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UTP and ATP increase extracellular signal-regulated kinase 1/2 phosphorylation in bovine chromaffin cells through epidermal growth factor receptor transactivation

UTP and ATP increase extracellular signal-regulated kinase 1/2 phosphorylation in bovine... Purinergic Signalling (2008) 4:323–330 DOI 10.1007/s11302-008-9098-y ORIGINAL ARTICLE UTP and ATP increase extracellular signal-regulated kinase 1/2 phosphorylation in bovine chromaffin cells through epidermal growth factor receptor transactivation Toni M. Luke & Terry D. Hexum Received: 25 April 2007 /Accepted: 14 March 2008 / Published online: 6 September 2008 Springer Science + Business Media B.V. 2008 Abstract Adenosine triphosphate (ATP) is coreleased with gest nucleotide-mediated ERK1/2 phosphorylation is medi- catecholamines from adrenal medullary chromaffin cells in ated by a P2Y or P2Y receptor, which stimulates 2 4 response to sympathetic nervous system stimulation and may metalloproteinase-dependent transactivation of the EGFR. regulate these cells in an autocrine or paracrine manner. Increases in extracellular signal-regulated kinase (ERK) 1/2 Keywords Extracellular signal-regulated kinase . . phosphorylation were observed in response to ATP stimula- Phosphorylation Epidermal growth factor receptor . . . tion of bovine chromaffin cells. The signaling pathway Transactivation P2Y receptor P2Y receptor UTP 2 4 involved in ATP-mediated ERK1/2 phosphorylation was investigated via Western blot analysis. ATP and uridine 5′- Abbreviations triphosphate (UTP) increased ERK1/2 phosphorylation po- BACC bovine adrenal chromaffin cells tently, peaking between 5 and 15 min. The mitogen-activated ERK1/2 extracellular signal-regulated kinase 1 and 2 protein kinase (MAPK/ERK)-activating kinase (MEK) in- EGF epidermal growth factor hibitor PD98059 blocked this response. UTP, which is EGFR EGF receptor selective for G-protein-coupled P2Y receptors, was the most MEK mitogen-activated protein kinase/ERK potent agonist among several nucleotides tested. Adenosine kinase 2+ 2+ 5′-O-(3-thio) triphosphate (ATPγS) and ATP were also [Ca ] cytosolic free Ca concentration potent agonists, characteristic of the P2Y or P2Y receptor PKC protein kinase C 2 4 subtypes, whereas agonists selective for P2X receptors or PKA protein kinase A 2+ other P2Y receptor subtypes were weakly effective. The CaMKII Ca /calmodulin-dependent protein kinase II receptor involved was further characterized by the nonspe- PI3K phosphoinositide-3 kinase cific P2 antagonists suramin and reactive blue 2, which each 2-MeSATP 2-methylthio ATP partially inhibited ATP-mediated ERK1/2 phosphorylation. α,β- α,β-methylene ATP Inhibitors of protein kinase C (PKC), protein kinase A meATP 2+ (PKA), Ca /calmodulin-dependent protein kinase II (CaM- HB-EGF heparin-binding EGF-like growth factor KII), and phosphoinositide-3 kinase (PI3K) had no effect on Pyk2 proline-rich tyrosine kinase ATP-mediated ERK1/2 phosphorylation. The Src inhibitor SH3 Src homology 3 PP2, epidermal growth factor receptor (EGFR) inhibitor AG1478, and metalloproteinase inhibitor GM6001 decreased ATP-mediated ERK1/2 phosphorylation. These results sug- Introduction T. M. Luke T. D. Hexum (*) Chromaffin cells are neuroendocrine cells that synthesize and University of Nebraska Medical Center, secrete catecholamines in response to sympathetic nervous 985800 Nebraska Medical Center, system stimulation and therefore participate in regulation of Omaha, NE 68198–5800, USA e-mail: thexum@unmc.edu stress-modified parameters such as heart rate and blood 324 Purinergic Signalling (2008) 4:323–330 pressure. A variety of additional agents are costored and were maintained on six-well plates at a density of 3×10 released along with the catecholamines from the chromaffin cells/well at 37°C with 5% CO . Viability and purity were granules, including neuropeptides such as the enkephalins and verified to be >95% by Trypan blue exclusion and neutral adenosine triphosphate (ATP) [1]. In addition to being re- red staining, respectively. leased into the circulation, these agents may regulate chro- maffin cell activity in an autocrine or a paracrine manner, Western blot analysis Cells were incubated with agonist for allowing the cells to adjust to varying levels of stimulation. 10 min, and inhibitor preincubations were 15 min, unless The role of ATP in chromaffin cell function has not been otherwise indicated. Cells were rinsed with phosphate well defined, even though it is secreted in high concen- buffered saline (PBS) and lysed with 200 μlof50 mM trations from chromaffin cells [1]. It has been suggested N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid that ATP regulates chromaffin cell secretion, either posi- (HEPES) (4-(2-hydroxyethyl)-1-piperazineethanesulfonic tively [2] or negatively [3, 4], and that ATP regulates the acid), pH 7.2, containing 1 mM ethylenediaminetetraacetate function of voltage-dependent calcium channels [5]. ATP (EDTA), 1 mM ethyleneglycoltetraacetic acid (EGTA), exerts its effects through either G-protein-coupled recep- 0.2% triton X-100, 10 mM β-glycerol 2-phosphate diso- tors, designated P2Y; or ion channels, designated P2X. dium salt, 1 mM sodium orthovanadate, 1 mM benzami- These receptor types are further divided into subtypes, dine, 4 μg/ml leupeptin, 1 μM microcystin-LR, and including P2Y , P2Y , P2Y ,P2Y , and P2Y for the 0.5 mM DTT. Lysates were centrifuged at 13,000 g, and 1 2 4 6 11–14 G-protein-coupled P2Y receptors and P2X for those that protein concentrations of supernatants were determined 1–7 activate ion channels [6]. Previous work with chromaffin with the Bio-Rad protein assay (Bio-Rad, Hercules, CA, cells indicated ATP stimulation results in increases in USA). Loading buffer (2X) was added to cell lysates, inositol phosphates [7], cyclic adenosine monophosphate consisting of 125 mM Tris(hydroxymethyl)aminomethane 2+ (cAMP) [8], and [Ca ] accumulation [7], likely via HCl (Research Organics, Cleveland, OH, USA), 4% activation of a P2Y or P2Y receptor, both of which are sodium dodecylsulfate (SDS) (Research Organics, Cleve- 2 4 present in bovine chromaffin cells (unpublished observa- land, OH, USA), 20% glycerol, and 0.02% bromophenol tions). The downstream effects of P2Y receptor stimulation blue. Samples were subsequently boiled and subjected to by ATP in chromaffin cells are not known. The observed SDS-polyacrylamide gel electrophoresis (PAGE) on 10% increases in signaling messengers may bring about the Tris-HCl Criterion gels (BioRad, Hercules, CA, USA). activation of multiple protein kinases or tyrosine kinases. Protein was transferred to fluorescent-polyvinylidene In several cell types, ATP signaling has been shown to fluoride (PVDF) membranes (Millipore, Billerica, MA, activate extracellular signal-regulated kinase 1 and 2 (ERK1/ USA). Membranes were next blocked in Odyssey blocking 2). P2Y receptors have been shown to couple to ERK1/2 buffer (LI-COR Biosciences, Lincoln, NE, USA) for 1 h, activation via activation of protein kinases such as phosphoi- then incubated in Odyssey blocking buffer containing 0.2% nositide-3 kinase (PI3K) [11] or protein kinase C (PKC) [10, Tween 20 (polyoxyethylene-sorbitan monolaurate) and 202 204 11]. P2Y receptor-mediated ERK1/2 has also been shown to mouse-anti-phospho-ERK 1/2 (Thr /Tyr ) and rabbit- be dependent on activation of tyrosine kinases such as Src or anti-ERK 1/2 (Cell Signaling Technology, Boston, MA, proline-rich tyrosine kinase (Pyk2) [9, 10] and/or on trans- USA) primary antibodies. Blots were then incubated in activation of the epidermal growth factor receptor (EGFR) Odyssey blocking buffer with 0.2% Tween 20 and 0.02% [9]. Additionally, increases in cAMP in response to ATP may SDS and with the secondary antibodies goat-anti-mouse result in activation of protein kinase A (PKA), which has immunoglobulin (Ig)G Alexa Fluor680 (Molecular Probes, 2+ been shown to activate ERK1/2 [12]. Increases in [Ca ] in Eugene, OR, USA) and goat-anti-rabbit IgG IR800 (Rock- 2+ response to ATP may result in activation of Ca /calmodulin- land Immunochemicals, Gilbertsville, PA, USA). Mem- dependent protein kinase II (CaMKII), which has also been branes were developed with the Odyssey Infrared Imaging shown to phosphorylate ERK1/2 [13]. Therefore, we elected System, which utilizes two infrared channels (700 nm and to examine ATP-mediated ERK1/2 phosphorylation and the 800 nm), allowing for detection of two target proteins receptor subtype and signaling mechanism present in bovine simultaneously, in this case phosphorylated and total chromaffin cells. ERK1/2. Statistics Band integrated intensities were determined with Materials and methods Odyssey Imaging software. Phospho-ERK1/2 intensities were divided by total ERK1/2 intensities and normalized to Chromaffin cell isolation and cell culture Bovine adrenal fold increases over control. Data were analyzed with chromaffin cells (BACC) were isolated using a collagenase GraphPad Prism software, and one-way analysis of vari- perfusion method as described previously [14, 15]. Cells ance (ANOVA) was utilized to determine statistical signif- Purinergic Signalling (2008) 4:323–330 325 pERK chlorophenyl)-7-(t-butyl)pyrazolo-D-3,4-pyrimidine], AG1478 [4-(3-Chloroanilino)-6,7-dimethoxyquinazoline], ERK and GM6001 [N-[(2R)-2-(hydroxamidocarbonylmethyl)-4- methylpentanoyl]-L-tryptophan methylamide] were pur- ATP-+ + -+-- - + + 2 min 5 min 10 min 15 min 30 min chased from Biomol (Philadelphia, PA, USA). Bis-I (bisindo- lylmaleimide-I), KN-92 [2-[N-(4-methoxybenzenesulfonyl)] pERK amino-N-(4-chlorocinnamyl)- N-methylbenzylamine], KN- ERK 93 [2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine], and UTP - + - + - + - + - + 2 min 5 min 10 min 15 min 30 min wortmannin were purchased from Merck-Calbiochem Bio- sciences (Darmstadt, Germany). H89 [N-[2-(p-Bromocinna- mylamino)ethyl]-5-isoquinolinesulfonamide] was obtained from Upstate (Charlottesville, VA, USA).Other reagents were obtained from either Sigma-Aldrich or Fisher Scientific. Results Western blot analyses with antibodies specific to activated 202 204 ERK1/2 phosphorylated at Thr /Tyr were utilized to examine the time course of ERK1/2 phosphorylation in response to nucleotide stimulation. Both ATP and UTP Fig. 1 Nucleotide-stimulated ERK1/2 phosphorylation is time depen- dent. BACCs were treated with ATP (100 μM) or UTP (100 μM) for 2 potently increased ERK1/2 phosphorylation, with a peak to 30 min. Blots are representative of three independent experiments between 5 and 15 min (Fig. 1). ERK1/2 has been shown to performed in triplicate (n=3); the upper band (phosphorylated or nonphosphorylated) is ERK1=44 kDa, and the lower band is ERK2= 42 kDa. Blot intensities were measured with the Odyssey Imaging Overlay Overlay System; values are phosphorylated ERK2 intensity divided by total ERK2 intensity. Points on the graph represent mean ± standard error pERK pERK of the mean ERK ERK - + - + icance. The decision was made to utilize ERK2 for UTP - + - + AT P No Inhibitor PD98059 graphical representations, as band intensities for phosphor- No Inhibitor PD98059 ylated ERK2 were stronger than phosphorylated ERK1, though results obtained were quantitatively similar for both. Chemicals Nucleotides and analogs were obtained from Sigma-Aldrich (St. Louis, MO, USA). PD98059 (2′-amino- 3′-methoxylflavone), PMA (phorbol 12-myristate 13-acetate), *** suramin, and reactive blue 2 (RB2) were also obtained from Sigma-Aldrich. NF279 [8,8′-[Carbonylbis(imino-4,1- *** phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis- 1,3,5-naphthalenetrisulfonic acid hexasodium salt] was pur- chased from Tocris (St. Louis, MO, USA). KT5720 Fig. 2 MEK inhibition decreases ATP- and UTP-mediated ERK1/2 [(9S,10S,12R)-2,3,9,10,11,12-Hexahydro-10-hydroxy-9- phosphorylation. BACCs were treated with PD98059 (10 μM) or methyl-1-oxo-9, 12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl] dimethylsulfoxide for 15 min, followed by a 10-min stimulation with pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ATP (100 μM) or UTP (100 μM). Blots are representative of three ester], Ro-31–8220 [2-[1-(3-(Amidinothio)propyl)-1H- independent experiments performed in triplicate (n=3); the upper band (phosphorylated or nonphosphorylated) is ERK1=44 kDa and indol-3-yl]-3-(1-methylindol-3-yl)maleimide methanesulfo- the lower band is ERK2=42 kDa. Blot intensities were measured with nate], LY294002 ]2-(4- morpholinyl)-8-phenyl-4H-1- the Odyssey Imaging System; values are phosphorylated ERK2 benzopyran-4-one], PP1 [4-amino-5-(4-methylphenyl)-7- intensity divided by total ERK2 intensity. Bars on the graph represent (t-butyl)pyrazolo-D-3,4-pyrimidine], PP2 [4-amino-5-(4- mean ± standard error of the mean. *** p<0.001 vs. stimulator alone 326 Purinergic Signalling (2008) 4:323–330 be catalyzed only by MEK; therefore, the MEK inhibitor receptor subtypes. Moreover, UTP is selective for P2Y PD98059 was utilized to confirm the immediate upstream receptors, precluding the involvement of a P2X receptor in signaling event responsible for nucleotide-mediated ERK1/ nucleotide-mediated ERK1/2 phosphorylation. 2 phosphorylation. PD98059 (10 μM) significantly de- ATP stimulation of chromaffin cells has been shown to 2+ creased both ATP-mediated (∼90%) and UTP-mediated increase inositol phosphates [7], cAMP [8], and [Ca ] (∼70%, Fig. 2, Table 1) ERK1/2 phosphorylation. accumulation [7], which may lead to activation of several Most studies designed to determine purinergic receptor protein kinases, which may in turn be involved in ATP- subtypes use ligand potency studies due to the lack of mediated phosphorylation of ERK1/2. Therefore, a variety available highly selective receptor subtype antagonists [16]. of inhibitors were used to examine which kinases are Correspondingly, we used a similar approach to character- involved in ATP-mediated ERK1/2 phosphorylation. The ize the receptor subtype involved in ERK1/2 phosphoryla- PKA inhibitors H89 (10 μM) and KT5720 (100 nM) had tion. Examination of several purine analogs revealed a rank no effect on ATP- or UTP-mediated ERK1/2 phosphoryla- order of potency of UTP (EC =1.6 μM)>ATPγS tion (Table 1). The broad-spectrum PKC inhibitors Ro-31– (6.5 μM)≥ATP (13 μM)>uridine diphosphate (UDP) 8220 (10 μM) and Bis-I (3.5 μM) also had no effect on (120 μM) = adenosine diphosphate (ADP) (220 μM)= 2- ATP- or UTP-mediated ERK1/2 phosphorylation, although methylthio (ATP)2-MeSATP (320 μM)>>α,β-methylene they were capable of blocking PMA-mediated ERK1/2 ATP (α,β-meATP) (Fig. 3), consistent with the involve- phosphorylation (∼60%, Table 1). Moreover, the CaMKII ment of a P2Y or P2Y receptor, as both UTP and ATP inhibitor KN93 (1 μM) and the PI3K inhibitors wortmannin 2 4 exhibit strong agonist action. (300 nM) and LY294002 (20 μM) had no effect on ATP- or The involvement of a P2 