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Endocrinology Vol. 139, No. 5 2622-2628
Copyright © 1998 by The Endocrine Society

ARTICLES

1,25-Dihydroxy Vitamin D3 Inhibits Adipocyte Differentiation and Gene Expression in Murine Bone Marrow Stromal Cell Clones and Primary Cultures1

Katherine A. Kelly and Jeffrey M. Gimble

Departments of Pathology (K.A.K., J.M.G.), Orthodontics (K.A.K.), and Surgery (J.M.G.), University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190; Immunobiology & Cancer (K.A.K., J.M.G.), Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104

Address all correspondence and requests for reprints to: Dr. Jeffrey M. Gimble, Department of Surgery, University of Oklahoma Health Sciences Center, Williams Pavilion 2140, WP2140, 920 Stanton L. Young Boulevard, Oklahoma City, Oklahoma 73190. E-mail: Jeffrey-Gimble{at}ouhsc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bone marrow stromal stem cells differentiate into adipocytes and osteoblasts. These two lineages are thought to be reciprocally related, in part due to the observation that the osteoblast-inducing factor, 1,25 dihydroxy vitamin D3 [1,25(OH)2D3], inhibited adipogenesis of rat femoral-derived stromal cell cultures. However, the literature is divided concerning the adipogenic effects of this steroid hormone. This work examined the effect of 1,25(OH)2D3 (10-12–10-8 M) on murine femoral-derived bone marrow stromal cell differentiation in response to adipogenic agonists employing two different classes of nuclear hormone receptors: the glucocorticoid receptor (hydrocortisone) or peroxisome proliferator-activated receptors (thiazolidinediones). Experiments used the multipotent murine bone marrow stromal cell line, BMS2, and its subclones, as well as primary-derived murine bone marrow stromal cell cultures. In all systems examined, 1,25(OH)2D3 blocked adipogenesis induced by hydrocortisone, methylisobutylxanthine, and indomethacin based on flow cytometric analysis of lipid accumulation. This correlated with reduced messenger RNA levels of the late adipocyte gene markers, aP2 and adipsin. In the BMS2 subclone no. 24, the 1,25(OH)2D3 actions were concentration dependent. Whereas 1,25(OH)2D3 partially inhibited thiazolidinedione-induced adipogenesis in the parental BMS2 cell line, it had minimal effect on the thiazolidinedione-induced differentiation of the BMS2 subclone and primary cultures. These findings indicate that 1,25(OH)2D3, at nanomolar concentrations, completely inhibits murine bone marrow stromal cell differentiation in response to glucocorticoid-based adipogenic agonists but is a less effective adipogenic antagonist following induction with thiazolidinediones. This work supports the conclusion that 1,25(OH)2D3 inhibits murine femoral-derived bone marrow stromal cell adipogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE BONE MARROW contains progenitors identified as stromal or mesenchymal stem cells that exhibit the ability to differentiate into adipocytes and osteoblasts (1, 2, 3, 4, 5). Steroids and other nuclear hormone receptor ligands play an important role in determining which of these terminal differentiation pathways the stromal stem cells will choose. It is well established that glucocorticoids, acting through the glucocorticoid receptor, and thiazolidinediones, acting through the peroxisome proliferator-activated receptors, induce bone marrow-derived stromal cells to undergo adipogenesis (4, 6). The resulting adipocytes are distinguished by their expression of distinct gene markers (lipoprotein lipase, aP2, adipsin) and their cytoplasmic accumulation of lipid vacuoles. Studies suggest that a reciprocal relationship exists between bone marrow stromal cell adipogenesis and osteogenesis (7, 8). The induction of adipocyte gene markers by glucocorticoids is accompanied by the reduced expression of distinctive osteoblast gene markers, like osteocalcin (7, 8). Experiments supporting this hypothesis included the observation that 1,25 dihydroxy vitamin D3 [1,25(OH)2D3], an osteoblast-inducing factor, antagonized the adipogenic actions of dexamethasone in primary stromal cultures derived from rat femoral bone marrow (7). However, the effects of 1,25(OH)2D3 on adipogenesis are controversial. 1,25(OH)2D3 has been reported to inhibit, enhance, or exhibit biphasic effects on adipocyte differentiation in a variety of cell lines and primary cultures examined in vitro (7, 9, 10, 11, 12, 13, 14, 15, 16, 17). These findings have led some authors to suggest that 1,25(OH)2D3 may positively regulate adipocyte differentiation in the bone marrow microenvironment (9, 10). We have previously demonstrated that the murine femoral bone marrow derived cell line, BMS2, can be used as an in vitro stromal stem cell model (4). These cells undergo adipocyte differentiation in response to hydrocortisone, methylisobutylxanthine, and indomethacin or to thiazolidinedione compounds (4, 6). Like other osteoblast cell lines and primary osteoblast cultures, BMS2 cells exhibit a dose-dependent induction of complement component C3 following exposure to 1,25(OH)2D3, indicating the presence of a functional vitamin D receptor (18, 19, 20, 21). In this study, we have examined the effect of 1,25(OH)2D3 on hydrocortisone and thiazolidinedione-induced adipogenesis using the parental BMS2 cell line, recently developed BMS2 subclones, and primary murine bone marrow stromal cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All reagents were obtained from Sigma Chemical Company (St. Louis, MO) and Fisher Scientific (Dallas, TX) unless otherwise noted.

