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Endocrinology, doi:10.1210/en.2003-0718
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Endocrinology Vol. 145, No. 1 87-94
Copyright © 2004 by The Endocrine Society

Effects of 1{alpha},25-Dihydroxyvitamin D3 and Growth Hormone on Apoptosis and Proliferation in UMR 106 Osteoblast-Like Cells

Orlando Morales, Magnus K. R. Samuelsson, Urban Lindgren and Lars-Arne Haldosén

Center for Surgical Sciences (O.M., M.K.R.S., U.L.), Division of Orthopedics, Karolinska Institutet, Huddinge University Hospital, S-141 86 Huddinge, Sweden; and Department of Medical Nutrition (L.-A.H.), Karolinska Institutet, Novum, S-141 86 Huddinge, Sweden

Address all correspondence and requests for reprints to: Lars-Arne Haldosén, Department of Medical Nutrition, Karolinska Institutet, Novum, S-141 86 Huddinge, Sweden. E-mail: lars-arne.haldosen{at}mednut.ki.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mechanisms maintaining a correct balance between bone-forming osteoblasts and bone-resorbing osteoclasts are essential for bone formation. Apoptosis has been proposed to play a key role in controlling osteoblast homeostasis. 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and GH, which are important regulators of bone growth and bone metabolism, also play pivotal roles in regulation of mitogenesis, differentiation, and apoptosis. We have recently shown that 1,25(OH)2D3 prolongs GH signaling via the Janus kinase 2 (JAK2)/STAT5 (signal transducer and activator of transcription 5) pathway in UMR 106 osteoblast-like cells. In the present study, we have investigated the effects of GH and 1,25(OH)2D3 on proliferation and apoptosis in UMR 106 cells. We found that 1,25(OH)2D3 and GH, separate or in combination, inhibited apoptosis. GH also had profound effects on cell cycle distribution and proliferation. In addition, pretreatment of cells with 1,25(OH)2D3 was necessary to detect GH-induced MAPK activation. We hypothesize that these hormones separately regulate the processes of apoptosis and proliferation, which may be important for maintaining osteoblast cell number.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE RATE OF bone formation and resorption is largely determined by the numbers of bone-forming (osteoblast) and bone-resorbing (osteoclast) cells present in the basic multicellular units responsible for the regeneration of the adult skeleton (1). Similarly to other regenerating tissues, the number of bone cells is controlled by changes not only in the production of mature cells but also in their survival. Recent evidence indicates that apoptosis (programmed cell death) represents the most common fate of osteoblasts during physiologic bone remodeling (2, 3) and agents that influence the rate of bone formation and bone mass control osteoblast apoptosis in vitro (4, 5).

Apoptosis is regulated by specific intracellular signaling pathways that ultimately induce cell self-destruction. The process of apoptosis is a highly ordered chain of events involving the sequential activation of different members of a family of aspartate-specific cystein proteases called caspases. These proteases are important effectors of apoptosis and are responsible for the proteolytic cleavage of selected cellular proteins, including proteins responsible for DNA repair and proteins involved in cell cycle regulation (6).

Members of the Bcl-2 protein family are proteins with the ability to modify the apoptotic pathway by affecting cytochrome c release from the mitochondrion. Some members of this protein family (for instance, Bax) act as promoters of apoptosis, and others (such as Bcl-2) as inhibitors of apoptosis (7).

GH and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] are important regulators of bone growth and bone metabolism. There is substantial experimental evidence to suggest that 1,25(OH)2D3 and GH play important roles in cellular mitogenesis, differentiation, and apoptosis (8). The most active form of vitamin D3, 1,25(OH)2D3 also plays a central role in the regulation of mineral homeostasis and exerts its effects via the vitamin D receptor (VDR), which is a member of the nuclear receptor superfamily. Upon ligand binding, VDR dimerizes with the retinoic X receptor and binds to promoter regions of responsive genes, where it can either activate or repress gene transcription (9); 1,25(OH)2D3 also plays an important role in bone metabolism and has been shown to increase bone mass in vivo (10, 11). Osteoblasts, the bone-forming cells, possess VDR and are considered the main target for 1,25(OH)2D3 action in bone. In cultured osteoblasts, 1,25(OH)2D3 has been shown to stimulate the production of several proteins involved in bone mineralization, including osteocalcin and alkaline phosphatase, thereby enhancing the functional activity of mature osteoblasts (12, 13).