receptor in ERK1/2 phosphor- UTP-mediated ERK1/2 phosphorylation (Table 1). ylation was further supported using the nonselective P2 In addition to activation of PKs, P2Y receptors may receptor antagonists suramin and RB2. Suramin (100 μM) utilize tyrosine kinases to activate ERK1/2 [10]. Therefore, significantly decreased ATP- or UTP-mediated ERK1/2 the tyrosine kinase inhibitor PP2 was used to examine the phosphorylation (∼60%, Fig. 4, Table 1). RB2 (100 μM) role of Src family members in ATP-mediated ERK1/2 also decreased the effect of ATP- or UTP-stimulation on phosphorylation. PP2 (1 μM) decreased ATP- or UTP- ERK1/2 phosphorylation (∼35%, Fig. 4, Table 1). The P2X- mediated ERK2 phosphorylation (∼40%, Fig. 5, Table 1). specific receptor agonist α,β-meATP had no effect on In addition to inhibiting Src family members, PP2 is also a ERK1/2 phosphorylation at concentrations up to 100 μM weak inhibitor of the EGFR. Also, G-protein-coupled (Fig. 3), eliminating the involvement of several of the P2X receptor activation of Src family members may result in Table 1 Involvement of signaling pathways in ATP- and UTP-mediated ERK1/2 phosphorylation Inhibitor/antagonist ATP UTP ERK1 ERK2 ERK1 ERK2 P2R Suramin (100 μM) 61.8±3.1*** 54.7±3.8*** 62.0±5.7*** 62.0±5.7*** Antagonists RB2 (100 μM) 32.3±4.5*** 29.5±4.5*** 44.9±10.7*** 44.9±9.1*** MEK inhibitor PD98059 (10 μM) 95.3±5.3*** 94.4±4.5*** 69.5±3.4*** 64.3±4.2*** PKC inhibitors Bis-I (3.5 μM) 12.7±20.5 4.6±7.9 5.2±12.9 −1.3±10.4 Bis-I-PMA 76.1±2.9*** 67.5±3.2*** –– Ro-31-8220 (10 μM) −40.0±28.8 −13.5±10.2 −13.4±14.9 −8.7±10.9 Ro-31-8220-PMA 66.6±2.6*** 54.4±2.3*** –– PKA inhibitors H89 (10 μM) −67.1±36.3 −42.4±14.4 −27.5±31.4 −16.4±16.5 KT5720 (100 nM) −1.5±13.3 −1.9±16.5 1.9±13.9 6.5±13.2 CaMKII inhibitor KN93 (1 μM) 22.5±27.2 7.1±12.7 −20.1±18.2 −14.2±15.4 PI3K inhibitors Wortmannin −10.0±34.2 −3.9±15.7 10.0±25.9 12.5±14.6 (300 nM) LY294002 (20 μM) −3.9±15.7 1.0±17.4 18.6±18.7 30.3±12.1 Src inhibitor PP2 (1 μM) 40.9±16.1* 39.5±9.1*** 46.0±8.1*** 32.4±9.6*** EGFR inhibitors AG1478 (2.6 μM) 73.7±2.0*** 71.1±2.3*** 62.9±7.2*** 69.6±6.7*** MMP inhibitors GM6001 (2.5 μM) 69.2±5.8** 60.8±7.1*** 66.7±10.1*** 62.0±10.4*** Cells were pretreated with inhibitors for 15 min then stimulated with 100 μM UTP (or 1 μM PMA) for 10 min. Values are percent inhibition of UTP-mediated ERK1/2 phosphorylation ± standard error of the mean for three experiments in triplicate. Bis-I-PMA and Ro-31-8220-PMA refer to using PMA as the stimulator rather than UTP *p<0.05 vs. stimulator alone,**p<0.01 vs. stimulator alone, **p<0.001 vs. stimulator alone Purinergic Signalling (2008) 4:323–330 327 ERK1/2 phosphorylation in response to ATP would allow Overlay the cells to respond quickly to varying levels of stimulation. Although the physiological effects of ERK1/2 phosphory- pERK lation in these cells are unknown, possible actions requiring a rapid response include either the acute activation of ERK proteins involved in catecholamine secretion and/or stimu- Con 100 nM 1 µM3 µM 10 µM 30 µM 100 µM 1 mM lation of protein expression important for exocytosis. ATP Ligand potency and inhibitor studies suggest either the P2Y or P2Y receptor subtype is responsible for nucleo- 2 4 tide-mediated ERK1/2 phosphorylation, similar to data obtained for increases in inositol phosphates (unpublished observations). Both of these receptor subtypes are present in chromaffin cells, based on reverse transcriptase real-time polymerase chain reaction (PCR) data for P2Y and P2Y , 2 4 and appear to be expressed in these cells according to Western blot analysis with specific antibodies (unpublished Overlay pERK Fig. 3 P2Y or P2Y receptor activation increases ERK1/2 phos- 2 4 ERK phorylation. BACCs were treated with increasing concentrations of -+ + -- + ATP nucleotides and analogs for 10 min. Blots are representative of three No Inhibitor Suramin RB2 independent experiments performed in triplicate (n=3); the upper band (phosphorylated or nonphosphorylated) is ERK1=44 kDa, and Overlay the lower band is ERK2=42 kDa. Blot intensities were measured with the Odyssey Imaging System; values are phosphorylated ERK2 intensity divided by total ERK2 intensity. Points on the graph pERK represent mean ± standard error of the mean. Con (control) refers to results obtained with unstimulated cells ERK -+ + -- UTP No Inhibitor Suramin RB2 activation of ERK1/2 via activation of Ras [17, 18] or via transactivation of the EGFR. Therefore, the involvement of EGFR in ATP-mediated ERK1/2 phosphorylation was determined by treating cells with the EGFR inhibitor AG1478 (2.6 μM), which decreased ATP- and UTP- *** mediated ERK2 phosphorylation by about 70% (Fig. 6a, *** Table 1). EGF-mediated ERK1/2 phosphorylation was *** *** completely blocked by AG1478 (100%, p<0.001, data not shown). Transactivation of the EGFR by G-protein-coupled receptors may be mediated by tyrosine kinases such as Src or via metalloproteinases, which release EGFR ligands such as heparin-binding EGF-like growth factor (HB-EGF) from Fig. 4 P2 receptor antagonists partially block ATP- and UTP- the cell membrane [19]. The metalloproteinase inhibitor mediated ERK1/2 phosphorylation. BACCs were pretreated with or GM6001 (2.5 μM) decreased ATP- and UTP-mediated without suramin (100 μM) or reactive blue 2 (RB2, 100 μM) for 15 ERK1/2 phosphorylation by about 65% (Fig. 6b, Table 1). min, followed by a 10-min stimulation with or without ATP (100 μM) or UTP (100 μM). Blots are representative of three independent experiments performed in triplicate (n=3); the upper band (phosphor- ylated or nonphosphorylated) is ERK1=44 kDa, and the lower band is Discussion ERK2=42 kDa. Blot intensities were measured with the Odyssey Imaging System; values are phosphorylated ERK2 intensity divided ATP and UTP potently increase ERK1/2 phosphorylation, by total ERK2 intensity. Bars on graph represent mean ± standard with a peak between 5 and 15 min. This rapid peak in error of the mean. *** p<0.001 vs. stimulator alone 328 Purinergic Signalling (2008) 4:323–330 Overlay Overlay P2Y-mediated ERK1/2 phosphorylation [10, 11]. In PC12 cells, ERK1/2 phosphorylation in response to P2Y pERK pERK receptor activation has been shown to be both dependent [9, 10] and independent [21] of the small tyrosine kinase ERK ERK Pyk2. P2Y receptors have also been shown to contain an - + - + 2 - + - + ATP UTP integrin-binding domain, arginine-glycine-aspartic acid No Inhibitor PP2 No Inhibitor PP2 (RGD), which is necessary for ERK1/2 activation in astrocytes [22, 23]. Additionally, P2Y receptors contain SH3-binding sites that associate with Src in astrocytoma cells [24] and astrocytes [23]. Also, in PC12 cells P2Y receptors have been shown to require EGFR transactivation *** *** to increase ERK1/2 phosphorylation [9]. Initially, we examined whether signaling pathways medi- ated by protein kinases were involved in ATP-mediated Overlay pERK Fig. 5 Tyrosine kinase inhibition decreases ATP- and UTP-mediated ERK1/2 phosphorylation. BACCs were pretreated with PP2 (1 μM) or ERK dimethylsulfoxide (DMSO) for 15 min, followed by a 10-min stimulation - ATP UTP - ATP UTP with or without ATP (100 μM) or UTP (100 μM). Blots are No Inhibitor AG1478 representative of three independent experiments performed in triplicate (n=3); the upper band (phosphorylated or nonphosphorylated) is ERK1 =44 kDa, and the lower band is ERK2=42 kDa. Blot intensities were measured with the Odyssey Imaging System; values