Cell culture: cell lines
BMS2, a murine bone marrow stromal cell line (22), was originally obtained from Drs. C. Pietrangeli and P. W. Kincade (Immunobiology & Cancer, Oklahoma Medical Research Foundation, Oklahoma City, OK) and maintained in modified DMEM (high glucose) supplemented with 10% (vol/vol) FBS (Defined; Hyclone, Logan, UT), 1 mM sodium pyruvate, 100 U penicillin per ml, 100 µg streptomycin per ml, and 50 µM ß-mercaptoethanol (referred to as supplemented DMEM) at 37 C in 7% CO2. The BMS2 cells were passaged every 7 days. In adipogenic studies, 4 x 104 BMS2 cells in a 2 ml vol were cultured in a 35-mm plate and grown for 6–7 days until confluent and quiescent. At that time, cells were induced with adipogenic factors and/or 1,25(OH)2D3 at the concentrations outlined belowed for a period of 3 days. At that time, the medium was replaced without any adipogenic agents. However, the fresh medium was supplemented with 1,25(OH)2D3 at the original concentration. Cultures were then maintained an additional 3 days, at which time they were harvested for RNA. Cell densities reached approximately 4.7 x 105 per 35-mm plate at the conclusion of these studies.

BMS2 subclones were developed from the parental stromal cell line by limiting dilution cloning (23). The parental cell line was serially diluted in supplemented DMEM to a concentration of approximately 0.5 cells per 200 µl volume. Two hundred-microliter volumes were aliquoted into individual wells of a 96-well plate. After 3 weeks in culture, 60 subclones were isolated, subpassaged, and characterized based on their ability to form fat in response to hydrocortisone, methylisobutylxanthine, and indomethacin as detected by flow cytometry. Subclones were maintained as described for the parental BMS2 cell line. In adipogenic assays, 2 x 104 cells in 0.5 ml vol were cultured in individual wells of a 24-well plate. After 3 days in culture, adipogenic factors and/or 1,25(OH)2D3 was introduced in fresh medium as outlined below.

Cell culture: primary bone marrow cells
Female Balb/c mice were obtained from the breeding colony housed at the Laboratory Animal Resource Center of the Oklahoma Medical Research Foundation. At 6 weeks of age, mice were euthanized by carbon dioxide asphyxiation according to protocols approved by the Institutional Animal Care and Use Committee. Femora and tibiae were aseptically harvested, and the whole bone marrow was flushed using supplemented DMEM in a 10-cc syringe and a 25-gauge needle. Suspended whole bone marrow was washed, the number of viable, nucleated cells determined by trypan blue/acetic acid staining and 1 x 107 cells plated in a 25-cm2 flask (Costar Corning, Corning, NY). Following 2 h incubation at 37 C in 7% CO2, humidified air, the nonadherent cells were removed from the cultures by gentle aspiration and the medium replaced with fresh supplemented DMEM. Cultures were fed weekly for 2 weeks with supplemented DMEM until greater than 80% confluent and quiescent, as previously described (6).