GH initiates a cascade of biological effects by binding to the GH receptor (GHR). GH binding results in GHR dimerization and activation of the receptor-bound tyrosine kinase Janus kinase 2 (JAK2), and, in turn, tyrosine phosphorylates GHR and several intracellular proteins, among them the signal transducers and activators of transcription (STAT) proteins (14). GH has also, in several cell types, been shown to activate the MAPK pathway. The MAPKs, ERK1, and ERK2 are mediators of cellular responses to extracellular signals and have an important role for the MAPK pathway in the GH proliferative response (14, 15).

We have recently shown that 1,25(OH)2D3 prolongs GH signaling via the JAK2/STAT5 pathway in UMR 106 osteoblast-like cells (16). The aim of the present study was to investigate the effects of GH and 1,25(OH)2D3 on proliferation and apoptosis in UMR 106 cells. We present data showing that 1,25(OH)2D3 and GH, separately or in combination, inhibit apoptosis. Furthermore, pretreatment of cells with 1,25(OH)2D3 was necessary to detect GH-induced MAPK activation. GH also exerted clear effects on cell cycle distribution and proliferation. We hypothesize that these hormones, separately regulating the processes of apoptosis and proliferation, are important for maintaining osteoblast cell number.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Rat osteosarcoma cells UMR 106 were cultured at 37 C in 5% CO2-air in MEM (Invitrogen, Life Technologies, Paisley, Scotland, UK), supplemented with 10% fetal bovine serum (FBS; Invitrogen), 1 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin (Invitrogen). FBS was reduced to 0.5% (vol/vol) when cells were treated with hormones.

Cell counting
For cell counting, 22 x 103 cells were seeded in six-well multidishes. Cells were treated with 10 nM 1,25(OH)2D3 (Calbiochem, Darmstadt, Germany) and/or 30 nM bovine GH (bGH) (American Cyanamid, Wayne, NJ) for 4 d in FBS-reduced (0.5%) medium; 1,25(OH)2D3 was solubilized in ethanol, stored at -80 C, and protected against light. Untreated cells were treated with vehicle (ethanol). Cells were harvested at 24-h intervals, for 4 d, by treatment with trypsin-EDTA (Life Technologies). Single-cell suspensions were prepared, and cells were counted in a Bürker chamber (Labasco, Stockholm, Sweden). Triplicates from each time point were analyzed.

Flow cytometric analysis
For flow cytometric analysis of cell cycle distribution, cells were treated with 10 nM 1,25(OH)2D3 and/or 30 nM bGH in FBS-reduced medium (0.5%) for 2 d, washed with PBS, fixed in cold (–20 C) 70% ethanol, and stored at -20 C. The samples were centrifuged, and the (pellets) were resuspended in sample buffer containing propidium iodide (PI) (50 µg/ml) (Caltag Laboratories, Burlingame, CA) and deoxyribonuclease-free ribonuclease A (50 µg/ml; Roche Molecular Biochemicals, Mannheim, Germany) for 1 h at room temperature while the tubes were agitated on a shaking platform. Ten thousand cells were analyzed by flow cytometry in a FACScan (Becton Dickinson and Co., Franklin Lakes, NJ) for cell cycle distribution. The number of cells in the different cell cycle stages were counted and then divided by the total number of cells analyzed; i.e. percentage of cells in each cell cycle stage is presented.

