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J. Biol. Chem., Vol. 277, Issue 20, 18191-18197, May 17, 2002

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Glucocorticoids Inhibit Cell Cycle Progression in Differentiating Osteoblasts via Glycogen Synthase Kinase-3^*

Elisheva Smith, Gerhard A. Coetzee^§, and Baruch Frenkel^¶

From the Departments of ^¶ Orthopedic Surgery,  Biochemistry and Molecular Biology, and ^§ Urology, Institute for Genetic Medicine, and the Norris Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, California 90033

Received for publication, October 9, 2001, and in revised form, February 27, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Differentiating osteoblasts in culture undergo a commitment stage, during which cobblestone-like cells grow to high density past confluency. In contrast to earlier proliferative stages, the cell cycle during this commitment stage is inhibited by glucocorticoids (GC). Chronic GC treatment also impedes mineral deposition if steroid administration commences early enough during commitment. This study defines a role for glycogen synthase kinase-3 (GSK3) and its target, c-Myc, in the GC-sensitive osteoblast persistent cell cycle. c-Myc levels decreased as cells reached confluence, but then increased during growth to high density. GC administration at this stage resulted in down-regulation of c-Myc. This was accompanied by GC-mediated attenuation of GSK3 Ser⁹ inhibitory phosphorylation and increased GSK3 kinase activity. Down-regulation of c-Myc was attributable to enhanced Thr⁵⁸ phosphorylation, leading to accelerated degradation. In contrast, GC did not inhibit the c-Myc synthesis rate or the level of -catenin, a transcriptional coactivator of c-myc. The attenuated cell cycle and the reduced c-Myc level were returned to control levels by specific inhibition of GSK3 using lithium chloride. These results suggest that tonal GSK3 repression at the cobblestone stage of osteoblast differentiation permits osteoblast growth to high density. GC interfere with this growth-permissive axis by GSK3 activation, resulting in c-Myc down-regulation and impediment of the G₁/S cell cycle transition.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Osteoporosis is a major adverse effect of glucocorticoid (GC)¹ treatment for the amelioration of symptoms of autoimmune and inflammatory diseases (1). The most important single mechanism contributing to bone loss in GC-treated patients is inhibition of osteoblastic bone formation (for review, see Refs. 2 and 3). We recently identified a commitment stage during osteoblast differentiation in culture (the "cobblestone" stage) in which GC exert their inhibitory effect (4). During this stage, proliferation persists, but is regulated uniquely compared with control of the cell cycle prior to confluence. At this commitment stage (but not earlier), GC inhibit cell cycle progression (4).

Cell growth to high density often reflects loss of cell cycle control. However, controlled growth to high density occurs during cell differentiation not only in cultured osteoblasts (4), but also in cultured adipocytes (5, 6), as well as during the early definition of skeletal elements in vivo (50). Differentiating nontransformed osteoblasts that grow to high density do exhibit growth inhibitory signals, e.g. increased levels of the cyclin-dependent kinase inhibitor p27^Kip1, decreased cyclin E, and reduced free E2F DNA-binding activity (4). To explain the persistent growth, we postulated that the Wnt signaling pathway, which links cell-cell and cell-matrix interactions to the cell cycle (7-10), may account for the post-confluent osteoblast persistent cell cycle and may be involved in the inhibition of this cell cycle by GC.

Wingless/Wnt-mediated cell-cell signaling molecules play a major role in organogenesis and are conserved among vertebrates, flies, and primitive multicellular organisms (11-13). Components of the Wingless/Wnt signaling pathway participate in cell-cell adhesion, body patterning, cell growth, and tumorigenesis (11, 12). Forced activation of the Wnt signaling pathway by overexpression of either Wnt1 (18) or its target, -catenin (19), results in cell growth to high density.

The -catenin pool relevant to Wnt signaling resides in the cytoplasm and is distinct from a membrane-associated pool that plays a role in cell-cell adherence. In response to Wnt signaling, cytoplasmic -catenin increases and enters the nucleus, where it cooperates with lymphoid enhancer factor/T-cell factor in the transcriptional activation of cell cycle stimulatory genes such as c-myc (9) and cyclin D1 (10). A major regulator of -catenin is glycogen synthase kinase-3 (GSK3). -Catenin and GSK3 are found in a cytoplasmic complex called the -catenin destruction complex, which also contains axin and adenomatous polyposis coli (25). The association of -catenin with GSK3 results in rapid degradation of -catenin, probably mediated by phosphorylation. Activation of the Wnt pathway results in decreased GSK3 intrinsic activity and, more importantly, disruption of the -catenin destruction complex, leading to increased stability and accumulation of -catenin.

GSK3 also plays a role in regulating growth signals elicited by molecules such as insulin, insulin-like growth factor I (14), epidermal growth factor (15), fibroblast growth factor-1,² and the fibronectin-activated integrin-linked kinase (17). These signals inhibit the intrinsic activity of GSK3, but do not usually lead to disruption of the -catenin destruction complex.