At this time, cultures were exposed to adipogenic agonists (MHI or 5 µM BRL 49653) and/or 10-8 M 1,25(OH)2D3. Cultures were maintained in the presence of MHI for 3 days, at which time the medium was replaced without MHI but with 1,25(OH)2D3 when present. Cultures were maintained in the presence of BRL 49653 throughout the 6-day experimental period when cells were harvested for total RNA or fixed for Oil Red O staining. Cell density was not determined at the time of RNA harvest. Flow cytometry analyses outlined in the following section determined that contaminating macrophages (Mac-1+) constituted greater than 3% of the final cell population in the primary stromal cultures.

Detection of contaminating macrophages
Cultured cells were suspended in 10 mM EDTA in PBS for 5 min at 22 C and fixed with 20% (vol/vol) paraformaldehyde at 4 C. Cells were resuspended in rat antimouse Mac-1 antibody (recognizing the {alpha}M integrin protein) (32) or control medium and incubated for 15 min on ice. Cells were washed and resuspended in goat antirat conjugated with DTAF secondary antibody or control medium for 15 min on ice before being centrifuged and resuspended in PBS for FACS analysis. The number of cells staining positive for antibody were quantitated as percentage of total number of cells.

Adipogenesis studies
The MHI cocktail consisted of 0.5 mM methylisobutyl xanthine, 0.5 µM hydrocortisone, and 60 µM indomethacin in DMSO (final concentration DMSO <0.1% (vol/vol) in fresh supplemented DMEM) and was added to cultures for a 3-day inductive period as previously described (6). The thiazolidinediones, BRL49653 and pioglitazone, were added at concentrations of 5 µM and 25 µM, respectively, in DMSO, using fresh supplemented DMEM as previously described (6). The thiazolidinedione compounds were generously supplied by Dr. D. Morris (GlaxoWellcome, Research Triangle Park, NC). Adipogenic inductions were performed in the absence or presence of 1,25(OH)2D3 (10-12–10-8 range). The 1,25(OH)2D3 was generously provided by Dr. M. Uskokovic (Hoffman-LaRoche, Nuttley, NJ). Whenever 1,25(OH)2D3 was introduced into the assay medium, the cultures were maintained continuously in the presence of 1,25(OH)2D3 throughout the experimental period. Cultures were harvested for Nile red staining and FACS, Oil Red O staining, or total RNA analysis 6 days after the initial addition of the adipogenic agonist. On average, 4.7 x 105 cells were harvested per 35-mm plate.

RNA analysis
Cultures were established at the initial plating density and treatments were performed as described above for parental BMS2 cells and primary bone marrow stromal cell cultures. Individual 35-mm plates of BMS2 cells or a single 25 cm2 flask of primary cultured cells were harvested for RNA following the modified method of Chomczynski and Sacchi (24) as previously described (8). Northern blots were run with approximately 10 µg total RNA per lane in a formaldehyde agarose gel (25), transferred to a MSI-NT nylon membrane (MSI, Westboro, MA), and UV cross-linked for 5 min with a UV transilluminator (UVP, Inc., San Gabriel, CA). Northern blots were prehybridized and hybridized with complementary DNA (cDNA) probes, in 500 mM sodium phosphate, pH 7.2, 7% SDS, and 1 mM EDTA at 55 C overnight (26). cDNA probes were labeled by the random-primer method using [{alpha}32P] dCTP (ICN, Irvine CA) (27). Following hybridization, blots were washed four times for 20 min in 40 mM NaHPO4, pH 7.2, 1% SDS, and 1 mM EDTA, at maximum stringency at 55 C and exposed to autoradiographic film (Eastman Kodak Co., Rochester, NY) for 1 to 7 days at -70 C with an enhancing screen. The following cDNA probes were used to hybridize Northern blots: lipoprotein lipase (LPL) (28); aP2 (H. Green, Harvard University, Cambridge, MA) (29); adipsin (B. Spiegelman and Wm. Wilkinson, Dana Farber, Boston, MA) (30); and actin (B. Spiegelman, Dana Farber, Boston, MA).