For flow cytometric analysis of apoptosis, cells were treated with 10 nM 1,25(OH)2D3 and/or 30 nM bGH or 10 µM U0126 (Cell Signaling, New England Biolabs, Inc., Beverly, MA) in FBS-reduced medium (0.5%) for 4 d, washed with PBS, and subsequently stained with Annexin-FITC (fluorescein isothiocyanate) and propidium iodide, using an Annexin V kit (Caltag Laboratories) according to the instructions from the manufacturer. Ten thousand cells were analyzed by flow cytometry in a FACScan (Becton Dickinson and Co.) for apoptosis. The number of cells in the different cell populations (Q3 = living cells; Q4 = early apoptotic; Q2 = late apoptotic and Q1 = necrotic cells) were counted and then divided by the total number of cells analyzed; i.e. percentage of cells in each population is presented.

Caspase-3, -8, and -9 -assay
Cells were grown in 6-cm plates to 80% confluence. Cells were nontreated or treated with 30 nM bGH and/or 10 nM 1,25(OH)2D3 in FBS-reduced (0.5%) medium for 48 h. To measure caspase-3, -8, and -9 activity, 1 x 106 cells were washed in PBS, pelleted in a microcentrifuge, and frozen at -80 C. Cells were thawed and resuspended in 50 µl PBS. The appropriate peptide substrate [DEVD-7-amido-4-methylcoumarin (AMC) for Caspase-3, IETD-AMC for Caspase-8, and LEHD–AMC for Caspase-9] was added according to instructions from the manufacturer (Calbiochem). Fluorescence was measured in a Fluoroscan plate reader (Labsystems, Stockholm, Sweden). Fluorescence units were converted to picomoles of AMC using a standard curve generated with free AMC.

Preparation of whole cell extract
UMR 106 cells were grown in 10-cm plates to 80% confluence and treated with 10 nM 1,25(OH)2D3 and/or 30 nM bovine GH in FBS-reduced medium for 24 and 48 h. Cells treated or untreated with 10 nM 1,25(OH)2D3, were also pretreated with U0126 for 30 min before addition of 30 nM bGH for 10 min. After hormone treatment, cells were rinsed with ice-cold PBS. Cells were then scraped into lysis buffer [10 mM Tris-HCl, 10 mM NaH2PO4/Na2HPO4 (pH 7.5), 130 mM NaCl, 1% Triton X-100, 10 mM NaPPi, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreithol, and 0.1 mM Na3VO4] and incubated on ice for 15 min. The supernatant obtained after centrifugation was used as whole-cell extract. Protein concentration was measured in triplicate by the Bradford method.

Western Blotting
SDS-solubilizing buffer was added to whole-cell extracts, and samples were boiled. Proteins were separated on 12% SDS-PAGE gels or 4–20% gradient gels (Bio-Rad Laboratories, Hercules, CA) and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA) by semidry blotting. The membrane was blocked for 1 h with a buffer containing 5% milk protein in Tris-buffered saline [TBS; 50 mM Tris-HCl (pH 7.5), and 150 mM NaCl]. After washing, the membrane was incubated overnight with rabbit anti-phospho-MAPK (Cell Signaling, New England Biolabs, Inc; diluted 1:1000), mouse anti-Bcl-2 (Upstate Biotechnology; Lake Placid, NY; diluted 1:500), or rabbit anti-Bax (Upstate Biotechnology; diluted 1:500) in a buffer containing 1% milk protein in TBS plus 0.05% Tween 20 (TTBS). The secondary antibody, goat antirabbit IgG or goat antimouse coupled with horseradish peroxidase, diluted 1:5000 in TTBS, was applied for 1 h after washing the membrane three times with TTBS. The membrane was then analyzed with the enhanced chemiluminescence method (ECL, Amersham Pharmacia Biotech, Uppsala, Sweden).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GH and 1,25(OH)2D3 decrease apoptosis and affect cell cycle distribution
To investigate the effects of 1,25(OH)2D3 and GH on growth of UMR 106 cells, cells were counted daily for 4 d (Fig. 1AGo). Cells were grown in serum-reduced medium (0.5% FBS) in the presence of 10 nM 1,25(OH)2D3 or 30 nM bGH or in a combination of both hormones. bGH was shown to induce a significant increase in the number of cells, an effect that was observed from d 1–4; 1,25(OH)2D3 did not have an effect on cell count, compared with untreated cells. Interestingly, treatment of cells with 1,25(OH)2D3 and bGH in combination resulted in significantly increased cell counts from d 2–4, compared with treatment of cells with bGH or 1,25(OH)2D3 alone.