In this study, we examined the involvement of the GSK3/-catenin/c-myc axis in GC inhibition of the persistent cell cycle in post-confluent differentiating osteoblasts. We found that GC-treated differentiating osteoblasts displayed increased GSK3 activity and decreased levels of c-Myc; however, c-Myc down-regulation by GSK3 was not mediated via -catenin, but rather by direct phosphorylation and enhanced degradation of c-Myc itself.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- The MC3T3-E1 osteoblastic cells used in this study were derived from a subclone recently isolated based on its robust mineralization potential (4). This subclone was expanded to ~10⁹ cells, at which time frozen stocks were prepared and defined as passage 1. Cells were maintained up to passage 12 in -minimal essential medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. All experiments were performed under differentiation conditions, i.e. in the presence of 50 µM ascorbic acid and 10 mM -glycerophosphate. SAOS-2 human osteosarcoma cells, stably transfected with either the wild-type or a dimerization-defective GC receptor, were a kind gift from Dr. M. J. Garabedian (New York University) (20). These cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 400 µg/ml Geneticin.

Cell Fractionation-- Nuclear and cytoplasmic fractions were prepared essentially according to Verona et al. (21). Cell pellets were resuspended in 2 packed cell volumes of hypotonic buffer containing 10 mM HEPES (pH 7.5), 10 mM KCl, 3 mM MgCl₂, 1 mM EDTA (pH 8), 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 mM NaF, and 0.1 mM Na₃VO₄. Cells were left to swell on ice for 10 min and then vortexed for 10 s and spun at 500 × g for 5 min. The supernatant (containing the cytoplasmic lysate) was supplemented with 0.33 volume of 80% glycerol and clarified by centrifugation at 20,000 × g for 30 min. The nuclear pellet was washed twice with hypotonic buffer and lysed in 2 nuclear pellet volumes of lysis buffer containing 100 mM HEPES (pH 7.4), 0.5 M KCl, 5 mM MgCl₂, 28% glycerol, and protease and phosphatase inhibitors as described above. The nuclear extracts were centrifuged at 20,000 × g for 1 h to remove cell debris.

Western Analysis-- Between 60 and 100 µg of protein from the nuclear extract, cytoplasmic lysate, or whole cell pellets were subjected to SDS-PAGE. Separated proteins were transferred to a 0.2-µm nitrocellulose membrane using a Mini Trans-Blot Transfer Cell (Bio-Rad), and immunodetection was performed using ECL (Amersham Biosciences) according to the manufacturers' recommendations, followed by exposure of the membrane to x-ray film. Results were quantitated by photodensitometry using an AlphaImager 2000 system (Alpha Innotech Corp.) and are expressed as means ± S.D. Differences were considered significant when p was 0.05 as determined by a paired t test.

GSK3 Immunoprecipitation and Kinase Assays-- These were performed essentially as described by Diehl et al. (22). Cells were lysed in 1% Triton X-100 buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 0.27 M sucrose, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.4 mM NaF, and 0.4 mM NaVO₄. GSK3 was immunoprecipitated from 200 µg of total protein extract using 1 µg of mouse monoclonal anti-GSK3 antibodies and protein A/G-agarose beads. The immunoprecipitate was resuspended in 24 µl of kinase buffer containing 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 1 mM EGTA, 14 µM ATP, 83 µM phosphoglycogen synthase peptide II, 10 µCi of [-³²P]ATP (6000 Ci/mmol), and protease and phosphatase inhibitors as described above. After 30 min at 30 °C, 8 µl of 4× loading buffer (final concentrations: 4% SDS, 12% glycerol, 50 mM Tris-HCl (pH 6.8), 2% -mercaptoethanol, and 0.01% Serva blue G) were added. The mixture was boiled for 5 min and loaded onto a Tricine-16.5% acrylamide gel (23), followed by autoradiography and quantitation as described above.

Flow Cytometry-- Cell cycle analysis was performed according to Darzynkiewicz et al. (24). Briefly, cells were lightly trypsinized, resuspended in Hanks' buffer, fixed in cold 70% ethanol, washed with Hanks' buffer, and suspended in 1 ml of Hanks' buffer containing 20 µg/ml propidium iodide and 5 Kunitz units of DNase-free RNase A. The percentage of cells in G₁, S, and G₂/M was determined using an EPICS^® Profile Analyzer.

