidence that implicates it in normal bio- brella cells was sufficient to block the majority of A AR- or stretch- logical phenomena (e.g., migration, adhesion, differentiation), as stimulated exocytosis in rat uroepithelium. well as in numerous pathologies (e.g., inflammation, multiple sclero - sis, diabetes, and kidney disorders; Plumb et al., 2006; Shah and A G → Gβγ → PLC → DAG → PKC pathway promotes Catt, 2006; Serino et al., 2007; Arribas and Esselens, 2009). None- ADAM17 activation theless, we still lack sufficient insight into the physiological cues that Although there are previous reports that adenosine can transacti- stimulate ADAM17 activity, the upstream signaling pathways that vate the EGFR, these studies did not identify the metalloproteinase 3804 | H. S. Prakasam et al. Molecular Biology of the Cell or upstream signaling pathways involved (Xie et al., 2009; Williams- traffic and/or “activation” of ADAM17 ligands (e.g., Dang et al., Pritchard et al., 2011). Furthermore, many studies of ADAM17 func- 2013). tion employ nonphysiological stimuli, such as PMA, and as such, the If phosphorylation of Ser-811 is physiologically relevant, then upstream signaling events that lead to ADAM17 activation are not how might it act? One possibility is that phosphorylation of this resi- always well understood. Work has shown roles for ERK, p38 MAPK, due alters the conformation of ADAM17, thus promoting its activity. and several classical PKC isoforms in ADAM17 function (Soond This could be a direct effect on ADAM17 or might be indirect; for et al., 2005; Bell and Gooz, 2010; Killock and Ivetic, 2010; Dang example, phosphorylation could act by modifying the association of et al., 2011, 2013; Hall and Blobel, 2012). For example, in lung cells, this proteinase with TIMP3, which may modulate ADAM17 activity PKCε-mediated ADAM17 activation was shown to be necessary for (Amour et al., 1998; Kwak et al., 2009; Xu et al., 2012). An additional premalignant changes after exposure to tobacco smoke (Lemjab- possibility is that phosphorylation or Ser-811 forms a docking site bar-Alaoui et al., 2011), whereas in glioblastoma cells, migration was for other proteins to bind and trigger ADAM17 activation or the as- initiated by PKCα-mediated activation and membrane translocation sociation of ADAM17 with its substrates. Potential interacting pro- of ADAM10 and the subsequent cleavage of N-cadherin (Kohutek teins include MAD-2, a component of mitotic spindle assembly et al., 2009). (Nelson et al., 1999), the protein tyrosine phosphatase-H1 (PTPH-1), Our studies showed that A AR-mediated apical exocytosis re- and SAP9. These last two interact with the COOH terminal of quired G and G , conr fi ming that the activation of ADAM17 occurs ADAM17 and negatively regulate its function (Zheng et al., 2002; i βγ downstream of an A AR, G -protein signaling event. Furthermore, Peiretti et al., 2003). Other potential interacting partners include 1 i we observed a requirement for PLC, which is known to generate IP3 Eve-1 (Tanaka et al., 2004) and the N-arginine dibasic convertase and DAG, a well-known activator of PKC. Finally, we observed a re- (nardilysin), which interacts with both HB-EGF and ADAM17 and quirement for PKC in apical exocytosis, noting that the selective regulates cleavage of the latter (Nishi et al., 2006). Obviously, more PKC inhibitor calphostin C inhibited adenosine-stimulated exocyto- work is needed to understand the mechanisms by which ADAM17 sis, whereas PMA stimulated it. We do not know whether there are activity is regulated. additional effectors in the pathway; however, our results are consis- In summary, we propose the following model for the role of tent with the hypothesis that a G → Gβγ → PLC → DAG → PKC ADAM17 and the role of Ser-811 phosphorylation in A AR-stimu- i 1 cascade promotes ADAM17 activation. lated exocytosis in umbrella cells. In the quiescent state, before ad- enosine stimulation (or stretch, in the case of rat umbrella cells), PKC may promote HB-EGF cleavage and apical exocytosis ADAM17 at the apical surface may be kept in its inactive, dimeric by phosphorylation of Ser-811 in the cytoplasmic domain of state by the inhibitory effects of TIMP3 (Figure 7A; Xu et al., 2012). ADAM17 As a result of its increased production (or decreased turnover), ad- An important but unresolved question concerns the mechanism(s) enosine binds to the A AR, triggering a G → G → PLC → PKC 1 i βγ by which upstream stimuli promote ADAM17-dependent func- pathway, which leads to phosphorylation at Ser-811 in the cytoplas- tion. One possible mechanism is phosphorylation, and several mic domain of ADAM17 (Figure 7B). This leads to activation of cytoplasmic targets have been described, including Tyr-702, Ser- ADAM17, possibly by altering its conformation and/or its associa- 791, Ser-829, and Thr-735 (Fan et al., 2003; Soond et al., 2005; tion with regulatory proteins (e.g., TIMP3) and/or its substrates. Niu et al., 2013). However, other reports show that ADAM17 Although not shown, signaling pathways downstream of the A AR lacking its cytoplasmic domain can still trigger stimulus-evoked are also likely to function by regulating other processes (e.g., “acti- S811D r shedding and EGFR transactivation (Doedens and Black, 2000; vation” of HB-EGF). This could explain why ADAM17 -HA was Black et al., 2003; Doedens et al., 2003; Le Gall et al., 2010), a unable to stimulate HB-EGF release in the absence of CCPA stimu- finding seemingly at odds with any role for phosphorylation in lation. Finally, in its activated state, ADAM17 cleaves and releases ADAM17 activation. HB-EGF, which binds to the EGFR, promoting autophosphorylation We observed that ADAM17 contains a canonical PKC phospho- of EGFR Y , ultimately leading to downstream ERK1/2 activation, rylation site (which includes Ser-811) that was phosphorylated in protein synthesis, and exocytosis (Figure 7B). response to A AR activation. Although we cannot completely rule out that PKC acts upstream of a different kinase, as reported for MATERIALS AND METHODS ERK-dependent phosphorylation of Thr-735 (Diaz-Rodriguez et al., Reagents and antibodies 2002), the most straightforward interpretation of our data is that Unless otherwise specified, all chemicals were obtained from PKC phosphorylates the residue directly. We further showed that Sigma-Aldrich (St. Louis, MO) and were of reagent grade or better. S811A the nonphosphorylatable mutant ADAM17 blocked CCPA- Adenosine was freshly prepared and dissolved in Krebs buffer induced HB-EGF shedding and hGH secretion. In contrast, (110 mM NaCl, 5.8 mM KCl, 25 mM NaHCO , 1.2 mM KH PO , 3 2 4 S811D ADAM17 (which also cannot be phosphorylated at Ser-811) 2.0 mM CaCl , 1.2 mM MgSO , 11.1 mM glucose, pH 7.4). The 2 4 was able to promote CCPA-stimulated HB-EGF cleavage and hGH following stock solutions were prepared in dimethyl sulfoxide: release. However, its inability to stimulate constitutive HB-EGF AG1478 (10 mM), calphostin C (250 μM), CCPA (10 mM), GM6001 cleavage likely indicates that phosphorylation of Ser-811 is neces- (15 mM), SB203580 (1 mM), Tapi-2 (15 mM; Tocris, Bristol, United sary but not sufficient to promote A AR-induced events. Finally, we Kingdom), and U0126 (10 mM). The following stocks were made in observed that a “tail-minus” construct was unable to stimulate molecular biology–grade water: pertussis toxin (100 μg/ml) and CCPA-induced HB-EGF-AP release. Thus our results are inconsis- M119K (10 mM; purchased from the National Cancer Institute, tent with the hypothesis that ADAM17 functions independently of Bethesda, MD). A 10 mM stock of PMA was prepared in ethanol. its cytoplasmic domain and phosphorylation. However, it is possible CRM197 (25 ng/ml) was dissolved directly in Krebs buffer. WGA- that the mechanism(s) of ADAM17 activation may depend on the FITC was purchased from Vector Labs (Burlingame, CA) and used stimulus, cell type, or growth conditions. Furthermore, some stimuli at a final concentration of 50 μg/ml. Beuthanasia-D was purchased may primarily work by directly affecting ADAM17 activity, whereas from Butler Schein (Dublin, OH). Lidocaine (LMX4) and isoflurane other stimuli may work through other mechanisms that affect the were purchased from Webster Veterinary (Webster, NY). The Volume 25 November 15, 2014 ADAM17-dependent exocytosis | 3805 FIGURE 6: Effect of Ser-811 phosphorylation on the function of ADAM17. (A) Top, alignment of the C-termini of ADAM17 from different species. Numbers indicate the amino acids involved. The canonical PKC phosphorylation site is shaded, and the position of the conserved Ser residue (Ser-811 in rat) is indicated in red. Bottom, domain structure of ADAM17. CR, cysteine-rich domain; C-tail, cytoplasmic domain; Dis, disintegrin domain; EL, EGF-like; MP, metalloproteinase domain; Pro, propeptide; TM, transmembrane domain. The consensus PKC phosphorylation motif is shown in an expanded view. Ser-811 is shaded, and the critical, flanking basic residues at the −3 and + 2 positions are r S811A r marked with arrows. (B) HEK-293FT cells expressing the A AR in combination with ADAM17-HA or ADAM17 -HA were labeled with P-orthophosphate and then left untreated or treated with CCPA (500 nM). HA-tagged ADAM17 3806 | H. S. Prakasam et al. Molecular Biology of the Cell isothiocyanate–labeled phalloidin and TO- PRO3 were from Molecular Probes/Invitro- gen (Grand Island, NY). Mouse monoclonal anti–uroplakin 3a antibody was described previously (Truschel et al., 1999). Animals Animals used in this study were female New Zealand white rabbits (3–4 kg; Covance) and female Sprague Dawley rats (250–300 g; Harlan Laboratories, Indianapolis, IN). Rab- bits were killed by intravenous injection of 300 mg of Buthensia D into the ear vein after the area was numbed using topical li- docaine ointment. After death, the bladders were rapidly excised and processed as de- scribed later. Rats were sedated by inhala- tion of isou fl rane and kept under sedation during the adenoviral transduction proce- dure by constant inhalation of isoflurane. At the end of the procedure, the rats were allowed to revive. At 24 h after infection, the rats were killed by inhalation of 100% CO , a thoracotomy was performed, and the bladder was excised. All animal studies were carried out with the approval of the University of Pittsburgh Animal Care and Use Committee. Cell culture HEK293FT cells were obtained from Invitro- gen. A HEK cell variant that has flattened morphology and increased viral transduc- FIGURE 7: Model for function of ADAM17 Ser-811 phosphorylation in A AR-stimulated EGFR 1 tion, HEK293A cells (Invitrogen), was used transactivation and exocytosis. See the text for description. to prepare ADAM17- or scrambled-shRNA viruses. The cells were grown in DMEM polyclonal anti-A AR rabbit antibody was obtained from Abcam purchased from Corning Cellgro (Corning, NY) supplemented with (ab82477; Cambridge, MA), anti-ADAM17 rabbit polyclonal anti- 10% (vol/vol) fetal bovine serum (GE Healthcare Life Sciences, body from EMD-Millipore (Billerica, MA), anti-EGFR and anti– Pittsburgh, PA), 1% (vol/vol) MEM nonessential amino acids (Life EGFR-phospho-Y-1173 rabbit polyclonal antibodies from Cell Technologies, Grand Island, NY), 2 mM glutamate (Sigma-Aldrich), Signaling Technology (Danvers, MA), anti-hemagglutinin (HA) rab- and 1% (vol/vol) penicillin/streptomycin (Lonza, Walkersville, MD). bit polyclonal antibody from Covance (Princeton, NJ), and anti– Cre8 cells were grown in DMEM purchased from Sigma-Aldrich HA-horseradish peroxidase (HRP) rabbit monoclonal antibody and supplemented with 10% (vol/vol) den fi ed fetal bovine serum from Roche (Mannheim, Germany); fluorophore- or HRP-conju - and 1% (vol/vol) penicillin/streptomycin. HEK293 cells were grown gated secondary antibodies were purchased from Jackson in DMEM supplemented with 10% (vol/vol) fetal bovine serum and Immuno Research (West Grove, PA), and tetramethylrhodamine 1% (vol/vol) penicillin/streptomycin. When producing viruses, cells constructs were immunoprecipitated and Western blots probed with an anti–HA-HRP- conjugated secondary antibody to detect total ADAM17 (middle) and subsequently exposed to a PhosphorImager screen to detect P-labeled ADAM17 (top). Total A AR was detected by Western blot (bottom). (C) Data (mean ± SEM; n = 3) were quantie fi d, and values significantly different from ADAM17-HA + CCPA are indicated with an asterisk. (D) HEK-293FT cells were r S811A r S811D r cotransfected with the A AR, HB-EGF-AP, and ADAM17-HA , ADAM17 -HA , or ADAM17 -HA . Fold stimulation of HB-EGF-AP release in CCPA-treated cells vs. control, untreated cells. Data are mean ± SEM, n = 5. Data signic fi antly different from HB-EGF-AP alone are indicated with an asterisk. (E) HEK-293FT cells were cotransfected with the A AR, HB-EGF-AP, and ADAM17-HA . Cells were pretreated with calphostin C (500 nM) for 60 min, and the fold stimulation of HB-EGF-AP release in CCPA-treated cells vs. control, untreated cells is reported. Data are mean ± SEM, n = 7. Values significantly different from the control, as assessed by ANOVA, are indicated (* p < 0.05). (F) Rat tissues were transduced r S811A r S811D in situ with hGH and ADAM17 shRNA alone or in combination with ADAM17-HA , ADAM17 -HA , or ADAM17 - HA . The excised bladder tissue was mounted in an Ussing stretch chamber, and CCPA (500 nM) was added to the mucosal hemichamber. After 60 min, the mucosal fluid was collected and concentrated, the tissues were lysed, and hGH detected using Western blot. (G) Quantification of hGH secretion. Data are mean ± SEM (n = 5). Values that are statistically different from ADAM17 shRNA alone (p < 0.05) are indicated with an asterisk. Volume 25 November 15, 2014 ADAM17-dependent exocytosis | 3807 were grown in their respective media but without penicillin/ the mucosal and serosal hemichambers bathing the tissue. How- streptomycin. ever, CCPA, CRM197, or PMA was added only to the mucosal hemi- chamber. The C and transepithelial resistance were measured as Immunou fl orescence labeling, image acquisition, described previously for a period up to 300 min (Wang et al., and colocalization analysis 2003a). Bladder tissue was fixed and processed as described previously Rat bladders were mounted in Ussing stretch chambers as de- (Truschel et al., 1999; Khandelwal et al., 2008). Briefly, tissues fixed scribed earlier (Khandelwal et al., 2008, 2010). Briey fl , the bladders with 4% PFA x fi ative (4% paraformaldehyde in 100 mM sodium were excised, cut open along one of the lateral veins and then care- cacodylate buffer, pH 7.4) were embedded in Optimal Cutting fully spread and pinned out on rubber dissection mats. The dis- Temperature (OCT; Sakura Finetek, Torrance, CA) medium and fro- sected bladders were then mounted on the pins of a plastic ring zen and stored at −80°C. Frozen sections, 4 m μ in thickness, were with an opening of 0.75 cm and the rings clamped between two obtained using a Leica CM1950 cryostat and adhered to Fisher Ussing stretch hemichambers. The chambers were filled with Krebs brand Superfrost slides. The sections were washed three times for buffer and equilibrated. To stretch the tissue, buffer was added to 5 min each with phosphate-buffered saline (PBS), and the fixation the mucosal hemichamber via Luer ports at a rate of 35 lμ /min using was stopped with quench buffer (20 mM glycine, 75 mM NH Cl, a syringe pump (New Era Pump Systems, Farmingdale, NY). Once dissolved in PBS) for 10 min. When staining for ADAM17, the sec- the chamber was lfi led, an additional 250 lμ was then pumped into tions were quenched for 7 min and subsequently treated with the chamber to stretch the tissue. quench buffer supplemented with 0.05% (wt/vol) SDS for an addi- tional 3 min. After quenching, the tissue was then washed with PBS Preparation of cell lysates and Western blot analysis and transferred to block buffer (7% [wt/vol] fish gelatin, 0.25% sa - To prepare lysates, bladder tissue was placed on a rubber dissec- ponin [vol/vol], and 0.05% NaN [wt/vol] made in PBS) for 1 h at tion mat with the mucosal surface facing up. The tissue was held room temperature. A 1:200 dilution of the antibodies was made in in place by pinning it at the four corners using 20-gauge ¾-inch the block buffer, applied to the sections, and incubated at room needles (BD Biosciences). An aliquot (35–50 μl) of SDS lysis buffer temperature for either 2 h (in the case of ADAM17 antibody) or (50 mM triethanolamine, pH 8.6, 100 mM NaCl, 5 mM EDTA, overnight at 4°C for the A AR or UP3a antibodies. The primary anti- 0.2% [wt/vol] NaN , 0.5% [wt/vol] SDS) containing a protease and 1 3 body was removed by three 5-min washes with PBS and detected phosphatase inhibitor cocktail (Cell Signaling Technologies, using Alexa 488– or Cy3–labeled secondary antibodies diluted in Boston, MA), as well as 10 mM 1,10-phenanthroline, was added block buffer. Additional washes with PBS were performed. Actin and to the mucosal surface and the outer uroepithelium recovered by nuclei were stained with either rhodamine or u fl orescein phalloidin gently scraping the cells using a rubber cell scraper (Sarstedt, and TO-PRO3, respectively. After staining, the samples were post- Newton, NC). The cell lysate was transferred to a 1.5-ml Eppen- x fi ed for 10 min with 4% PFA x fi ative and then mounted in medium dorf tube, vortex shaken at 4°C for 10 min using a model 5432 containing 1% (wt/vol) phenylenediamine, 90% (vol/vol) glycerol, vortex mixer (Eppendorf, Hauppauge, NY), and centrifuged at and 20 mM Tris, pH 8.0. 13,000 rpm for 10 min at 4°C in a table-top model 5415D micro- Images were captured using a 63×/1.2 numerical aperture glyc- centrifuge (Eppendorf). The clear supernatant was collected, flash erol objective and the appropriate laser lines of a Leica TCS SP5 frozen, and stored at −80°C. Before use, the protein concentra- CW-STED confocal microscope (in normal confocal mode). The tion was quantified using the bicinchoninic acid assay (Pierce, photomultipliers were set at 900–1200 V, and images were col- Rockford, IL). Equal amounts of protein from the bladder lysates lected using an average of six line scans. Serial 0.25-μm Z-sections were resolved by SDS–PAGE on 4–15% polyacrylamide gradient were acquired. The images were imported into Volocity 4-D soft- gels (Bio-Rad, Hercules, CA) at 200 V and constant current for ware (PerkinElmer, Waltham, MA) and, after image reconstruction 30 min. For ADAM17 and hGH, proteins were transferred to Im- and contrast correction, exported as TIFF lfi es. Composite images mobilon P membranes (Millipore) at 375 mA constant current us- were prepared in Illustrator CS5 (Adobe, San Jose, CA). Colocaliza- ing a 100 mM 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS), tion analysis and measurements of fluorescence intensity were per - pH 11.0, transfer buffer. In the case of the EGFR, proteins were formed as described previously, using a x fi ed threshold of 40 transferred to nitrocellulose membranes (GE Healthcare Life Sci- (Khandelwal et al., 2008, 2010). ences) at 375 mA constant current using a Tris-glycine running buffer (25 mM Tris, 190 mM glycine) containing 0.01% (wt/vol) Mounting rabbit uroepithelium or rat bladders in Ussing SDS for 2 h. In either case, the membrane was blocked for 30 min stretch chambers and measurement of C with 5% (wt/vol) nonfat milk made in TBST buffer (Tris-buffered Isolation of rabbit uroepithelium from the underlying muscle layers saline + 0.1% Tween 20). After the blocking step, the membrane and mounting in Ussing stretch chambers was performed as de- was incubated with primary antibodies overnight at 4°C. After scribed earlier (Wang et al., 2003a). Briefly, rabbit bladders were several washes with TBST buffer, the membrane was incubated excised, slit open vertically along one of the lateral veins, and spread with goat anti-rabbit-HRP or goat anti-mouse-HRP secondary on a custom-made Teflon rack with the uroepithelium facing down. antibodies for 1 h at room temperature and washed with TBST The muscles were carefully removed with the pair of tweezers and buffer. The bands were detected by incubating the membrane sharp scissors. The remaining tissue containing the intact uroepithe- with enhanced chemiluminescence (ECL) solution for 2 min lium was mounted on the pins at the outer edges of a plastic ring (Pierce), followed by film capture on Carestream Kodak Biomax with an opening of 2 cm . The rings were locked between two films (Carestream, Rochester, NY). Data were quantified using Ussing stretch hemichambers, which were clamped into position on QuantityOne quantification software (Bio-Rad) a Teflon base. Warm Krebs buffer was simultaneously added to the mucosal (apical facing) and serosal (muscle facing) chambers. The Generation of adenoviruses encoding ADAM17 shRNA tissue was gassed with 95% air/5% CO during a 30- to 45-min The iRNAi software (Nucleobytes.com) was used to search the equilibration period and treated with the indicated drug added to Rat ADAM17 cDNA (PubMed accession number NM_020306) for 3808 | H. S. Prakasam et al. Molecular Biology of the Cell r S811D r optimal targets containing the sequence AA(N19). Four shRNA se- HA (TCA to GCC) and pADLOX-ADAM17 -HA (TCA to GAC). quences were selected. The top and the bottom strands were indi- In addition, we also generated a tail-minus construct, pADLOX- vidually synthesized (IDT, Coralville, IA) and annealed by mixing the ADAM17-∆CT-HA . This was made by deleting the last 127 amino two strands in equal amounts, heating to 94°C, and cooling gradu- acids of the ADAM17 backbone, leaving two cytoplasmic amino ac- ally to room temperature. The annealed shRNA sequences were ids that were fused in-frame to an HA tag. The constructs (3 μg) were then ligated into the linearized pU6/ENTR vector (Life Technolo- preincubated with Lipofectamine 2000 Opti-MEM media for 30 min, gies). The ability of the different shRNA sequences to silence mixed with 3 μg of Ψ5 adenoviral genomic DNA (Ad5 strain), and ADAM17 was determined by cotransfecting SV40 large T antigen– added to Cre8 cells (Hardy et al., 1997; Khandelwal et al., 2008). expressing HEK293FT cells with the shRNA-pU6/ENTR-shRNA vec- Production of adenoviruses was performed as described, except tors and rat ADAM17 cDNA. At 24 h later, cells were lysed, the that Cre8 cells were used throughout. proteins were resolved by SDS–PAGE, and ADAM17 was detected by Western blotting using the techniques described earlier. Of the In situ adenoviral transduction and detection of hGH release four, the sequence that had maximum silencing efficiency ( >80%; In situ transduction of rat bladder uroepithelium and measurement 5′-GGATTAGCTTACGTTGGTTCT-3′) was selected and recombined of hGH release were performed as described (Khandelwal et al., into the pBLOCKiT Adenovirus System vector (Invitrogen) using an 2008). Briefly, rats were sedated with isoflurane, and a Jelco IV cath - in vitro Clonase-mediated recombination reaction according to the eter (Smith Medicals, Southington, CT) was introduced into the vendor’s protocol. The recombined pBLOCKiT vector was linearized bladder via the urethra. The bladder was rinsed with PBS and lfi led by Pac1 restriction digestion and transfected into 293A cells using with 400 μl of 0.1% (wt/vol) n-dodecyl-β-d -maltoside dissolved in Lipofectamine 2000 reagent (Life Technologies). At 11 d posttrans- PBS. The urethra was clamped, and after 5 min, it was unclamped to fection, when the cytopathic effect was >75%, the cells in this first allow the detergent to void. The latter step was facilitated by apply- P1 generation were harvested by tituration and lysed by freezing the ing slight pressure to the lower abdomen. The bladder was filled cells at −70°C and thawing them for 5–10 min in a 37°C water bath. with 400 μl of PBS containing adenoviruses encoding hGH alone or Three freeze–thaw cycles were typically sufficient to lyse the cells. in combination with adenoviruses encoding scrambled-shRNA, The lysate, containing released virus, was used to infect a new round ADAM17-shRNA, or the ADAM17-HA constructs described earlier of cells (P2), a process that was repeated one additional time (P3). To (2.5 × 10 infectious virus particles, typically in a volume of 2–10 μl). produce large quantities of virus, 10 15-cm Petri dishes (BD Falcon, The bladder was then clamped. After 30 min, the clamp was re- San Jose, CA) of 293A cells were infected with virus-containing moved, and the virus solution was allowed to void. The bladder was lysate from P3. On the third day, when the cells showed >85% cyto- rinsed with PBS, anesthesia was discontinued, and the rats were al- pathic effect, they were recovered by tituration, pooled, centrifuged lowed to