are phosphorylated ERK2 intensity divided by total ERK2 intensity. Bars on the graphs *** represent mean ± standard error of the mean. *** p<0.001 vs. *** stimulator alone observations.) Several lines of evidence suggest P2X receptors are not involved in the increase in ERK1/2 in Overlay Overlay response to nucleotide stimulation. First, UTP does not pERK activate P2X ion channels but potently increases ERK1/2 pERK phosphorylation. Additionally, α,βmeATP, an agonist se- ERK ERK lective for several P2X receptor subtypes, had no effect on UTP - + - + - + - + ATP ERK1/2 phosphorylation. The P2Y receptor involved is No Inhibitor GM6001 No Inhibitor GM6001 most likely either P2Y or P2Y . UTP is highly selective 2 4 for two P2Y receptor subtypes P2Y and P2Y and weakly 2 4 effective on the P2Y receptor. The P2Y subtype can be 6 6 ruled out because of the subtypes activated by UTP; only the P2Y and P2Y subtypes are also strongly activated by *** 2 4 *** ATP. The weak effect of ADP, UDP and 2-MeSATP confirms this designation, as these agonists are specific for P2 receptor subtypes other than the P2Y or P2Y 2 4 subtypes [6]. There are no available agonists or antagonists Fig. 6 Nucleotide-mediated ERK1/2 phosphorylation is dependent on EGFR transactivation. BACCs were pretreated with or without to distinguish between the P2Y and P2Y receptors. Even 2 4 AG1478 (2.6 μM) (a), GM6001 (2.5 μM) (b) and dimethylsulfoxide so, suramin and RB2 are commonly used to characterize (DMSO) for 15 min, followed by a 10-min stimulation with or without these receptors in a given cell type, and their partial ATP (100 μM) or UTP (100 μM). Blots are representative of three effectiveness is not contradictory to results found in other independent experiments performed in triplicate (n=3); the upper band (phosphorylated or nonphosphorylated) is ERK1=44 kDa ,and cell types for P2Y or P2Y receptors [20]. 2 4 the lower band is ERK2=42 kDa. Blot intensities were measured with Nucleotides utilize multiple signaling pathways in the Odyssey Imaging System; values are phosphorylated ERK2 different cell types to bring about increases in ERK1/2 intensity divided by total ERK2 intensity. Bars on the graphs represent phosphorylation. PKC and PI3K have been implicated in mean ± standard error of the mean. *** p<0.001 vs. stimulator alone Purinergic Signalling (2008) 4:323–330 329 ERK1/2 phosphorylation. Previous studies determined that mediated by metalloproteinases in response to ATP may be ATP-mediated stimulation of bovine chromaffin cells results responsible for transactivation of the EGFR and subse- 2+ in increases in inositol phosphates [7], cAMP [8], and [Ca ] quently stimulation of ERK1/2 phosphorylation. accumulation [7]. P2Y or P2Y receptors couple to G As expected, the MEK inhibitor PD98059 blocked ATP- 2 4 q to increase activation of PKC. Additionally, the observed and UTP-mediated ERK1/2 phosphorylation, confirming increases in cAMP may result in activation of PKA, whereas that MEK, the only known kinase upstream of ERK1/2, 2+ increased [Ca ] may result in activation of CaMKII. contributes to ERK1/2 phosphorylation. PD98059 blocked However, inhibitors of each of these protein kinases had no ATP-mediated ERK1/2 phosphorylation (∼90%) to a effect on ATP- or UTP-mediated ERK1/2 phosphorylation, greater extent than UTP-mediated ERK1/2 phosphorylation including the PKA inhibitors H89 and KT5720, PKC (∼70%). This may be due to the fact that UTP is a more inhibitors Bis-I and Ro-81–3220, or the CaMKII inhibitor potent agonist and elicited a larger response than ATP for KN93. The PKC inhibitors Bis-I and Ro-81–3220 were ERK1/2 phosphorylation. For the other inhibitors, the capable of blocking PMA-mediated ERK1/2 phosphoryla- responses were very similar; however, none of the other tion, suggesting PKC can couple to ERK1/2 phosphorylation inhibitors had such a pronounced effect on ATP-mediated in these cells, and yet confirming the lack of involvement of ERK1/2 phosphorylation. Alternatively, ATP and UTP may this pathway in ATP-mediated ERK1/2 phosphorylation. We activate multiple receptors with distinct signaling pathways also examined the PI3K inhibitors wortmannin and that are variously more specific for ATP or UTP. Activation LY294002, as this kinase has been implicated in P2Y- of ERK1/2 independent of MEK1 may involve inhibition of mediated ERK1/2 phosphorylation. These inhibitors also phosphatases. proved to be ineffective, suggesting the protein kinases Further studies are necessary to determine the conse- examined were not responsible for ATP-mediated ERK1/2 quence(s) of ERK1/2 phosphorylation in response to ATP phosphorylation. stimulation of chromaffin cells. As chromaffin cells are We next examined the involvement of tyrosine kinases nonproliferating, the stimulation of ERK1/2 phosphoryla- in ATP-mediated ERK1/2 phosphorylation, as these kinases tion by ATP and UTP may couple to regulation of gene have been shown to be involved in P2Y-mediated ERK1/2 transcription essential to exocytosis. phosphorylation. PP2 (1 μM) significantly decreased ATP- To our knowledge this is the first study demonstrating and UTP-mediated ERK1/2 phosphorylation. The reported phosphorylation of ERK1/2 in response to ATP or UTP IC s for PP2-mediated inhibition of Src family members stimulation in bovine chromaffin cells. Our data show that are in the nanomolar range (http://www.biomol.com), the ERK1/2 phosphorylation response to ATP is mediated whereas the dose used in these studies is reported to cause by either a P2Y or P2Y receptor. Protein kinases are not 2 4 weak inhibition of the EGFR [25]. Lower doses of PP2 had involved in nucleotide-mediated ERK1/2 phosphorylation, no effect on ATP- or UTP-mediated ERK1/2 phosphoryla- but rather, metalloproteinase-dependent transactivation of tion (data not shown.) Therefore, at the effective dose used, the EGFR is necessary for ATP-mediated ERK1/2 phos- it is not possible to conclude whether Src or EGFR phorylation. inhibition was responsible for the decrease in ATP- Acknowledgment We thank Matthew Beaver for excellent technical mediated ERK1/2 phosphorylation. Additionally, activation assistance and Robin Taylor for expert assistance with graphics. This of Src family members by G-protein-coupled receptors may work was supported by a grant to Terry D. Hexum from the American increase ERK1/2 phosphorylation via activation of the Heart Association (#0550030Z). renin angiotensin system (Ras) [17, 18] or via trans- activation of the EGFR [26]. Thus, the involvement of EGFR in ATP-mediated ERK1/2 phosphorylation was References investigated further. Inhibition of the EGFR with the specific inhibitor AG1478 decreased ATP- and UTP- 1. Viveros OH, Diliberto EJ Jr, Daniels AJ (1983) Biochemical and mediated ERK1/2 phosphorylation, strongly suggesting functional evidence for the cosecretion of multiple messengers EGFR transactivation is important for ATP-mediated from single and multiple compartments. Fed Proc 42:2923–2928 2. Reichsman F, Santos S, Westhead EW (1995) Two distinct ATP ERK1/2 phosphorylation. receptors activate calcium entry and internal calcium release in G-protein-coupled receptors may transactivate the EGFR bovine chromaffin cells. J Neurochem 65:2080–2086 via activation of tyrosine kinases such as Src, or via 3. Ennion SJ, Powell AD, Seward EP (2004) Identification of the activation of metalloproteinases to generate EGFR ligands P2Y(12) receptor in nucleotide inhibition of exocytosis from bovine chromaffin cells. Mol Pharmacol 66:601–611 such as HB-EGF [19]. The role of metalloproteinases in 4. Chen XK, Wang LC, Zhou Y, Cai Q, Prakriya M, Duan KL, ATP-mediated ERK1/2 phosphorylation was investigated Sheng ZH, Lingle C, Zhou Z (2005) Activation of GPCRs with broad-spectrum inhibitor GM6001, which significantly modulates quantal size in chromaffin cells through G(betagamma) decreased the response, suggesting that HB-EGF cleavage and PKC. Nat Neurosci 8:1160–1168 330 Purinergic Signalling (2008) 4:323–330 5. Ohta T, Kai T, Ito S (2004) Evidence for paracrine modulation of C (PLC)-beta1 via Galphaq/11 and to PLC-beta3 via Gbetagam- voltage-dependent calcium channels by amperometric analysis in mai3. J Biol Chem 273:4695–4704 cultured porcine adrenal chromaffin cells. Brain Res 1030:183– 17. 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UTP and ATP increase extracellular signal-regulated kinase 1/2 phosphorylation in bovine chromaffin cells through epidermal growth factor receptor transactivation