Reagents-- Tissue culture reagents were purchased from Invitrogen. Antibodies against GSK3 (G22320) and -catenin (C19220) were purchased from Transduction Laboratories. Anti-phospho-Ser⁹ GSK3 (catalog no. 9336) and anti-phospho-Thr⁵⁸ c-Myc (catalog no. 9401) antibodies were from Cell Signaling Technology (Beverly, MA). Anti-c-Myc antibodies (SC-764) and protein A/G-agarose beads were from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphoglycogen synthase peptide II was from Upstate Biotechnology, Inc. (Lake Placid, NY). MG132 was from Calbiochem. Protein concentration was determined using a micro BCA protein assay kit (Pierce).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

GC Activate GSK3 by Attenuating Its Ser⁹ Inhibitory Phosphorylation-- We have recently shown that GC inhibit persistent cell cycle progression at a commitment stage during osteoblast differentiation characterized by a cobblestone appearance (4). We postulated that components of the Wnt signaling pathway might play a role during this commitment stage in mediating cell cycle persistence and its inhibition by GC. One of these components is GSK3, a cell cycle guardian that is inactivated by a variety of growth stimuli. We evaluated GSK3 in non-treated and GC-treated MC3T3-E1 cells at and immediately after the cobblestone stage. As determined by Western analysis, DEX treatment of cells that had just become confluent resulted in a small but consistent increase in the total cellular levels of GSK3 (Fig. 1A). Because nuclear localization of GSK3 has recently been proposed to play a cell cycle-related role (22), we also performed Western analysis on nuclear extracts and found that the increase in GSK3 levels was even more pronounced in the nuclear fraction of DEX-treated cells (Fig. 1A). Notably, this effect was attenuated in cultures that had already grown to high density (Fig. 1A, Day 9). Accordingly, we thereafter confined our work specifically to cells that just initiate growth to high density.

View larger version (38K): [in this window] [in a new window]   Fig. 1.   GC activate GSK3 in differentiating osteoblasts. A: MC3T3-E1 cells were maintained under the differentiation protocol and treated with 1 µM DEX either at the cobblestone stage (days 5-7) or thereafter (days 7-9). Whole cell Triton X-100 extracts (WCE) and nuclear extracts (Nuc) were prepared and analyzed by Western blotting using anti-GSK3 antibody. B: upper left panel, the Triton X-100 extracts from the day 7 cultures in A were subjected to GSK3 kinase activity assay. GSK3 was immunoprecipitated and incubated with [-32P]ATP and phosphoglycogen synthase peptide II (32P-GS-II), followed by electrophoresis and autoradiography. Middle left panel, SDS whole cell extracts were prepared from non-treated and DEX-treated cultures on day 7 and subjected to Western blot analysis using anti-phospho-Ser9 GSK3 antibody ([pSer9]GSK3). Lower left panel, Western analysis of total GSK3 was performed on the same membrane used in the middle panel. Right panel, shown is a graph representing the DEX/control ratios (mean ± S.D., n = 3) for Ser9-phosphorylated GSK3 (pGSK), total GSK3 (GSK), and the relative GSK3 Ser9 phosphorylation level (pGSK/GSK). The latter value was calculated for each experiment by dividing the Ser9-phosphorylated GSK3 ratio by the total GSK3 ratio. C: nuclear and cytoplasmic (Cyt) extracts of cells harvested on day 7 were subjected to Western blot analysis using anti--catenin antibody. D: differentiating MC3T3-E1 cells were treated with 1 µM DEX on day 5 and harvested 20 h later for Western analysis of c-Myc in SDS whole cell extracts.

GSK3 activity is subject to functionally critical post-translational modifications. We therefore measured GSK3 activity by immunoprecipitation and kinase assays using a glycogen synthase-derived peptide as substrate. As exemplified in Fig. 1B (upper left panel), DEX significantly increased GSK3 activity, and this increase amounted to 1.77 ± 0.1-fold in three independent experiments.

An important post-translational modification of GSK3 is the phosphorylation of Ser⁹, which results in GSK3 inhibition, such as seen following growth factor stimulation (14, 15).² We therefore evaluated the effect of GC on GSK3 Ser⁹ phosphorylation. Indeed, as shown in Fig. 1B, DEX decreased the level of Ser⁹-phosphorylated GSK3 (middle left panel) while increasing total GSK3 (lower left panel). In three independent experiments, DEX induced an average 2.4-fold decrease in the level of Ser⁹-phosphorylated GSK3 (Fig. 1B, right panel, pGSK). When corrected for the total levels of GSK3, DEX reduced by 3-fold the relative level of GSK3 Ser⁹ phosphorylation (Fig. 1B, right panel, pGSK/GSK). These results suggest that GC attenuate the inhibitory phosphorylation of GSK3 mediated by growth stimulatory signals. Higher activity of GSK3, a cell cycle inhibitor, might contribute to the antimitogenic effect of GC in post-confluent MC3T3-E1 osteoblasts (4).

GC Down-regulate c-Myc in MC3T3-E1 Cells in a -Catenin-independent Fashion-- An important downstream target of the Wnt signaling pathway is -catenin, which participates in the transcriptional activation of cell cycle stimulatory genes such as c-myc (9) and cyclin D1 (10). GSK3-mediated phosphorylation promotes the degradation of -catenin. We therefore examined whether -catenin is down-regulated in GC-treated MC3T3-E1 cells. However, as shown in Fig. 1C, DEX did not decrease either the nuclear or the cytoplasmic -catenin level, suggesting that -catenin is resistant to GC-mediated activation of GSK3 in MC3T3-E1 osteoblasts. This is consistent with the notion that some GSK3 exists in pools that are physically and functionally distinct from those found complexed with adenomatous polyposis coli, axin, and -catenin (for review, see Ref. 25).