revive. At 30 h postinfection, the animals were killed by at 3500 × g (Eppendorf 5810 R) for 14 min at 4°C, and mixed in 7 ml inhalation of CO , and the bladder was immediately excised, slit of resuspension buffer (100 mM Tris, pH 7.4, 10 mM EDTA). The open, and mounted on tissue rings as described. After 90 min of concentrated cell suspension was lysed by repeated freeze–thaw equilibration, the buffer bathing the apical surface was isovolumetri- cycles as described. The lysate was separated from the cell debris cally replaced with fresh buffer and then treated or not with 500 nM by centrifuging at 5000 × g (Eppendorf 5810 R) for 15 min at 4°C. CCPA. At 60 min later, the apical buffer was removed and concen- The supernatant was carefully removed and applied to the top of a trated using a 10K molecular weight cutoff Amicon Centricon step gradient containing 2.5 ml of 1.25 g/ml CsCl solution, which (Millipore) to a volume of 250 μl. The corresponding tissue was un - was layered on top of 2.5 ml of 1.4 g/ml CsCl loaded into clear mounted from its tissue ring, and a lysate was prepared before 13-ml PET ultracentrifugation tubes (Thermo Scientific). The sam - Western blot analysis to detect hGH. The fraction of hGH secreted ples were centrifuged at 35,000 rpm for 1 h at 4°C using a Beckman was calculated as secreted hGH/(secreted hGH + cell-associated Coulter centrifuge (Brea, CA) and an SW-41 swinging bucket rotor. hGH in lysate). The concentrated virus, which appeared as an off-white band at the interface of the two CsCl layers, was collected by piercing the Endocytosis of WGA-FITC side wall of the tube with an 18-gauge needle and aspirating it into Rat bladders were mounted in Ussing chambers, and after equilibra- a connected 5-cc syringe (BD Biosciences). The viruses were further tion, they were incubated with 25 μg/ml WGA-FITC for 2 h with or purified by passage through a PE10 gel filtration column (GE Health - without 500 nM CCPA added to the mucosal hemichamber. The tis- care) equilibrated with virus suspension buffer (PBS containing 10% sues were then unmounted from the chambers, washed with ice- [vol/vol] glycerol). The virus-containing fractions were detected by cold 100 mM N-acetyl-d -glucosamine four times for 20 min, and monitoring the A , pooled, and stored at −70°C in small aliquots, then washed with ice-cold PBS three times for 15 min and then fixed which were thawed in a 37°C water bath just before use. with 4% PFA fixative for 30 min at 37°C. The tissue was stained, and the images were captured and processed as described. Because Generation of adenoviruses encoding ADAM17-HA , some WGA-FITC remained bound to the apical surface of the um- S811A r S811D r r ADAM17 -HA , ADAM17 -HA , or ADAM17-∆CT-HA brella cells even after washing with cognate sugar, the apicalmost ADAM17 rat cDNA (from Addgene plasmid 19141; Lemieux et al., sections (∼1–1.5 m μ ) in the confocal stacks were excluded from the 2007) was cloned into pADLOX vector, and an oligonucleotide en- quantitation. coding the HA tag (YPYDVPDYA) was added in-frame to the C-ter- minus of ADAM17 after a gap of two amino acids. A silent mutation P-orthophosphate labeling of ADAM17 was engineered into the ADAM17 sequence (5′-GGATTAGCG- HEK293FT cells were cotransfected with 3 μg of pcDNA-A AR and r S811A TACGTTG GTTCT) using the QuikChange XL mutagenesis kit 3 μg of either pADLOX-ADAM17-HA or pADLOX-ADAM17 - (Agilent, Santa Clara, CA), making the construct resistant to the HA using Lipofectamine 2000 reagent. At 48 h posttransfection, the ADAM17 shRNA. This resulting construct, called pADLOX-ADAM17- cells were washed two times with PO efflux buffer (140 mM NaCl, HA , was further mutagenized to convert serine at position 811 to an 2 mM KCl, 1 mM MgSO , 1 mM CaCl , 10 mM glucose, 10 mM 4 2 S811A alanine or aspartate residue, generating pADLOX-ADAM17 - 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, titrated to pH 7.4 Volume 25 November 15, 2014 ADAM17-dependent exocytosis | 3809 using 1 M Tris-base) and then incubated with PO efflux buffer CCPA was normalized to reactions in which CCPA treatment was supplemented with 300 μCi of P-orthophosphate (5 mCi/ml; MP omitted. Each experiment was repeated at least five times. No AP Biomedicals; Santa Ana, CA) for 2 h at 37°C. During the last 15 min activity was present in the supernatant of nontransfected cells. of the incubation, the cells were treated or not with 500 nM CCPA. Subsequently, the efflux buffer was aspirated, the cells were washed Statistical analysis two times with Tris-buffered saline (pH 7.5), and the cells were dis- Statistical significance between means was determined by Stu - solved in 500 μl of RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM dent’s t test or, in the case of multiple comparisons, by analysis of NaCl, 50 mM NaF, 0.1% [wt/vol] SDS, 1% [wt/vol] sodium deoxy- variance (ANOVA). If a significant difference in the means was cholate, 1% [vol/vol] Triton X-100, 1 mM EDTA, 1 mM phenylmeth- detected by ANOVA, multiple comparisons were performed us- ylsulfonyl fluoride, 1 mM orthovanadate, 0.1 mg/ml aprotinin, 1 mM ing Dunnett’s posttest correction. Statistical analyses were per- ethylene glycol tetraacetic acid, and 10 mM 1,10-phenanthroline). formed using Prism 5 software (GraphPad, La Jolla, CA). In the The cell lysate was collected, placed in 1.5-ml Eppendorf tubes, and figure legends, n refers to the number of individual animals or incubated on ice for 30 min to complete the cell lysis. The lysate was experiments. centrifuged at 16,000 × g at 4°C using a Marathon 16km table-top centrifuge (Fisher Scientific, Waltham, MA) for 1 h, and the resulting supernatant was collected in a fresh tube. The volume in each tube was brought up to 900 μl with RIPA lysis buffer, and then 7 μl of rab - ACKNOWLEDGMENTS bit anti-HA antibody (Covance) and 40 lμ of 10% SDS were added This work was supported by National Institutes of Health Grants to each sample. After a 1-h incubation at 4°C with constant rotation, R37-DK54425, R01-DK077777, and P30-DK079307 to G.A. and 100 lμ of a 20% (wt/vol) slurry of protein G-Sepharose beads (GE R01-DK075048 to K.R.H. and the Cellular Physiology and Kidney Healthcare) was added to each sample and incubated at 4°C over- Imaging Cores of the Pittsburgh Center for Kidney Research night with constant rotation. The beads were washed with RIPA lysis (P30-DK079307). buffer three times, resuspended in 40 μl of 2 × Laemmli sample buf- fer, heated for 15 min at 65°C, and centrifuged at 16,000 × g for 10 min, and then the proteins in the supernatant were resolved by REFERENCES SDS–PAGE. The proteins were transferred to nitrocellulose mem- Amour A, Slocombe PM, Webster A, Butler M, Knight CG, Smith BJ, Stephens PE, Shelley C, Hutton M, Knauper V, et al. (1998). TNF-alpha branes as described and the immobilized HA-labeled proteins re- converting enzyme (TACE) is inhibited by TIMP-3. 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A1 adenosine receptor–stimulated exocytosis in bladder umbrella cells requires phosphorylation of ADAM17 Ser-811 and EGF receptor transactivation

Molecular Biology of the Cell , Volume 25 (23) – Nov 15, 2014

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© 2014 Prakasam et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
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1939-4586
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10.1091/mbc.E14-03-0818
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Abstract

MB oC | ARTICLE A adenosine receptor–stimulated exocytosis in bladder umbrella cells requires phosphorylation of ADAM17 Ser-811 and EGF receptor transactivation H. Sandeep Prakasam, Luciana I. Gallo, Hui Li, Wily G. Ruiz, Kenneth R. Hallows, and Gerard Apodaca Departments of Medicine and Cell Biology, University of Pittsburgh, Pittsburgh, PA 15261 ABSTRACT Despite the importance of ADAM17-dependent cleavage in normal biology and Monitoring Editor Keith E. Mostov disease, the physiological cues that trigger its activity, the effector pathways that promote its University of California, function, and the mechanisms that control its activity, particularly the role of phosphorylation, San Francisco remain unresolved. Using native bladder epithelium, in some cases transduced with adenovi- ruses encoding small interfering RNA, we observe that stimulation of apically localized A 1 Received: Mar 20, 2014 adenosine receptors (A ARs) triggers a G -Gβγ-phospholipase C-protein kinase C (PKC) cas- Revised: Sep 10, 2014 1 i Accepted: Sep 11, 2014 cade that promotes ADAM17-dependent HB-EGF cleavage, EGFR transactivation, and apical exocytosis. We further show that the cytoplasmic tail of rat ADAM17 contains a conserved serine residue at position 811, which resides in a canonical PKC phosphorylation site, and is phosphorylated in response to A AR activation. Preventing this phosphorylation event by S811A expression of a nonphosphorylatable ADAM17 mutant or expression of a tail-minus con- struct inhibits A AR-stimulated, ADAM17-dependent HB-EGF cleavage. Furthermore, ex- S811A pression of ADAM17 in bladder tissues impairs A AR-induced apical exocytosis. We con- clude that adenosine-stimulated exocytosis requires PKC- and ADAM17-dependent EGFR transactivation and that the function of ADAM17 in this pathway depends on the phosphory- lation state of Ser-811 in its cytoplasmic domain. INTRODUCTION Protein ectodomain shedding, a process regulated by proteolysis, factors, and cell adhesion molecules (Reiss and Saftig, 2009) and is is a fundamental mechanism for the release of cytokines, growth altered in cancer, autoimmune and inflammatory diseases, cardio - vascular disease, and neurodegeneration (Murphy, 2008). The best- understood sheddases include the “a disintegrin and a metallopro- This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E14-03-0818) on September 17, 2014. teinase” (ADAM) family members ADAM10 and ADAM17 (also H.S.P., L.I.G., W.G.R., and H.L. designed and executed the experiments and as- known as TACE), both of which shed a variety of substrates, includ- sisted in the writing of the manuscript. K.R.H. and G.A. assisted in the design of ing the transmembrane ligands for the epidermal growth factor re- the experiments and wrote and prepared the manuscript. ceptor (EGFR). Whereas ADAM10 targets betacellulin, EGF, and The authors have no conflicts of interest to report. Address correspondence to: Gerard Apodaca (gla6@pitt.edu). neuregulin, ADAM17 is the principal sheddase for transforming Abbreviations used: A AR, A adenosine receptor; ADAM, a disintegrin and a growth factor (TGF) α, amphiregulin, epiregulin, epigen, and hepa- 1 1 metalloproteinase; CCPA, 2-chloro-N6-cyclopentyladenosine; DFV, discoidal rin-binding (HB) EGF (Jackson et al., 2003; Sahin et al., 2004; Sahin and/or fusiform-shaped vesicle; EGFR, epidermal growth factor receptor; ERK, and Blobel, 2007; Blobel, 2005; Horiuchi et al., 2005; Sternlicht extracellular signal-regulated kinase; GPCR, G protein–coupled receptor; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; PLC, phospholipase C; et al., 2005; Hassemer et al., 2010; Luo et al., 2011). The physiologi- PMA, phorbol-12-myristate-13-acetate; TIMP, tissue inhibitor of metalloprotei- cal cues that trigger shedding remain to be specified; however, nase. ADAM17-elicited shedding occurs in response to some G protein– © 2014 Prakasam et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is avail- coupled receptor (GPCR) ligands as well as treatment with ionomy- able to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported cin or phorbol-12-myristate-13-acetate (PMA), a diacylglycerol Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). ® ® (DAG) mimic and activator of classical protein kinase C (PKC) iso- “ASCB ,” “The American Society for Cell Biology ,” and “Molecular Biology of the Cell ” are registered trademarks of The American Society for Cell Biology. forms (Horiuchi et al., 2007; Le Gall et al., 2009; Dang et al., 2011). 