Fluorescence-activated cell sorting by Nile Red lipophilic stain
Stromal cell cultures were resuspended in 0.25% trypsin/ 1 mM EDTA (Gibco BRL, Gaithersburg, MD), and fixed with 0.35% (vol/vol) paraformaldehyde. Neutral lipids of adipocytes were stained by adding 88 ng/ml Nile Red to the cell suspension and the gold fluorescent emission was detected between 564 and 604 nm with a bandpass filter using a FACScan (Becton Dickinson, San Jose, CA) (8, 31).

Oil Red O
Adipogenic cultures were fixed with periodate-lysine-paraformaldehyde pH 7.4 for 5 min at room temperature and rinsed in distilled water. Working stock solution of Oil Red O was prepared from 1% (wt/vol) Oil Red O in 99% isopropanol and diluted to 0.3% (vol/vol) with distilled water (33). Cultures were stained with Oil Red O working solution for 15 min with gentle rocking, rinsed with distilled water, stored under 50% glycerol, and photographed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Vitamin D3 effects on adipogenesis by BMS2 stromal cells
The initial studies were designed to determine the effect of 1,25(OH)2D3 on bone marrow adipogenesis. The preadipocyte cell line BMS2 was induced with two different adipogenic agents containing either glucocorticoids in combination with methylisobutylxanthine and indomethacin (MHI) or thiazolidinediones alone. As seen in Fig. 1Go, BMS2 cells underwent adipogenesis in response to treatment with the MHI cocktail (39% adipogenesis) or thiazolidinediones (31–35% adipogenesis), as measured by Nile Red staining on FACS analysis. The concurrent addition of 1,25(OH)2D3 (10-8 M) with the MHI cocktail effectively blocked adipocyte differentiation, reducing it to below 10% of the maximal levels. In contrast, the simultaneous addition of 1,25(OH)2D3 with either thiazolidinedione, BRL 49653, or pioglitazone decreased adipogenesis to 30% of the maximal induced level. In control experiments where 1,25(OH)2D3 alone was added to the preadipocytes, the cells did not accumulate lipid vacuoles based on Nile Red staining or visual inspection.



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  		Figure 1. Parental BMS2 adipogenesis-FACS analysis. The parental BMS2 cells were grown under adipogenic conditions in the absence and presence of 1,25(OH)2D3 (vitamin D) as described in Materials and Methods, then fixed with paraformaldehyde and stained with Nile Red. The number of cells emitting fluorescence at gold emission (565–604 nm) were measured on FACS and reported as a percentage of total stromal cells. A representative tracing from a single experiment is shown. The values reported in the figure are the mean ± SD of the percentage adipogenesis from four separate experiments, each performed in duplicate.

 
Additional studies determined the level of adipocyte gene markers expressed under these culture conditions (Fig. 2Go). Northern blots were probed with cDNA probes for lipoprotein lipase (LPL), an early marker of adipogenesis, and adipsin and aP2, later markers of adipocyte commitment. Actin was used to monitor relative total RNA loading between lanes as was ethidium bromide staining of the gel. The presence of 1,25(OH)2D3 blocked expression of aP2 and adipsin genes following induction with MHI but not thiazolidinediones. The messenger RNA (mRNA) levels of both aP2 and adipsin correlated with the degree of Nile Red staining as described in Fig. 1Go. In contrast, treatment with 1,25(OH)2D3 did not markedly inhibit the mRNA level of the early adipogenic marker, LPL. The actin signal was relatively decreased under conditions corresponding to a high percentage of adipocyte differentiation. This is consistent with earlier reports in the 3T3 preadipocyte model where cytoskeletal gene mRNAs (actin, tubulin) were reduced following adipogenesis (34).