Purinergic Signalling , Volume 4 (4) – Sep 6, 2008

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© Springer Science+Business Media B.V. 2008
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Abstract

Purinergic Signalling (2008) 4:323–330 DOI 10.1007/s11302-008-9098-y ORIGINAL ARTICLE UTP and ATP increase extracellular signal-regulated kinase 1/2 phosphorylation in bovine chromaffin cells through epidermal growth factor receptor transactivation Toni M. Luke & Terry D. Hexum Received: 25 April 2007 /Accepted: 14 March 2008 / Published online: 6 September 2008 Springer Science + Business Media B.V. 2008 Abstract Adenosine triphosphate (ATP) is coreleased with gest nucleotide-mediated ERK1/2 phosphorylation is medi- catecholamines from adrenal medullary chromaffin cells in ated by a P2Y or P2Y receptor, which stimulates 2 4 response to sympathetic nervous system stimulation and may metalloproteinase-dependent transactivation of the EGFR. regulate these cells in an autocrine or paracrine manner. Increases in extracellular signal-regulated kinase (ERK) 1/2 Keywords Extracellular signal-regulated kinase . . phosphorylation were observed in response to ATP stimula- Phosphorylation Epidermal growth factor receptor . . . tion of bovine chromaffin cells. The signaling pathway Transactivation P2Y receptor P2Y receptor UTP 2 4 involved in ATP-mediated ERK1/2 phosphorylation was investigated via Western blot analysis. ATP and uridine 5′- Abbreviations triphosphate (UTP) increased ERK1/2 phosphorylation po- BACC bovine adrenal chromaffin cells tently, peaking between 5 and 15 min. The mitogen-activated ERK1/2 extracellular signal-regulated kinase 1 and 2 protein kinase (MAPK/ERK)-activating kinase (MEK) in- EGF epidermal growth factor hibitor PD98059 blocked this response. UTP, which is EGFR EGF receptor selective for G-protein-coupled P2Y receptors, was the most MEK mitogen-activated protein kinase/ERK potent agonist among several nucleotides tested. Adenosine kinase 2+ 2+ 5′-O-(3-thio) triphosphate (ATPγS) and ATP were also [Ca ] cytosolic free Ca concentration potent agonists, characteristic of the P2Y or P2Y receptor PKC protein kinase C 2 4 subtypes, whereas agonists selective for P2X receptors or PKA protein kinase A 2+ other P2Y receptor subtypes were weakly effective. The CaMKII Ca /calmodulin-dependent protein kinase II receptor involved was further characterized by the nonspe- PI3K phosphoinositide-3 kinase cific P2 antagonists suramin and reactive blue 2, which each 2-MeSATP 2-methylthio ATP partially inhibited ATP-mediated ERK1/2 phosphorylation. α,β- α,β-methylene ATP Inhibitors of protein kinase C (PKC), protein kinase A meATP 2+ (PKA), Ca /calmodulin-dependent protein kinase II (CaM- HB-EGF heparin-binding EGF-like growth factor KII), and phosphoinositide-3 kinase (PI3K) had no effect on Pyk2 proline-rich tyrosine kinase ATP-mediated ERK1/2 phosphorylation. The Src inhibitor SH3 Src homology 3 PP2, epidermal growth factor receptor (EGFR) inhibitor AG1478, and metalloproteinase inhibitor GM6001 decreased ATP-mediated ERK1/2 phosphorylation. These results sug- Introduction T. M. Luke T. D. Hexum (*) Chromaffin cells are neuroendocrine cells that synthesize and University of Nebraska Medical Center, secrete catecholamines in response to sympathetic nervous 985800 Nebraska Medical Center, system stimulation and therefore participate in regulation of Omaha, NE 68198–5800, USA e-mail: thexum@unmc.edu stress-modified parameters such as heart rate and blood 324 Purinergic Signalling (2008) 4:323–330 pressure. A variety of additional agents are costored and were maintained on six-well plates at a density of 3×10 released along with the catecholamines from the chromaffin cells/well at 37°C with 5% CO . Viability and purity were granules, including neuropeptides such as the enkephalins and verified to be >95% by Trypan blue exclusion and neutral adenosine triphosphate (ATP) [1]. In addition to being re- red staining, respectively. leased into the circulation, these agents may regulate chro- maffin cell activity in an autocrine or a paracrine manner, Western blot analysis Cells were incubated with agonist for allowing the cells to adjust to varying levels of stimulation. 10 min, and inhibitor preincubations were 15 min, unless The role of ATP in chromaffin cell function has not been otherwise indicated. Cells were rinsed with phosphate well defined, even though it is secreted in high concen- buffered saline (PBS) and lysed with 200 μlof50 mM trations from chromaffin cells [1]. It has been suggested N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid that ATP regulates chromaffin cell secretion, either posi- (HEPES) (4-(2-hydroxyethyl)-1-piperazineethanesulfonic tively [2] or negatively [3, 4], and that ATP regulates the acid), pH 7.2, containing 1 mM ethylenediaminetetraacetate function of voltage-dependent calcium channels [5]. ATP (EDTA), 1 mM ethyleneglycoltetraacetic acid (EGTA), exerts its effects through either G-protein-coupled recep- 0.2% triton X-100, 10 mM β-glycerol 2-phosphate diso- tors, designated P2Y; or ion channels, designated P2X. dium salt, 1 mM sodium orthovanadate, 1 mM benzami- These receptor types are further divided into subtypes, dine, 4 μg/ml leupeptin, 1 μM microcystin-LR, and including P2Y , P2Y , P2Y ,P2Y , and P2Y for the 0.5 mM DTT. Lysates were centrifuged at 13,000 g, and 1 2 4 6 11–14 G-protein-coupled P2Y receptors and P2X for those that protein concentrations of supernatants were determined 1–7 activate ion channels [6]. Previous work with chromaffin with the Bio-Rad protein assay (Bio-Rad, Hercules, CA, cells indicated ATP stimulation results in increases in USA). Loading buffer (2X) was added to cell lysates, inositol phosphates [7], cyclic adenosine monophosphate consisting of 125 mM Tris(hydroxymethyl)aminomethane 2+ (cAMP) [8], and [Ca ] accumulation [7], likely via HCl (Research Organics, Cleveland, OH, USA), 4% activation of a P2Y or P2Y receptor, both of which are sodium dodecylsulfate (SDS) (Research Organics, Cleve- 2 4 present in bovine chromaffin cells (unpublished observa- land, OH, USA), 20% glycerol, and 0.02% bromophenol tions). The downstream effects of P2Y receptor stimulation blue. Samples were subsequently boiled and subjected to by ATP in chromaffin cells are not known. The observed SDS-polyacrylamide gel electrophoresis (PAGE) on 10% increases in