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  		FIG. 1. Effect of 1,25(OH)2D3 and GH on apoptosis and proliferation of UMR 106 osteosarcoma cells. UMR 106 cells were treated with 10 nM 1,25(OH)2D3 and 30 nM bGH, separate or in combination. A, Cells were treated for 4 d. Every 24 h, cell number was determined by counting cells in a Bürker chamber. All experiments were performed in triplicate at least three times; ±SD and significant differences, compared with untreated cells, are indicated in the figure (*, P < 0.05; ANOVA and Turkey’s post hoc test were used for statistical analysis with Sigma Stat software, Jandel Corp., Sausalito, CA). B, Cells were treated for 4 d, and apoptosis was measured by flow cytometric analysis after staining with Annexin V-FITC and propidium iodide (PI) as described in Materials and Methods. Apoptotic cells are present in the Q2 and Q4 populations. This experiment was repeated twice. C, Cells were treated with hormones for 2 d, and cell cycle distribution was measured by FACS analysis after staining with PI as described in Materials and Methods. Indicated in the figure are the G1 and the S/G2/M populations. This experiment was repeated three times.

 
The number of cells in a tissue or in a culture dish is dependent on mitogenesis and apoptosis. To further investigate the mechanism behind the increased number of UMR 106 cells observed after 1,25(OH)2D3 and bGH treatment, we analyzed cell cycle distribution and apoptosis by flow cytometric analysis. Percentage of early apoptotic cells, the Q4 population with high Annexin V and low propidium iodide staining, was decreased from 20% to 10% after 4 d of treatment with 10 nM 1,25(OH)2D3, compared with the control (Fig. 1BGo). Also, the population of late apoptotic/necrotic cells, Q2, with high Annexin V and high propidium iodide staining, was reduced from 26% to 9%. Similarly, 4 d of treatment with 30 nM bGH reduced the population of early apoptotic cells to 17% and the population of late apoptotic/necrotic cells to 11%. Compared with treatment with 1,25(OH)2D3 alone, cotreatment of cells with bGH and 1,25(OH)2D3 did not further decrease the early apoptotic (9%) nor the late apoptotic/necrotic populations (12%), indicating that 1,25(OH)2D3 was the major antiapoptotic stimuli in this setting. By staining the cells with propidium iodide, we also measured cell cycle distribution after 2 d of treatment with 1,25(OH)2D3 and bGH, alone or in combination. The results show that a redistribution of cells from G1 (33.8%) to S/G2/M-phases (55.4%) occurred after bGH treatment, compared with the control cells, where the G1 population was 59.4% and the S/G2/M population was 29.3% (Fig. 1CGo). These data indicate an increased proliferation after bGH treatment. In contrast, treatment with 1,25(OH)2D3 increased the number of cells in the G1-phase (from 59.4 to 65%) as compared with control. The G1-accumulating effect of 1,25(OH)2D3 was also observed during cotreatment with bGH (G1 = 45.7%), given that an intermediate response was seen as compared with untreated (G1 = 59.4%) and bGH (G1 = 33.8%) treated cells, respectively. In summary, these data indicate that decreased apoptosis and altered cell cycle distribution are important processes by which 1,25(OH)2D3 and GH regulate the number of UMR 106 cells.