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  		Figure 2. Parental BMS2 adipogenesis-Northern blot analysis. The parental BMS2 cells were grown under adipogenic conditions as described and harvested for total RNA. Northern blots were generated with 10 µg total RNA per lane and blots were probed with the following cDNA probes: adipsin, AP2, LPL, actin. A photograph of the ethidium bromide stained gel is included to assess relative RNA loading between lanes. Data shown are representative of three individual experiments.

 
A limitation of the parental BMS2 cell line is that, at most, only 50% of the cells differentiate into adipocytes. Therefore, subclones of the parental BMS2 cell line with the capacity for higher levels of adipogenesis were developed by limiting dilution and screening following exposure to MHI. Over 60 BMS2 subclones were categorized based on their percentage of adipogenesis. Overall, the subclones exhibited an average of 40% adipogenesis, which was comparable with that of the parental BMS2 cells. Two subclones with the greatest adipogenic potential were used to further investigate the effect of 1,25(OH)2D3 (Table 1Go). Subclones no. 24 and no. 39 were induced with adipogenic agonists (MHI cocktail, BRL or Pioglitazone) for 3 days; vitamin D (10-8 M), when present, was added during this period and for the remaining 3 days of culture. Addition of 1,25(OH)2D3 by itself had minimal adipogenic agonist activity. The most effective agonist was the MHI cocktail, which induced adipogenesis in 78% of subclone no. 24 and 81% in subclone no. 39 cells. Induction by the thiazolidinediones was less pronounced; the subclone no. 24 cells responded with levels of 28% adipocytes to both compounds, whereas the subclone no. 39 cells increased adipocyte numbers to 19% and 33% with BRL49653 and pioglitazone, respectively. The addition of 1,25(OH)2D3 reduced MHI induction to between 1 and 2%, levels comparable with the control preadipocyte cells. In contrast, the effect of 1,25(OH)2D3 on thiazolidinediones was less pronounced. The maximal inhibition obtained with addition of 1,25(OH)2D3 to either subclone in the presence of thiazolidinediones was a level of 19% adipogenesis. In one case (subclone no. 39 induced with BRL49653), the level of adipogenesis actually rose in the presence of 1,25(OH)2D3.


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  		Table 1. BMS2 subclones FACS analysis: response to MHI cocktail or thiazolidinediones and 1,25(OH)2D3

 
To determine if 1,25(OH)2D3 actions were dose dependent and/or biphasic, subclone no. 24 was induced with MHI or BRL49653 in the presence of increasing concentrations of 1,25(OH)2D3 (Fig. 3Go). In the presence of 1,25(OH)2D3 alone, the subclone no. 24 cells retained a fibroblast-like morphology. Adipogenesis in response to MHI showed a dose-dependent inhibition by 1,25(OH)2D3 over a concentration range from 0.1–10 nM; the inhibition was statistically significant (P values < 0.01) relative to control (MHI alone) values, with greater than a 90% reduction in adipocyte numbers. These same concentrations of 1,25(OH)2D3 did not significantly inhibit BRL49653-induced adipogenesis relative to control (BRL alone) values.



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  		Figure 3. BMS2 Subclone no. 24 dose-dependent effects of 1,25(OH)2D3 on adipogenesis. BMS2 Subclone #24 was treated with MHI cocktail or BRL as described in Materials and Methods, in the presence of increasing doses of 1,25(OH)2D3 (0 to 10 -8M). Cells staining with Nile Red are detected and measured on FACS analysis and reported as the percentage of total stromal cells. The tracing shown is that of a representative experiment. The numerical values reported represent the mean ± SD for three experiments, each performed in duplicate.