3798 | H. S. Prakasam et al. Molecular Biology of the Cell 2+ In contrast, ADAM10-dependent shedding responds to Ca iono- (A AR). Finally, PKC likely promotes the phosphorylation of a previ- phores (e.g., ionomycin) but generally not PMA (Horiuchi et al., ously unreported canonical PKC site centered at Ser-811 in the cyto- 2007; Le Gall et al., 2009; Dang et al., 2011). In addition, our under- plasmic domain of rat ADAM17. This phosphorylation event is re- standing of the signaling and associated effector pathways that act quired for adenosine-induced and ADAM17-dependent HB-EGF downstream of these stimuli remains incomplete. The extracellular cleavage, EGFR transactivation, and exocytosis. These studies lend signal-regulated kinase (ERK), as well as the related p38 mitogen- support to the hypothesis that posttranslational modification of activated protein kinase (MAPK), PKCα, PKCδ, and the PKC-regu- ADAM17—phosphorylation in particular—can control its activity in lated protein phosphatase inhibitor 14D, has also been linked to a physiologically relevant setting. ADAM17-dependent shedding, and PKCδ is required for shedding of neuregulin (Bell and Gooz, 2010; Killock and Ivetic, 2010; Dang RESULTS et al., 2011, 2013; Hall and Blobel, 2012). A AR-stimulated apical exocytosis occurs through An additional unresolved question is how these effectors pro- transactivation of the EGFR mote ADAM-dependent shedding of EGFR ligands. Data indicate Late-phase exocytosis is triggered when uroepithelial tissue is maxi- that they work through multiple mechanisms and perhaps in a cell- mally stretched (Balestreire and Apodaca, 2007; Yu et al., 2009), but and stimulus-dependent manner (Fan et al., 2003; Mifune et al., it is unknown whether other stimuli promote a similar response. Be- 2005; Xu and Derynck, 2010). Potential regulatory steps include cause adenosine release is significantly increased as the bladder membrane trafficking of the ADAM or its ligands (Soond et al., reaches its capacity (Prakasam et al., 2012), and because extracel- 2005), effects on the ADAM17 dimer–monomer equilibrium, and lular adenosine also triggers exocytosis (Yu et al., 2006), we tested association with tissue inhibitor of metalloproteinases (TIMP) 3 (Xu the hypothesis that adenosine stimulates exocytosis by way of EGFR et al., 2012) or changes in the redox potential of the extracellular transactivation. Adenosine can stimulate exocytosis when added to protein disulfide isomerase (Willems et al., 2010), which is hypoth- either the serosal or mucosal surfaces of isolated uroepithelium; esized to alter ADAM17 activity by rearrangement of its disuld fi e however, in this study, we focused our attention on the mucosal bonds. An additional regulatory mechanism may be phosphoryla- events, as these are primarily mediated by the A AR, and EGFR tion of EGFR receptor substrates (Dang et al., 2013) or the ADAM transactivation occurs at this surface (Yu et al., 2006; Balestreire and metalloproteinase itself. For example, phosphorylation of ADAM17 Apodaca, 2007). Consistent with our previous reports (Yu et al., cytoplasmic residues Tyr-702, Ser-791, Ser-819, and Thr-735 have 2011; Prakasam et al., 2012), the A AR was localized to the apical been reported (Fan et al., 2003; Hall and Blobel, 2012; Niu et al., pole of rat umbrella cells (as well as in the underlying lamina propria; 2013). Tyr-702 is reportedly phosphorylated by the Src kinase when Figure 1A). When adenosine was added to the mucosal surface of myogenic precursor cells are stretched (Niu et al., 2013). In contrast, isolated rabbit tissue, it stimulated increased tissue capacitance (C ; Ser-791 is phosphorylated before stimulation, and mutations of Ser- 1 μF ≈ 1 cm ; Figure 1B), which in this tissue correlates well with 819 do not appear to affect shedding (Fan et al., 2003). In the case other measures of apical exocytosis (Truschel et al., 2002; Wang of Thr-735, very high doses of PMA (1 μM) are reported to promote et al., 2003a). Similar results were obtained when the highly selec- ERK-dependent phosphorylation of this residue (Soond et al., 2005). tive and high-affinity A AR agonist 2-chloro-N6-cyclopentyladenos- However, others report that phosphorylation of this residue is medi- ine (CCPA) was used. Unlike adenosine, CCPA is not rapidly con- ated by the p38 MAPK and is required for ADAM17-dependent verted to inosine or to AMP (Manjunath and Sakhare, 2009; Prakasam shedding in response to various forms of stress but not PMA (Xu and et al., 2012) and was thus used in our subsequent studies. We next Derynck, 2010). A significant argument against the role for phos - determined whether A AR-stimulated exocytosis, like that observed phorylation relies on reports that show that stimulus-evoked shed- in response to excess stretch, is dependent on protein synthesis or ding of ADAM17 occurs in cells expressing truncated versions of secretion. Indeed, treatment with cycloheximide or brefeldin A sig- this metalloproteinase that lack a C-terminus (Reddy et al., 2000; Le nificantly blocked CCPA-mediated exocytosis (Figure 1C). The ef - Gall et al., 2010; Hall and Blobel, 2012). fect of the latter was particularly pronounced. A potentially useful and physiologically relevant model system in We also assessed whether CCPA triggered apical exocytosis by which to study the signals, effector pathways, and mode of ADAM transactivating the EGFR. We r fi st tested the effect of treating the activation is the uroepithelium, a tissue that can be studied both ex mucosal surface of the tissue with CRM197, a mutant version of vivo and in vivo (Khandelwal et al., 2008, 2010, 2013). Bladder lfi ling diphtheria toxin that binds selectively and with high affinity to mem - triggers the exocytosis of an abundant subapical pool of discoidal- brane-bound HB-EGF and prevents its cleavage (Uchida et al., and/or fusiform-shaped vesicle (DFVs) in the outer umbrella cell 1973). Indeed, we observed that CRM197 almost completely layer (Wang et al., 2003a,b). Stretch-induced exocytosis progresses blocked CCPA-mediated apical exocytosis (Figure 2A). Next we in two phases. “Early-phase” exocytosis occurs during bladder fill - pretreated tissue with AG1478, a small-molecule inhibitor of EGFR, ing, as the epithelium is bowing outward, and is triggered by apical which also significantly blocked CCPA-mediated apical exocytosis 2+ Ca entry, likely conducted by a nonselective cation channel (Yu (Figure 2B). As further evidence that the EGFR was transactivated, et al., 2009). In contrast, “late-phase” exocytosis is initiated once we generated lysates from CCPA-treated epithelium and then the tissue is maximally bowed outward (i.e., in response to a full probed Western blots with an antibody that detects phosphoryla- bladder) and requires metalloproteinase-dependent cleavage of tion of the EGFR at Tyr-1173. This residue promotes assembly of a HB-EGF, leading to “transactivation” of apical EGFRs (Balestreire MAPK signaling cascade downstream of other GPCRs (Balestreire and Apodaca, 2007). The latter initiates a downstream ERK pathway and Apodaca, 2007). Phosphorylation of Tyr-1173 was maximally that culminates in protein synthesis and exocytosis (Balestreire and stimulated 10 min after continuous treatment with CCPA, decreased Apodaca, 2007). We now report that adenosine, which is released at 30 min posttreatment, and returned to control levels by 60 min from the epithelium in response to stress (Prakasam et al., 2012), (Figure 2, C and D). The Tyr-1173 phosphorylation was prevented spurs a late phase–like response in umbrella cells. We also find that when the tissue was pretreated with the EGFR inhibitor AG1478 a G -, Gβγ-, phospholipase C (PLC)-, and PKC-dependent effector before CCPA treatment (Figure 2, C and D), confirming that phos - cascade is initiated downstream of the adenosine A receptor phorylation of Tyr-1173 likely results from autophosphorylation. Volume 25 November 15, 2014 ADAM17-dependent exocytosis | 3799 DFVs and the apical surface of umbrella cells (Figure 3C; Apodaca, 2004). Consistent with a role for ADAM17 in DFV trafc fi king, we observed that the broad-spectrum metallo- proteinase inhibitor GM6001 and the ADAM17-selective inhibitor Tapi-2 (Bal- estreire and Apodaca, 2007; Kveiborg et al., 2011) both significantly inhibited the CCPA- mediated increases in C (Figure 3D). More- over, Tapi-2 blocked CCPA-mediated phos- phorylation of EGFR Tyr-1173 (Figure 2, C and D). To provide further evidence that ADAM17 is required for EGFR transactiva- tion, we exploited our previously de- scribed in situ viral transduction approach (Khandelwal et al., 2008, 2010), in this case to express ADAM17-specific short hairpin RNAs (shRNAs) or scrambled shR- NAs in the rat bladder uroepithelium. We used rat bladders in these studies because the volume capacity of the bladder, and therefore the number of virus particles FIGURE 1: A AR-stimulated exocytosis is dependent on protein synthesis and secretion. needed, was relatively small (∼500 μl) (A) Cryosection of rat bladder epithelium labeled with an A AR-specic fi antibody (green), compared with the volumes required to fill rhodamine–phalloidin to label the actin cytoskeleton (red), and TOPRO-3 (blue) to label the the rabbit bladder (∼60–100 ml). This tech- nucleus. Right, merge. An umbrella cell is marked with an asterisk and the white arrows mark the nique targets the umbrella cell layer and apicolateral borders of the cell. Scale bar, 12 m μ . (B) Rabbit uroepithelium was mounted in Ussing chambers and after equilibration left untreated (no drug), or 1 μM adenosine or 500 nM achieves transduction efficiencies of 70– CCPA was added to the mucosal hemichamber. Percentage changes in C were monitored. 95% (Khandelwal et al., 2008, 2010). In- (C) Rabbit tissue was left untreated, pretreated with 5 g μ /ml brefeldin A (BFA) for 30 min, or deed, we were able to achieve >90% pretreated with 100 ng/ml cyclohexamide for 60 min. CCPA (500 nM) was then added to the knockdown of ADAM17 expression (Figure mucosal hemichamber and C recorded. Data for CCPA alone are reproduced from B. 4, A and B). On examining the expression (B, C) Mean changes in C ± SEM (n ≥ 4). Significant differences ( p < 0.05), relative to no drug in and distribution of ADAM17 in cross sec- A or CCPA in B, are indicated with an asterisk. tions of uroepithelium, we observed that knockdown of ADAM17 was largely con- Finally, we observed that U0126, an inhibitor of MEK activity, also fined to the umbrella cells, and expression of ADAM17 in the caused a significant decrease in CCPA-stimulated changes in C underlying cell layers was largely undisturbed (Figure 4C). (Figure 2A). Next we used ADAM17-specific shRNAs to test whether ADAM17 Taken together, these data indicate that, like the previously de- was required for CCPA-stimulated EGFR transactivation and exocy- scribed late-phase response (Balestreire and Apodaca, 2007), A AR tosis in rat epithelium. Treatment with ADAM17-shRNA, but not activation stimulates apical exocytosis by promoting transactivation scrambled shRNA, decreased EGFR phosphorylation (Figure 4, D of the EGFR downstream of HB-EGF cleavage, leading to MEK/ERK and E). We show later in Figure 6, F and G, that the effects of the activation and protein synthesis. ADAM17-shRNA are specific, and ADAM17 function is restored when the shRNA is coexpressed with shRNA-resistant variant of wild- ADAM17 is localized to the apical surface of umbrella cells, type ADAM17. We also attempted to measure changes in C in the where it stimulates CCPA-induced EGFR transactivation rat bladders treated with apical CCPA; however, we did not observe and exocytosis a response. Because C is dependent on the rates of membrane ad- In vivo studies implicate ADAM17 as the physiologically relevant dition and removal, we reasoned that one possible explanation was sheddase in HB-EGF–mediated transactivation (Jackson et al., 2003; that CCPA stimulated both exocytosis and endocytosis in rat epithe- Sahin et al., 2004). Consistent with these observations, we observed lium, thus obviating any change in apparent C . Indeed, we observed that ADAM17 was localized to small vesicular elements under the that wheat germ agglutinin–fluorescein isothiocyanate (WGA-FITC), apical surface of the rat umbrella cells (Figure 3A), the likely site of added to the apical hemichamber of mounted rat bladders, was en- HB-EGF cleavage and EGFR transactivation during the late-phase docytosed in CCPA-treated tissue but much less so in control, un- response (Balestreire and Apodaca, 2007). Rat tissues were used in treated tissue (Figure 4F). To circumvent the effects of endocytosis, these experiments because our antibody was produced in rabbits. we instead measured