GH and 1,25(OH)2D3 decrease caspase-3, -8, and -9 activity in UMR 106 cells
To investigate the mechanism and to further verify the effects of 1,25(OH)2D3 and GH on apoptosis, we measured the activity of caspase-3, -8, and -9 after treatment of UMR 106 cells for 2 d with 1,25(OH)2D3 and bGH (Fig. 2Go). Caspase-3 activity was significantly decreased after 2 d of treatment with 1,25(OH)2D3 and bGH, both alone or in combination, compared with untreated cells (Fig. 2AGo). However, the inhibition of caspase-3 was most clearly seen in the presence of 1,25(OH)2D3, further verifying a major role for this hormone in the observed antiapoptotic effect. Because caspase-3 is activated by two partially different pathways, one involving caspase-8 and one involving caspase-9, it was relevant to investigate the effect of 1,25(OH)2D3 and GH on these caspases as well. Results presented in Fig. 2BGo show that caspase-8 activity was inhibited by 1,25(OH)2D3 and bGH, both individually and in combination. Similarly, inhibitory effects by 1,25(OH)2D3 and bGH were observed when measuring caspase-9 activity (Fig. 2CGo). These data suggest that the 1,25(OH)2D3- and GH-mediated decrease in caspase-3 activity may be carried out through a mechanism involving inhibition of both caspase-8 and caspase-9 proteolytic activity.



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  		FIG. 2. Effects of 1,25(OH)2D3 and GH on caspase-3, -8, and -9 activity. UMR 106 cells were treated for 2 d with 10 nM 1,25(OH)2D3 and 30 nM bGH, separate or in combination. For each analysis, extracts were prepared from 1 x 106 cells and incubated with caspase-3 (A), -8 (B), or -9 (C) fluorogenic substrates as described in Materials and Methods. Fluorescence was measured as released picomoles of AMC in a spectrofluorometer. Triplicate samples were compared with untreated cell lysates. This experiment was repeated three times; ±SD and significant differences are indicated in the figure (*, P < 0.05; ANOVA and Turkey’s post hoc test was used for statistical analysis with Sigma Stat software).

 
Potential targets for the regulation of caspase-9 activity are members of the Bcl-2 family. We therefore performed immunoblot analysis of Bcl-2 and Bax (Fig. 3Go) with whole-cell extracts from cells treated 1 or 2 d with 10 nM 1,25(OH)2D3 and 30 nM bGH, alone or in combination. The results show that expression of the antiapoptotic protein Bcl-2 was slightly increased after 24 h of treatment with 1,25(OH)2D3, bGH, or a combination of both, compared with untreated cells (Fig. 3AGo; lanes 1, 3, 5, and 7). The induced expression was less prominent after 48 h of treatment. Expression of the proapoptotic protein Bax was increased after 24 h treatment with bGH, compared with untreated cells (Fig. 3BGo, lanes 1 and 3); but after 48 h of treatment with bGH, the level of Bax expression was reduced (Fig. 3BGo, lanes 2 and 4). Treatment with 1,25(OH)2D3 for 24 and 48 h, alone or in combination with bGH, reduced the levels of Bax as compared with untreated cells (Fig. 3BGo; lanes 1, 2, 5, 6, 7, and 8).



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  		FIG. 3. Effects of 1,25(OH)2D3 and GH on expression of Bcl-2 and Bax proteins. UMR 106 cells were grown in the absence or presence of 10 nM 1,25-(OH)2D3 and 30 nM bGH, alone or in combination, for 24 h and 48 h. Whole-cell extracts (50 µg protein in each lane) were separated by SDS-PAGE and analyzed by Western blotting with antibodies recognizing Bcl-2 (A) and Bax (B). One representative of three experiments is shown.