 
1,25(OH)2D3 effects on adipogenesis in primary bone marrow cultures
The findings from the BMS2 parental cell line and subclones are of interest but may not reflect the behavior of bone marrow stromal cells in vivo. To more accurately assess the cells in their native state, additional studies were performed using primary murine bone marrow stromal cell cultures. Unlike the stromal cell lines, primary cultures are heterogeneous, containing macrophages and monocytes in addition to fibroblast-like stromal cells. The level of contaminating myeloid cells in 2-week-old primary cultures was determined by FACS analysis with the Mac-1 antibody, a specific macrophage/monocyte marker; fewer than 3% of the adherent cell population stained Mac-1 positive (data not shown). These primary cultures were induced with adipogenic agonists and/or 1,25(OH)2D3 in a manner identical to the parental BMS2 cell line. Adipocyte differentiation was assessed by staining fixed cultures with Oil Red O and light microscopic examination (Fig. 4Go). Adipocytes were present 6 days following exposure to BRL49653 (in the presence or absence of 1,25(OH)2D3) or MHI cocktail alone. No adipocytes were detected in the presence of 1,25(OH)2D3 alone. Northern blot analysis determined that 1,25(OH)2D3 blocked the induction of the late adipocyte-specific genes, aP2 and adipsin, in MHI-induced cultures but not in thiazolidinedione-induced cultures (Fig. 5Go). The mRNA levels of the early adipocyte marker, LPL, were relatively unchanged in response to 1,25(OH)2D3. The level of actin signal was reduced in the presence of BRL 49653, correlating with increased adipogenesis (34).



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  		Figure 4. Primary adipogenic bone marrow cultures: Oil red O stain. Whole bone marrow cultures were grown under adipogenic conditions as described in Materials and Methods. Following 6 days of treatment with MHI cocktail or BRL in the presence or absence of 1,25(OH)2D3, cultures were fixed and stained with Oil red O, visualized, and photographed. Pictures shown are representative of two experiments performed in duplicate.

 


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  		Figure 5. Primary adipogenic bone marrow cultures: Northern blot analysis. Whole bone marrow was cultured for 2 weeks before addition of MHI cocktail or BRL in the presence or absence of 1,25(OH)2D3. Following 6 days of adipogenic/1,25(OH)2D3 treatment, cultures were harvested for total RNA. Northern blots generated were probed with cDNA probes for adipsin, aP2, LPL, and actin. A photograph of the ethidium bromide stained gel is included to assess relative RNA loading between lanes. Northern blots shown are representative of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The bone marrow stromal cell plays a critical role in bone formation, blood cell formation, and mineral homeostasis. In the adult bone marrow, the adipocyte is the most abundant stromal cell. The function of adipocytes and the mechanisms leading to their formation remain controversial (4). Agents known to influence adipocyte formation through nuclear hormone receptors include hydrocortisone, thiazolidinediones, and 1,25(OH)2D3. Glucocorticoids and thiazolidinediones are well documented as agonists for adipocyte formation (4). Stromal cells and preadipocytes express functional vitamin D3 receptors (11, 18, 19, 20, 21, 35, 36). However, 1,25(OH)2D3 has been reported as both an adipogenic agonist and antagonist in a variety of systems. Vu et al. (10), working with the murine embryonic fibroblast cell line 3T3-L1, found that 1,25(OH)2D3 alone induced an adipocyte morphology and the expression of both LPL and aP2 mRNAs. Likewise, Bellows et al. (9) found 1,25(OH)2D3 to induce adipocyte differentiation of rat fetal calvarial derived osteoblasts. Dace, Lenoir, and their colleagues (16, 17), using the Ob17 cell line derived from the epididymal fat pad of the ob/ob mouse (37), found that calcitriol exhibited a biphasic effect on adipogenesis under serum free conditions. At subnanomolar concentrations (0.25 nM), calcitriol acted in synergy with triodothyronine and insulin to induce adipogenesis. At higher concentrations (>1 nM), calcitriol inhibited adipocyte differentiation. In contrast, multiple groups have reported that 1,25(OH)2D3 inhibited 3T3-L1 adipogenesis driven by glucocorticoids and insulin (11, 13, 14, 15). Similar inhibitory actions of 1,25(OH)2D3 at nanomolar concentrations have been reported in the murine mammary gland-derived ST13 cell line (11, 38), the murine bone marrow derived MC3T3-G2/PA6 cell line (12), and primary bone marrow stromal cultures derived from the rat femur (7).