GSK3 can down-regulate cell cycle regulatory factors such as c-Myc and cyclin D1 in both -catenin-dependent and -independent manners. The -catenin-independent pathway entails direct phosphorylation of c-Myc at Thr⁵⁸, followed by ubiquitination and proteasomal degradation (26, 27). Indeed, despite the persistence of -catenin in GC-treated MC3T3-E1 cells, Western blot analysis of c-Myc revealed reduced levels in DEX-treated compared with non-treated cultures (Fig. 1D). In three independent experiments, c-Myc levels were significantly decreased by 2.3 ± 0.2-fold.

Osteoblast Growth to High Density Is Associated with c-Myc Up-regulation-- c-Myc has been previously found to express in osteoblasts and chondrocytes in vivo (28, 29). We therefore analyzed c-Myc by Western blot analysis during MC3T3-E1 osteoblast differentiation in vitro. Expectedly, as cells reached confluence (Fig. 2A, Day 5), c-Myc levels decreased (Fig. 2B, Day 5 versus Day 3). The same was observed in NIH3T3 cells under similar culture conditions (data not shown). However, 2 days after the cobblestone stage (Fig. 2A, Day 7) c-Myc was up-regulated back to levels observed in pre-confluent cultures (Fig. 2B, Day 7). This was not seen with the NIH3T3 cells (data not shown). In contrast to c-Myc, cyclin A, which is down-regulated by GC in post-confluent persistently cycling osteoblasts (4), was expressed at steady levels during pre-confluence and confluence and until at least 2 days after confluence (Fig. 2C). Taken together, this expression pattern and the effect of DEX (Fig. 1D) suggest a role for c-Myc in driving the post-confluent osteoblast persistent cell cycle.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Up-regulation of c-Myc in post-confluent differentiating osteoblasts. A, shown is the morphology of MC3T3-E1 osteoblasts during the GC-sensitive commitment (cobblestone) stage. Left panel, cells on day 3, just prior to confluence; middle panel, cells with a cobblestone appearance (day 5); right panel, cell growth to high density (day 7). Magnification ×100. B and C, cells were harvested at the three developmental stages represented in A, and Western blot analysis was performed using either anti-c-Myc (B) or anti-cyclin A (C) antibody.

GC Promote Thr⁵⁸ Phosphorylation and Enhanced Degradation of c-Myc-- Based on the activation of GSK3 (Fig. 1B), without an accompanying decrease in -catenin (Fig. 1C), the decreased c-Myc level in GC-treated MC3T3-E1 cells (Fig. 1D) is attributable to protein instability. We therefore studied c-Myc synthesis and degradation in GC-treated cultures. Initially, we evaluated c-Myc synthesis rates by blocking degradation using the proteasomal inhibitor MG132; this was immediately followed by concomitant MG132 withdrawal and administration of cycloheximide to block protein synthesis during the measurement of the c-Myc degradation rate. As shown in Fig. 3, which represents one of three experiments with similar results, DEX did not inhibit c-Myc synthesis. However, c-Myc degradation was significantly accelerated in DEX-treated cells (Fig. 3, A and B). Half-life values can only be approximated in these experiments because proteasomal recovery following MG132 withdrawal is not instantaneous. Assuming complete recovery at 1.5 h post-MG132 withdrawal, c-Myc was degraded in the DEX-treated cells over the following hour with an apparent half-life value 3.2 ± 0.7 times greater than that measured in the non-treated cells (p = 0.02; n = 3).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   GC accelerate c-Myc degradation. A, post-confluent MC3T3-E1 cells were treated on day 6 with either 1 µM DEX or vehicle (control (CONT)). At 16 h of DEX treatment, 50 µM MG132 in 0.1% Me2SO (final concentrations) was administered, and cells were collected for c-Myc Western blot analysis after 0, 1, 2, 3, or 4 h. At the 4-h time point, MG132 was washed, and cells were refed with fresh medium containing 50 µM cycloheximide. Cultures were again harvested at 1, 1.5, 2, 2.5, and 3 h following MG132 withdrawal for Western analysis of c-Myc. B, shown is a graph illustrating the degradation of c-Myc in treated () versus non-treated () cells. Values were derived by densitometry of the autoradiogram shown in A (right panels). Similar results were obtained in three independent experiments. C, Western analysis was carried out on 4-h MG132-treated cells using antibody that recognize either all forms or just the Thr58-phosphorylated form of c-Myc ([pThr58]c-MYC).