 
Inhibition of MAPK signaling induces apoptosis in UMR 106 cells
Reduced MAPK activity has been associated with increased apoptosis in different cell types (17). To investigate the importance of MAPK in transmitting antiapoptotic signals in osteoblast-like cells, cells were treated with 10 µM U0126, a specific inhibitor of MAPK kinase (MEK), the upstream activating dual-specificity kinase of ERK 1 and 2 (Fig. 4Go, A and B). UMR 106 cells, grown for 48 h with or without 10 nM 1,25(OH)2D3, were stimulated with 30 nM bGH for 10 min in the absence or presence of 10 µM U0126. Whole-cell extracts were analyzed by Western blot using an antibody that recognizes active phosphorylated ERK 1 and 2. In unstimulated cells, a weak signal for active ERK1 and 2 was detected, which disappeared after treatment with U0126 for 30 min (Fig. 4AGo, lanes 1 and 2). Treatment of cells with 1,25(OH)2D3 alone did not significantly increase activation of ERK 1 and 2; and also in this setting, ERK 1 and 2 were inhibited by U0126 (Fig. 4AGo, lanes 3 and 4). In the absence of 1,25(OH)2D3, stimulation of cells with bGH weakly activated ERK1 and 2, an effect which was inhibited by U0126 (Fig. 4AGo, lanes 5 and 6). Interestingly, pretreatment with 1,25(OH)2D3 resulted in a significantly increased activation of ERK1 and 2 (Fig. 4AGo, lane 7). This increased activation was abolished when cells were pretreated for 30 min with U0126 (Fig. 4AGo, lane 8).



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  		FIG. 4. Effects of the MEK inhibitor U0126 on MAPK activation and apoptosis of UMR 106 cells. A, UMR 106 cells were untreated or pretreated with 10 nM 1,25(OH)2D3 for 48 h. Then, bGH (30 nM) was added for 10 min to some cells, as indicated in this figure (lanes 5–8). Some cells were also treated with 10 µM U0126 for 30 min before addition of bGH (lanes 2, 4, 6, and 8). Whole-cell extracts (50 µg protein in each lane) were separated by SDS-PAGE and analyzed by Western blotting with an antibody recognizing phospho-MAPK. This experiment was repeated twice. B, Cells were treated with 10 µM U0126 for 1 d, and apoptosis was detected by FACS analysis after staining with Annexin V-FITC and propidium iodide (PI). Apoptotic cells are present in the Q2 and Q4 populations. This experiment was repeated at least three times. P-ERK 1,2, Phospho-ERK 1,2.

 
To detect whether U0126 also could influence apoptosis, cells were treated for 1 d with U0126 and thereafter analyzed by flow cytometry. As can be seen in Fig. 4BGo, nearly all cells were detected in the Q4 population; i.e. they were in the early apoptosis stage. This illustrates the importance of MAPK for the survival of UMR 106 cells. Pretreatment with 10 nM 1,25(OH)2D3 and 30 nM bGH, separate or in combination, did not protect the cells from apoptosis induced by U0126 (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have investigated the effects of 1,25(OH)2D3 and GH on apoptosis and proliferation in UMR 106 osteoblast-like cells. The UMR 106 is a rat clonal cell line with osteoblast-like phenotypic properties, responsive to both GH and 1,25(OH)2D3 treatment (18, 19). We found that 1,25(OH)2D3 and GH in combination significantly increased the number of cells, compared with cells treated with the hormones separately. This effect was associated with decreased apoptosis and altered cell cycle distribution. Inhibition of apoptosis was mainly the effect of 1,25(OH)2D3, and increased proliferation was the main effect of GH (Fig. 1Go, B and C). This is in agreement with a model where 1,25(OH)2D3 and GH, in an additive manner, increase the number of osteoblast-like cells by inhibition of apoptosis and stimulation of proliferation, respectively. Previous reports have indicated that 1,25(OH)2D3 and GH have effects on apoptosis in several cell types and tissues. 1,25(OH)2D3 has mainly been shown to have proapoptotic effects on tumor cells from different origins (20, 21). Furthermore, 1,25(OH)2D3 has been implicated to increase apoptosis in mouse hypertrophy chondrocytes, where VDR deficiency decreased apoptosis (22). However, in the present study, we present data that clearly show an antiapoptotic effect of 1,25(OH)2D3 on UMR 106 osteoblast-like cells (Fig. 1BGo). This is in line with earlier data obtained from osteosarcoma cells (23) and may provide one explanation for the well-established anabolic effects of 1,25(OH)2D3 on bone (10, 11). Earlier studies have also described antiproliferative effects of 1,25(OH)2D3 in other cell models (24, 25), mainly by inhibiting the G1-S phase transition in the cell cycle (24). Indeed, our data support a G1-accumulating effect of 1,25(OH)2D3 in osteoblast-like cells as well (Fig. 1CGo). This effect is, however, likely to be compensated for by the antiapoptotic effect exerted by this hormone, because no decrease in cell number was observed (Fig. 1AGo).