release of exogenously expressed human ADAM17 was also detected in the intermediate and basal cell layers growth hormone (hGH), which we and others previously showed is of the uroepithelium, as well as in cells in the underlying lamina packaged into DFVs and released from the luminal surface of the propria (Figure 3, A and C). The signal for ADAM17 was diminished bladder into the urinary space (Kerr et al., 1998; Khandelwal et al., when the anti-ADAM17 antibody was preincubated with immuniz- 2008). Because of rapid dilution, there is little secreted hGH that is ing peptide, confirming the specificity of the antibody (Figure 3B). endocytosed. Compared to control bladders, CCPA stimulated a ADAM17 showed a high degree of colocalization with uroplakin 3a large increase in the mucosal release of hGH from the tissue (Figure (Manders coefc fi ient of colocalization, 0.92); this is associated with 4, G and H). Moreover, expression of ADAM-17-specific shRNA, but 3800 | H. S. Prakasam et al. Molecular Biology of the Cell compared with tissues transduced with scrambled shRNA (Figure 4, G and H). Taken together, our results provide strong evi- dence that in rat tissues, ADAM17 is critical for CCPA- and stretch-induced EGFR trans- activation and exocytosis. G , PLC, and PKC act upstream of ADAM17 and HB-EGF to promote A AR-mediated EGFR transactivation We next addressed how ADAM17 activity is coupled to A AR activation. Previous stud- ies showed that A AR signals through Gα 1 i to inhibit the activity of adenylyl cyclase, whereas the βγ subunits of G increase the activity of phospholipase C-β (PLCβ), which hydrolyzes phosphatidylinositol 4,5-bispho- sphate to generate inositol trisphosphate (IP3) and diacylgycerol (Freund et al., 1994; Bucheimer and Linden, 2004; Chang et al., 2008). The latter stimulates the activity of PKC, a well-known regulatory kinase that was previously implicated in ADAM17 acti- vation (Dang et al., 2011; Kveiborg et al., 2011; Lemjabbar-Alaoui et al., 2011). We found that pertussis toxin–mediated inhibi- tion of G or inhibition of G subunit activity i βγ by the inhibitor M119K (Kirui et al., 2010) significantly impaired CCPA-mediated api - cal exocytosis in rabbit bladder umbrella cells (Figure 5A). Furthermore, the PLC-se- lective antagonist U73122 caused marked inhibition of CCPA-induced changes in C (Figure 5A). Thus ADAM17 activation down- stream of A AR likely occurs by way of a classical G -stimulated signaling cascade in- volving G and PLCβ. βγ Next we examined whether PKC is im- portant for A AR-dependent EGFR transac- tivation and exocytosis. Strikingly, the PKC FIGURE 2: The A AR transactivates the EGF receptor. (A) The mucosal surface of rabbit uroepithelium was pretreated with 25 ng/ml CRM197 for 25 min or with 10 M μ U0126 for inhibitor calphostin C caused a marked de- 60 min. CCPA (500 nM) was then added to the mucosal hemichamber, and C was recorded. crease in CCPA-stimulated changes in C (B) Rabbit uroepithelium was pretreated with 1 μM AG1478 for 30 min, and then CCPA (500 nM) and EGFR transactivation (Figures 2, C and was added to the mucosal hemichamber and C was recorded. (A, B) CCPA control data are D, and 5B). In contrast, treatment with PMA, reproduced from Figure 1B. Mean changes in C ± SEM (n ≥ 3). Statistically significant an activator of classical PKCs (Nishizuka, differences (p < 0.05), relative to CCPA treatment alone, are marked with an asterisk. 1992), caused robust stimulation of exocyto- (C, D) Rabbit uroepithelium was either left untreated (left) or treated with AG1478 (1 μM) for sis in the absence of CCPA (Figure 5B). Of 25 min, Tapi-2 (15 M μ ) for 90 min, or calphostin C (500 nM) for 60 min (right). CCPA (500 nM) interest, the kinetics of PMA-mediated exo- was then added to the mucosal hemichamber. Left, cells were lysed at the indicated time points. cytosis were faster than those mediated by Right, cells were lysed at the 10-min time point. Equal amounts of proteins were resolved by CCPA, particularly during the first 30 min, SDS–PAGE and immunoblots probed with a rabbit anti–EGFR-phospho-Y antibody or rabbit anti-EGFR antibody. (D) Quantification of Y phosphorylation. Data (mean ± SEM, n ≥ 3) are and then appeared to increase at a similar reported as fold increase above untreated tissue samples at t = 0. Statistically signic fi ant values rate to CCPA-treated tissue. This may indi- (p < 0.05) above t = 0 are marked by an asterisk. cate that PKC stimulates not only late-phase- like responses, but perhaps early-phase not scrambled shRNA, caused a large inhibition (>90%) in mucosal ones as well. To conr fi m that PMA acted by way of ADAM17, we hGH release (Figure 4, G and H). treated the tissue with Tapi-2, which significantly inhibited PMA- Finally, because stretch-mediated, late-phase exocytosis is de- mediated apical exocytosis (Figure 5C). We also observed that pendent on EGFR transactivation and sensitive to metalloproteinase AG1478 impaired PMA-mediated exocytosis (Figure 5C). Our data inhibitors (Balestreire and Apodaca, 2007), we determined whether pointed to the possibility that PKC acted upstream of ADAM17, ADAM17 also played a role in stretch-mediated exocytosis. We which acted before HB-EGF cleavage. We reasoned, therefore, that observed that shRNA-mediated ADAM17 knockdown signic fi antly the calphostin C–or TAPI-2–mediated inhibition of C should be reduced the stretch-induced apical release of hGH by ∼80% relieved by addition of HB-EGF. To test this possibility, we pretreated Volume 25 November 15, 2014 ADAM17-dependent exocytosis | 3801 requires G , Gβγ, PLC, PKC, and ADAM17, and these effectors likely act upstream of HB-EGF release and EGFR transactivation. CCPA-stimulated HB-EGF shedding and exocytosis are dependent on phosphorylation of ADAM17 Ser-811 To explore how PKC might act to stimu- late ADAM17 activity, we compared the amino acid sequences from multiple ver- tebrate species using the proteomics tool Scansite and identified a conserved, canonical PKC phosphorylation motif (X-R/K-X-X-S/T-X-R/K-X; Pearson and Kemp, 1991; Nishikawa et al., 1997) in the cytoplas- mic tail of the protein centered at Ser-811 (equivalent to Ser-808 in the human protein; Figure 6A). To our knowledge, a functional role for this residue was not explored previ- ously. We r fi st conr fi med that ADAM17 was phosphorylated in response to CCPA by ex- pressing the A AR in combination with epitope-tagged ADAM17-HA (this variant of ADAM17 is resistant to shRNA; see Figure 6F) in P-orthophosphate–labeled HEK cells (Figure 6B). HEK cells were used in these assays because these experiments were difficult to perform in whole tissues. On addition of CCPA, we observed that phosphorylation of both the “immature” proform of ADAM17-HA (∼120 kDa) and the “mature” cleaved form of the enzyme (∼93 kDa) was stimulated approximately threefold (Figure 6, B and C). In contrast, CCPA had no significant effect when wild- type ADAM17-HA was substituted with a nonphosphorylatable mutant of ADAM17 in which Ser-811 was changed to an Ala resi- S811A r due (ADAM17 -HA ; Figure 6, B and C). These results indicate that in response to A AR activation, Ser-811 may be a major site of ADAM17 phosphorylation. Because the physiological role of ADAM17 phosphorylation is a matter of some controversy (Reddy et al., 2000; Horiuchi et al., 2007; Le Gall et al., 2010; Dang et al., 2013), we next determined FIGURE 3: ADAM17 expression in the uroepithelium. (A–C) Cryosections of rat bladder whether phosphorylation of Ser-811 was bi- uroepithelium were reacted with antibodies specic fi for ADAM17 (A), a mixture of antibody and ologically relevant. First, we measured shed- inhibitory peptide (B), or antibodies specific for ADAM17 and uroplakin 3a (C). After incubation ding of a chimeric protein in which alkaline with fluorophore-labeled secondary antibodies, the samples were analyzed using confocal phosphatase (AP) was fused to the extracel- microscopy. Where indicated, actin was labeled with phalloidin and nuclei were labeled with lular domain of HB-EGF (HB-EGF-AP; Sahin TOPRO-3. The umbrella cells are marked with the asterisk in the merged images and the et al., 2004; Uttarwar et al., 2011), an apicolateral junction is indicated by arrowheads; scale bar, 10 m μ . (D) Rabbit uroepithelium was ADAM17 substrate that was coexpressed in pretreated with Tapi-2 (15 μM) or GM6001 (15 μM) for 90 min, CCPA (500 nM) was added, and C was recorded. Control CCPA data are reproduced from Figure 1B. Data are mean ±SEM (n ≥ 3), HEK cells along with the A AR and ADAM17- and values significantly different ( p < 0.05) from CCPA alone are marked with an asterisk. HA (Figure 6D). In response to CCPA, we observed a significant ∼70% increase in HB- cells with calphostin C or TAPI-2, added CCPA at t = 0, and then EGF-AP release. In contrast, when we substituted ADAM17-HA S811A r added HB-EGF 2 h later (Figure 5, D and E). In both cases, we ob- with ADAM17 -HA , there was no significant increase in CCPA- served that HB-EGF signic fi antly stimulated exocytosis, even in the stimulated HB-EGF-AP release. We also tested a mutant in which S811D r presence of the PKC or ADAM17 inhibitor. Together our data are Ser-811 was mutated to an Asp residue (ADAM17 -HA ). In the consistent with a model in which A AR-mediated transactivation absence of CCPA, this variant did not significantly affect constitutive 3802 | H. S. Prakasam et al. Molecular Biology of the Cell HG-EGF-AP release. However, AD- S811D r AM17 -HA retained the ability to pro- mote HB-EGF-AP release in response to CCPA (Figure 6D), indicating that under these conditions it acted as a “phosphomi- metic.” In addition, we determined whether HB-EGF-AP release was stimulated in cells expressing a mutant of ADAM17 that lacked all but two of its cytoplasmic amino acids (ADAM17-∆CT), This construct was previ- ously used to show that activation of ADAM17-dependent shedding occurred in- dependent of its cytoplasmic domain (Le Gall et al., 2010; Hall and Blobel, 2012). However, this mutant was unable to stimu- late HB-EGF-AP release in CCPA-treated cells (Figure 6D). In addition, we assessed whether inhibition of PKC affected HB-EGF release. As predicted, calphostin C caused significant inhibition of CCPA-stimulated HB-EGF-AP release (Figure 6E). We also tested whether phosphory lation of Ser-811 might be critical for CCPA-induced exocytosis in umbrella cells. We silenced endogenous ADAM17 by transducing rat bladders in situ with adenovirus encoding shRNA and then expressed hGH in con- junction with shRNA-resistant ADAM17- r S811A r S811D HA , ADAM17 -HA , or ADAM17 - HA . The expression levels of the three ADAM constructs were approximately equal (e.g., see Figure 6F) and similar to the expression levels of the endogenous pro- tein (∼50–90% of endogenous levels). The exogenous expression of ADAM17-HA re- stored CCPA-induced hGH secretion in the shRNA background (Figure 6, F and G). In S811A r contrast, expression of ADAM17 -HA caused a significant, ∼80% reduction in hGH release, again indicating a critical role for this amino acid in A AR-mediated acti- vation of ADAM17 (Figure 6, F and G). S811D r Finally, ADAM17 -HA was able to res- FIGURE 4: A AR-stimulated exocytosis is dependent on ADAM17. (A) Uroepithelial lysates obtained from rat tissues transduced in situ with scrambled shRNA or ADAM17 shRNA were cue shRNA-mediated inhibition of hGH resolved by SDS–PAGE and Western blots probed with rabbit-anti ADAM17 antibody (top) or secretion (Figure 6, F and G). mouse anti–β-actin (bottom). (B) Quantic fi ation of ADAM17 expression in rat tissue treated with In sum, our data indicate that activation scrambled shRNA or ADAM17 shRNA. Data are mean ± SEM (n ≥ 4). The means of the two of the A AR stimulates phosphorylation of treatment groups are significantly different ( p < 0.05). (C) Immunolocalization of ADAM17 ADAM17 Ser-811, and this phosphorylation expression (red) in rat tissue treated with scrambled or ADAM17-specific shRNAs. Nuclei (blue) event is likely necessary for A AR-induced are labeled with TOPRO-3. Scale bar, 13 μm. The cell junctions are marked with arrows and the HB-EGF cleavage and exocytosis in um- umbrella cells with an asterisk. (D) CCPA (500 nM) was added to rat tissue treated with brella cells. scrambled or ADAM17 shRNA and total EGFR or receptor phosphorylated at Y was detected by Western