GSK3 has been shown to target c-Myc for proteasomal degradation by phosphorylation at Thr⁵⁸ (26, 27). We therefore tested the c-Myc Thr⁵⁸ phosphorylation status in DEX-treated versus non-treated cells. Samples from 4-h MG132-treated cultures (as in Fig. 3A) were subjected to Western analysis using anti-phospho-Thr⁵⁸ c-Myc antibody. As shown in Fig. 3C, DEX increased the c-Myc Thr⁵⁸ phosphorylation level. Corrected for total c-Myc, the relative DEX-mediated increases in c-Myc Thr⁵⁸ phosphorylation were 1.7- and 2.6-fold in two independent experiments. These results are consistent with the insensitivity of -catenin to GC (Fig. 1C) and strongly suggest that increased c-Myc phosphorylation by GSK3 in GC-treated osteoblasts leads to accelerated degradation and reduced c-Myc steady-state levels.

GC Effects on GSK3 and c-Myc Precede Alterations in the Cell Cycle Profile-- The increased GSK3 activity in GC-treated cells (Fig. 1B) may be a result, not a cause, of cell cycle attenuation (30). We therefore tested GSK3 activity following short periods of GC treatment (before an effect on the cell cycle is mounted). Triton X-100 extracts of 2- and 5-h treated cultures were subjected to GSK3 immunoprecipitation and kinase assays. These short treatment periods did not alter the distribution of cells among the phases of the cell cycle (data not shown). As shown in Fig. 4 (upper panel), DEX activated GSK3 within as little as 2 h of treatment, with somewhat lesser activation observed at 5 h.

View larger version (78K): [in this window] [in a new window]   Fig. 4.   DEX-mediated GSK3 activation precedes cell cycle attenuation. MC3T3-E1 cells were treated on day 6 with either 1 µM DEX or vehicle and harvested after either 2 or 5 h for preparation of Triton X-100 whole cell extracts. The same extracts were subjected to either GSK3 kinase assays (upper panel), as described for Fig. 1B, or Western blot analysis using the indicated antibodies (lower three panels). [pSer9]GSK3, anti-phospho-Ser9 GSK3 antibody.

Next, Triton X-100 extracts as described above were subjected to Western blot analysis using either anti-GSK3 or anti-phospho-Ser⁹ GSK3 antibody. As shown in Fig. 4, 2-h DEX treatment significantly reduced Ser⁹-phosphorylated GSK3, and this reduction amounted to 47 ± 5% in three independent experiments. Corrected for total GSK3, the change in the relative Ser⁹ phosphorylation level (calculated, as in Fig. 1B, as the ratio between phospho-GSK3 (DEX)/phospho-GSK3 (control) and GSK3 (DEX)/GSK3 (control)) was significantly reduced within the 2-h treatment period by 46 ± 20% (n = 3). The GC effect on the relative Ser⁹ phosphorylation of GSK3 was similar at the 5-h time point, amounting to a significant 50 ± 18% reduction (n = 3). Because Ser⁹ phosphorylation is a major (albeit not the only (31)) determinant influencing GSK3 activity, our data suggest that the fast decrease in GSK3 Ser⁹ phosphorylation following 2 h of treatment may be the initial event leading to GSK3 activation, c-Myc down-regulation, and cell cycle attenuation. Consistent with this, a reduction in c-Myc levels was apparent at the 5-h (although not at the 2-h) treatment time point (Fig. 4, lower panel).

GC Attenuate GSK3 Ser⁹ Phosphorylation That Follows Serum Stimulation-- c-Myc plays a critical role in the response of the cell cycle machinery to growth signals. To examine the c-Myc immediate response to serum stimulation, MC3T3-E1 cells were incubated in serum-free medium for 48 h and then refed with medium containing 10% serum. DEX was administered 2 h prior to and again during serum stimulation. Following serum stimulation, c-Myc immediately began to accumulate in the non-treated cells (Fig. 5). In three independent experiments, c-Myc levels significantly increased by 2.5 ± 1.1-fold within 20 min of serum stimulation. In contrast, c-Myc accumulation was blunted in the DEX-treated cells, where an insignificant 27 ± 25% increase was measured following serum stimulation. The antimitogenic effect of DEX in serum-stimulated cells was confirmed by flow cytometry analysis at 12 h following serum stimulation with or without DEX (Fig. 5B).

View larger version (34K): [in this window] [in a new window]   Fig. 5.   GC attenuate serum-induced GSK3 Ser9 phosphorylation. A, post-confluent differentiating cells were deprived of serum for 48 h and then refed with serum-containing medium. DEX treatment (1 µM) was initiated 2 h prior to serum stimulation. SDS cell extracts were prepared at 0, 10, and 20 min of serum stimulation, and Western blot analysis was performed using the indicated antibodies. [pSer9]GSK3, anti-phospho-Ser9 GSK3 antibody. B, parallel DEX-treated and non-treated cultures were harvested 12 h following serum stimulation, fixed, and stained with propidium iodide for flow cytometry analysis. The percentage of cells in the S + G2 + M phases of the cell cycle is depicted (mean ± S.D., n = 3).