Several studies have elucidated the proliferative effects of GH on osteoblasts (19) as well as the anabolic effects on bone (26). In agreement with this, results presented in this study show an increased amount of cells in the S/G2/M phase after GH treatment (Fig. 1CGo), clearly implicating a proliferative effect. Modulation of apoptosis by GH has also been studied in different cell types and tissues. GH has, for instance, been shown to prevent apoptosis of cells in the immune system (27, 28). However, little is known about the apoptotic properties of GH in osteoblasts. Results presented in this study clearly show an antiapoptotic effect of GH on osteoblast-like cells (Fig. 1BGo), which may act in concert with the proliferative effect resulting in increased cell number.

To investigate the mechanism behind the antiapoptotic effects exerted by 1,25(OH)2D3 and GH, the activities of caspase-3, -8, and -9 were measured. Caspases have been identified as essential effectors of apoptosis, exerting their effects in a cascade involving both receptor-dependent and mitochondria-dependent pathways. Data presented in this study clearly show that caspase-3, a down-stream effector caspase, is inhibited in cells treated with 1,25(OH)2D3 and GH (Fig. 2AGo). The inhibition of caspase-3 was seen after 48 h treatment with 1,25(OH)2D3 and/or GH. In addition, both the receptor-mediated caspase-8 and the mitochondria-dependent caspase-9 were inhibited after 48 h with the same hormonal treatment (Fig. 2Go, B and C). Although 1,25(OH)2D3 and GH have a relatively equal capacity to inhibit the initiator caspases, caspase-8 and -9, 1,25(OH)2D3 elicited a more pronounced inhibition than GH of the effector caspase, caspase-3 (Fig. 2AGo). Thus, the total activity of caspase-8 and -9 cannot fully explain the effect on caspase-3 activity, indicative of alternative pathways participating in the regulation of this caspase. Such mechanisms are indeed described in the literature, one example involving inhibition of caspase activity by specific proteins called inhibitors of apoptosis proteins (29). However, the exact mechanism for the antiapoptotic effects of 1,25(OH)2D3 and GH in UMR 106 cells needs to be further explored. Despite this, a likely mechanism for the antiapoptotic effect of 1,25(OH)2D3 and GH involves regulation of caspase activity, both by mitochondria and receptor mediated pathways.

Proteins belonging to the Bcl-2 family are known regulators of mitochondrial-dependent caspase-9 and, subsequently, caspase-3 activity (30). Pro- and antiapoptotic Bcl family proteins are expressed in cartilage and osteoblasts (31), although the functional consequences of the expression of these proteins in osteoblasts have not yet been determined. Therefore, the up- and down-regulation of these proteins are potentially mediating the observed inhibitory effects of 1,25(OH)2D3 and GH on apoptosis in UMR 106 osteoblast-like cells. Interestingly, our data show that both the antiapoptotic Bcl-2 and the proapoptotic Bax are regulated by treatment with 1,25(OH)2D3 and GH. Bcl-2 expression is increased after 24 h of 1,25(OH)2D3 and GH treatment, separate or in combination (Fig. 3AGo). Although Bax expression was transiently increased by GH after 24 h, a down-regulation of this protein was observed after 48 h. 1,25(OH)2D3 down-regulated Bax alone or in combination with GH after 24 and 48 h, compared with untreated cells (Fig. 3BGo). Thus, the ratio of Bcl-2/Bax at this time point is in favor of antiapoptosis. We therefore speculate that down-regulation of caspase-3, -8, and -9 activity, through regulation of pro- and antiapoptotic members of the Bcl-2 family, contributes to the antiapoptotic phenotype observed after 1,25(OH)2D3 and GH treatment in osteoblast-like cells.