Our findings are in agreement with those studies indicating that 1,25(OH)2D3 inhibits adipogenesis. By itself, 1,25(OH)2D3 at subnanomolar or nanomolar concentrations did not induce adipocyte differentiation during a 6-day period in the parental BMS2 cell line. However, when added in the presence of the hydrocortisone and other factors (MHI), 1,25(OH)2D3 inhibited adipogenesis. This was accompanied by a reduction in the mRNA levels of late adipocyte gene markers (aP2, adipsin). However, the mRNA levels of the early adipocyte marker, LPL, were not reduced. Similar results were observed using primary cultures derived from murine femoral bone marrow. This suggests that the 1,25(OH)2D3 cells do not progress beyond an early step in the adipocytic pathway. Similar observations have been made in 3T3-derived preadipocyte models. In this system, blockade of the adipocyte transcriptional regulator, the CCAAT/enhancer binding protein (C/EBP), by antisense constructs prevented the appearance of lipid droplets in response to glucocorticoids but did not block the expression of LPL mRNA (39).

Several possible explanations may account for the discrepant adipogenic effects by 1,25(OH)2D3 reported in the literature. One is that the tissue site of origin for each of the preadipocyte cell models influences the response to 1,25(OH)2D3. All of the data in the literature indicates that 1,25(OH)2D3 inhibits adipogenesis in femoral bone marrow derived cells, both primary cultures and cell lines, independent of the species of origin (7, 12). Our current findings on murine femoral-derived stromal cells are consistent with those reported by Beresford et al. (7), using rat femoral-derived primary cultures. However, these results differ from those reported by Bellows et al. (9), who found that 1,25(OH)2D3 induced adipogenesis in rat calvarial-derived cells. There are important differences in the isolation procedures and embryonic origin of femoral and calvarial stromal cells that may explain the discrepancies in these independent observations. Femoral stromal cells are isolated by flushing the marrow directly from the adult femur, an endochondral bone, whereas calvarial osteoblasts are isolated by collagenase digestion of the neonatal calvarium, an intramembranous bone. Consequently, these studies may focus on distinct populations of mesenchymal precursor cells that both exhibit osteoblastic characteristics. Differences in the behavior of cell lines might also be traced to the murine strain from which they originated. The Ob17 cell line was derived from the ob/ob mouse (37); this strain is prone to obesity due to a mutation of its leptin gene (40). Thus, the failure to synthesize leptin may alter the behavior of the Ob17 preadipocyte model relative to cell lines derived from other murine strains. Likewise, the 3T3-L1 cell line has been passaged extensively over the 20+ years since its first description (41). Therefore, variation between 3T3-L1 sublines could account for the independent reports that 1,25(OH)2D3 acted as both an adipogenic agonist and antagonist when added to 3T3-L1 cells (10, 11, 13, 14, 15). Independent of the explanation, the current work demonstrates that 1,25(OH)2D3 antagonizes glucocorticoid-induced adipogenesis in murine femoral-derived bone marrow stromal cells.


   Acknowledgments
 
We wish to thank: P. W. Kincade, L. Thompson, M. R. Hill, N. Nadon, C. Webb, X. Wu, and C. E. Robinson for helpful discussions; P. Anderson, J. Young, and J. Mowdy for editorial and photographic assistance; and B. R. Rodriguez for technical assistance.


   Footnotes
 
1 This work was supported in part by NIDR Grant K-15DE-360 (to K.A.K.), NIH Grant CA-50898 (to J.M.G.), and the Oklahoma Medical Research Foundation. These studies were submitted in partial fulfillment of the Ph.D. dissertation requirement by K.A.K. in the Department of Pathology, University of Oklahoma Health Sciences Center. Back

Received October 17, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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