signaling messengers may bring about the Tris-HCl Criterion gels (BioRad, Hercules, CA, USA). activation of multiple protein kinases or tyrosine kinases. Protein was transferred to fluorescent-polyvinylidene In several cell types, ATP signaling has been shown to fluoride (PVDF) membranes (Millipore, Billerica, MA, activate extracellular signal-regulated kinase 1 and 2 (ERK1/ USA). Membranes were next blocked in Odyssey blocking 2). P2Y receptors have been shown to couple to ERK1/2 buffer (LI-COR Biosciences, Lincoln, NE, USA) for 1 h, activation via activation of protein kinases such as phosphoi- then incubated in Odyssey blocking buffer containing 0.2% nositide-3 kinase (PI3K) [11] or protein kinase C (PKC) [10, Tween 20 (polyoxyethylene-sorbitan monolaurate) and 202 204 11]. P2Y receptor-mediated ERK1/2 has also been shown to mouse-anti-phospho-ERK 1/2 (Thr /Tyr ) and rabbit- be dependent on activation of tyrosine kinases such as Src or anti-ERK 1/2 (Cell Signaling Technology, Boston, MA, proline-rich tyrosine kinase (Pyk2) [9, 10] and/or on trans- USA) primary antibodies. Blots were then incubated in activation of the epidermal growth factor receptor (EGFR) Odyssey blocking buffer with 0.2% Tween 20 and 0.02% [9]. Additionally, increases in cAMP in response to ATP may SDS and with the secondary antibodies goat-anti-mouse result in activation of protein kinase A (PKA), which has immunoglobulin (Ig)G Alexa Fluor680 (Molecular Probes, 2+ been shown to activate ERK1/2 [12]. Increases in [Ca ] in Eugene, OR, USA) and goat-anti-rabbit IgG IR800 (Rock- 2+ response to ATP may result in activation of Ca /calmodulin- land Immunochemicals, Gilbertsville, PA, USA). Mem- dependent protein kinase II (CaMKII), which has also been branes were developed with the Odyssey Infrared Imaging shown to phosphorylate ERK1/2 [13]. Therefore, we elected System, which utilizes two infrared channels (700 nm and to examine ATP-mediated ERK1/2 phosphorylation and the 800 nm), allowing for detection of two target proteins receptor subtype and signaling mechanism present in bovine simultaneously, in this case phosphorylated and total chromaffin cells. ERK1/2. Statistics Band integrated intensities were determined with Materials and methods Odyssey Imaging software. Phospho-ERK1/2 intensities were divided by total ERK1/2 intensities and normalized to Chromaffin cell isolation and cell culture Bovine adrenal fold increases over control. Data were analyzed with chromaffin cells (BACC) were isolated using a collagenase GraphPad Prism software, and one-way analysis of vari- perfusion method as described previously [14, 15]. Cells ance (ANOVA) was utilized to determine statistical signif- Purinergic Signalling (2008) 4:323–330 325 pERK chlorophenyl)-7-(t-butyl)pyrazolo-D-3,4-pyrimidine], AG1478 [4-(3-Chloroanilino)-6,7-dimethoxyquinazoline], ERK and GM6001 [N-[(2R)-2-(hydroxamidocarbonylmethyl)-4- methylpentanoyl]-L-tryptophan methylamide] were pur- ATP-+ + -+-- - + + 2 min 5 min 10 min 15 min 30 min chased from Biomol (Philadelphia, PA, USA). Bis-I (bisindo- lylmaleimide-I), KN-92 [2-[N-(4-methoxybenzenesulfonyl)] pERK amino-N-(4-chlorocinnamyl)- N-methylbenzylamine], KN- ERK 93 [2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine], and UTP - + - + - + - + - + 2 min 5 min 10 min 15 min 30 min wortmannin were purchased from Merck-Calbiochem Bio- sciences (Darmstadt, Germany). H89 [N-[2-(p-Bromocinna- mylamino)ethyl]-5-isoquinolinesulfonamide] was obtained from Upstate (Charlottesville, VA, USA).Other reagents were obtained from either Sigma-Aldrich or Fisher Scientific. Results Western blot analyses with antibodies specific to activated 202 204 ERK1/2 phosphorylated at Thr /Tyr were utilized to examine the time course of ERK1/2 phosphorylation in response to nucleotide stimulation. Both ATP and UTP Fig. 1 Nucleotide-stimulated ERK1/2 phosphorylation is time depen- dent. BACCs were treated with ATP (100 μM) or UTP (100 μM) for 2 potently increased ERK1/2 phosphorylation, with a peak to 30 min. Blots are representative of three independent experiments between 5 and 15 min (Fig. 1). ERK1/2 has been shown to performed in triplicate (n=3); the upper band (phosphorylated or nonphosphorylated) is ERK1=44 kDa, and the lower band is ERK2= 42 kDa. Blot intensities were measured with the Odyssey Imaging Overlay Overlay System; values are phosphorylated ERK2 intensity divided by total ERK2 intensity. Points on the graph represent mean ± standard error pERK pERK of the mean ERK ERK - + - + icance. The decision was made to utilize ERK2 for UTP - + - + AT P No Inhibitor PD98059 graphical representations, as band intensities for phosphor- No Inhibitor PD98059 ylated ERK2 were stronger than phosphorylated ERK1, though results obtained were quantitatively similar for both. Chemicals Nucleotides and analogs were obtained from Sigma-Aldrich (St. Louis, MO, USA). PD98059 (2′-amino- 3′-methoxylflavone), PMA (phorbol 12-myristate 13-acetate), *** suramin, and reactive blue 2 (RB2) were also obtained from Sigma-Aldrich. NF279 [8,8′-[Carbonylbis(imino-4,1- *** phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis- 1,3,5-naphthalenetrisulfonic acid hexasodium salt] was pur- chased from Tocris (St. Louis, MO, USA). KT5720 Fig. 2 MEK inhibition decreases ATP- and UTP-mediated ERK1/2 [(9S,10S,12R)-2,3,9,10,11,12-Hexahydro-10-hydroxy-9- phosphorylation. BACCs were treated with PD98059 (10 μM) or methyl-1-oxo-9, 12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl] dimethylsulfoxide for 15 min, followed by a 10-min stimulation with pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ATP (100 μM) or UTP (100 μM). Blots are representative of three ester], Ro-31–8220 [2-[1-(3-(Amidinothio)propyl)-1H- independent experiments performed in triplicate (n=3); the upper band (phosphorylated or nonphosphorylated) is ERK1=44 kDa and indol-3-yl]-3-(1-methylindol-3-yl)maleimide methanesulfo- the lower band is ERK2=42 kDa. Blot intensities were measured with nate], LY294002 ]2-(4- morpholinyl)-8-phenyl-4H-1- the Odyssey Imaging System; values are phosphorylated ERK2 benzopyran-4-one], PP1 [4-amino-5-(4-methylphenyl)-7- intensity divided by total ERK2 intensity. Bars on the graph represent (t-butyl)pyrazolo-D-3,4-pyrimidine], PP2 [4-amino-5-(4- mean ± standard error of the mean. *** p<0.001 vs. stimulator alone 326 Purinergic Signalling (2008) 4:323–330 be catalyzed only by MEK; therefore, the MEK inhibitor receptor subtypes. Moreover, UTP is selective for P2Y PD98059 was utilized to confirm the immediate upstream receptors, precluding the involvement of a P2X receptor in signaling event responsible for nucleotide-mediated ERK1/ nucleotide-mediated ERK1/2 phosphorylation. 2 phosphorylation. PD98059 (10 μM) significantly de- ATP stimulation of chromaffin cells has been shown to 2+ creased both ATP-mediated (∼90%) and UTP-mediated increase inositol phosphates [7], cAMP [8], and [Ca ] (∼70%, Fig. 2, Table 1) ERK1/2 phosphorylation. accumulation [7], which may lead to activation of several Most studies designed to determine purinergic receptor protein kinases, which may in turn be involved in ATP- subtypes use ligand potency studies due to the lack of mediated phosphorylation of ERK1/2. Therefore, a variety available highly selective receptor subtype antagonists [16]. of inhibitors were used to examine which kinases are Correspondingly, we used a similar approach to character- involved in ATP-mediated ERK1/2 phosphorylation. The ize the receptor subtype involved in ERK1/2 phosphoryla- PKA inhibitors H89 (10 μM) and KT5720 (100 nM) had tion. Examination of several purine analogs revealed a rank no effect on ATP- or UTP-mediated ERK1/2 phosphoryla- order of potency of UTP (EC =1.6 μM)>ATPγS tion (Table 1). The broad-spectrum PKC inhibitors Ro-31– (6.5 μM)≥ATP (13 μM)>uridine diphosphate (UDP) 8220 (10 μM) and Bis-I (3.5 μM) also had no effect on (120 μM) = adenosine diphosphate (ADP) (220 μM)= 2- ATP- or UTP-mediated ERK1/2 phosphorylation, although methylthio (ATP)2-MeSATP (320 μM)>>α,β-methylene they were capable of blocking PMA-mediated ERK1/2 ATP (α,β-meATP) (Fig. 3), consistent with the involve- phosphorylation (∼60%, Table 1). Moreover, the CaMKII ment of a P2Y or P2Y receptor, as both UTP and ATP inhibitor KN93 (1 μM) and the PI3K inhibitors wortmannin 2 4 exhibit strong agonist action. (300 nM) and LY294002 (20 μM) had no effect on ATP- or The involvement of a P2 receptor in ERK1/2 phosphor- UTP-mediated ERK1/2 phosphorylation (Table 1). ylation was further supported using the nonselective P2 In addition to activation of PKs, P2Y receptors may receptor antagonists suramin and RB2. Suramin (100 μM) utilize tyrosine kinases to activate ERK1/2 [10]. Therefore, significantly decreased ATP- or UTP-mediated ERK1/2 the tyrosine kinase inhibitor PP2 was used to examine the phosphorylation (∼60%, Fig. 4, Table 1). RB2 (100 μM) role of Src family members in ATP-mediated ERK1/2 also decreased the effect of ATP- or UTP-stimulation on phosphorylation. PP2 (1 μM) decreased ATP- or UTP- ERK1/2 phosphorylation (∼35%, Fig. 4, Table 1). The P2X- mediated ERK2 phosphorylation (∼40%, Fig. 5, Table 1). specific receptor agonist α,β-meATP had no effect on In addition to inhibiting Src family members, PP2 is also a ERK1/2 phosphorylation at concentrations up to 100 μM weak inhibitor of the EGFR. Also, G-protein-coupled (Fig. 3), eliminating the involvement of several of the P2X receptor activation of Src family members may result in Table 1 Involvement of signaling pathways in ATP- and UTP-mediated ERK1/2 phosphorylation Inhibitor/antagonist ATP UTP ERK1 ERK2 ERK1 ERK2 P2R Suramin (100 μM) 61.8±3.1*** 54.7±3.8*** 62.0±5.7*** 62.0±5.7*** Antagonists RB2 (100 μM) 32.3±4.5*** 29.5±4.5*** 44.9±10.7*** 44.9±9.1*** MEK inhibitor PD98059 (10 μM) 95.3±5.3*** 94.4±4.5*** 69.5±3.4*** 64.3±4.2*** PKC inhibitors Bis-I (3.5 μM) 12.7±20.5 4.6±7.9 5.2±12.9 −1.3±10.4 Bis-I-PMA 76.1±2.9*** 67.5±3.2*** –– Ro-31-8220 (10 μM) −40.0±28.8 −13.5±10.2 −13.4±14.9 −8.7±10.9 Ro-31-8220-PMA 66.6±2.6*** 54.4±2.3*** –– PKA inhibitors H89 (10 μM) −67.1±36.3 −42.4±14.4 −27.5±31.4 −16.4±16.5 KT5720 (100 nM) −1.5±13.3 −1.9±16.5 1.9±13.9 6.5±13.2 CaMKII inhibitor KN93 (1 μM) 22.5±27.2 7.1±12.7 −20.1±18.2 −14.2±15.4 PI3K inhibitors Wortmannin −10.0±34.2 −3.9±15.7 10.0±25.9 12.5±14.6 (300 nM) LY294002 (20 μM) −3.9±15.7 1.0±17.4 18.6±18.7 30.3±12.1 Src inhibitor PP2 (1 μM) 40.9±16.1* 39.5±9.1*** 46.0±8.1*** 32.4±9.6*** EGFR inhibitors AG1478 (2.6 μM) 73.7±2.0*** 71.1±2.3*** 62.9±7.2*** 69.6±6.7*** MMP inhibitors GM6001 (2.5 μM) 69.2±5.8** 60.8±7.1*** 66.7±10.1*** 62.0±10.4*** Cells were pretreated with inhibitors for 15 min then stimulated with 100 μM UTP (or 1 μM PMA) for 10 min. Values are percent inhibition of UTP-mediated ERK1/2 phosphorylation ± standard error of the mean for three experiments in triplicate. Bis-I-PMA and Ro-31-8220-PMA refer to using PMA as the stimulator rather than UTP *p<0.05 vs. stimulator alone,**p<0.01 vs. stimulator alone, **p<0.001 vs. stimulator alone Purinergic Signalling (2008) 4:323–330 327 ERK1/2 phosphorylation in response to ATP would allow Overlay the cells to respond quickly to varying levels of stimulation. Although the physiological effects of ERK1/2 phosphory- pERK lation in these cells are unknown, possible actions requiring a rapid response include either the acute activation of ERK proteins involved in catecholamine secretion and/or stimu- Con 100 nM 1 µM3 µM 10 µM 30 µM 100 µM 1 mM lation of protein expression important for exocytosis. ATP Ligand potency and inhibitor studies suggest either the P2Y or P2Y receptor subtype is responsible for nucleo- 2 4 tide-mediated ERK1/2 phosphorylation, similar to data obtained for increases in inositol phosphates (unpublished observations). Both of these receptor subtypes are present in chromaffin cells, based on reverse transcriptase real-time polymerase chain reaction (PCR) data for P2Y and P2Y , 2 4 and appear to be expressed in these cells according to Western blot analysis with specific antibodies (unpublished Overlay pERK Fig. 3 P2Y or P2Y receptor activation increases ERK1/2 phos- 2 4 ERK phorylation. BACCs were treated with increasing concentrations of -+ + -- + ATP nucleotides and analogs for 10 min. Blots are representative of three No Inhibitor Suramin RB2 independent experiments performed in triplicate (n=3); the upper band (phosphorylated or nonphosphorylated) is ERK1=44 kDa, and Overlay the lower band is ERK2=42 kDa. Blot intensities were measured with the Odyssey Imaging System; values are phosphorylated ERK2 intensity divided by total ERK2 intensity. Points on the graph pERK represent mean ± standard error of the mean. Con (control) refers to results obtained with unstimulated cells ERK -+ + -- UTP No Inhibitor Suramin RB2 activation of ERK1/2 via activation of Ras [17, 18] or via transactivation of the EGFR. Therefore, the involvement of EGFR in ATP-mediated ERK1/2 phosphorylation was determined by treating cells with the EGFR inhibitor AG1478 (2.6 μM), which decreased ATP- and UTP- *** mediated ERK2 phosphorylation by about 70% (Fig. 6a, *** Table 1). EGF-mediated ERK1/2 phosphorylation was *** *** completely blocked by AG1478 (100%, p<0.001, data not shown). Transactivation of the EGFR by G-protein-coupled receptors may be mediated by tyrosine kinases such as Src or via metalloproteinases, which release EGFR ligands such as heparin-binding EGF-like growth factor (HB-EGF) from Fig. 4 P2 receptor antagonists partially block ATP- and UTP- the cell membrane [19]. The metalloproteinase inhibitor mediated ERK1/2 phosphorylation. BACCs were pretreated with or GM6001 (2.5 μM) decreased ATP- and UTP-mediated without suramin (100 μM) or reactive blue 2 (RB2, 100 μM) for 15 ERK1/2 phosphorylation by about 65% (Fig. 6b, Table 1). min, followed by a 10-min stimulation with or without ATP (100 μM) or UTP (100 μM). Blots are representative of three independent experiments performed in triplicate (n=3); the upper band (phosphor- ylated or nonphosphorylated) is ERK1=44 kDa, and the lower band is Discussion ERK2=42 kDa. Blot intensities were measured with the Odyssey Imaging System; values are phosphorylated ERK2 intensity divided ATP and UTP potently increase ERK1/2 phosphorylation, by total ERK2 intensity. Bars on graph represent mean ± standard with a peak between 5 and 15 min. This rapid peak in error of the mean. *** p<0.001 vs. stimulator alone 328 Purinergic Signalling (2008) 4:323–330 Overlay Overlay P2Y-mediated ERK1/2 phosphorylation [10, 11]. In PC12 cells, ERK1/2 phosphorylation in response to P2Y pERK pERK receptor activation has been shown to be both dependent [9, 10] and independent [21] of the small tyrosine kinase ERK ERK Pyk2. P2Y receptors have also been shown to contain an - + - + 2 - + - + ATP UTP integrin-binding domain, arginine-glycine-aspartic acid No Inhibitor PP2 No Inhibitor PP2 (RGD), which is necessary for ERK1/2 activation in astrocytes [22, 23]. Additionally, P2Y receptors contain SH3-binding sites that associate with Src in astrocytoma cells [24] and astrocytes [23]. Also, in PC12 cells P2Y receptors have been shown to require EGFR transactivation *** *** to increase ERK1/2 phosphorylation [9]. Initially, we examined whether signaling pathways medi- ated by protein kinases were involved in ATP-mediated Overlay pERK Fig. 5 Tyrosine kinase inhibition decreases ATP- and UTP-mediated ERK1/2 phosphorylation. BACCs were pretreated with PP2 (1 μM) or ERK dimethylsulfoxide (DMSO) for 15 min, followed by a 10-min stimulation - ATP UTP - ATP UTP with or without ATP (100 μM) or UTP (100 μM). Blots are No Inhibitor AG1478 representative of three independent experiments performed in triplicate (n=3); the upper band (phosphorylated or nonphosphorylated) is ERK1 =44 kDa, and the lower band is ERK2=42 kDa. Blot intensities were measured with the Odyssey Imaging System; values are phosphorylated ERK2 intensity divided by total ERK2 intensity. Bars on the graphs *** represent mean ± standard error of the mean. *** p<0.001 vs. *** stimulator alone observations.) Several lines of evidence suggest P2X receptors are not involved in the increase in ERK1/2 in Overlay Overlay response to nucleotide stimulation. First, UTP does not pERK activate P2X ion channels but potently increases ERK1/2 pERK phosphorylation. Additionally, α,βmeATP, an agonist se- ERK ERK lective for several P2X receptor subtypes, had no effect on UTP - + - + - + - + ATP ERK1/2 phosphorylation. The P2Y receptor involved is No Inhibitor GM6001 No Inhibitor GM6001 most likely either P2Y or P2Y . UTP is highly selective 2 4 for two P2Y receptor subtypes P2Y and P2Y and weakly 2 4 effective on the P2Y receptor. The P2Y subtype can be 6 6 ruled out because of the subtypes activated by UTP; only the P2Y and P2Y subtypes are also strongly activated by *** 2 4 *** ATP. The weak effect of ADP, UDP and 2-MeSATP confirms this designation, as these agonists are specific for P2 receptor subtypes other than the P2Y or P2Y 2 4 subtypes [6]. There are no available agonists or antagonists Fig. 6 Nucleotide-mediated ERK1/2 phosphorylation is dependent on EGFR transactivation. BACCs were pretreated with or without to distinguish between the P2Y and P2Y receptors. Even 2 4 AG1478 (2.6 μM) (a), GM6001 (2.5 μM) (b) and dimethylsulfoxide so, suramin and RB2 are commonly used to characterize (DMSO) for 15 min, followed by a 10-min stimulation with or without these receptors in a given cell type, and their partial ATP (100 μM) or UTP (100 μM). Blots are representative of three effectiveness is not contradictory to results found in other independent experiments performed in triplicate (n=3); the upper band (phosphorylated or nonphosphorylated) is ERK1=44 kDa ,and cell types for P2Y or P2Y receptors [20]. 2 4 the lower band is ERK2=42 kDa. Blot intensities were measured with Nucleotides utilize multiple signaling pathways in the Odyssey Imaging System; values are phosphorylated ERK2 different cell types to bring about increases in ERK1/2 intensity divided by total ERK2 intensity. Bars on the graphs represent phosphorylation. PKC and PI3K have been implicated in mean ± standard error of the mean. *** p<0.001 vs. stimulator alone Purinergic Signalling (2008) 4:323–330 329 ERK1/2 phosphorylation. Previous studies determined that mediated by metalloproteinases in response to ATP may be ATP-mediated stimulation of bovine chromaffin cells results responsible for transactivation of the EGFR and subse- 2+ in increases in inositol phosphates [7], cAMP [8], and [Ca ] quently stimulation of ERK1/2 phosphorylation. accumulation [7]. P2Y or P2Y receptors couple to G As expected, the MEK inhibitor PD98059 blocked ATP- 2 4 q to increase activation of PKC. Additionally, the observed and UTP-mediated ERK1/2 phosphorylation, confirming increases in cAMP may result in activation of PKA, whereas that MEK, the only known kinase upstream of ERK1/2, 2+ increased [Ca ] may result in activation of CaMKII. contributes to ERK1/2 phosphorylation. PD98059 blocked However, inhibitors of each of these protein kinases had no ATP-mediated ERK1/2 phosphorylation (∼90%) to a effect on ATP- or UTP-mediated ERK1/2 phosphorylation, greater extent than UTP-mediated ERK1/2 phosphorylation including the PKA inhibitors H89 and KT5720, PKC (∼70%). This may be due to the fact that UTP is a more inhibitors Bis-I and Ro-81–3220, or the CaMKII inhibitor potent agonist and elicited a larger response than ATP for KN93. The PKC inhibitors Bis-I and Ro-81–3220 were ERK1/2 phosphorylation. For the other inhibitors, the capable of blocking PMA-mediated ERK1/2 phosphoryla- responses were very similar; however, none of the other tion, suggesting PKC can couple to ERK1/2 phosphorylation inhibitors had such a pronounced effect on ATP-mediated in these cells, and yet confirming the lack of involvement of ERK1/2 phosphorylation. Alternatively, ATP and UTP may this pathway in ATP-mediated ERK1/2 phosphorylation. We activate multiple receptors with distinct signaling pathways also examined the PI3K inhibitors wortmannin and that are variously more specific for ATP or UTP. Activation LY294002, as this kinase has been implicated in P2Y- of ERK1/2 independent of MEK1 may involve inhibition of mediated ERK1/2 phosphorylation. These inhibitors also phosphatases. proved to be ineffective, suggesting the protein kinases Further studies are necessary to determine the conse- examined were not responsible for ATP-mediated ERK1/2 quence(s) of ERK1/2 phosphorylation in response to ATP phosphorylation. stimulation of chromaffin cells. As chromaffin cells are We next examined the involvement of tyrosine kinases nonproliferating, the stimulation of ERK1/2 phosphoryla- in ATP-mediated ERK1/2 phosphorylation, as these kinases tion by ATP and UTP may couple to regulation of gene have been shown to be involved in P2Y-mediated ERK1/2 transcription essential to exocytosis. phosphorylation. PP2 (1 μM) significantly decreased ATP- To our knowledge this is the first study demonstrating and UTP-mediated ERK1/2 phosphorylation. The reported phosphorylation of ERK1/2 in response to ATP or UTP IC s for PP2-mediated inhibition of Src family members stimulation in bovine chromaffin cells. Our data show that are in the nanomolar range (http://www.biomol.com), the ERK1/2 phosphorylation response to ATP is mediated whereas the dose used in these studies is reported to cause by either a P2Y or P2Y receptor. Protein kinases are not 2 4 weak inhibition of the EGFR [25]. Lower doses of PP2 had involved in nucleotide-mediated ERK1/2 phosphorylation, no effect on ATP- or UTP-mediated ERK1/2 phosphoryla- but rather, metalloproteinase-dependent transactivation of tion (data not shown.) Therefore, at the effective dose used, the EGFR is necessary for ATP-mediated ERK1/2 phos- it is not possible to conclude whether Src or EGFR phorylation. inhibition was responsible for the decrease in ATP- Acknowledgment We thank Matthew Beaver for excellent technical mediated ERK1/2 phosphorylation. Additionally, activation assistance and Robin Taylor for expert assistance with graphics. This of Src family members by G-protein-coupled receptors may work was supported by a grant to Terry D. 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Purinergic SignallingPubmed Central

Published: Sep 6, 2008

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