Signaling by GH and other cytokines leads to the activation of the Ras-MAPK signaling pathway (14). ERK1/ERK2, down-stream kinases in the MAPK pathway, regulate the activity of various transcription factors, which are subsequently involved in regulation of proliferation, apoptosis, and differentiation (32). Antiapoptotic effects of the activated MAPK pathway have been implicated in several cell systems (17, 33). In support of an important antiapoptotic function, the MEK has been shown to be cleaved in a caspase-dependent manner, leading to inactivation of down-stream ERK1/2 during apoptosis (21). The critical involvement of this MAPK pathway in protection against apoptosis was indeed found in the present study, where the MEK inhibitor, U0126, effectively inhibited the phosphorylation of ERK1/2 and strongly induced apoptosis in UMR 106 osteoblast-like cells (Fig. 4Go, A and B). Furthermore, GH-induced MAPK activity was potentiated by 1,25(OH)2D3 treatment (Fig. 4AGo), supporting a model where MAPK signaling is involved in the antiapoptotic response of these hormones. Interestingly, ERK1 and ERK2 have been implicated in up-regulation of the antiapoptotic Bcl-2 family member Bcl-xL, a mechanism that may explain the involvement of these kinases in protecting against apoptosis (34). Furthermore, a functional STAT5 responsive element has been described in the Bcl-xL promoter (35). It cannot be excluded that GH-activated STAT5 contributed to antiapoptosis by up-regulation of Bcl-xL.

However, mechanisms independent of MAPK activity are also likely to play a role, because 1,25(OH)2D3, which in itself has a strong antiapoptotic potential, alone did not promote a clear MAPK activation. It is also important to stress that, despite the synergistic effects of 1,25(OH)2D3 and GH on MAPK activity and cell number, no synergism was observed either on apoptosis or cell cycle distribution, suggesting separate mechanisms regulating these processes.

In conclusion, this study presents data supporting a model where 1,25(OH)2D3 and GH, in combination, increase the amount of osteoblast-like cells. This is mediated by an antiapoptotic effect exerted mainly by 1,25(OH)2D3 but also by GH, as well as a proliferative effect of GH. We hypothesize that these hormones, separately regulating the processes of apoptosis and proliferation, are important for maintaining osteoblast cell number.

Osteoporosis is characterized by low bone mass and microarchitectural deterioration of the skeleton, leading to an increased risk of fracture after minimal trauma (1). Therapeutic agents that increase the number of osteoblasts could improve bone mass and decrease the risk of fractures. Controlling cell proliferation, differentiation, and apoptosis is also important for the production of preosteoblasts and osteoblast from mesenchymal stem cells. Ultimately, these cells could be used in the reconstruction of bone defects. It is possible that new hormonal regimens including 1,25(OH)2D3 and GH in the future will be of benefit for treatment of osteoporosis and production of bone-forming cells from mesenchymal stem cells.


   Footnotes
 
Abbreviations: AMC, 7-Amido-4-methylcoumarin; bGH, bovine GH; FBS, fetal bovine serum; GHR, GH receptor; JAK, Janus kinase; MEK, MAPK kinase; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; PI, propidium iodine; STAT, signal transducer and activator of transcription; TTBS, TBS plus 0.05% Tween 20; VDR, vitamin D receptor.

Received June 9, 2003.

Accepted for publication September 22, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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D. J. DiGirolamo, A. Mukherjee, K. Fulzele, Y. Gan, X. Cao, S. J. Frank, and T. L. Clemens
Mode of Growth Hormone Action in Osteoblasts
J. Biol. Chem., October 26, 2007; 282(43): 31666 - 31674.
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