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Nucleoplasmic Calcium Is Required for Cell Proliferation *

Nucleoplasmic Calcium Is Required for Cell Proliferation * THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 23, pp. 17061–17068, June 8, 2007 © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Received for publication, January 17, 2007, and in revised form, April 6, 2007 Published, JBC Papers in Press, April 9, 2007, DOI 10.1074/jbc.M700490200 ‡§ ‡ § ¶ ¶ ¶ Michele A. Rodrigues , Dawidson A. Gomes , M. Fatima Leite , Wayne Grant , Lei Zhang , Wing Lam , ¶ ¶ ‡1 Yung-Chi Cheng , Anton M. Bennett , and Michael H. Nathanson ‡ ¶ From the Departments of Medicine and Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8019 and the Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil 31270-901 Ca signals regulate cell proliferation, but the spatial and ture Collection (Manassas, VA) and were used for all experi- temporal specificity of these signals is unknown. Here we use ments. The cells were grown at 37 °C with 5% CO :95% O in 2 2 selective buffers of nucleoplasmic or cytoplasmic Ca to deter- Dulbecco’s modified Eagle’s medium supplemented with 1% mine that cell proliferation depends upon Ca signals within penicillin-streptomycin and 10% heat-inactivated fetal bovine the nucleus rather than in the cytoplasm. Nuclear Ca signals serum, all from Invitrogen. The cells were grown on glass cov- stimulate cell growth rather than inhibit apoptosis and specifi- erslips overnight in the absence of serum before infection with cally permit cells to advance through early prophase. Selective each parvalbumin (PV) construct. buffering of nuclear but not cytoplasmic Ca signals also Generation of Parvalbumin Constructs and Adenoviral impairs growth of tumors in vivo. These findings reveal a major Infection—Constructs encoding red fluorescence protein physiological and potential pathophysiological role for nucleo- (DsRed) from Clontech (Mountain View, CA) and targeted PV plasmic Ca signals and suggest that this information can be proteins (PV-NLS, PV-NES, and PV-NLS-CD) were PCR-am- used to design novel therapeutic strategies to regulate condi- plified and subcloned into pShuttle-CMV (kindly provided by tions of abnormal cell growth. Bert Vogelstein, Johns Hopkins) by restriction digestion with XhoI and XbaI to generate pShuttle-CMV-PVNLS-DSR, pShuttle-CMV-PVNES-DSR, and pShuttle-CMV-PVNLS-CD- Ca is a ubiquitous second messenger that mediates a wide DSR. Recombinant adenoviruses were generated by transfor- range of cellular responses such as contraction, fluid and elec- mation of pShuttle-CMV-PV-NLS-DSR into AdEasier-1 cells, a trolyte secretion, exocytosis, gene transcription, and apoptosis derivative of BJ5183 bacteria already containing the adenovirus (1). This ability to simultaneously control multiple processes backbone plasmid pAdEasy-1. Positive recombinant adenovi- occurs by careful modulation of Ca signals, not only over ruses (pAd-PV-NLS-DSR, etc.) were screened for resistance to time but in different subcellular regions as well (2). For exam- kanamycin (Invitrogen) and restriction enzyme analysis using ple, polarized Ca waves direct apical secretion in epithelia (3), the PacI enzyme. Parvalbumin adenoviruses were amplified whereas presynaptic increases in Ca trigger neurotransmit- using HEK-293 cells and then purified (10, 17). ter release (4) and mitochondrial increases in Ca regulate Measurement of BrdUrd Incorporation—Cell proliferation apoptosis (5, 6). Ca signals also can be regulated independ- was measured by BrdUrd incorporation using an enzyme- ently in the nucleoplasm relative to the cytoplasm (7, 8), but the linked immunosorbent assay (Roche Applied Science) accord- physiological significance of this aspect of spatial control is not ing to the manufacturer’s instructions. SKHep1 cells infected entirely understood. Nucleoplasmic Ca signals have distinct with parvalbumin constructs or HepG2 cells stably transduced effects on activation of transcription factors (9, 10) and kinases with inducible parvalbumin constructs were plated 48 h later in (11, 12), but it is not known whether nuclear Ca signals also 96-well culture plates (BD Biosciences, San Diego, CA) and regulate more global aspects of cell function. Because cell pro- treated for 2 h with BrdUrd labeling solution. The cells were liferation (13, 14) and progression through the cell cycle (15, 16) then fixed, and anti-BrdUrd antibody was added. A colorimet- are Ca -dependent, we investigated the relative roles of ric substrate was used, and BrdUrd incorporation was meas- nuclear and cytoplasmic Ca on cell growth. ured with a multiplate reader. Cell Counting Assay—SKHep1 cells (10 cells/ml) were EXPERIMENTAL PROCEDURES plated on 35-mm dishes without serum and infected with tar- Materials, Reagents, and Cell Lines—SKHep1, HepG2, and geted PV constructs. The cells were counted with trypan blue HEK-293 cell lines were obtained from the American Type Cul- (Invitrogen) 24, 48, and 72 h after infection. Apoptosis Assay—Two methods were used for measuring apoptosis. Caspase-3 enzyme activity was assayed using a * This work was supported by National Institutes of Health Grants DK57751, DK34989, and DK45710 and by grants from Howard Hughes Medical Insti- caspase-3 activity kit with colorimetric detection (BD Bio- tute, Conselho Nacional de Desenvolvimento Cientifico e Tecnologico; sciences) according to the manufacturer’s instructions. Alter- and Fundacao de Amparo a Pesquisa do Estado de Minas Gerais. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: PV, parvalbumin; BrdUrd, bromodeoxyuri- To whom correspondence should be addressed: Digestive Diseases, Rm. dine; PBS, phosphate-buffered saline; ERK, extracellular signal-regu- TAC S241D, Yale University School of Medicine, New Haven, CT 06520- lated kinase; CREB, cAMP-responsive element-binding protein; NLS, 8019. Tel.: 203-785-7312; Fax: 203-785-4306; E-mail: michael.nathanson@ nuclear localization sequence; NES, nuclear export sequence; InsP3, yale.edu. inositol 1,4,5-trisphosphate. JUNE 8, 2007• VOLUME 282 • NUMBER 23 JOURNAL OF BIOLOGICAL CHEMISTRY 17061 This is an Open Access article under the CC BY license. 2 Nuclear Ca and Cell Growth natively, the cells were loaded with 0.2 g/ml of the nuclear doxycycline was added to the drinking water. Tumor measure- stain Hoechst 33258 (Molecular Probes, Eugene, OR), and then ment began when the tumor dimension reached 5  4mm chromatin condensation and nuclear fragmentation were (15 days after injection of the cells). HepG2 tumors were assessed by confocal microscopy. Apoptosis was induced using measured daily using a caliper, and the body weights of the mice 500 nM staurosporine (Sigma-Aldrich) as a positive control. were monitored as well. Tumor volume V was estimated by Cell Cycle Analysis—Cells infected with targeted PV con- the formula V  (length)  (width) /2 (20, 21). 