Following serum stimulation, Ser⁹-phosphorylated GSK3 consistently accumulated in the non-treated cultures (Fig. 5); the accumulation rate in the DEX-treated cultures was reduced by an average of 27% as compared with the control cultures, but this difference did not reach statistical significance (p = 0.15; n = 3). However, when corrected for the respective total GSK3 values, the increase in relative GSK3 Ser⁹ phosphorylation was significantly attenuated in the DEX-treated cultures to levels 63% lower than the respective control values (p = 0.03; n = 3). Thus, GC attenuate the Ser⁹ inhibitory phosphorylation of GSK3 in serum-stimulated MC3T3-E1 cells, potentially blunting the c-Myc accumulation.

Lithium Protects the MC3T3-E1 Cell Cycle from GC-- If GSK3 plays a role in the antimitogenic effect of GC, then inhibition of GSK3 may ameliorate the inhibitory effect of GC on cell cycle progression. We therefore tested the antimitogenic effect of DEX on the post-confluent osteoblast cell cycle in the presence of lithium, a specific GSK3 inhibitor (32). As shown in Fig. 6A, exposure to lithium accelerated cell cycle progression in both DEX-treated and non-treated post-confluent osteoblast cultures. However, the lithium effect on the DEX-treated cells was more pronounced, bringing, within 9 h, the cells in S + G₂ + M to virtually the same percentage as measured in the DEX-free cultures. Fig. 6B shows representative cell cycle profiles from this 9-h time point. As shown, a 9-h treatment was clearly sufficient for DEX to induce a G₁/S block; and more importantly, lithium completely equalized the cell cycle profile in non-treated and DEX-treated cells. This could be explained by lithium counteracting the DEX-mediated c-Myc down-regulation within as early as 4 h (Fig. 6B, inset). Interestingly, the GC inhibition of cyclin A (4) was not rescued by lithium at this time point (Fig. 6B, inset). By 18 h of treatment, DEX further inhibited cell cycle progression (Fig. 6A). Again, lithium almost entirely counteracted the decrease in the percentage of cells in S + G₂ + M (Fig. 6A). It should be noted that the distribution of cells between S and G₂/M in the DEX-treated cultures was different from that observed in the absence of DEX (data not shown), possibly reflecting effects of DEX and lithium on other cell cycle transitions (33). As opposed to lithium, potassium did not reverse the inhibitory effect of DEX on the G₁/S transition (Fig. 6A). In conclusion, our results indicate that GSK3 is a critical regulator of the post-confluent cell cycle and that GSK3 activation contributes to the antimitogenic effect of GC in differentiating osteoblasts.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Inhibition of GSK3 by lithium rescues the GC-inhibited cell cycle. Post-confluent MC3T3-E1 cells were treated with either 0.1 µM DEX or vehicle, together with 40 mM LiCl (a specific GSK3 inhibitor) or KCl as a control. Cells were collected after either 9 h (only the lithium group) or 18 h (lithium and potassium treatments). Cells were ethanol-fixed and stained with propidium iodide, and the cell cycle profile was analyzed by flow cytometry. A, percentage of cells in S + G2 + M is presented for each experimental group. Bars represent mean ± S.D. of three plates, except for the KCl-treated groups, for which the bars represent the mean of duplicate measurements, which differed by <5%. B, representative cell cycle profiles are shown for the 9-h DEX-treated and non-treated cells in the absence and presence of lithium. The percentages of cells in G1, S, and G2/M are depicted underneath each histogram. The inset shows the results of Western analysis of c-Myc and cyclin A (cycA) in 18-h DEX-treated cells to which lithium was administered 4 h prior to collection (D+L). The results of Western analysis of c-Myc and cyclin A in DEX-treated cells in the absence of lithium (D) and in non-treated cells (control (C)) are shown for comparison.

GC Inhibition of GSK3 Ser⁹ Phosphorylation Requires Receptor Dimerization-- As a first step toward elucidating mechanisms by which GC activate GSK3, we investigated whether GC receptor dimerization is required for this activation. Receptor dimerization is a prerequisite for DNA binding and classical transcriptional activation, but is not required for DNA binding-independent action such as AP-1 trans-repression (34). We employed SAOS-2 human osteosarcoma cell lines (originally GC receptor-negative) that had been stably transfected with either the wild-type or a dimerization-defective (R479D/D481R) rat GC receptor (35). Each of these cell lines was treated with DEX, and GSK3 Ser⁹ phosphorylation status was evaluated by Western blot analysis. Similar to MC3T3-E1 osteoblasts (Fig. 1B), DEX significantly inhibited GSK3 Ser⁹ phosphorylation in SAOS-2 cells expressing the wild-type GC receptor (Fig. 7). In contrast, DEX did not inhibit GSK3 Ser⁹ phosphorylation in SAOS-2 cells expressing the dimerization-defective GC receptor (Fig. 7). These results indicate that DEX-mediated GSK3 activation requires receptor dimerization, suggesting that it occurs via classical transcriptional activation of genes yet to be identified.