blot. (E) Quantic fi ation of effects of scrambled or ADAM17-specic fi shRNA treatment on EGFR activation. Data are mean ± SEM (n = 4). The two treatment groups were significantly different ( p < 0.05). (F) WGA-FITC (50 μg/ml) was added to the mucosal mucosal hemichamber. The mucosal u fl id was hemichamber of untreated rat tissue (control) or that treated with 500 nM CCPA. After 120 min, collected and concentrated, the tissues were the tissue was incubated at 4°C with N-acetylglucosamine to remove surface lectin, fixed, and lysed, and hGH was detected in the secreted then processed for immunofluorescence. In these whole-mounted tissues, internalized WGA- fraction (1/20 of total) or tissue lysates FITC is shown in green, phalloidin-labeled actin in red, and TOPRO-3-labeled nuclei in blue. Bar, (1/13 of total) using Western blot. 15 μm. Quantification of the fluorescence intensity of WGA-FITC below the apical membrane. (H) Quantification of effects of ADAM17- Data are mean ± SEM (n ≥ 8). The two treatment groups were significantly different ( p < 0.05). specic fi shRNAs on hGH secretion. Data are (G) Rat tissues were transduced with hGH alone (control) or transduced with hGH and either mean ± SEM (n ≥ 3). Statistically signic fi ant scrambled or ADAM17 shRNAs. The excised bladder tissue was mounted in an Ussing stretch effects (p < 0.05) are indicated with an chamber and left untreated (control), treated with CCPA (500 nM), or stretched by lfi ling the asterisk. Volume 25 November 15, 2014 ADAM17-dependent exocytosis | 3803 promote ADAM17 activation, or the mecha- nisms that control ADAM17-dependent sheddase activity. Our studies show that 1) like stretch, the A AR stimulates umbrella cell exocytosis by way of EGFR transactiva- tion and ADAM17 is the physiologically rel- evant proteinase; 2) an A AR → G → G 1 αi βγ → PLC → PKC signaling cascade likely acts upstream of ADAM17 to promote HB-EGF cleavage; and 3) phosphorylation of Ser-811 in the cytoplasmic domain of ADAM17 ap- pears to be required for A AR-stimulated HB-EGF shedding and exocytosis. A AR-mediated exocytosis requires ADAM17 and EGFR transactivation Similar to the effects of maximal stretch (Bal- estreire and Apodaca, 2007), we found that activation of the A AR stimulated a slow and gradual increase in apical exocytosis, which required metalloproteinase-dependent HB- EGF cleavage, EGFR transactivation, protein synthesis, and secretion. Although these two stimuli appear dissimilar at first blush, adenosine is released from the uroepithe- lium in response to maximal stretch (Yu et al., 2006; Prakasam et al., 2012) and can func- tion as a stress hormone in some settings (Fredholm et al., 2001; Hasko et al., 2008; Wilson and Mustafa, 2009). Thus EGFR transactivation may be a common pathway the uroepithelium uses to cope with stressful stimuli. A critical step in transactivation is the cleavage of EGFR substrates by a metallo- proteinase, whose identity in umbrella cells was previously unknown. We focused our studies on ADAM17 in part because it is the FIGURE 5: Role for G , Gβγ, PLC, and PKC in A AR-stimulated exocytosis. (A) Rabbit tissue was major sheddase for several EGFR ligands, i 1 left untreated or pretreated with 100 ng/ml pertussis toxin (PTX) for 90 min, 10 μM M119K for including HB-EGF (Sahin et al., 2004; Blobel, 60 min, or 10 M μ U73122 for 60 min. CCPA (500 nM) was added to the mucosal hemichamber, 2005; Hassemer et al., 2010). In addition, we and C was recorded. (B) Rabbit tissue was left untreated or pretreated with calphostin C found that in umbrella cells, ADAM17 was (500 nM) for 90 min. Subsequently, tissue was treated with CCPA (500 nM) or PMA (10 nM). localized to uroplakin 3a–positive DFVs, the (C) Rabbit tissue was pretreated with 15 μM Tapi-2 for 90 min or 1 μM AG1478 for 30 min and major apically directed vesicle population in then treated with 10 nM PMA. The data for PMA treatment alone were reproduced from B. these cells. Of importance, these vesicles (A–C) Data for CCPA treatment alone were reproduced from Figure 1B. (D, E) Rabbit tissue was position ADAM17 at or near the apical sur- pretreated with calphostin C (D) or Tapi-2 (E) for 90 min, and then at t = 0, CCPA was added to face of the umbrella cells, which we previ- the mucosal hemichamber. After 120 min, HG-EGF (1 nM) was added to the mucosal hemichamber (indicated with an arrow), and the tissue was incubated for additional 180 min. In ously showed is a primary site for EGFR- and D, data for calphostin C + CCPA are reproduced from B. (A–E) Data are presented as mean ± HB-EGF-dependent receptor transactivation SEM (in A–C, n ≥ 3; in D and E, n ≥ 6). In A–C, values that are significantly different from CCPA (Balestreire and Apodaca, 2007). Further- alone (p < 0.05) are marked with an asterisk. In D and E, values that are significantly different more, our use of adenovirally mediated ex- from calphostin C + CCPA or Tapi-2 + CCPA (p < 0.05) are marked with an asterisk. pression of shRNAs allowed us to signifi - cantly decrease ADAM17 expression in the DISCUSSION uroepithelium, which caused >90% decrease in CCPA-induced hGH ADAM17 is expressed in a variety of tissues, including heart, lungs, release. Although we cannot rule out a role for other ADAMs in brain, kidney, skeletal muscles, and the bladder (Gooz, 2010), and A AR-mediated exocytosis, the knockdown of ADAM17 in rat um- there exists a large body of evidence that implicates it in normal bio- brella cells was sufficient to block the majority of A AR- or stretch- logical phenomena (e.g., migration, adhesion, differentiation), as stimulated exocytosis in rat uroepithelium. well as in numerous pathologies (e.g., inflammation, multiple sclero - sis, diabetes, and kidney disorders; Plumb et al., 2006; Shah and A G → Gβγ → PLC → DAG → PKC pathway promotes Catt, 2006; Serino et al., 2007; Arribas and Esselens, 2009). None- ADAM17 activation theless, we still lack sufficient insight into the physiological cues that Although there are previous reports that adenosine can transacti- stimulate ADAM17 activity, the upstream signaling pathways that vate the EGFR, these studies did not identify the metalloproteinase 3804 | H. S. Prakasam et al. Molecular Biology of the Cell or upstream signaling pathways involved (Xie et al., 2009; Williams- traffic and/or “activation” of ADAM17 ligands (e.g., Dang et al., Pritchard et al., 2011). Furthermore, many studies of ADAM17 func- 2013). tion employ nonphysiological stimuli, such as PMA, and as such, the If phosphorylation of Ser-811 is physiologically relevant, then upstream signaling events that lead to ADAM17 activation are not how might it act? One possibility is that phosphorylation of this resi- always well understood. Work has shown roles for ERK, p38 MAPK, due alters the conformation of ADAM17, thus promoting its activity. and several classical PKC isoforms in ADAM17 function (Soond This could be a direct effect on ADAM17 or might be indirect; for et al., 2005; Bell and Gooz, 2010; Killock and Ivetic, 2010; Dang example, phosphorylation could act by modifying the association of et al., 2011, 2013; Hall and Blobel, 2012). For example, in lung cells, this proteinase with TIMP3, which may modulate ADAM17 activity PKCε-mediated ADAM17 activation was shown to be necessary for (Amour et al., 1998; Kwak et al., 2009; Xu et al., 2012). An additional premalignant changes after exposure to tobacco smoke (Lemjab- possibility is that phosphorylation or Ser-811 forms a docking site bar-Alaoui et al., 2011), whereas in glioblastoma cells, migration was for other proteins to bind and trigger ADAM17 activation or the as- initiated by PKCα-mediated activation and membrane translocation sociation of ADAM17 with its substrates. Potential interacting pro- of ADAM10 and the subsequent cleavage of N-cadherin (Kohutek teins include MAD-2, a component of mitotic spindle assembly et al., 2009). (Nelson et al., 1999), the protein tyrosine phosphatase-H1 (PTPH-1), Our studies showed that A AR-mediated apical exocytosis re- and SAP9. These last two interact with the COOH terminal of quired G and G , conr fi ming that the activation of ADAM17 occurs ADAM17 and negatively regulate its function (Zheng et al., 2002; i βγ downstream of an A AR, G -protein signaling event. Furthermore, Peiretti et al., 2003). Other potential interacting partners include 1 i we observed a requirement for PLC, which is known to generate IP3 Eve-1 (Tanaka et al., 2004) and the N-arginine dibasic convertase and DAG, a well-known activator of PKC. Finally, we observed a re- (nardilysin), which interacts with both HB-EGF and ADAM17 and quirement for PKC in apical exocytosis, noting that the selective regulates cleavage of the latter (Nishi et al., 2006). Obviously, more PKC inhibitor calphostin C inhibited adenosine-stimulated exocyto- work is needed to understand the mechanisms by which ADAM17 sis, whereas PMA stimulated it. We do not know whether there are activity is regulated. additional effectors in the pathway; however, our results are consis- In summary, we propose the following model for the role of tent with the hypothesis that a G → Gβγ → PLC → DAG → PKC ADAM17 and the role of Ser-811 phosphorylation in A AR-stimu- i 1 cascade promotes ADAM17 activation. lated exocytosis in umbrella cells. In the quiescent state, before ad- enosine stimulation (or stretch, in the case of rat umbrella cells), PKC may promote HB-EGF cleavage and apical exocytosis ADAM17 at the apical surface may be kept in its inactive, dimeric by phosphorylation of Ser-811 in the cytoplasmic domain of state by the inhibitory effects of TIMP3 (Figure 7A; Xu et al., 2012). ADAM17 As a result of its increased production (or decreased turnover), ad- An important but unresolved question concerns the mechanism(s) enosine binds to the A AR, triggering a G → G → PLC → PKC 1 i βγ by which upstream stimuli promote ADAM17-dependent func- pathway, which leads to phosphorylation at Ser-811 in the cytoplas- tion. One possible mechanism is phosphorylation, and several mic domain of ADAM17 (Figure 7B). This leads to activation of cytoplasmic targets have been described, including Tyr-702, Ser- ADAM17, possibly by altering its conformation and/or its associa- 791, Ser-829, and Thr-735 (Fan et al., 2003; Soond et al., 2005; tion with regulatory proteins (e.g., TIMP3) and/or its substrates. Niu et al., 2013). However, other reports show that ADAM17 Although not shown, signaling pathways downstream of the A AR lacking its cytoplasmic domain can still trigger stimulus-evoked are also likely to function by regulating other processes (e.g., “acti- S811D r shedding and EGFR transactivation (Doedens and Black, 2000; vation” of HB-EGF). This could explain why ADAM17 -HA was Black et al., 2003; Doedens et al., 2003; Le Gall et al., 2010), a unable to stimulate HB-EGF release in the absence of CCPA stimu- finding seemingly at odds with any role for phosphorylation in lation. Finally, in its activated state, ADAM17 cleaves and releases ADAM17 activation. HB-EGF, which binds to the EGFR, promoting autophosphorylation We observed that ADAM17 contains a canonical PKC phospho- of EGFR Y , ultimately leading to downstream ERK1/2 activation, rylation site (which includes Ser-811) that was phosphorylated in protein synthesis, and exocytosis (Figure 7B). response to A AR activation. Although we cannot completely rule out that PKC acts upstream of a different kinase, as reported for MATERIALS AND METHODS ERK-dependent phosphorylation of Thr-735 (Diaz-Rodriguez et al., Reagents and antibodies 2002), the most straightforward interpretation of our data is that Unless otherwise specified, all chemicals were obtained from PKC phosphorylates the residue directly. We further showed that Sigma-Aldrich (St. Louis, MO) and were of reagent grade or better. S811A the nonphosphorylatable mutant ADAM17 blocked CCPA- Adenosine was freshly prepared and dissolved in Krebs buffer induced HB-EGF shedding and hGH secretion. In contrast, (110 mM NaCl, 5.8 mM KCl, 25 mM NaHCO , 1.2 mM KH PO , 3 2 4 S811D ADAM17 (which also cannot be phosphorylated at Ser-811) 2.0 mM CaCl , 1.2 mM MgSO , 11.1 mM glucose, pH 7.4). The 2 4 was able to promote CCPA-stimulated HB-EGF cleavage and hGH following stock solutions were prepared in dimethyl sulfoxide: release. However, its inability to stimulate constitutive HB-EGF AG1478 (10 mM), calphostin C (250 μM), CCPA (10 mM), GM6001 cleavage likely indicates that phosphorylation of Ser-811 is neces- (15 mM), SB203580 (1 mM), Tapi-2 (15 mM; Tocris, Bristol, United sary but not sufficient to promote A AR-induced events. Finally, we Kingdom), and U0126 (10 mM). The following stocks were made in observed that a “tail-minus” construct was unable to stimulate molecular biology–grade water: pertussis toxin (100 μg/ml) and CCPA-induced HB-EGF-AP release. Thus our results are inconsis- M119K (10 mM; purchased from the National Cancer Institute, tent with the hypothesis that ADAM17 functions independently of Bethesda, MD). A 10 mM stock of PMA was prepared in ethanol. its cytoplasmic domain and phosphorylation. However, it is possible CRM197 (25 ng/ml) was dissolved directly in Krebs buffer. WGA- that the mechanism(s) of ADAM17 activation may depend on the FITC was purchased from Vector Labs (Burlingame, CA) and used stimulus, cell type, or growth conditions. Furthermore, some stimuli at a final concentration of 50 μg/ml. Beuthanasia-D was purchased may primarily work by directly affecting ADAM17 activity, whereas from Butler Schein (Dublin, OH). Lidocaine (LMX4) and isoflurane other stimuli may work through other mechanisms that affect the were purchased from Webster Veterinary (Webster, NY). The Volume 25 November 15, 2014 ADAM17-dependent exocytosis | 3805 FIGURE 6: Effect of Ser-811 phosphorylation on the function of ADAM17. (A) Top, alignment of the C-termini of ADAM17 from different species. Numbers indicate the amino acids involved. The canonical PKC phosphorylation site is shaded, and the position of the conserved Ser residue (Ser-811 in rat) is indicated in red. Bottom, domain structure of ADAM17. CR, cysteine-rich domain; C-tail, cytoplasmic domain; Dis, disintegrin domain; EL, EGF-like; MP, metalloproteinase domain; Pro, propeptide; TM, transmembrane domain. The consensus PKC phosphorylation motif is shown in an expanded view. Ser-811 is shaded, and the critical, flanking basic residues at the −3 and + 2 positions are r S811A r marked with arrows. (B) HEK-293FT cells expressing the A AR in combination with ADAM17-HA or ADAM17 -HA were labeled with P-orthophosphate and then left untreated or treated with CCPA (500 nM). HA-tagged ADAM17 3806 | H. S. Prakasam et al. Molecular Biology of the Cell isothiocyanate–labeled phalloidin and TO- PRO3 were from Molecular Probes/Invitro- gen (Grand Island, NY). Mouse monoclonal anti–uroplakin 3a antibody was described previously (Truschel et al., 1999). Animals Animals used in this study were female New Zealand white rabbits (3–4 kg; Covance) and female Sprague Dawley rats (250–300 g; Harlan Laboratories, Indianapolis, IN). Rab- bits were killed by intravenous injection of 300 mg of Buthensia D into the ear vein after the area was numbed using topical li- docaine ointment. After death, the bladders were rapidly excised and processed as de- scribed later. Rats were sedated by inhala- tion of isou fl rane and kept under sedation during the adenoviral transduction proce- dure by constant inhalation of isoflurane. At the end of the procedure, the rats were allowed to revive. At 24 h after infection, the rats were killed by inhalation of 100% CO , a thoracotomy was performed, and the bladder was excised. All animal studies were carried out with the approval of the University of Pittsburgh Animal Care and Use Committee. Cell culture HEK293FT cells were obtained from Invitro- gen. A HEK cell variant that has flattened morphology and increased viral transduc- FIGURE 7: Model for function of ADAM17 Ser-811 phosphorylation in A AR-stimulated EGFR 1 tion, HEK293A cells (Invitrogen), was used transactivation and exocytosis. See the text for description. to prepare ADAM17- or scrambled-shRNA viruses. The cells were grown in DMEM polyclonal anti-A AR rabbit antibody was obtained from Abcam purchased from Corning Cellgro (Corning, NY) supplemented with (ab82477; Cambridge, MA), anti-ADAM17 rabbit polyclonal anti- 10% (vol/vol) fetal bovine serum (GE Healthcare Life Sciences, body from EMD-Millipore (Billerica, MA), anti-EGFR and anti– Pittsburgh, PA), 1% (vol/vol) MEM nonessential amino acids (Life EGFR-phospho-Y-1173 rabbit polyclonal antibodies from Cell Technologies, Grand Island, NY), 2 mM glutamate (Sigma-Aldrich), Signaling Technology (Danvers, MA), anti-hemagglutinin (HA) rab- and 1% (vol/vol) penicillin/streptomycin (Lonza, Walkersville, MD). bit polyclonal antibody from Covance (Princeton, NJ), and anti– Cre8 cells were grown in DMEM purchased from Sigma-Aldrich HA-horseradish peroxidase (HRP) rabbit monoclonal antibody and supplemented with 10% (vol/vol) den fi ed fetal bovine serum from Roche (Mannheim, Germany); fluorophore- or HRP-conju - and 1% (vol/vol) penicillin/streptomycin. HEK293 cells were grown gated secondary antibodies were purchased from Jackson in DMEM supplemented with 10% (vol/vol) fetal bovine serum and Immuno Research (West Grove, PA), and tetramethylrhodamine 1% (vol/vol) penicillin/streptomycin. When producing viruses, cells constructs were immunoprecipitated and Western blots probed with an anti–HA-HRP- conjugated secondary antibody to detect total ADAM17 (middle) and subsequently exposed to a PhosphorImager screen to detect P-labeled ADAM17 (top). Total A AR was detected by Western blot (bottom). (C) Data (mean ± SEM; n = 3) were quantie fi d, and values significantly different from ADAM17-HA + CCPA are indicated with an asterisk. (D) HEK-293FT cells were r S811A r S811D r cotransfected with the A AR, HB-EGF-AP, and ADAM17-HA , ADAM17 -HA , or ADAM17 -HA . Fold stimulation of HB-EGF-AP release in CCPA-treated cells vs. control, untreated cells. Data are mean ± SEM, n = 5. Data signic fi antly different from HB-EGF-AP alone are indicated with an asterisk. (E) HEK-293FT cells were cotransfected with the A AR, HB-EGF-AP, and ADAM17-HA . Cells were pretreated with calphostin C (500 nM) for 60 min, and the fold stimulation of HB-EGF-AP release in CCPA-treated cells vs. control, untreated cells is reported. Data are mean ± SEM, n = 7. Values significantly different from the control, as assessed by ANOVA, are indicated (* p < 0.05). (F) Rat tissues were transduced r S811A r S811D in situ with hGH and ADAM17 shRNA alone or in combination with ADAM17-HA , ADAM17 -HA , or ADAM17 - HA . The excised bladder tissue was mounted in an Ussing stretch chamber, and CCPA (500 nM) was added to the mucosal hemichamber. After 60 min, the mucosal fluid was collected and concentrated, the tissues were lysed, and hGH detected using Western blot. (G) Quantification of hGH secretion. Data are mean ± SEM (n = 5). Values that are statistically different from ADAM17 shRNA alone (p < 0.05) are indicated with an asterisk. Volume 25 November 15, 2014 ADAM17-dependent exocytosis | 3807 were grown in their respective media but without penicillin/ the mucosal and serosal hemichambers bathing the tissue. How- streptomycin. ever, CCPA, CRM197, or PMA was added only to the mucosal hemi- chamber. The C and transepithelial resistance were measured as Immunou fl orescence labeling, image acquisition, described previously for a period up to 300 min (Wang et al., and colocalization analysis 2003a). Bladder tissue was fixed and processed as described previously Rat bladders were mounted in Ussing stretch chambers as de- (Truschel et al., 1999; Khandelwal et al., 2008). Briefly, tissues fixed scribed earlier (Khandelwal et al., 2008, 2010). Briey fl , the bladders with 4% PFA x fi ative (4% paraformaldehyde in 100 mM sodium were excised, cut open along one of the lateral veins and then care- cacodylate buffer, pH 7.4) were embedded in Optimal Cutting fully spread and pinned out on rubber dissection mats. The dis- Temperature (OCT; Sakura Finetek, Torrance, CA) medium and fro- sected bladders were then mounted on the pins of a plastic ring zen and stored at −80°C. Frozen sections, 4 m μ in thickness, were with an opening of 0.75 cm and the rings clamped between two obtained using a Leica CM1950 cryostat and adhered to Fisher Ussing stretch hemichambers. The chambers were filled with Krebs brand Superfrost slides. The sections were washed three times for buffer and equilibrated. To stretch the tissue, buffer was added to 5 min each with phosphate-buffered saline (PBS), and the fixation the mucosal hemichamber via Luer ports at a rate of 35 lμ /min using was stopped with quench buffer (20 mM glycine, 75 mM NH Cl, a syringe pump (New Era Pump Systems, Farmingdale, NY). Once dissolved in PBS) for 10 min. When staining for ADAM17, the sec- the chamber was lfi led, an additional 250 lμ was then pumped into tions were quenched for 7 min and subsequently treated with the chamber to stretch the tissue. quench buffer supplemented with 0.05% (wt/vol) SDS for an addi- tional 3 min. After quenching, the tissue was then washed with PBS Preparation of cell lysates and Western blot analysis and transferred to block buffer (7% [wt/vol] fish gelatin, 0.25% sa - To prepare lysates, bladder tissue was placed on a rubber dissec- ponin [vol/vol], and 0.05% NaN [wt/vol] made in PBS) for 1 h at tion mat with the mucosal surface facing up. The tissue was held room temperature. A 1:200 dilution of the antibodies was made in in place by pinning it at the four corners using 20-gauge ¾-inch the block buffer, applied to the sections, and incubated at room needles (BD Biosciences). An aliquot (35–50 μl) of SDS lysis buffer temperature for either 2 h (in the case of ADAM17 antibody) or (50 mM triethanolamine, pH 8.6, 100 mM NaCl, 5 mM EDTA, overnight at 4°C for the A AR or UP3a antibodies. The primary anti- 0.2% [wt/vol] NaN , 0.5% [wt/vol] SDS) containing a protease and 1 3 body was removed by three 5-min washes with PBS and detected phosphatase inhibitor cocktail (Cell Signaling Technologies, using Alexa 488– or Cy3–labeled secondary antibodies diluted in Boston, MA), as well as 10 mM 1,10-phenanthroline, was added block buffer. Additional washes with PBS were performed. Actin and to the mucosal surface and the outer uroepithelium recovered by nuclei were stained with either rhodamine or u fl orescein phalloidin gently scraping the cells using a rubber cell scraper (Sarstedt, and TO-PRO3, respectively. After staining, the samples were post- Newton, NC). The cell lysate was transferred to a 1.5-ml Eppen- x fi ed for 10 min with 4% PFA x fi ative and then mounted in medium dorf tube, vortex shaken at 4°C for 10 min using a model 5432 containing 1% (wt/vol) phenylenediamine, 90% (vol/vol) glycerol, vortex mixer (Eppendorf, Hauppauge, NY), and centrifuged at and 20 mM Tris, pH 8.0. 13,000 rpm for 10 min at 4°C in a table-top model 5415D micro- Images were captured using a 63×/1.2 numerical aperture glyc- centrifuge (Eppendorf). The clear supernatant was collected, flash erol objective and the appropriate laser lines of a Leica TCS SP5 frozen, and stored at −80°C. Before use, the protein concentra- CW-STED confocal microscope (in normal confocal mode). The tion was quantified using the bicinchoninic acid assay (Pierce, photomultipliers were set at 900–1200 V, and images were col- Rockford, IL). Equal amounts of protein from the bladder lysates lected using an average of six line scans. Serial 0.25-μm Z-sections were resolved by SDS–PAGE on 4–15% polyacrylamide gradient were acquired. The images were imported into Volocity 4-D soft- gels (Bio-Rad, Hercules, CA) at 200 V and constant current for ware (PerkinElmer, Waltham, MA) and, after image reconstruction 30 min. For ADAM17 and hGH, proteins were transferred to Im- and contrast correction, exported as TIFF lfi es. Composite images mobilon P membranes (Millipore) at 375 mA constant current us- were prepared in Illustrator CS5 (Adobe, San Jose, CA). Colocaliza- ing a 100 mM 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS), tion analysis and measurements of fluorescence intensity were per - pH 11.0, transfer buffer. In the case of the EGFR, proteins were formed as described previously, using a x fi ed threshold of 40 transferred to nitrocellulose membranes (GE Healthcare Life Sci- (Khandelwal et al., 2008, 2010). ences) at 375 mA constant current using a Tris-glycine running buffer (25 mM Tris, 190 mM glycine) containing 0.01% (wt/vol) Mounting rabbit uroepithelium or rat bladders in Ussing SDS for 2 h. In either case, the membrane was blocked for 30 min stretch chambers and measurement of C with 5% (wt/vol) nonfat milk made in TBST buffer (Tris-buffered Isolation of rabbit uroepithelium from the underlying muscle layers saline + 0.1% Tween 20). After the blocking step, the membrane and mounting in Ussing stretch chambers was performed as de- was incubated with primary antibodies overnight at 4°C. After scribed earlier (Wang et al., 2003a). Briefly, rabbit bladders were several washes with TBST buffer, the membrane was incubated excised, slit open vertically along one of the lateral veins, and spread with goat anti-rabbit-HRP or goat anti-mouse-HRP secondary on a custom-made Teflon rack with the uroepithelium facing down. antibodies for 1 h at room temperature and washed with TBST The muscles were carefully removed with the pair of tweezers and buffer. The bands were detected by incubating the membrane sharp scissors. The remaining tissue containing the intact uroepithe- with enhanced chemiluminescence (ECL) solution for 2 min lium was mounted on the pins at the outer edges of a plastic ring (Pierce), followed by film capture on Carestream Kodak Biomax with an opening of 2 cm . The rings were locked between two films (Carestream, Rochester, NY). Data were quantified using Ussing stretch hemichambers, which were clamped into position on QuantityOne quantification software (Bio-Rad) a Teflon base. Warm Krebs buffer was simultaneously added to the mucosal (apical facing) and serosal (muscle facing) chambers. The Generation of adenoviruses encoding ADAM17 shRNA tissue was gassed with 95% air/5% CO during a 30- to 45-min The iRNAi software (Nucleobytes.com) was used to search the equilibration period and treated with the indicated drug added to Rat ADAM17 cDNA (PubMed accession number NM_020306) for 3808 | H. S. Prakasam et al. Molecular Biology of the Cell r S811D r optimal targets containing the sequence AA(N19). Four shRNA se- HA (TCA to GCC) and pADLOX-ADAM17 -HA (TCA to GAC). quences were selected. The top and the bottom strands were indi- In addition, we also generated a tail-minus construct, pADLOX- vidually synthesized (IDT, Coralville, IA) and annealed by mixing the ADAM17-∆CT-HA . This was made by deleting the last 127 amino two strands in equal amounts, heating to 94°C, and cooling gradu- acids of the ADAM17 backbone, leaving two cytoplasmic amino ac- ally to room temperature. The annealed shRNA sequences were ids that were fused in-frame to an HA tag. The constructs (3 μg) were then ligated into the linearized pU6/ENTR vector (Life Technolo- preincubated with Lipofectamine 2000 Opti-MEM media for 30 min, gies). The ability of the different shRNA sequences to silence mixed with 3 μg of Ψ5 adenoviral genomic DNA (Ad5 strain), and ADAM17 was determined by cotransfecting SV40 large T antigen– added to Cre8 cells (Hardy et al., 1997; Khandelwal et al., 2008). expressing HEK293FT cells with the shRNA-pU6/ENTR-shRNA vec- Production of adenoviruses was performed as described, except tors and rat ADAM17 cDNA. At 24 h later, cells were lysed, the that Cre8 cells were used throughout. proteins were resolved by SDS–PAGE, and ADAM17 was detected by Western blotting using the techniques described earlier. Of the In situ adenoviral transduction and detection of hGH release four, the sequence that had maximum silencing efficiency ( >80%; In situ transduction of rat bladder uroepithelium and measurement 5′-GGATTAGCTTACGTTGGTTCT-3′) was selected and recombined of hGH release were performed as described (Khandelwal et al., into the pBLOCKiT Adenovirus System vector (Invitrogen) using an 2008). Briefly, rats were sedated with isoflurane, and a Jelco IV cath - in vitro Clonase-mediated recombination reaction according to the eter (Smith Medicals, Southington, CT) was introduced into the vendor’s protocol. The recombined pBLOCKiT vector was linearized bladder via the urethra. The bladder was rinsed with PBS and lfi led by Pac1 restriction digestion and transfected into 293A cells using with 400 μl of 0.1% (wt/vol) n-dodecyl-β-d -maltoside dissolved in Lipofectamine 2000 reagent (Life Technologies). At 11 d posttrans- PBS. The urethra was clamped, and after 5 min, it was unclamped to fection, when the cytopathic effect was >75%, the cells in this first allow the detergent to void. The latter step was facilitated by apply- P1 generation were harvested by tituration and lysed by freezing the ing slight pressure to the lower abdomen. The bladder was filled cells at −70°C and thawing them for 5–10 min in a 37°C water bath. with 400 μl of PBS containing adenoviruses encoding hGH alone or Three freeze–thaw cycles were typically sufficient to lyse the cells. in combination with adenoviruses encoding scrambled-shRNA, The lysate, containing released virus, was used to infect a new round ADAM17-shRNA, or the ADAM17-HA constructs described earlier of cells (P2), a process that was repeated one additional time (P3). To (2.5 × 10 infectious virus particles, typically in a volume of 2–10 μl). produce large quantities of virus, 10 15-cm Petri dishes (BD Falcon, The bladder was then clamped. After 30 min, the clamp was re- San Jose, CA) of 293A cells were infected with virus-containing moved, and the virus solution was allowed to void. The bladder was lysate from P3. On the third day, when the cells showed >85% cyto- rinsed with PBS, anesthesia was discontinued, and the rats were al- pathic effect, they were recovered by tituration, pooled, centrifuged lowed to revive. At 30 h postinfection, the animals were killed by at 3500 × g (Eppendorf 5810 R) for 14 min at 4°C, and mixed in 7 ml inhalation of CO , and the bladder was immediately excised, slit of resuspension buffer (100 mM Tris, pH 7.4, 10 mM EDTA). The open, and mounted on tissue rings as described. After 90 min of concentrated cell suspension was lysed by repeated freeze–thaw equilibration, the buffer bathing the apical surface was isovolumetri- cycles as described. The lysate was separated from the cell debris cally replaced with fresh buffer and then treated or not with 500 nM by centrifuging at 5000 × g (Eppendorf 5810 R) for 15 min at 4°C. CCPA. At 60 min later, the apical buffer was removed and concen- The supernatant was carefully removed and applied to the top of a trated using a 10K molecular weight cutoff Amicon Centricon step gradient containing 2.5 ml of 1.25 g/ml CsCl solution, which (Millipore) to a volume of 250 μl. The corresponding tissue was un - was layered on top of 2.5 ml of 1.4 g/ml CsCl loaded into clear mounted from its tissue ring, and a lysate was prepared before 13-ml PET ultracentrifugation tubes (Thermo Scientific). The sam - Western blot analysis to detect hGH. The fraction of hGH secreted ples were centrifuged at 35,000 rpm for 1 h at 4°C using a Beckman was calculated as secreted hGH/(secreted hGH + cell-associated Coulter centrifuge (Brea, CA) and an SW-41 swinging bucket rotor. hGH in lysate). The concentrated virus, which appeared as an off-white band at the interface of the two CsCl layers, was collected by piercing the Endocytosis of WGA-FITC side wall of the tube with an 18-gauge needle and aspirating it into Rat bladders were mounted in Ussing chambers, and after equilibra- a connected 5-cc syringe (BD Biosciences). The viruses were further tion, they were incubated with 25 μg/ml WGA-FITC for 2 h with or purified by passage through a PE10 gel filtration column (GE Health - without 500 nM CCPA added to the mucosal hemichamber. The tis- care) equilibrated with virus suspension buffer (PBS containing 10% sues were then unmounted from the chambers, washed with ice- [vol/vol] glycerol). The virus-containing fractions were detected by cold 100 mM N-acetyl-d -glucosamine four times for 20 min, and monitoring the A , pooled, and stored at −70°C in small aliquots, then washed with ice-cold PBS three times for 15 min and then fixed which were thawed in a 37°C water bath just before use. with 4% PFA fixative for 30 min at 37°C. The tissue was stained, and the images were captured and processed as described. Because Generation of adenoviruses encoding ADAM17-HA , some WGA-FITC remained bound to the apical surface of the um- S811A r S811D r r ADAM17 -HA , ADAM17 -HA , or ADAM17-∆CT-HA brella cells even after washing with cognate sugar, the apicalmost ADAM17 rat cDNA (from Addgene plasmid 19141; Lemieux et al., sections (∼1–1.5 m μ ) in the confocal stacks were excluded from the 2007) was cloned into pADLOX vector, and an oligonucleotide en- quantitation. coding the HA tag (YPYDVPDYA) was added in-frame to the C-ter- minus of ADAM17 after a gap of two amino acids. A silent mutation P-orthophosphate labeling of ADAM17 was engineered into the ADAM17 sequence (5′-GGATTAGCG- HEK293FT cells were cotransfected with 3 μg of pcDNA-A AR and r S811A TACGTTG GTTCT) using the QuikChange XL mutagenesis kit 3 μg of either pADLOX-ADAM17-HA or pADLOX-ADAM17 - (Agilent, Santa Clara, CA), making the construct resistant to the HA using Lipofectamine 2000 reagent. At 48 h posttransfection, the ADAM17 shRNA. This resulting construct, called pADLOX-ADAM17- cells were washed two times with PO efflux buffer (140 mM NaCl, HA , was further mutagenized to convert serine at position 811 to an 2 mM KCl, 1 mM MgSO , 1 mM CaCl , 10 mM glucose, 10 mM 4 2 S811A alanine or aspartate residue, generating pADLOX-ADAM17 - 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, titrated to pH 7.4 Volume 25 November 15, 2014 ADAM17-dependent exocytosis | 3809 using 1 M Tris-base) and then incubated with PO efflux buffer CCPA was normalized to reactions in which CCPA treatment was supplemented with 300 μCi of P-orthophosphate (5 mCi/ml; MP omitted. Each experiment was repeated at least five times. No AP Biomedicals; Santa Ana, CA) for 2 h at 37°C. During the last 15 min activity was present in the supernatant of nontransfected cells. of the incubation, the cells were treated or not with 500 nM CCPA. Subsequently, the efflux buffer was aspirated, the cells were washed Statistical analysis two times with Tris-buffered saline (pH 7.5), and the cells were dis- Statistical significance between means was determined by Stu - solved in 500 μl of RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM dent’s t test or, in the case of multiple comparisons, by analysis of NaCl, 50 mM NaF, 0.1% [wt/vol] SDS, 1% [wt/vol] sodium deoxy- variance (ANOVA). If a significant difference in the means was cholate, 1% [vol/vol] Triton X-100, 1 mM EDTA, 1 mM phenylmeth- detected by ANOVA, multiple comparisons were performed us- ylsulfonyl fluoride, 1 mM orthovanadate, 0.1 mg/ml aprotinin, 1 mM ing Dunnett’s posttest correction. Statistical analyses were per- ethylene glycol tetraacetic acid, and 10 mM 1,10-phenanthroline). formed using Prism 5 software (GraphPad, La Jolla, CA). In the The cell lysate was collected, placed in 1.5-ml Eppendorf tubes, and figure legends, n refers to the number of individual animals or incubated on ice for 30 min to complete the cell lysis. The lysate was experiments. centrifuged at 16,000 × g at 4°C using a Marathon 16km table-top centrifuge (Fisher Scientific, Waltham, MA) for 1 h, and the resulting supernatant was collected in a fresh tube. The volume in each tube was brought up to 900 μl with RIPA lysis buffer, and then 7 μl of rab - ACKNOWLEDGMENTS bit anti-HA antibody (Covance) and 40 lμ of 10% SDS were added This work was supported by National Institutes of Health Grants to each sample. After a 1-h incubation at 4°C with constant rotation, R37-DK54425, R01-DK077777, and P30-DK079307 to G.A. and 100 lμ of a 20% (wt/vol) slurry of protein G-Sepharose beads (GE R01-DK075048 to K.R.H. and the Cellular Physiology and Kidney Healthcare) was added to each sample and incubated at 4°C over- Imaging Cores of the Pittsburgh Center for Kidney Research night with constant rotation. The beads were washed with RIPA lysis (P30-DK079307). buffer three times, resuspended in 40 μl of 2 × Laemmli sample buf- fer, heated for 15 min at 65°C, and centrifuged at 16,000 × g for 10 min, and then the proteins in the supernatant were resolved by REFERENCES SDS–PAGE. The proteins were transferred to nitrocellulose mem- Amour A, Slocombe PM, Webster A, Butler M, Knight CG, Smith BJ, Stephens PE, Shelley C, Hutton M, Knauper V, et al. (1998). TNF-alpha branes as described and the immobilized HA-labeled proteins re- converting enzyme (TACE) is inhibited by TIMP-3. 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