2 2 structs were trypsinized, washed in phosphate-buffered saline Ca Measurements—Nuclear and cytosolic Ca were (PBS), fixed overnight with ice-cold 70% EtOH at 4 °C, and then monitored in SKHep1 cells using time lapse confocal micros- washed in staining buffer (Dulbecco’s PBS with 2% fetal calf copy (8, 12). SKHep1 cells were cultured on glass coverslips, serum and 0.01% NaN ). After centrifugation the cells were incubated for 30 min in the presence of 6 M Fluo-4/AM to supplemented with RNase (100 g/ml) for 30 min at 37 °C and monitor nuclear or cytosolic Ca , and then transferred to a stained with propidium iodide (50 g/ml) at room temperature perfusion chamber on the stage of a Zeiss LSM 510 confocal in the dark. DNA content was determined using a FACSCalibur microscope (Thornwood, NY). The cells were maintained in a (BD Biosciences), and the data were analyzed using Flowjo soft- HEPES-buffered solution during experiments and observed ware version 5.5. using a 63, 1.4 numerical aperture objective lens. The 488-nm Immunoblotting—Standard methods were used for immuno- line of a krypton/argon laser was used to excite the dye, and blots (8). Briefly, cells grown in 35-mm dishes were washed emission signals between 505 and 550 nm were collected. The three times with PBS and solubilized in 80 l of Nonidet P-40 cells were stimulated with 100 nM vasopressin (Sigma), and the containing a protease inhibitor mixture (Roche Applied Sci- images were acquired at a rate of 2–10 frames/s. Neither ence). The protein concentration was determined spectropho- autofluorescence nor background signals were detectable at the tometrically, and 30 g of protein was separated by electro- machine settings used. phoresis in a 10% polyacrylamide gel and then transferred to a Statistical Analysis—Changes in proliferation were deter- polyvinylidene difluoride membrane. The membrane was mined by one-way analysis of variance. Significance of changes blocked with 5% skim milk in PBST (PBS plus 0.1% Tween 20) in treatment groups relative to controls were determined by t for 60 min and then incubated with primary antibody (1:500). test. Significance of changes in the size of individual tumors Commercially available antibodies for cyclin D1 (Stressgen, over time was determined by two-way analysis of variance. The Victoria, Canada), cyclin E, B1, phospho-cdk1 and -cdk1 (BD data are represented as the means  S.E. Biosciences), p15, p21, p27, cdK4, phospho-cdc25c, and total RESULTS cdc25c (Cell Signaling, Danvers, MA) and p-ERK, total ERK, ERK-5, total AKT, p-AKT, total CREB, and p-CREB (Cell Sig- Selective Buffering of Nuclear or Cytoplasmic Ca —The rel- naling) were used. Incubations were carried out overnight. ative role of nuclear and cytoplasmic Ca signals in specific After three washes with TBST, the membranes were incu- cell functions can be determined by selectively attenuating bated with peroxidase-conjugated secondary antibody Ca increases in either the nucleus or cytoplasm. The first (1:5000) for1hat room temperature. The bands were studies in this area used microinjection of dextran-linked Ca revealed by enhanced chemiluminescence (ECL plus; Amer- buffers into either the nucleus or cytoplasm (9, 22), but a more sham Biosciences). The films were scanned with a GS-700 efficient approach was subsequently developed using targeted imaging densitometer (Bio-Rad). expression of Ca buffering proteins such as PV or calretinin Mitotic Index Measurements and Immunofluorescence—The in these compartments (10, 23). This approach used constructs cells were labeled for phospho-histone-3 (Upstate Biotechnol- encoding PV targeted to the nucleus with a nuclear localization ogy, Inc., Chicago, IL) and -tubulin (Sigma-Aldrich) by immu- sequence (PV-NLS) or to the cytoplasm with a nuclear export nofluorescence and then examined using a Zeiss LSM 510 con- sequence (PV-NES). To examine the effects of targeted Ca focal microscope (8, 12). Mitotic figures were scored for cells in buffering in entire cell populations, we delivered these con- each phase of mitosis according to the phospho-histone-3 dis- structs to SKHep1 cells using an adenovirus vector to achieve tribution and DNA condensation (18, 19). high transfection efficiencies. The PV constructs were fused to Establishment of Inducible Tet-On HepG2 Cell Line—The DsRed to monitor expression and subcellular localization, inducible Tet-On HepG2 cell line system was established by which demonstrated the efficacy of the adenoviral vector (Fig. using the PV-NLS and PV-NES constructs previously reported 1A). Consistent with previously published data (10, 23) time (20, 21), along with the Tet-On Gene expression system lapse confocal imaging confirmed that expression of PV-NLS (Invitrogen). and PV-NES was able to locally attenuate by50% nuclear and Animal Studies—Male 4-week-old NCR nude mice (average cytoplasmic Ca signals, respectively, in response to stimula- body weight, 20 g) were obtained from Taconic Farms (Hudson, tion with vasopressin (Fig. 1B). NY) and acclimated to laboratory conditions 1 week before Nuclear Ca Controls Cell Proliferation—To determine the tumor implantation. Nude mice were maintained in accord- involvement of nuclear and cytoplasmic Ca in cell prolifera- ance with the Institutional Animal Care and Use Committee tion, SKHep1 cells were synchronized in G and then infected procedures and guidelines. HepG2 tumor xenografts were with the targeted PV constructs. The cells were stimulated to established by subcutaneous injection of 5  10 HepG2 cells/ re-enter the cell cycle by the addition of serum. BrdUrd uptake site in each mouse (pcDNA5-TO-PV-NLS-DsRed, PV-NES- was reduced by 63  17% in cells expressing PV-NLS, com- DsRed or DsRed). To express PV constructs, 100 g/ml of pared with infected and uninfected cells (p  0.001), whereas 17062 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 23 •JUNE 8, 2007 2 Nuclear Ca and Cell Growth FIGURE 2. Cell growth depends on nuclear rather than cytosolic Ca . A, BrdUrd incorporation is decreased in SKHep1 cells expressing PV in the nucleus but not the cytoplasm. *, p  0.001. BrdUrd incorporation decreases by an intermediate amount in cells expressing the CD mutant of PV in the nucleus, in which one of the two Ca -binding sites is disrupted. BrdUrd uptake is not decreased in DsRed adenoviral infection controls. The results are representative of what was observed in three separate experiments. B, cell growth curves of SKHep1 cells 24, 48, and 72 h after infection with targeted PV constructs confirm that proliferation is reduced by expression of PV-NLS but not PV-NES. no reduction in BrdUrd uptake was seen in cells expressing PV-NES or in adenoviral infection controls (Fig. 2A). BrdUrd uptake was reduced to an intermediate degree (28  17%; p 0.001) in cells infected with a mutant PV-NLS in which one of two Ca -binding sites was disrupted (Fig. 2A). This PV-NLS-CD mutant is known to have an intermediate effect on inhibition of nuclear Ca signals, relative to PV-NLS (10). Cell proliferation in response to serum stimulation in PV-NLS- and expression and subcellular location. An adenoviral delivery system is used for each construct. Confocal microscopy demonstrates very high transfection rates and that PV-NLS-DsRed is expressed in the nucleus, PV-NES-DsRed is expressed in the cytoplasm, and DsRed alone, which serves as a transfection control, is expressed in both compartments. In each image, red indicates DsRed, blue indicates the nuclear stain Hoechst, and purple indicates co-local- ization of the two labels. B, representative tracings of nuclear and cytoplasmic Ca signals in single SKHep1 cells stimulated with vasopressin (100 nM). The tracings represent the percentage of increase in fluorescence relative to base line in each compartment (F 100% (F F )/F ). The cells were examined 0 0 48 h after infection with the respective adenoviral constructs. Ca was mon- itored with fluo-4 using time lapse confocal microscopy (8). PV-NLS-DsRed attenuates vasopressin-induced increases in nucleoplasmic but not cytoplas- mic Ca , whereas PV-NES-DsRed attenuates vasopressin-induced increases 2 2 in cytoplasmic but not nucleoplasmic Ca .Ca signals were not attenuated FIGURE 1. Adenoviral delivery of the Ca buffer protein PV targeted to in either compartment in cells expressing DsRed alone. Similar results were the nucleus or cytoplasm. A, subcellular localization of targeted PV con- observed in at least 11 cells in each group (p  0.005; data not shown), con- structs. NLS or NES sequences are used to target PV to the nucleus or cyto- firming the effects of these constructs on Ca signaling that have been plasm, respectively, and each construct is tagged with DsRed to monitor reported previously (10). JUNE 8, 2007• VOLUME 282 • NUMBER 23 JOURNAL OF BIOLOGICAL CHEMISTRY 17063 2 Nuclear Ca and Cell Growth ied 48 h later. In populations expressing PV-NLS, there was a significant (p  0.001) reduction in the fraction of cells in G phase (58  2%), but an increase in the fraction of cells in the S (14  1%) and G /M (27  1%) phases relative to uninfected controls (82  0.3%, 5  0.1%, and 12  0.4% in the G ,S,and G /M phases, respectively). No such change was observed in cells expressing PV-NES or in infected controls (Fig. 4A). These findings suggest that buffering nuclear Ca specifically blocks either the G /M transition or mitosis (25, 26). Progression through the cell cycle is regulated at various checkpoints by activation of specific complexes of cyclins and cyclin-dependent kinases (27, 28). These checkpoint proteins ensure that events comprising each phase of the cell cycle have completed before the next phase is initiated (27). To determine whether expression of PV-NLS was specifically affecting the G /M transition, we examined expression of the G /M check- 2 2 point proteins cyclin B1 and cdk1. The expression of total cyclin B1 and cdk1 were unchanged, but phosphorylation of cdk1 was decreased (Fig. 4B). Because dephosphorylation activates rather than inhibits cdk1, these results suggest that the inhibi- tory effects of PV-NLS occur after rather than at the G /M transition. To confirm that PV-NLS expression does not inhibit progression through earlier phases of the cell cycle, we investi- gated expression of the G /S and S/G checkpoint proteins cyc- 1 2 lins D1/cdk4 and cyclin E/cdk2, respectively. Buffering nuclear Ca did not alter expression of any of these checkpoint pro- teins (Fig. 4, B and C). Similarly, buffering nuclear Ca was associated with no change in the expression of the cyclin D1/cdk4 inhibitor p15 and decreased expression of the cyclin E/cdk2 inhibitor p27 and the cyclin B1/cdk1 inhibitor p21 (Fig. 4C). Interestingly, both expression and phosphorylation of the cdk1 phosphatase cdc25c was decreased in cells expressing PV- NLS (Fig. 4C), even though the net effect in these cells was FIGURE 3. Buffering nuclear or cytoplasmic Ca does not induce apopto- sis. A, expression of the indicated PV constructs does not induce apoptosis, as dephosphorylation of cdk1 (Fig. 4B), which perhaps reflects measured by caspase-3 activity. Caspase-3 activity was measured in control that multiple factors contribute to the regulation of cdk1 activ- and infected SKHep1 cells. The cells were treated with staurosporine (ST; 500 ity (29, 30). As an additional way to examine the effects of nM) as a positive control. *, p 0.001 relative to every other group; n 4 for all groups. B, SKHep1 cells were loaded with the nuclear dye Hoechst 33258 (0.2 nuclear Ca on cell proliferation, the effect of PV-NLS on the g/ml), and then chromatin condensation and nuclear fragmentation were growth-related kinases ERK1/2, ERK5, and AKT and the tran- assessed by confocal microscopy. Hoechst nuclear labeling confirms that scription factor CREB were examined. Buffering cytoplasmic apoptosis is not induced by buffering nuclear Ca (n 4 for each condition). Staurosporine (500 nM) was used as a positive control. *, p 0.001 relative to rather than nuclear Ca decreased the level of p-ERK1/2, and every other group. PV-NLS had no effect on total expression of ERK1/2, ERK5, or AKT (Fig. 4D). However, expression of PV-NLS decreased PV-NES-expressing cells was also measured directly by cell p-CREB (Fig. 4D), consistent with previous reports that this counting. Like BrdUrd uptake, cell proliferation was reduced by transcription factor is activated by nuclear rather than cytosolic 2 2 buffering nuclear but not cytoplasmic Ca (Fig. 2B). To deter- Ca (9). Together, these observations suggest that nuclear mine whether the apparent decrease in cell proliferation was Ca acts during rather than before mitosis to regulate cell due in part to an increase in cell death, apoptosis was monitored proliferation. using caspase-3 activation (24) (Fig. 3A) as well as Hoechst Nuclear Ca Controls Progression through Mitosis—To labeling of the nucleus (19) (Fig. 3B). No increase in apoptosis investigate the effects of nuclear and cytoplasmic Ca on was observed in any infected group (Fig. 3B). Together, these mitosis, cells were labeled with the mitotic marker phospho- findings demonstrate that cell proliferation is dependent upon histone-3 and the centrosome marker -tubulin 48 h after increases in Ca that occur in the nucleus but not the infection with the various PV constructs. Labeled cells were cytoplasm. examined by confocal microscopy (Fig. 5A) to determine the Buffering Nuclear Ca Alters Cell Cycle Kinetics—We used fraction of cells in mitosis. The mitotic index was doubled in flow cytometry analysis to understand which phases of the cell cells in which nuclear Ca was buffered (p 0.001) and was no 2 2 cycle are regulated by nuclear Ca . The cells were synchro- different from controls in cells in which cytoplasmic Ca was nized in G , then released into the cell cycle by addition of buffered (Fig. 5B). Examination of the phospho-histone-3 pat- serum, infected with adenoviral PV constructs, and then stud- tern in cells expressing PV-NLS furthermore suggested that 17064 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 23 •JUNE 8, 2007 2 Nuclear Ca and Cell Growth we examined the effects of nuclear and cytoplasmic Ca signals on the growth of tumors implanted into nude mice. The HepG2 tumor cell line was stably transfected with PV- NLS-DsRed, PV-NES-DsRed, or DsRed alone, each under the control of a doxycycline-sensitive promoter (Fig. 6A). BrdUrd uptake in these cells was reduced by over 60% (p 0.001) by expression of PV in the nucleus but not the cytosol (Fig. 6B), similar to what was observed in SkHep1 cells (Fig. 2A). These HepG2 cells were implanted into the subcutaneous space, and then tumor growth was measured over a two-week period with or without administration of doxycycline in the drinking water (20, 21). Immuno- blot analysis of tumors removed 2 weeks after implantation confirmed expression of the targeted PV con- structs in doxycycline-treated ani- mals and not in untreated controls (Fig. 6C). Tumor growth was signif- icantly impaired by expression of PV-NLS-DsRed (p  0.001) but not by expression of PV-NES-DsRed or DsRed alone (Fig. 7). Direct exami- nation of resected tumors (not shown) suggested an even greater inhibition of tumor growth by PV- NLS-DsRed than was appreciated FIGURE 4. Buffering nuclear Ca suggests a block in late G or M phase. A, fluorescence activated cell sorting analysis of DNA content performed 48 h after infection with the indicated PV constructs or controls. In by in vivo measurements, perhaps cells expressing PV-NLS-DsRed, there is an 8 and 16% increase in the fraction of cells in S and G phase, because caliper measurements in- respectively, and a 24% decrease in cells in G , suggesting a block in late G or M phase. Cell cycle profiles are 1 2 not changed in cells expressing PV-NES-DsRed. For G , p  0.001 for control or DsRed versus NLS. For S, p  clude overlying skin and associated 0.001 for control or DsRed versus NLS. For G2, p 0.001 for control or DsRed versus NLS; n 6 experiments for tissues as well. These findings con- each group. B, immunoblot analysis of cells infected with the indicated PV constructs demonstrates decreased firm the role of nuclear rather than phospho-cdk1 but no change in expression of cell cycle checkpoint proteins cdk1/cdk2 and cyclins B1, D1, and E. -Actin loading controls were the same for each column. C, immunoblot analysis demonstrates no change in cytoplasmic Ca signals in the reg- expression of the cyclin dependent kinase inhibitor p15 or cdk4 but a decrease in p21, p27, cdc25C, and ulation of cell growth. phospho-cdc25c in cells infected with PV-NLS. D, immunoblot analysis demonstrates that expression of PV- NES decreases phosphorylation of ERK1/2, whereas PV-NLS decreases phosphorylation of CREB. DISCUSSION It is well appreciated that increases in Ca occur in both the cytoplasm and the nucleus. these cells were in early prophase, and -tubulin staining simi- Initial evidence suggested that nucleoplasmic Ca passively larly demonstrated that only a single centrosome was present in these cells (Fig. 5C). Analysis of the phospho-histone-3 staining follows changes in cytoplasmic Ca (31, 32). However, subse- pattern in all mitotic cells within each treatment group con- quent work established that the nucleus contains distinct Ca firmed that expression of PV-NLS induced a block in early stores (7), including a nucleoplasmic reticulum (12), plus phos- phatidylinositol (4,5)biphosphate (33), phospholipase C (34), prophase (Fig. 5D). Specifically, 27  2% of cells expressing PV-NLS were in prophase, and none were in metaphase or and InsP3 receptors (12, 35). Together, these factors provide anaphase, whereas in control cells 12  1% were in prophase, the machinery necessary to permit nucleoplasmic Ca to be 2  0.3% were in metaphase, and 3  0.3% were in anaphase regulated independently from cytoplasmic Ca (8). The signif- icance of this machinery within the nucleus has long been a (p  0.001). These results demonstrate that nuclear Ca is necessary specifically for centrosome separation and progres- question. Both growth factors (12) and integrins (36) can pref- sion through early prophase. erentially increase nucleoplasmic Ca , consistent with the 2 2 Nuclear Ca Is Needed for Tumor Growth in Vivo—To idea that nucleoplasmic Ca would be more important than understand the potential significance of these findings in vivo, cytoplasmic Ca to regulate cell proliferation. Increases in JUNE 8, 2007• VOLUME 282 • NUMBER 23 JOURNAL OF BIOLOGICAL CHEMISTRY 17065 2 Nuclear Ca and Cell Growth 17066 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 23 •JUNE 8, 2007 2 Nuclear Ca and Cell Growth FIGURE 6. Regulated expression of targeted PV constructs in a tumor cell line. A, immunoblot analysis of the engineered HepG2 stable cell lines after treatment using the indicated concentrations (62.5–1000 ng/ml) of doxycy- cline to induce expression of PV-NLS or PV-NES. B, BrdUrd incorporation is decreased in HepG2 cells expressing PV in the nucleus but not the cytoplasm. *, p 0.001. BrdUrd uptake is not decreased in controls that inducibly express DsRed. The results are representative of what was observed in three separate experiments. C, the phenotype of the engineered HepG2 cells is retained after implantation into nude mice. Immunoblot analysis of tumors removed 2 FIGURE 7.NuclearCa regulatescellgrowth in vivo. Nuclear but not cytoplas- weeks after implantation confirms parvalbumin expression is preserved in mic Ca buffers retard tumor growth. HepG2 tumor cells expressing PV-NES- PV-NLS and PV-NES but not DsRed tumors and only in tumors from animals DsRed, PV-NLS-DsRed, or DsRed under the control of a doxycycline-sensitive pro- treated with doxycycline. The slight difference in molecular weight between moter were implanted subcutaneously into nude mice. Serial measurements of PV-NLS and PV-NES tumors reflects differences in weight caused by the tar- tumor volume in the presence or absence of doxycycline (100 g/ml, added to geting sequences. the drinking water) were obtained, starting when tumor volume reached 5 4 mm . The data represent the means S.E. of tumor volume over time (n 5–15 mice in each group), normalized with respect to the initial volume. *, p 0.001. InsP3 (16, 37), plus temporally related increases in Ca in the perinuclear region (26, 38, 39), have been associated with breakdown of the nuclear envelope and entry into mitosis. In activates other transcription factors such as SRE (9), plus phos- addition, nuclear Ca activates transcription factors such as phatases such as calcineurin that are important for transcrip- CREB (9, 11), Elk-1 (10), and DREAM (40), but cytosolic Ca tion (41). Therefore, the relative importance of nuclear and FIGURE 5. Nuclear Ca regulates progression through early prophase. A, representative images of SKHep1 cells in early and late prophase, pro-metaphase, metaphase, and anaphase. These confocal immunofluorescence images were obtained after staining with anti-phospho-histone-3 (green) and anti--tubulin (pink)to label each phase of mitosis, plus Hoechst (blue) to label the nucleus. B, the mitotic index is increased in cells in which nuclear Ca is buffered. *, p 0.001. Mitotic SKHep-1 cells were identified by histone-3 labeling, measured 48 h after infection with the indicated PV constructs. A total of 200 cells were examined in each experiment, and each experiment was conducted six times. C, representative image of a mitotic SKHep1 cell in which nuclear Ca is buffered with PV-NLS-DsRed. Serial confocal sections (one of which is shown here) reveal a single centrosome by -tubulin staining, and mitotic figures illustrative of early prophase by histone-3 staining. D, mitotic index, subdivided to demonstrate the fraction of cells in each phase of mitosis, demonstrates that cells in which nuclear Ca is buffered do not progress beyond prophase. *, p 0.001. A total of 200 cells were examined in each experiment, and the experiments were performed in quadruplicate. JUNE 8, 2007• VOLUME 282 • NUMBER 23 JOURNAL OF BIOLOGICAL CHEMISTRY 17067 2 Nuclear Ca and Cell Growth 10. Pusl, T., Wu, J. J., Zimmerman, T. L., Zhang, L., Ehrlich, B. 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Med. 111, 127–148 17068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 23 •JUNE 8, 2007 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 23, pp. 17061–17068, June 8, 2007 © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Received for publication, January 17, 2007, and in revised form, April 6, 2007 Published, JBC Papers in Press, April 9, 2007, DOI 10.1074/jbc.M700490200 ‡§ ‡ § ¶ ¶ ¶ Michele A. Rodrigues , Dawidson A. Gomes , M. Fatima Leite , Wayne Grant , Lei Zhang , Wing Lam , ¶ ¶ ‡1 Yung-Chi Cheng , Anton M. Bennett , and Michael H. Nathanson ‡ ¶ From the Departments of Medicine and Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8019 and the Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil 31270-901 Ca signals regulate cell proliferation, but the spatial and ture Collection (Manassas, VA) and were used for all experi- temporal specificity of these signals is unknown. Here we use ments. The cells were grown at 37 °C with 5% CO :95% O in 2 2 selective buffers of nucleoplasmic or cytoplasmic Ca to deter- Dulbecco’s modified Eagle’s medium supplemented with 1% mine that cell proliferation depends upon Ca signals within penicillin-streptomycin and 10% heat-inactivated fetal bovine the nucleus rather than in the cytoplasm. Nuclear Ca signals serum, all from Invitrogen. The cells were grown on glass cov- stimulate cell growth rather than inhibit apoptosis and specifi- erslips overnight in the absence of serum before infection with cally permit cells to advance through early prophase. Selective each parvalbumin (PV) construct. buffering of nuclear but not cytoplasmic Ca signals also Generation of Parvalbumin Constructs and Adenoviral impairs growth of tumors in vivo. These findings reveal a major Infection—Constructs encoding red fluorescence protein physiological and potential pathophysiological role for nucleo- (DsRed) from Clontech (Mountain View, CA) and targeted PV plasmic Ca signals and suggest that this information can be proteins (PV-NLS, PV-NES, and PV-NLS-CD) were PCR-am- used to design novel therapeutic strategies to regulate condi- plified and subcloned into pShuttle-CMV (kindly provided by tions of abnormal cell growth. Bert Vogelstein, Johns Hopkins) by restriction digestion with XhoI and XbaI to generate pShuttle-CMV-PVNLS-DSR, pShuttle-CMV-PVNES-DSR, and pShuttle-CMV-PVNLS-CD- Ca is a ubiquitous second messenger that mediates a wide DSR. Recombinant adenoviruses were generated by transfor- range of cellular responses such as contraction, fluid and elec- mation of pShuttle-CMV-PV-NLS-DSR into AdEasier-1 cells, a trolyte secretion, exocytosis, gene transcription, and apoptosis derivative of BJ5183 bacteria already containing the adenovirus (1). This ability to simultaneously control multiple processes backbone plasmid pAdEasy-1. Positive recombinant adenovi- occurs by careful modulation of Ca signals, not only over ruses (pAd-PV-NLS-DSR, etc.) were screened for resistance to time but in different subcellular regions as well (2). For exam- kanamycin (Invitrogen) and restriction enzyme analysis using ple, polarized Ca waves direct apical secretion in epithelia (3), the PacI enzyme. Parvalbumin adenoviruses were amplified whereas presynaptic increases in Ca trigger neurotransmit- using HEK-293 cells and then purified (10, 17). ter release (4) and mitochondrial increases in Ca regulate Measurement of BrdUrd Incorporation—Cell proliferation apoptosis (5, 6). Ca signals also can be regulated independ- was measured by BrdUrd incorporation using an enzyme- ently in the nucleoplasm relative to the cytoplasm (7, 8), but the linked immunosorbent assay (Roche Applied Science) accord- physiological significance of this aspect of spatial control is not ing to the manufacturer’s instructions. SKHep1 cells infected entirely understood. Nucleoplasmic Ca signals have distinct with parvalbumin constructs or HepG2 cells stably transduced effects on activation of transcription factors (9, 10) and kinases with inducible parvalbumin constructs were plated 48 h later in (11, 12), but it is not known whether nuclear Ca signals also 96-well culture plates (BD Biosciences, San Diego, CA) and regulate more global aspects of cell function. Because cell pro- treated for 2 h with BrdUrd labeling solution. The cells were liferation (13, 14) and progression through the cell cycle (15, 16) then fixed, and anti-BrdUrd antibody was added. A colorimet- are Ca -dependent, we investigated the relative roles of ric substrate was used, and BrdUrd incorporation was meas- nuclear and cytoplasmic Ca on cell growth. ured with a multiplate reader. Cell Counting Assay—SKHep1 cells (10 cells/ml) were EXPERIMENTAL PROCEDURES plated on 35-mm dishes without serum and infected with tar- Materials, Reagents, and Cell Lines—SKHep1, HepG2, and geted PV constructs. The cells were counted with trypan blue HEK-293 cell lines were obtained from the American Type Cul- (Invitrogen) 24, 48, and 72 h after infection. Apoptosis Assay—Two methods were used for measuring apoptosis. Caspase-3 enzyme activity was assayed using a * This work was supported by National Institutes of Health Grants DK57751, DK34989, and DK45710 and by grants from Howard Hughes Medical Insti- caspase-3 activity kit with colorimetric detection (BD Bio- tute, Conselho Nacional de Desenvolvimento Cientifico e Tecnologico; sciences) according to the manufacturer’s instructions. Alter- and Fundacao de Amparo a Pesquisa do Estado de Minas Gerais. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: PV, parvalbumin; BrdUrd, bromodeoxyuri- To whom correspondence should be addressed: Digestive Diseases, Rm. dine; PBS, phosphate-buffered saline; ERK, extracellular signal-regu- TAC S241D, Yale University School of Medicine, New Haven, CT 06520- lated kinase; CREB, cAMP-responsive element-binding protein; NLS, 8019. Tel.: 203-785-7312; Fax: 203-785-4306; E-mail: michael.nathanson@ nuclear localization sequence; NES, nuclear export sequence; InsP3, yale.edu. inositol 1,4,5-trisphosphate. JUNE 8, 2007• VOLUME 282 • NUMBER 23 JOURNAL OF BIOLOGICAL CHEMISTRY 17061 This is an Open Access article under the CC BY license. 2 Nuclear Ca and Cell Growth natively, the cells were loaded with 0.2 g/ml of the nuclear doxycycline was added to the drinking water. Tumor measure- stain Hoechst 33258 (Molecular Probes, Eugene, OR), and then ment began when the tumor dimension reached 5  4mm chromatin condensation and nuclear fragmentation were (15 days after injection of the cells). HepG2 tumors were assessed by confocal microscopy. Apoptosis was induced using measured daily using a caliper, and the body weights of the mice 500 nM staurosporine (Sigma-Aldrich) as a positive control. were monitored as well. Tumor volume V was estimated by Cell Cycle Analysis—Cells infected with targeted PV con- the formula V  (length)  (width) /2 (20, 21). 2 2 structs were trypsinized, washed in phosphate-buffered saline Ca Measurements—Nuclear and cytosolic Ca were (PBS), fixed overnight with ice-cold 70% EtOH at 4 °C, and then monitored in SKHep1 cells using time lapse confocal micros- washed in staining buffer (Dulbecco’s PBS with 2% fetal calf copy (8, 12). SKHep1 cells were cultured on glass coverslips, serum and 0.01% NaN ). After centrifugation the cells were incubated for 30 min in the presence of 6 M Fluo-4/AM to supplemented with RNase (100 g/ml) for 30 min at 37 °C and monitor nuclear or cytosolic Ca , and then transferred to a stained with propidium iodide (50 g/ml) at room temperature perfusion chamber on the stage of a Zeiss LSM 510 confocal in the dark. DNA content was determined using a FACSCalibur microscope (Thornwood, NY). The cells were maintained in a (BD Biosciences), and the data were analyzed using Flowjo soft- HEPES-buffered solution during experiments and observed ware version 5.5. using a 63, 1.4 numerical aperture objective lens. The 488-nm Immunoblotting—Standard methods were used for immuno- line of a krypton/argon laser was used to excite the dye, and blots (8). Briefly, cells grown in 35-mm dishes were washed emission signals between 505 and 550 nm were collected. The three times with PBS and solubilized in 80 l of Nonidet P-40 cells were stimulated with 100 nM vasopressin (Sigma), and the containing a protease inhibitor mixture (Roche Applied Sci- images were acquired at a rate of 2–10 frames/s. Neither ence). The protein concentration was determined spectropho- autofluorescence nor background signals were detectable at the tometrically, and 30 g of protein was separated by electro- machine settings used. phoresis in a 10% polyacrylamide gel and then transferred to a Statistical Analysis—Changes in proliferation were deter- polyvinylidene difluoride membrane. The membrane was mined by one-way analysis of variance. Significance of changes blocked with 5% skim milk in PBST (PBS plus 0.1% Tween 20) in treatment groups relative to controls were determined by t for 60 min and then incubated with primary antibody (1:500). test. Significance of changes in the size of individual tumors Commercially available antibodies for cyclin D1 (Stressgen, over time was determined by two-way analysis of variance. The Victoria, Canada), cyclin E, B1, phospho-cdk1 and -cdk1 (BD data are represented as the means  S.E. Biosciences), p15, p21, p27, cdK4, phospho-cdc25c, and total RESULTS cdc25c (Cell Signaling, Danvers, MA) and p-ERK, total ERK, ERK-5, total AKT, p-AKT, total CREB, and p-CREB (Cell Sig- Selective Buffering of Nuclear or Cytoplasmic Ca —The rel- naling) were used. Incubations were carried out overnight. ative role of nuclear and cytoplasmic Ca signals in specific After three washes with TBST, the membranes were incu- cell functions can be determined by selectively attenuating bated with peroxidase-conjugated secondary antibody Ca increases in either the nucleus or cytoplasm. The first (1:5000) for1hat room temperature. The bands were studies in this area used microinjection of dextran-linked Ca revealed by enhanced chemiluminescence (ECL plus; Amer- buffers into either the nucleus or cytoplasm (9, 22), but a more sham Biosciences). The films were scanned with a GS-700 efficient approach was subsequently developed using targeted imaging densitometer (Bio-Rad). expression of Ca buffering proteins such as PV or calretinin Mitotic Index Measurements and Immunofluorescence—The in these compartments (10, 23). This approach used constructs cells were labeled for phospho-histone-3 (Upstate Biotechnol- encoding PV targeted to the nucleus with a nuclear localization ogy, Inc., Chicago, IL) and -tubulin (Sigma-Aldrich) by immu- sequence (PV-NLS) or to the cytoplasm with a nuclear export nofluorescence and then examined using a Zeiss LSM 510 con- sequence (PV-NES). To examine the effects of targeted Ca focal microscope (8, 12). Mitotic figures were scored for cells in buffering in entire cell populations, we delivered these con- each phase of mitosis according to the phospho-histone-3 dis- structs to SKHep1 cells using an adenovirus vector to achieve tribution and DNA condensation (18, 19). high transfection efficiencies. The PV constructs were fused to Establishment of Inducible Tet-On HepG2 Cell Line—The DsRed to monitor expression and subcellular localization, inducible Tet-On HepG2 cell line system was established by which demonstrated the efficacy of the adenoviral vector (Fig. using the PV-NLS and PV-NES constructs previously reported 1A). Consistent with previously published data (10, 23) time (20, 21), along with the Tet-On Gene expression system lapse confocal imaging confirmed that expression of PV-NLS (Invitrogen). and PV-NES was able to locally attenuate by50% nuclear and Animal Studies—Male 4-week-old NCR nude mice (average cytoplasmic Ca signals, respectively, in response to stimula- body weight, 20 g) were obtained from Taconic Farms (Hudson, tion with vasopressin (Fig. 1B). NY) and acclimated to laboratory conditions 1 week before Nuclear Ca Controls Cell Proliferation—To determine the tumor implantation. Nude mice were maintained in accord- involvement of nuclear and cytoplasmic Ca in cell prolifera- ance with the Institutional Animal Care and Use Committee tion, SKHep1 cells were synchronized in G and then infected procedures and guidelines. HepG2 tumor xenografts were with the targeted PV constructs. The cells were stimulated to established by subcutaneous injection of 5  10 HepG2 cells/ re-enter the cell cycle by the addition of serum. BrdUrd uptake site in each mouse (pcDNA5-TO-PV-NLS-DsRed, PV-NES- was reduced by 63  17% in cells expressing PV-NLS, com- DsRed or DsRed). To express PV constructs, 100 g/ml of pared with infected and uninfected cells (p  0.001), whereas 17062 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 23 •JUNE 8, 2007 2 Nuclear Ca and Cell Growth FIGURE 2. Cell growth depends on nuclear rather than cytosolic Ca . A, BrdUrd incorporation is decreased in SKHep1 cells expressing PV in the nucleus but not the cytoplasm. *, p  0.001. BrdUrd incorporation decreases by an intermediate amount in cells expressing the CD mutant of PV in the nucleus, in which one of the two Ca -binding sites is disrupted. BrdUrd uptake is not decreased in DsRed adenoviral infection controls. The results are representative of what was observed in three separate experiments. B, cell growth curves of SKHep1 cells 24, 48, and 72 h after infection with targeted PV constructs confirm that proliferation is reduced by expression of PV-NLS but not PV-NES. no reduction in BrdUrd uptake was seen in cells expressing PV-NES or in adenoviral infection controls (Fig. 2A). BrdUrd uptake was reduced to an intermediate degree (28  17%; p 0.001) in cells infected with a mutant PV-NLS in which one of two Ca -binding sites was disrupted (Fig. 2A). This PV-NLS-CD mutant is known to have an intermediate effect on inhibition of nuclear Ca signals, relative to PV-NLS (10). Cell proliferation in response to serum stimulation in PV-NLS- and expression and subcellular location. An adenoviral delivery system is used for each construct. Confocal microscopy demonstrates very high transfection rates and that PV-NLS-DsRed is expressed in the nucleus, PV-NES-DsRed is expressed in the cytoplasm, and DsRed alone, which serves as a transfection control, is expressed in both compartments. In each image, red indicates DsRed, blue indicates the nuclear stain Hoechst, and purple indicates co-local- ization of the two labels. B, representative tracings of nuclear and cytoplasmic Ca signals in single SKHep1 cells stimulated with vasopressin (100 nM). The tracings represent the percentage of increase in fluorescence relative to base line in each compartment (F 100% (F F )/F ). The cells were examined 0 0 48 h after infection with the respective adenoviral constructs. Ca was mon- itored with fluo-4 using time lapse confocal microscopy (8). PV-NLS-DsRed attenuates vasopressin-induced increases in nucleoplasmic but not cytoplas- mic Ca , whereas PV-NES-DsRed attenuates vasopressin-induced increases 2 2 in cytoplasmic but not nucleoplasmic Ca .Ca signals were not attenuated FIGURE 1. Adenoviral delivery of the Ca buffer protein PV targeted to in either compartment in cells expressing DsRed alone. Similar results were the nucleus or cytoplasm. A, subcellular localization of targeted PV con- observed in at least 11 cells in each group (p  0.005; data not shown), con- structs. NLS or NES sequences are used to target PV to the nucleus or cyto- firming the effects of these constructs on Ca signaling that have been plasm, respectively, and each construct is tagged with DsRed to monitor reported previously (10). JUNE 8, 2007• VOLUME 282 • NUMBER 23 JOURNAL OF BIOLOGICAL CHEMISTRY 17063 2 Nuclear Ca and Cell Growth ied 48 h later. In populations expressing PV-NLS, there was a significant (p  0.001) reduction in the fraction of cells in G phase (58  2%), but an increase in the fraction of cells in the S (14  1%) and G /M (27  1%) phases relative to uninfected controls (82  0.3%, 5  0.1%, and 12  0.4% in the G ,S,and G /M phases, respectively). No such change was observed in cells expressing PV-NES or in infected controls (Fig. 4A). These findings suggest that buffering nuclear Ca specifically blocks either the G /M transition or mitosis (25, 26). Progression through the cell cycle is regulated at various checkpoints by activation of specific complexes of cyclins and cyclin-dependent kinases (27, 28). These checkpoint proteins ensure that events comprising each phase of the cell cycle have completed before the next phase is initiated (27). To determine whether expression of PV-NLS was specifically affecting the G /M transition, we examined expression of the G /M check- 2 2 point proteins cyclin B1 and cdk1. The expression of total cyclin B1 and cdk1 were unchanged, but phosphorylation of cdk1 was decreased (Fig. 4B). Because dephosphorylation activates rather than inhibits cdk1, these results suggest that the inhibi- tory effects of PV-NLS occur after rather than at the G /M transition. To confirm that PV-NLS expression does not inhibit progression through earlier phases of the cell cycle, we investi- gated expression of the G /S and S/G checkpoint proteins cyc- 1 2 lins D1/cdk4 and cyclin E/cdk2, respectively. Buffering nuclear Ca did not alter expression of any of these checkpoint pro- teins (Fig. 4, B and C). Similarly, buffering nuclear Ca was associated with no change in the expression of the cyclin D1/cdk4 inhibitor p15 and decreased expression of the cyclin E/cdk2 inhibitor p27 and the cyclin B1/cdk1 inhibitor p21 (Fig. 4C). Interestingly, both expression and phosphorylation of the cdk1 phosphatase cdc25c was decreased in cells expressing PV- NLS (Fig. 4C), even though the net effect in these cells was FIGURE 3. Buffering nuclear or cytoplasmic Ca does not induce apopto- sis. A, expression of the indicated PV constructs does not induce apoptosis, as dephosphorylation of cdk1 (Fig. 4B), which perhaps reflects measured by caspase-3 activity. Caspase-3 activity was measured in control that multiple factors contribute to the regulation of cdk1 activ- and infected SKHep1 cells. The cells were treated with staurosporine (ST; 500 ity (29, 30). As an additional way to examine the effects of nM) as a positive control. *, p 0.001 relative to every other group; n 4 for all groups. B, SKHep1 cells were loaded with the nuclear dye Hoechst 33258 (0.2 nuclear Ca on cell proliferation, the effect of PV-NLS on the g/ml), and then chromatin condensation and nuclear fragmentation were growth-related kinases ERK1/2, ERK5, and AKT and the tran- assessed by confocal microscopy. Hoechst nuclear labeling confirms that scription factor CREB were examined. Buffering cytoplasmic apoptosis is not induced by buffering nuclear Ca (n 4 for each condition). Staurosporine (500 nM) was used as a positive control. *, p 0.001 relative to rather than nuclear Ca decreased the level of p-ERK1/2, and every other group. PV-NLS had no effect on total expression of ERK1/2, ERK5, or AKT (Fig. 4D). However, expression of PV-NLS decreased PV-NES-expressing cells was also measured directly by cell p-CREB (Fig. 4D), consistent with previous reports that this counting. Like BrdUrd uptake, cell proliferation was reduced by transcription factor is activated by nuclear rather than cytosolic 2 2 buffering nuclear but not cytoplasmic Ca (Fig. 2B). To deter- Ca (9). Together, these observations suggest that nuclear mine whether the apparent decrease in cell proliferation was Ca acts during rather than before mitosis to regulate cell due in part to an increase in cell death, apoptosis was monitored proliferation. using caspase-3 activation (24) (Fig. 3A) as well as Hoechst Nuclear Ca Controls Progression through Mitosis—To labeling of the nucleus (19) (Fig. 3B). No increase in apoptosis investigate the effects of nuclear and cytoplasmic Ca on was observed in any infected group (Fig. 3B). Together, these mitosis, cells were labeled with the mitotic marker phospho- findings demonstrate that cell proliferation is dependent upon histone-3 and the centrosome marker -tubulin 48 h after increases in Ca that occur in the nucleus but not the infection with the various PV constructs. Labeled cells were cytoplasm. examined by confocal microscopy (Fig. 5A) to determine the Buffering Nuclear Ca Alters Cell Cycle Kinetics—We used fraction of cells in mitosis. The mitotic index was doubled in flow cytometry analysis to understand which phases of the cell cells in which nuclear Ca was buffered (p 0.001) and was no 2 2 cycle are regulated by nuclear Ca . The cells were synchro- different from controls in cells in which cytoplasmic Ca was nized in G , then released into the cell cycle by addition of buffered (Fig. 5B). Examination of the phospho-histone-3 pat- serum, infected with adenoviral PV constructs, and then stud- tern in cells expressing PV-NLS furthermore suggested that 17064 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 23 •JUNE 8, 2007 2 Nuclear Ca and Cell Growth we examined the effects of nuclear and cytoplasmic Ca signals on the growth of tumors implanted into nude mice. The HepG2 tumor cell line was stably transfected with PV- NLS-DsRed, PV-NES-DsRed, or DsRed alone, each under the control of a doxycycline-sensitive promoter (Fig. 6A). BrdUrd uptake in these cells was reduced by over 60% (p 0.001) by expression of PV in the nucleus but not the cytosol (Fig. 6B), similar to what was observed in SkHep1 cells (Fig. 2A). These HepG2 cells were implanted into the subcutaneous space, and then tumor growth was measured over a two-week period with or without administration of doxycycline in the drinking water (20, 21). Immuno- blot analysis of tumors removed 2 weeks after implantation confirmed expression of the targeted PV con- structs in doxycycline-treated ani- mals and not in untreated controls (Fig. 6C). Tumor growth was signif- icantly impaired by expression of PV-NLS-DsRed (p  0.001) but not by expression of PV-NES-DsRed or DsRed alone (Fig. 7). Direct exami- nation of resected tumors (not shown) suggested an even greater inhibition of tumor growth by PV- NLS-DsRed than was appreciated FIGURE 4. Buffering nuclear Ca suggests a block in late G or M phase. A, fluorescence activated cell sorting analysis of DNA content performed 48 h after infection with the indicated PV constructs or controls. In by in vivo measurements, perhaps cells expressing PV-NLS-DsRed, there is an 8 and 16% increase in the fraction of cells in S and G phase, because caliper measurements in- respectively, and a 24% decrease in cells in G , suggesting a block in late G or M phase. Cell cycle profiles are 1 2 not changed in cells expressing PV-NES-DsRed. For G , p  0.001 for control or DsRed versus NLS. For S, p  clude overlying skin and associated 0.001 for control or DsRed versus NLS. For G2, p 0.001 for control or DsRed versus NLS; n 6 experiments for tissues as well. These findings con- each group. B, immunoblot analysis of cells infected with the indicated PV constructs demonstrates decreased firm the role of nuclear rather than phospho-cdk1 but no change in expression of cell cycle checkpoint proteins cdk1/cdk2 and cyclins B1, D1, and E. -Actin loading controls were the same for each column. C, immunoblot analysis demonstrates no change in cytoplasmic Ca signals in the reg- expression of the cyclin dependent kinase inhibitor p15 or cdk4 but a decrease in p21, p27, cdc25C, and ulation of cell growth. phospho-cdc25c in cells infected with PV-NLS. D, immunoblot analysis demonstrates that expression of PV- NES decreases phosphorylation of ERK1/2, whereas PV-NLS decreases phosphorylation of CREB. DISCUSSION It is well appreciated that increases in Ca occur in both the cytoplasm and the nucleus. these cells were in early prophase, and -tubulin staining simi- Initial evidence suggested that nucleoplasmic Ca passively larly demonstrated that only a single centrosome was present in these cells (Fig. 5C). Analysis of the phospho-histone-3 staining follows changes in cytoplasmic Ca (31, 32). However, subse- pattern in all mitotic cells within each treatment group con- quent work established that the nucleus contains distinct Ca firmed that expression of PV-NLS induced a block in early stores (7), including a nucleoplasmic reticulum (12), plus phos- phatidylinositol (4,5)biphosphate (33), phospholipase C (34), prophase (Fig. 5D). Specifically, 27  2% of cells expressing PV-NLS were in prophase, and none were in metaphase or and InsP3 receptors (12, 35). Together, these factors provide anaphase, whereas in control cells 12  1% were in prophase, the machinery necessary to permit nucleoplasmic Ca to be 2  0.3% were in metaphase, and 3  0.3% were in anaphase regulated independently from cytoplasmic Ca (8). The signif- icance of this machinery within the nucleus has long been a (p  0.001). These results demonstrate that nuclear Ca is necessary specifically for centrosome separation and progres- question. Both growth factors (12) and integrins (36) can pref- sion through early prophase. erentially increase nucleoplasmic Ca , consistent with the 2 2 Nuclear Ca Is Needed for Tumor Growth in Vivo—To idea that nucleoplasmic Ca would be more important than understand the potential significance of these findings in vivo, cytoplasmic Ca to regulate cell proliferation. Increases in JUNE 8, 2007• VOLUME 282 • NUMBER 23 JOURNAL OF BIOLOGICAL CHEMISTRY 17065 2 Nuclear Ca and Cell Growth 17066 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 23 •JUNE 8, 2007 2 Nuclear Ca and Cell Growth FIGURE 6. Regulated expression of targeted PV constructs in a tumor cell line. A, immunoblot analysis of the engineered HepG2 stable cell lines after treatment using the indicated concentrations (62.5–1000 ng/ml) of doxycy- cline to induce expression of PV-NLS or PV-NES. B, BrdUrd incorporation is decreased in HepG2 cells expressing PV in the nucleus but not the cytoplasm. *, p 0.001. BrdUrd uptake is not decreased in controls that inducibly express DsRed. The results are representative of what was observed in three separate experiments. C, the phenotype of the engineered HepG2 cells is retained after implantation into nude mice. Immunoblot analysis of tumors removed 2 FIGURE 7.NuclearCa regulatescellgrowth in vivo. Nuclear but not cytoplas- weeks after implantation confirms parvalbumin expression is preserved in mic Ca buffers retard tumor growth. HepG2 tumor cells expressing PV-NES- PV-NLS and PV-NES but not DsRed tumors and only in tumors from animals DsRed, PV-NLS-DsRed, or DsRed under the control of a doxycycline-sensitive pro- treated with doxycycline. The slight difference in molecular weight between moter were implanted subcutaneously into nude mice. Serial measurements of PV-NLS and PV-NES tumors reflects differences in weight caused by the tar- tumor volume in the presence or absence of doxycycline (100 g/ml, added to geting sequences. the drinking water) were obtained, starting when tumor volume reached 5 4 mm . The data represent the means S.E. of tumor volume over time (n 5–15 mice in each group), normalized with respect to the initial volume. *, p 0.001. InsP3 (16, 37), plus temporally related increases in Ca in the perinuclear region (26, 38, 39), have been associated with breakdown of the nuclear envelope and entry into mitosis. In activates other transcription factors such as SRE (9), plus phos- addition, nuclear Ca activates transcription factors such as phatases such as calcineurin that are important for transcrip- CREB (9, 11), Elk-1 (10), and DREAM (40), but cytosolic Ca tion (41). Therefore, the relative importance of nuclear and FIGURE 5. Nuclear Ca regulates progression through early prophase. A, representative images of SKHep1 cells in early and late prophase, pro-metaphase, metaphase, and anaphase. These confocal immunofluorescence images were obtained after staining with anti-phospho-histone-3 (green) and anti--tubulin (pink)to label each phase of mitosis, plus Hoechst (blue) to label the nucleus. B, the mitotic index is increased in cells in which nuclear Ca is buffered. *, p 0.001. Mitotic SKHep-1 cells were identified by histone-3 labeling, measured 48 h after infection with the indicated PV constructs. A total of 200 cells were examined in each experiment, and each experiment was conducted six times. C, representative image of a mitotic SKHep1 cell in which nuclear Ca is buffered with PV-NLS-DsRed. Serial confocal sections (one of which is shown here) reveal a single centrosome by -tubulin staining, and mitotic figures illustrative of early prophase by histone-3 staining. D, mitotic index, subdivided to demonstrate the fraction of cells in each phase of mitosis, demonstrates that cells in which nuclear Ca is buffered do not progress beyond prophase. *, p 0.001. A total of 200 cells were examined in each experiment, and the experiments were performed in quadruplicate. JUNE 8, 2007• VOLUME 282 • NUMBER 23 JOURNAL OF BIOLOGICAL CHEMISTRY 17067 2 Nuclear Ca and Cell Growth 10. Pusl, T., Wu, J. J., Zimmerman, T. L., Zhang, L., Ehrlich, B. 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Journal

Journal of Biological ChemistryAmerican Society for Biochemistry and Molecular Biology

Published: Jun 8, 2007

References