View larger version (35K): [in this window] [in a new window]   Fig. 7.   GC-mediated attenuation of GSK3 Ser9 phosphorylation is dependent on receptor dimerization. SAOS-2 osteosarcoma cells expressing either the wild-type (wt) or a dimerization mutant form (dim) of the rat GC receptor (GR) were treated for 48 h with 1 µM DEX and subjected to Western analysis using antibody against either Ser9-phosphorylated GSK3 ([pSer9]GSK3) or total GSK3, as indicated. A, autoradiograms from a representative experiment; B, graph representing the DEX/control ratios (mean ± S.D., n = 3) for Ser9-phosphorylated GSK3 (pGSK), total GSK3 (GSK), and the relative GSK3 Ser9 phosphorylation level (pGSK/GSK). The latter value was calculated for each experiment by dividing the Ser9-phosphorylated GSK3 ratio by the total GSK3 ratio.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell proliferation and differentiation are traditionally perceived as reciprocal processes, cell cycle withdrawal being a prerequisite for terminal differentiation. However, stages that precede expression of an ultimate cellular phenotype may occur as cells are cycling, such as during the differentiation of lymphocytes (36), adipocytes (5, 6), and osteoblasts (4). Our earlier work with osteoblasts suggests that the differentiation-related cell cycle, occurring in cultures that have reached confluence, is regulated in a unique manner, rendering it susceptible to inhibition by GC (4).

This study suggests that c-Myc plays a crucial role in the persistent proliferation of post-confluent osteoblasts in culture. c-Myc steady-state levels decrease as osteoblasts reach confluence, similar to what is observed in non-osteoblastic cells. However, soon thereafter, during osteoblast differentiation, c-Myc rises back to levels observed prior to confluence, and the cells grow to high density. GC decrease the c-Myc serum response and steady-state levels and attenuate the persistent cell cycle in the post-confluent differentiating cells.

The increase in c-Myc levels in post-confluent differentiating osteoblasts may explain the persistent cell cycle despite high levels of the cyclin-dependent kinase inhibitor p27^Kip1 (4, 37) and even when p27^Kip1 is forcibly elevated further using viral transduction (4). The role of c-Myc in osteoblast differentiation may be to delay exit from the cell cycle, thus allowing differentiation steps, which require cell division. Alternatively, c-Myc may play a role in regulating differentiation-related genes, with the post-confluent cell cycle being a nonessential accompanying event. It is difficult to identify a differentiation stage in vivo, which may be analogous to the post-confluent cell cycle observed in vitro. However, in favor of a physiological role for c-Myc in mineralizing tissues, this proto-oncogenic transcription factor has been shown to express in osteoblasts and in hypertrophic chondrocytes in vivo (28, 29). These observations and the inhibitory effects of GC on c-Myc and on the osteoblast phenotype suggest a role for c-Myc and possibly the persistent cell cycle in osteoblast differentiation.

The increase in c-Myc levels in post-confluent differentiating osteoblasts may also shed light on mechanisms of osteosarcoma development. It is intriguing to speculate that physiological up-regulation of c-Myc at a specific stage of osteoblast differentiation provides a window of opportunity for the action of osteoblast-transforming agents such as Fos (38, 39). In this context, the c-Myc up-regulation is consistent with E2F4-p130 being the predominant and functionally critical E2F-pocket complex in post-confluent differentiating osteoblasts (4) because both c-Myc (40) and E2F4 (41) cooperate with Ras in cellular transformation.

How do GC exert their inhibitory effects on c-Myc and on the osteoblast persistent cell cycle? In search for upstream effectors, it is important to remember that the osteoblast cell cycle becomes sensitive to GC only at confluence. It therefore seemed logical that the GC inhibition may involve the Wnt signaling pathway, which bridges cell-cell and cell-matrix interactions with the cell cycle machinery. Interestingly, what we observed in GC-treated cultures is increased intrinsic activity of GSK3, but not decreased nuclear -catenin levels, suggesting that GC primarily affect GSK3 in the growth factor pathway. However, we feel it is premature to rule out an effect of GC on GSK3 in the Wnt pathway. In fact, GC decreased reporter activity in cells transiently transfected with a lymphoid enhancer factor/T-cell factor-controlled luciferase construct (data not shown). Thus, we believe that GC affect GSK3 in both the Wnt and growth factor pathways. Importantly, inhibition of GSK3 by lithium neutralizes the inhibitory effects of GC, indicating a causal relationship between GSK3 activation and cell cycle attenuation.

The lack of a GC-mediated decrease in nuclear -catenin is consistent with the unaffected c-Myc synthesis rate, which is regulated by -catenin (9, 42, 43). However, GSK3 may work directly on c-Myc, phosphorylating and accelerating its degradation (26, 27). This mechanism clearly contributes to the antimitogenic effect of GC in differentiating osteoblasts, as indicated by the increased Thr⁵⁸ phosphorylation and the accelerated degradation of c-Myc in response to GC treatment. The preferential effect of GC on GSK3 in the growth factor pathway versus the Wnt pathway may be explained by selective requirements for substrate priming (44, 45).

GSK3 is a cell cycle guardian that is inactivated in response to growth factors, typically by Ser⁹ phosphorylation. Our results show that GC attenuate this inactivating phosphorylation. GC decreased GSK3 Ser⁹ phosphorylation in cultures 2 h (Fig. 4) as well as 48 h (Fig. 1B) after being fed fresh medium with or without DEX. The GC effect on GSK3 Ser⁹ phosphorylation was also apparent during the immediate response to serum stimulation (Fig. 5). Thus, GC activate GSK3 by prevention of the serum-induced Ser⁹ inhibitory phosphorylation, which is sustained for as long as 48 h.

A limitation of our study is that it employs cell lines, either transformed (SAOS-2) or nontransformed (MC3T3-E1), that may be regulated differently than osteoblasts in vivo. Preliminary results with primary murine bone marrow-derived mesenchymal stem cells indicated that, as in the MC3T3-E1 cultures, DEX inhibited both condensation and calcium deposition (data not shown). Also, GSK3 Ser⁹ phosphorylation was decreased in the DEX-treated cultures, but this effect did not reach statistical significance. It is possible that a DEX effect on the bone marrow-derived osteoblasts was diluted by different responses of non-osteoblasts and/or osteoblasts at DEX-insensitive stages of differentiation. Thus, the role of GSK3 in osteoblast differentiation and in GC-induced osteoporosis remains to be confirmed in primary cultures and in vivo.

Among the cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors that were analyzed, the most significant effect of GC in MC3T3-E1 differentiating osteoblasts is down-regulation of cyclin A and its dissociation from E2F4-p130 complexes (4). It is possible that GC down-regulate c-Myc and cyclin A via independent mechanisms and that both of these effects contribute to attenuation of the cell cycle. However, this study demonstrated rapid inhibitory effects of DEX on c-Myc, which occurred prior to any significant effect on cyclin A (data not shown). Furthermore, rescue of the GC-inhibited cell cycle by lithium was accompanied by restoration of c-Myc (but not cyclin A) levels (Fig. 6B). These results suggest a more crucial role for c-Myc in GC inhibition of the osteoblast persistent cell cycle. However, <2 h of treatment was insufficient for DEX to mount any effect on c-Myc or GSK3 (data not shown). This and the requirement for GC receptor dimerization for the Ser⁹ dephosphorylation of GSK3 (Fig. 7) suggest that GC-mediated GSK3 activation occurs via transcriptional stimulation of an intermediary gene(s).

Are our results with lithium relevant to bone and mineral metabolism in lithium-treated psychiatric patients? Limited work in this controversial field indicates lithium-induced hyperparathyroidism, but no hypercalcemia or bone loss in these patients (46); one study reported increased levels of alkaline phosphatase (47), a serum marker of bone formation. Preserved bone mass in the face of hyperparathyroidism has been attributed to enhancement of renal calcium reabsorption (46, 48). Based on our in vitro studies, it is interesting to speculate that enhanced osteoblast function may also contribute to bone preservation in lithium-treated patients with hyperparathyroidism.

Maintenance of bone mass throughout life requires that bone formation equals bone resorption. GC impair this balance primarily by inhibiting osteoblastic bone formation. The present study demonstrates that GC activate GSK3 in osteoblasts. This may interfere with important differentiation cues generated by cell-cell contacts; cell-matrix interactions; or growth factors signaling through GSK3, in particular insulin-like growth factor I, known to play a role in osteoblast growth and differentiation (49). Our current findings suggest that impediment of osteoblast function by GSK3 activation may be mediated by the down-regulation of c-Myc, potentially via inhibition of a differentiation-related cell cycle. Thus, GC activation of osteoblast GSK3 may play an important role in the pathogenesis of and potentially future therapies for GC-induced osteoporosis.

	ACKNOWLEDGEMENTS

We are grateful to M. J. Garabedian (New York University), J. R. Woodgett, and A. Ali (Ontario Cancer Institute) for suggestions and reagents; Abel Valdovinos (University of Southern California) for technical assistance; and M. J. Roberts (University of Southern California) for preparation of primary mesenchymal stem cell cultures from murine bone marrow.

	FOOTNOTES

^* This work were supported by Grants RO1-AR47052 and T32-CA09659 from the National Institutes of Health and by grants from the Arthritis Foundation and the Zumberge Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Inst. for Genetic Medicine, University of Southern California Keck School of Medicine, 2250 Alcazar St., CSC/IGM240, Los Angeles, CA 90033. Tel.: 323-442-1322; Fax: 323-442-2764; E-mail: frenkel@hsc.usc.edu.

Published, JBC Papers in Press, February 27, 2002, DOI 10.1074/jbc.M109708200

² M. Hashimoto, Y. Sagara, D. Langford, I. P. Everall, M. Mallory, A. Everson, M. Digicaylioglu, and E. Masliah, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: GC, glucocorticoid(s); GSK3, glycogen synthase kinase-3; DEX, dexamethasone; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES