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Endocrinology Vol. 140, No. 7 3245-3254
Copyright © 1999 by The Endocrine Society

ARTICLES

Thiazolidinedione Effects on Glucocorticoid Receptor-Mediated Gene Transcription and Differentiation in Osteoblastic Cells

Timothy E. Johnson1, Robert Vogel, Su Jane Rutledge, Gideon Rodan and Azriel Schmidt

Department of Bone Biology and Osteoporosis Research, Merck Research Laboratories, West Point, Pennsylvania 19486

Address all correspondence and requests for reprints to: Azriel Schmidt, Ph.D., Merck Research Laboratories, WP 26A-1000, West Point, Pennsylvania 19486.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The glucocorticoid receptor (GR) and peroxisome proliferator-activated receptors (PPARs) play important roles in the differentiation of mesenchymal cells. Glucocorticoids acting via the GR promote osteoblastic differentiation of bone marrow stromal cells, whereas PPAR ligands induce these cells to become adipocytes. To explore potential interactions between PPAR and GR pathways in osteoblasts, we studied the interaction between PPAR subtype-selective ligands and dexamethasone (DEX) in a murine calvaria-derived osteoblastic cell line (MB 1.8) that expresses endogenous GR and PPARs. In ligand-dependent transcription assays, the PPAR{gamma}-selective ligand TZD [(5-(4-N-methyl-N(2-pyridyl)amino)ethoxy)benzyl)thiazolidine-2,4-dione], a thiazolidinedione antidiabetic, enhanced the effect of DEX to stimulate transcription of a glucocorticoid-inducible reporter gene (mouse mammary tumor virus-luciferase). No effect was seen with PPAR{alpha}- or hNUC1/PPAR{delta}-selective ligands. The GR antagonist RU-486 inhibited the DEX and TZD responses, suggesting that the effects were mediated through endogenous GR. TZD also enhanced glucocorticoid-mediated transcription in SaOS-2/B10 human osteosarcomatous cells, but not in CV-1 cells, even though both cell lines were transfected with GR plasmid and expressed significant levels of endogenous PPAR{gamma} messenger RNA. In MB 1.8 cells, TZD decreased alkaline phosphatase activity and the expression of osteoblast-associated genes while it up-regulated the adipocyte fatty acid-binding protein. DEX counteracted the effects of TZD on alkaline phosphatase enzyme activity and osteoblastic gene expression, but enhanced the actions of TZD on adipocyte fatty acid-binding protein. Interestingly, TZD inhibited in vitro bone nodule formation and mineralization, and DEX counteracted this effect. Thus, depending on the promoter context, TZD and DEX can oppose or enhance each other’s actions on gene transcription. Collectively, these results point to a complex interaction between PPAR and GR signaling pathways that regulates the effects of TZD and DEX on osteoblastic differentiation. The mechanism of this interaction is still under investigation, but might involve PPAR-dependent and -independent pathways. As thiazolidinediones represent an important new class of drugs, our findings also raise the need for further studies in bone.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OSTEOBLASTS arise from a pool of progenitor stem cells in the bone marrow stroma. Depending upon the stimulus, these uncommitted stem cells can differentiate into a variety of cell lineages, including osteoblast, chondrocyte, myoblast, fibroblast, and adipocyte (1). Ligand-activated nuclear receptors play a crucial role in this process by binding to hormone response elements on gene promoters and modulating the transcription of growth/differentiation regulatory genes. In bone, glucocorticoids, acting via the glucocorticoid receptor (GR), promote the differentiation of preosteoblasts from the bone marrow stroma into mature osteoblastic cells that have the potential to mineralize. The actions of glucocorticoids in osteoblasts are complex and depend on the maturation stage and the species of the osteoblastic population. In vitro studies have found that glucocorticoids can have biphasic effects on osteoblast-related gene expression and differentiation (2, 3, 4, 5). The concentration of glucocorticoids and the length of exposure are also important factors. Although physiological concentrations of glucocorticoids appear to promote osteoblastic differentiation, pharmacological doses inhibit bone growth, and chronic treatment with supraphysiological concentrations can result in osteopenia and osteoporosis (6).

In addition to GR, another nuclear receptor family, the peroxisome proliferator-activated receptors (PPARs) might also be involved in regulating osteoblastic differentiation. PPARs are nuclear transcription factors that modulate the expression of a variety of genes involved in lipid metabolism and fat storage (7). The PPAR family consists of three subtypes, PPAR{alpha}, PPAR{delta} (also known as hNUC1 and FAAR), and PPAR{gamma}. PPAR ligands include fatty acids, eicosanoids, nonsteroidal antiinflammatory agents, and a diverse class of chemicals known as peroxisome proliferators, e.g. WY-14643 (8, 9, 10, 11). Ligand-activated PPARs regulate gene transcription by binding in conjunction with the retinoic X receptor to a peroxisome proliferator response element (PPRE).

All three PPAR subtypes have been identified in osteoblasts in vivo by in situ hybridization (12), but their function in regulating genes involved in osteoblastic differentiation has not been well characterized. With aging, there is an increase in the number of adipocytes in the bone marrow with a concomitant loss of bone, suggesting a potential conversion from osteoblasts into adipocytes (13). Several studies have implicated PPARs in regulating adipocyte differentiation (14, 15, 16). We have recently reported that the PPAR{gamma} ligand TZD [(5-(4-N-methyl-N(2-pyridyl)amino)ethoxy)benzyl)thiazolidine-2,4-dione], a thiazolidinedione that is being evaluated as a new therapeutic for noninsulin-dependent diabetes, promoted the conversion of an osteoblastic cell line into adipocyte-like cells (17). These findings suggest that PPARs can inhibit osteoblastic differentiation by promoting an adipocyte differentiation program in osteoblastic cells. Interestingly, glucocorticoids were reported in vitro and in vivo to up-regulate the expression of PPAR{alpha} in rat liver (18, 19, 20). Taken together, these studies raise the possibility of an interaction between GR and PPAR signaling pathways that could potentially influence osteoblastic differentiation.

Therefore, we investigated the effect of PPAR subtype-selective ligands on glucocorticoid-mediated transcription and osteoblastic differentiation in a murine calvaria-derived osteoblastic cell line (MB 1.8). We found that the thiazolidinedione TZD, which is selective for PPAR{gamma}, enhanced glucocorticoid-mediated transcription specifically in osteoblasts, and this response was dependent on GR. Moreover, the effect of TZD on gene expression and osteoblastic differentiation was modulated by glucocorticoids.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Dexamethasone (DEX) was obtained from Sigma Chemical Co. (St. Louis, MO). WY-14643 was purchased from Chemsyn Sciences (Lenexa, KS). TZD was a gift from Dr. G. Berger of Merck & Co., Inc. (Rahway, NJ), and L-631,033 was obtained from the Merck Chemical Collection. RU-486 was provided by Roussel-UCLAF (Romainville, France). Chemicals were dissolved in dimethylsulfoxide (DMSO) as 1000- or 500-fold stock solutions and diluted 1000- or 500-fold into culture medium. The concentration of DMSO in the cultures did not exceed 0.2%.

Cell culture
Mouse calvaria-derived osteoblastic cells (MB 1.8) were cultured in MEM{alpha} with ribonucleosides (Life Technologies, Grand Island, NY). SaOS-2/B10 cells were isolated from a human osteosarcoma as described previously (21) and were grown in RPMI 1640 medium. Monkey kidney epithelium-derived CV-1 cells were obtained from American Type Culture Collection (Manassas, VA) and were cultured in DMEM. Osteoblast-enriched primary cells were isolated from timed pregnant 19-day-old Swiss-Webster mice (Taconic Farms, Inc., Germantown, NY) as described previously (22) and were cultured in F-12 medium. Each medium was supplemented with 10% FBS [BioWhittaker, Inc. (Walkersville, MD), or Sigma Chemical Co.], 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine (Life Technologies). All cells were cultured in a humidified atmosphere of 5% CO2-95% air at 37 C.

Ligand-dependent transcription assays
Cells were plated in 96-well dishes (Wallac, Inc., Gaithersburg, MD) at 8 x 103 cells/well·0.2 ml growth medium. The receptor constructs and the reporter gene [mouse mammary tumor virus-luciferase (MMTV-luc)] have been described previously (23). Briefly, the reporter gene MMTV-luc was the plasmid pJA358 in which the firefly luciferase gene is regulated by an inducible MMTV promoter that contains tandem repeats of a glucocorticoid hormone response element. The chimeric receptor constructs GR/PPAR{alpha}, GR/PPAR{gamma}, GR/NUC1, and GR/PR contained the amino-terminal domain and the DNA-binding domain of GR fused to the ligand-binding domains of PPAR{alpha}, PPAR{gamma}, hNUC1/PPAR{delta}, and progesterone receptor (PR), respectively. For MB 1.8 and CV-1 cells, a calcium phosphate precipitate containing 10 µg/ml of the receptor plasmids, where appropriate, and 10 µg/ml of the MMTV-luc reporter plasmid was diluted 100-fold and then added to the cells. In experiments evaluating MMTV-luc transcription in the absence of nuclear receptors, 10 µg/ml carrier plasmid (PSV2Neo) were also transfected to equalize the DNA content. SaOS-2/B10 cells were transfected using Dosper, a lipofectamine (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s instructions. Osteoblast-enriched primary cells were transfected by the calcium phosphate precipitation method followed by glycerol shock as described previously (24).

About 24 h after transient transfection, the cells were washed twice with Dulbecco’s PBS and then refed with culture medium containing 5% activated charcoal-stripped serum (HyClone Laboratories, Inc., Logan, UT). The cells were then treated with test agents in the presence or the absence of 10–100 nM DEX for 48 h, except in time-course experiments. At harvest, the cell monolayers were washed twice with Dulbecco’s PBS and then lysed in luciferase lysis buffer (Promega Corp., Madison, WI) directly in the 96-well plate. Extracts were assayed using the luciferase assay system (Promega Corp.). Samples were analyzed in a Microlumat LB 96 P luminometer (Wallac, Inc.). Each experiment was repeated two to five times, with similar results obtained in each experiment. The treatments at the concentrations used in these studies caused no apparent change in cellular morphology or reduction in monolayer confluence over the 48-h treatment period compared with the concurrent control.

Alkaline phosphatase
Cells were plated in 96-well dishes at 8 x 103 cells/well·0.2 ml medium and grown until they were about 80–90% confluent (usually about 2 days). The cells were then treated with TZD in the presence and absence of DEX for 48 h. The cells were lysed in cell lysis buffer [10 mM Tris (pH 7.5), 0.5 mM MgCl2, and 0.1% Triton X-100) for 15 min at room temperature. Alkaline phosphatase was measured using sodium-nitrophenyl phosphate as described previously (25). Absorbance was determined at 405 nm using a 96-well plate reader (Bio-Tek Instruments, Inc., Winooski, VT). The protein concentration of each sample was measured using the Coomassie Plus Protein Assay Reagent kit (Pierce Chemical Co., Rockford, IL) according to the manufacturer’s instructions, and the amount of protein in micrograms per ml was calculated from a standard curve. Alkaline phosphatase was normalized to protein concentration for each sample.

RNA isolation and Northern hybridizations
Cells were plated in 150-cm2 dishes (Nunc, Roskilde, Denmark) and grown until they were about 80% confluent. Cells were then treated with TZD, with or without DEX, for 24 h. Total RNA was isolated using Tri-reagent (Sigma Chemical Co.), following the instructions provided by the manufacturer. Twenty micrograms of total RNA were electrophoresed on 0.9% agarose gels containing 6% formaldehyde. The RNA was transferred to positively charged nylon filters and UV cross-linked. Complementary DNA (cDNA) probes were 32P labeled to high specific activity using the Redi-Prime kit (Amersham, Aylesbury, UK). Probes used included cDNAs for alkaline phosphatase collagen type 1{alpha}1 and the adipocyte fatty acid-binding protein, aP2 (26, 27, 28). Filters were hybridized with cDNA probes overnight, washed, and autoradiographed as described previously (17).

In vitro bone mineralization
Cells were plated in 12-well cluster dishes (Costar, Cambridge, MA) at about 3 x 104 cells/well·ml growth medium the day before treatment. Cells were refed with medium containing 10% serum, 10 mM ß-glycerol phosphate, and 50 µg/ml ascorbic acid (mineralization medium) and then treated with TZD at the indicated concentrations in the presence or absence of DEX for 21 days. Fresh mineralization medium and test agents were added every 2–3 days. On day 21, the cells were fixed in 70% ethanol and then stained using the Von Kossa technique as described previously (29). Each well was divided into four equal parts, and the number of mineralized nodules was determined in each region by counting under a dissecting microscope at x20. The number of mineralized nodules present in each well was determined in three replicate plates.

Statistical analysis
At selected points, statistical analysis was performed using Student’s t test (two-sample test, assuming equal variances). The level of statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of PPAR ligands on glucocorticoid-mediated transcription in osteoblastic cells
In our studies we used a mouse osteoblastic cell line (MB 1.8) that was derived from calvaria and is spontaneously immortalized but not tumorigenic (30). MB 1.8 cells express several osteoblastic markers, including alkaline phosphatase, collagen type 1, osteopontin, and osteocalcin. In addition, these cells support the differentiation of mouse bone marrow progenitor cells into mature osteoclasts, which can resorb bone in vivo (30). Thus, these cells represent a relatively homogeneous osteoblastic cell population compared with primary mouse calvaria cells, which are a more heterogeneous population of cells that can be induced to differentiate into a variety of cell types. We previously found that MB 1.8 cells express low levels of hNUC1/PPAR{delta} and trace amounts of PPAR{gamma} messenger RNA (mRNA), but have no detectable expression of PPAR{alpha} (17). These cells also express low levels of endogenous GR (data not shown). To study the effects of PPARs on glucocorticoid-mediated transcription, we used the PPAR ligands WY-14643, L-631,033, and TZD, which we had previously shown in COS-7 cells to be selective for PPAR{alpha}, hNUC1/PPAR{delta}, and PPAR{gamma}, respectively (31). To determine whether a similar activation selectivity by these ligands was seen in osteoblastic cells, MB 1.8 cells were cotransfected with the glucocorticoid-inducible luciferase reporter gene MMTV-luc and the chimeric PPAR receptors GR/PPAR{alpha}, GR/PPAR{gamma}, and GR/NUC1. These receptors contained the amino-terminal and DNA-binding domains of GR fused to the ligand binding of the respective PPAR receptor and thus bind to the MMTV-luc reporter gene when activated by ligand. The cells were then treated with the ligands for 48 h. WY-14643 activated GR/PPAR{alpha} at 10 and 50 µM, but was a weak activator of GR/NUC1 or GR/PPAR{gamma} compared with L-631,033 (GR/NUC1) and TZD (GR/PPAR{gamma}; Fig. 1Go). In contrast, L-631,033 appeared more selective for GR/NUC1 then the other two PPAR subtypes, whereas TZD was clearly selective for GR/PPAR{gamma} (Fig. 1Go). Thus, the profile of PPAR activation by these ligands in MB 1.8 cells is similar to that in COS-7 cells.



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  		Figure 1. Activation of PPARs by ligands in MB 1.8 osteoblastic cells. Cells were transiently transfected with MMTV-luc and GR/PPAR{alpha}, GR/NUC1, or GR/PPAR{gamma} and then treated with ligands for 48 h. Cells were harvested and analyzed for luciferase activity as described in Materials and Methods. Shown is the mean fold stimulation ± SEM of four replicate wells for each treatment from a single experiment. The data shown are representative of three independent experiments.

 
To study the action of endogenous PPARs on glucocorticoid-mediated transcription, MB 1.8 cells were transiently transfected with the MMTV-luc reporter gene and then treated with the PPAR subtype-selective ligands with or without the synthetic glucocorticoid DEX. Treatment of MB 1.8 cells with 50 nM DEX led to about a 5-fold stimulation of MMTV-luc over the DMSO control value (Fig. 2AGo). This supports the Northern hybridization data and demonstrates that the GR is endogenously expressed in these cells and is functional. None of the PPAR ligands significantly stimulated MMTV-luc in the absence of DEX, but the PPAR{gamma}-selective ligand TZD enhanced the effect of DEX on luciferase expression by about 6-fold (range, 2- to 6-fold depending upon the experiment). In contrast, neither the hNUC1/PPAR{delta} activator L-631,033 nor the PPAR{alpha}-selective agonist WY-14643 significantly modulated DEX-mediated transcription (Fig. 2AGo). Similar results were seen when full-length GR plasmid was cotransfected with MMTV-luc (data not shown). To determine whether this response also occurred in primary cells, osteoblast-enriched cells were isolated from mouse calvaria and transfected with MMTV-luc. As seen in MB 1.8 cells, TZD alone had no effect on MMTV-luc activity, but significantly enhanced the action of DEX on luciferase expression (Fig. 2BGo). Because similar results were seen in primary cells, further experimentation was limited to osteoblastic cell lines, as these cells are proliferation competent and have higher transfection efficiencies.



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  		Figure 2. Enhancement of glucocorticoid-mediated transcription by TZD. A, MB 1.8 cells were transiently transfected with the MMTV-luc reporter gene. Cells were treated with 50 µM TZD, WY-14643, or L-631,033 in the presence or absence of 50 nM DEX for 48 h. B, Osteoblast-enriched primary cells were transfected as described in A, then treated with 50 µM TZD with or without 25 nM DEX for 48 h. Cells were harvested and analyzed for luciferase activity as described in Materials and Methods. Shown is the mean fold stimulation ± SEM of four replicate wells for each treatment from a single experiment. The data shown are representative of two to five independent experiments. *, Significantly different (P < 0.05) from DEX-treated cells.

 
Studies were conducted in MB 1.8 cells to examine the dose dependency and the kinetics of the DEX and TZD effects on MMTV-luc transcription. Figure 3aGo shows that DEX stimulated MMTV-luc transcription in MB 1.8 cells in a dose-dependent manner, and that the response was saturable, with maximal stimulation seen at 50 nM. No enhancement was seen at 1 µM TZD (data not shown), but concentrations of 10 µM or more increased the affinity for the DEX effect and the extent of stimulation (Fig. 3AGo). A time-course study showed that DEX stimulated MMTV-luc within 3 h of administration, and the response reached a plateau of about 6-fold at approximately 6 h (Fig. 3BGo). Enhancement by TZD was apparent as early as 6 h and continued to increase up to 48 h. These results suggest that TZD enhances glucocorticoid-mediated transcription in MB 1.8 cells in a concentration- and time-dependent manner.



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  		Figure 3. TZD modulates glucocorticoid-mediated transcription in a concentration- and time-dependent manner. A, MB 1.8 cells were transiently transfected with the MMTV-luc reporter gene and then treated with TZD at the indicated concentrations in the presence and absence of increasing concentrations of DEX for 48 h. B, Cells were transfected as described in A, then treated with 50 µM TZD, with or without 25 nM DEX, and harvested at the indicated times. Cells were harvested and analyzed for luciferase activity as described in Materials and Methods. Shown is the mean fold stimulation of four replicate wells for each treatment from a single experiment. The data shown are representative of two independent experiments. SEs (not shown) were ±10%. *, Significantly different (P < 0.05) from DEX-treated cells.

 
Effect of TZD on stimulation of DEX-induced MMTV-luc transcription is mediated by GR
To confirm that the action of TZD on DEX-induced transcription of the MMTV-luc reporter gene was mediated by endogenous GR present in MB 1.8 cells, we tested the effect of the GR antagonist RU 486. This compound at both 1 and 10 µM significantly inhibited the stimulation of transcription of MMTV-luc by DEX and completely suppressed the enhancement action of TZD (Fig. 4AGo), suggesting that GR must be activated and bound by agonist for the enhancement by TZD to occur. Treatment of cells with 1 µM, but not 10 µM, RU-486 stimulated transcription of MMTV-luc but at much lower levels than that seen with DEX or DEX and TZD. This effect is consistent with a previous report that in the absence of DEX, RU-486 can act as a partial agonist (32).



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  		Figure 4. GR is required for the enhancement of glucocorticoid-mediated transcription by TZD. A, MB 1.8 cells were transiently transfected with the MMTV-luc reporter gene and then were treated with 50 nM DEX, 50 µM TZD, or a combination of both agents with or without RU-486 for 48 h. B, MB 1.8 cells were cotransfected with MMTV-luc and GR/PR and then treated with 50 µM TZD in the absence and presence of progesterone for 48 h. Cells were harvested and analyzed for luciferase activity as described in Materials and Methods. Shown is the mean fold stimulation ± SEM of four replicate wells for each treatment from a single experiment. The data shown are representative of two independent experiments. *, Significantly different (P < 0.05) from no RU-486 treatment.

 
To further test the requirement for GR in this response, we cotransfected cells with MMTV-luc and the chimeric receptor GR/PR, which contains the amino-terminal and DNA-binding domain of GR fused to the ligand-binding domain of the PR. These cells were then treated with the cognate hormone progesterone. Progesterone should activate GR/PR by binding to the ligand-binding domain of PR, which will, in turn, bind to the glucocorticoid response element on MMTV-luc and stimulate the reporter gene. As shown in Fig. 4BGo, progesterone at both doses tested stimulated MMTV-luc transcription in a dose-dependent manner, and TZD failed to modulate this effect. Taken together, these results strongly suggest that the ligand-binding domain of GR is necessary for the action of DEX and TZD on MMTV-luc transcription.

The TZD effect on glucocorticoid-mediated transcription is present in osteoblastic cells, but not in CV-1 kidney cells
To test the cell type specificity of this TZD response, SaOS-2/B10 human osteosarcomatous cells and CV-1 monkey kidney cells were transfected with the MMTV-luc reporter gene and then treated with TZD, with or without DEX. Both cell lines express significant levels of endogenous PPAR{gamma} mRNA compared with MB 1.8 cells (Fig. 5AGo), but have little or no endogenous GR (33) (data not shown). As expected, in SaOS-2/B10 cells transfected with MMTV-luc alone, DEX at up to 25 nM had little or no effect on stimulating the MMTV-luc reporter gene. However, in the presence of transfected GR, DEX stimulated the MMTV-luc reporter gene by about 13-fold over the control level, and TZD significantly enhanced DEX-mediated transcription by about 2- to 3-fold at 10 and 50 µM (Fig. 5BGo), confirming the requirement for GR in this response. As seen in SaOS-2/B10 cells, CV-1 kidney cells that were transiently transfected with MMTV-luc were not stimulated by DEX or DEX and TZD, except at 100 nM DEX, where there was about a 3-fold increase in reporter activity (Fig. 5CGo). However, in cells cotransfected with MMTV-luc and GR, DEX increased luciferase activity in a concentration-dependent manner. Surprisingly, TZD did not enhance MMTV-luc transcription at any DEX concentration (Fig. 5CGo). These results suggest that enhancement of glucocorticoid-mediated transcription by TZD not only requires GR, but is also dependent on an additional factor(s) present in SaOS-2/B10, but not in CV-1, cells. Furthermore, these findings raise the interesting possibility that PPAR{gamma} does not entirely mediate this response, because both cell lines express significant levels of PPAR{gamma} mRNA.



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  		Figure 5. Cell type-specific actions of TZD on glucocorticoid-mediated transcription. A, Endogenous levels of PPAR{gamma} present in untreated CV-1, SaOS-2/B10, and MB 1.8 cells. Total RNA was isolated and analyzed (20 µg/lane) by hybridization with the cDNA (ligand-binding coding region) of PPAR{gamma}. B, Effects of TZD on glucocorticoid-mediated transcription in SaOS-2/B10 osteosarcomatous cells. Cells were transiently transfected with the MMTV-luc reporter gene or cotransfected with MMTV-luc and GR, then treated with DEX with or without 10 or 50 µM TZD for 48 h. Shown is the mean fold stimulation ± SEM of four replicate wells for each treatment from a single experiment. The data shown are representative of two independent experiments. *, Significantly different (P < 0.05) from DEX treatment. C, CV-1 kidney cells were transfected with MMTV-luc (solid symbols) or MMTV-luc and GR (open symbols) and then treated with increasing concentrations of DEX in the presence (circles) or absence (diamonds) of 50 µM TZD. Shown is the mean fold stimulation of four replicate wells for each treatment from a single experiment. The data shown are representative of two independent experiments. SEs (not shown) were ±10%.

 
Effect of DEX and TZD to modulate osteoblastic gene expression and alkaline phosphatase activity
The ability of TZD to alter glucocorticoid-mediated transcription could result in modulation of osteoblastic differentiation. To determine whether TZD altered the expression of endogenous genes, MB 1.8 cells were treated with TZD in the absence or presence of DEX for 24 h. Total RNA was isolated, and Northern analysis was carried out using cDNA probes for the osteoblast-associated genes alkaline phosphatase and collagen type 1{alpha}1 and for the adipocyte differentiation marker aP2. Results are shown in the autoradiogram of Fig. 6AGo and normalized to actin (Fig. 6BGo). TZD inhibited the expression of alkaline phosphatase and collagen type 1. DEX, while having no effect on its own, counteracted the suppression of these genes by TZD. In contrast, DEX and TZD on their own stimulated aP2 mRNA levels in both MB 1.8 (Fig. 6Go, A and B) and osteoblast-enriched primary cells (Fig. 6CGo), and the combination of DEX and TZD appeared additive. Although the expression levels of ß-actin varied slightly between treatments, this was not indicative of regulation by the chemical treatments, because ethidium bromide staining of the 28S and 18S bands in the gel showed a similar pattern. Thus, DEX and TZD appear to have opposing or enhancing actions on gene expression in osteoblastic cells.



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  		Figure 6. Effect of TZD and DEX on osteoblastic gene expression. A, MB 1.8 cells were treated with 50 µM TZD, 10 nM DEX, or DEX plus TZD for 24 h. Total RNA was isolated as described in Materials and Methods. Twenty micrograms of each RNA sample were fractionated on a 0.9% agarose gel containing 6% formaldehyde and then transferred to a nylon filter. Northern hybridizations were carried out as described inMaterials and Methods using cDNA probes for alkaline phosphatase (ALP), collagen type 1 (COL 1), aP2, and ß-actin. B, The relative mRNA concentrations from the autoradiogram in A were quantitated in a densitometer and normalized to concurrent ß-actin levels. Results are shown for TZD and DEX treatments and are expressed as a percentage of the concurrent DMSO control value. Data are representative of two independent experiments. C, Calvaria-derived osteoblast-enriched primary cells were treated, processed, electrophoresed, and hybridized with aP2 cDNA as described in A. The 28S and 18S ribosomal RNA are shown as a reference.

 
To determine whether the effects of TZD seen on gene expression also occurred at the protein level, alkaline phosphatase activity was measured, as the levels of this enzyme are an indicator of the differentiation state of the osteoblastic culture. MB 1.8 cells were treated with TZD for 48 h, with and without DEX, and alkaline phosphatase activity was measured. Consistent with the effects of TZD on alkaline phosphatase gene expression, TZD caused a dose-dependent decrease in alkaline phosphatase activity, and DEX counteracted this inhibition at the lower doses (Fig. 7AGo).



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  		Figure 7. A, Effect of TZD and DEX on alkaline phosphatase (ALP) activity. MB 1.8 cells were treated with TZD at the indicated concentrations, with and without 10 nM DEX, for 48 h. Alkaline phosphatase activity was measured as described inMaterials and Methods. Shown is the alkaline phosphatase activity, normalized to protein and expressed as a percentage of the concurrent control of four replicate wells for each treatment. The data shown are representative of two independent experiments. The SEs (not shown) were 5% or less. B, Effect of TZD and DEX on modulating bone nodule formation and mineralization. MB 1.8 cells were treated for 21 days with TZD at the indicated concentrations with or without 10 nM DEX in medium containing 10% serum, 10 mM ß-glycerol phosphate, and 50 µg/ml ascorbic acid. Fresh mineralization medium and test agents were added every 2–3 days. Cells were fixed in 75% ethanol and then stained for mineralization using the Von Kossa technique. Shown is the mean number of mineralized nodules of three replicate wells for each treatment. The data shown are representative of four independent experiments. SEs are shown. *, Significantly different (P < 0.05) from DMSO treatment.

 
Effect of DEX and TZD to modulate in vitro bone mineralization
As terminal osteoblastic differentiation results in the formation of mineralized bone nodules, we studied the ability of TZD to modulate nodule formation and bone mineralization in MB 1.8 cells. Mineralization was induced by growing the cells in medium containing ß-glycerol phosphate and ascorbic acid (29), with or without DEX and TZD, for 21 days. TZD inhibited the number of mineralized nodules formed in a dose-dependent manner (Fig. 7BGo). DEX alone did not increase the number of mineralized nodules formed, although the individual size of the nodules appeared larger. However, DEX significantly counteracted the inhibition of mineralization by TZD at all three dose levels tested (Fig. 7BGo). The suppression of mineralization caused by TZD was not the result of overt toxicity, because cell morphology remained normal, and there was no evidence of decreased cell monolayer confluence in cells treated for up to 21 days with TZD in either the presence or absence of DEX.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we observed a complex interaction between DEX and the PPAR{gamma} ligand, and thiazolidinedione (TZD) in transient transfection experiments and in the control of endogenous gene expression and mineralization in osteoblastic cells.

Enhancement of glucocorticoid-mediated transcription in osteoblasts
At the transcriptional level, we found that TZD enhanced the effect of DEX on the glucocorticoid-inducible MMTV-luc reporter gene in MB 1.8 osteoblastic cells and in osteoblast-enriched primary cells isolated from mouse calvaria. Several lines of evidence point to the involvement of GR in the DEX and TZD response on MMTV-luc. First, the response occurred in MB 1.8 osteoblastic cells, which have detectable expression levels of endogenous GR. Second, both the stimulation of the MMTV-luc reporter gene by DEX and the enhancement of the DEX effect by TZD were significantly inhibited in the presence of the GR antagonist RU-486. Third, TZD did not enhance the effect of progesterone on stimulating MMTV-luc via activation of the GR/PR chimeric receptor in cells transiently transfected with GR/PR and MMTV-luc. Fourth, TZD enhanced DEX-mediated MMTV-luc activity in SaOS-2/B10 osteosarcomatous cells, only when GR was cotransfected with MMTV-luc. Collectively, these experiments strongly suggest that the effects of DEX and TZD on MMTV-luc are mediated through endogenous GR present in MB 1.8 cells.

Although GR is required for the DEX and TZD response on glucocorticoid-mediated transcription, our experiments in SaOS-2/B10 and CV-1 cells suggest that it might not be sufficient. As expected, no effect of DEX and TZD on MMTV-luc transcription was seen in either SaOS-2/B10 or CV-1 cells in the absence of transfected GR, because these cell lines express little or no endogenous GR. However, cotransfection of GR plasmid with MMTV-luc restored the DEX and TZD response in SaOS-2/B-10 cells, but not in CV-1 cells. This result cannot be attributed to differences in transfection efficiency or a lower amount of expressed GR protein in transfected CV-1 compared with SaOS-2/B10 cells because DEX significantly stimulated MMTV-luc in both cell lines after transfection with the GR plasmid. Moreover, both cell lines express significant levels of endogenous PPAR{gamma} mRNA. Thus, in addition to GR, the TZD enhancement of glucocorticoid-mediated transcription appears to require other factors that are present in MB 1.8, SaOS-2/B10 cells and osteoblast-enriched primary cells, but not in CV-1 cells.

These results support the hypothesis that TZD acts to up-regulate or activate factors that modulate GR activity and/or bind to the MMTV promoter and enhance transcription in a GR-dependent manner. Consistent with this hypothesis, we found that TZD alone did not stimulate the MMTV-luc reporter gene and that TZD enhanced the effect of DEX on MMTV-luc in a time-dependent manner, with maximal enhancement seen 24–48 h after the beginning of treatment. Interestingly, a NF-1/CTF family transcription factor was recently identified that binds to the 5'-end of the MMTV long terminal repeat and regulates transcription of MMTV only in the presence of glucocorticoids (34). Several other transcription factors, including octamer transcription factor-1 and activating protein-2 (AP-2), have also been shown to regulate MMTV transcription (35, 36). As there is no apparent PPAR-binding site on the MMTV long terminal repeat, it is interesting to speculate that TZD might enhance glucocorticoid-mediated transcription by up-regulating and/or activating an accessory transcription factor(s).

Interaction between GR and TZD signaling pathways modulates gene expression and osteoblastic differentiation
The inhibition of alkaline phosphatase and collagen type 1 gene expression by TZD suggests that this compound may suppress osteoblastic differentiation. These genes are positively correlated with the osteoblastic phenotype and appear to increase during bone nodule formation and mineralization (25, 37, 38). In contrast, TZD up-regulated the expression of aP2 in both MB 1.8 and osteoblast-enriched primary cells. This gene is thought to be a relatively late marker of adipocyte differentiation and was previously shown to be induced by TZD and other thiazolidinediones in 3T3-L1 preadipocytes (16). These findings indicate that TZD might be promoting an adipocyte differentiation program in these cells. Supporting our hypothesis, TZD was reported to induce adipogenesis in bone marrow stromal cells (39), and we recently showed that TZD and fatty acids isolated from rabbit serum can increase the differentiation of several osteoblastic cell lines into adipocyte-like cells (17).

DEX alone did not affect alkaline phosphatase or collagen type 1 gene expression at the time point studied. This could be attributed to the complex nature of glucocorticoids in regulating osteoblastic differentiation, which appears to be dependent on the state of differentiation and the type of osteoblastic cell studied (2, 6). However, DEX clearly counteracted the suppression of these genes by TZD, indicating that the interaction between DEX and TZD signaling pathways regulates osteoblastic gene expression. Interestingly, DEX up-regulated aP2 gene expression, and the combination of DEX and TZD appeared additive. Notably, unsaturated fatty acids were reported to potentiate glucocorticoid-mediated transcription and enhance the effect of DEX on aP2 expression (40, 41), suggesting that fatty acids and TZD may act through a similar mechanism.

Supporting our findings on osteoblastic gene expression, we found that TZD dose dependently inhibited alkaline phosphatase activity and bone mineralization in MB 1.8 cells, and DEX counteracted this effect. Compared with other mineralization studies in cells derived from mouse calvaria, which show a dependency for glucocorticoids to mineralize (29, 42, 43), we did not find that DEX was required for mineralization or significantly increased the number of mineralized nodules in MB 1.8 cells, although the overall size of the nodules appeared increased. A predominant role for glucocorticoids is to recruit preosteoblastic cells from the heterogeneous stromal environment in the bone marrow. In human stromal cells that were induced to differentiate into osteoblast-like cells it was found that removal of DEX after 2–3 days of treatment had no effect on alkaline phosphatase activity or the cAMP response to PTH compared with those in cultures that received DEX for 23 days. Similarly, removal of DEX after 7 days of treatment had no effect on mineralization (25, 37). As MB 1.8 cells represent a relatively homogeneous and mature osteoblastic cell population (indicative of the expression of osteopontin and osteocalcin, which are usually expressed at the latter stages of osteoblastic differentiation), they may already be past the recruitment stage and thus might not need DEX to form mineralized bone nodules. However, DEX counteracted the inhibition of alkaline phosphatase activity and mineralization by TZD, suggesting that DEX can maintain and/or promote an osteoblastic phenotype in this cell system under certain conditions.

Potential mechanisms for the effect of TZD and DEX in osteoblasts
One potential mechanism for the observed actions of TZD on gene transcription and osteoblastic differentiation might be through activation of PPAR{gamma}, as it is known that TZD is a ligand for this receptor (16), and we have shown that TZD activates GR/PPAR{gamma} at 10–50 µM in MB 1.8 cells, with maximal activation seen at 50 µM. The inability of the PPAR{alpha} ligand WY-14643 (8) and the hNUC1/PPAR{delta} agonist L-631,033 (31) to enhance glucocorticoid-mediated transcription also supports the hypothesis that the TZD response might be predominately mediated through PPAR{gamma}, although the involvement of the other PPAR subtypes cannot be excluded. PPAR{gamma} could potentially bind directly to GR or interact with AP-1, a transcription factor that associates with GR and modulates gene transcription (44). Interestingly, TZD and other thiazolidinediones were shown to inhibit macrophage activation at similar concentrations through a mechanism that involved PPAR{gamma} and other transcription factors, e.g. AP-1, nuclear factor-{kappa}-B, and STAT (signal transducer and activator of transcription) (45, 46). To date, a PPRE has not been reported in either the alkaline phosphatase or collagen type 1 gene promoters, but it is possible that an unidentified PPRE or similar sequence could be present.

An alternative mechanism is that the effect of TZD is independent of PPARs. Supporting this hypothesis, we found only trace amounts of endogenous PPAR{gamma} and low levels of hNUC1/PPAR{delta} mRNA in MB 1.8 cells. Moreover, TZD did not enhance the effect of DEX on MMTV-luc transcription in CV-1 cells, even though these cells were transfected with GR and expressed significantly higher levels of endogenous PPAR{gamma} than those seen in MB 1.8 cells. Studies by Nordeen and colleagues have demonstrated that protein kinase A activators, e.g. forskolin and 8-bromoadenosine cAMP, and the protein kinase C activator 12-O-tetraphorbol 12-myristate 13-acetate modulated the effect of DEX on a MMTV-luc integrated expression vector in T47D breast carcinoma cells. (47, 48). These elegant studies demonstrated that the actions of glucocorticoids on gene transcription could be regulated by multiple cell signaling pathways. As TZD is similar in structure to fatty acids and PGs, it could potentially modulate cell signaling pathways by modulating and/or substituting for signal transduction molecules. We are currently testing this hypothesis; however, we believe that it is likely that the effect of TZD in osteoblastic cells is mediated through both PPAR-dependent and -independent pathways.

The enhancement of TZD on MMTV-luc transcription in transient transfection assays appears to contrast with the opposing effects of DEX and TZD on gene expression and osteoblastic differentiation. The transient transfection experiments examined the interaction of DEX and TZD on an artificially constructed promoter that contains two copies of a glucocorticoid response element. Studies investigating differences between a transiently transfected MMTV plasmid (naked DNA) and a stably integrated construct have shown that plasmid DNA was more responsive to glucocorticoids because it was not bound by histones and thus was more accessible to basal and accessory transcription factors (49). Furthermore, the transient transfection assays in our studies examined the response on a single gene promoter. Therefore, it is not surprising that the results did not correlate with the effect of DEX and TZD on endogenous genes. However, these experiments support the hypothesis that there is an interaction between GR and TZD that regulates gene transcription in osteoblasts. The additive effects of DEX and TZD on aP2 gene expression further support this argument.

The observed differences after DEX and TZD treatment in gene expression on alkaline phosphatase and collagen type 1 (opposing) and on aP2 (enhancing) suggest that the effects of these agents may depend on the context of the gene promoter. Promoters can be simple or complex, i.e. they can be composed of a single hormone response element (simple) or have multiple response elements (composite) that bind to different transcription factors. Transcription factor binding to composite response elements can be distinct or overlapping, resulting in either cooperative activation or transcriptional gene repression. For example, studies with the phosphoenol pyruvate carboxykinase gene have shown that PPAR{gamma} and GR bind to the same 5'-flanking region of the promoter in 3T3-F442A adipocytes (50). Activation of GR by glucocorticoids repressed transcription of phosphoenol pyruvate carboxykinase induced by TZD. In contrast, a composite response element in the c-fos promoter was recently identified that contained binding sites for the retinoid X receptor and vitamin D nuclear receptors and the CCAAT-binding transcription factor/nuclear factor-1 transcription factor. These factors were found to cooperatively mediate the effects of vitamin D on c-fos transcription (51). In addition, DEX- and TZD-bound receptors could associate with different coregulator molecules (coactivators and corepressors) through protein-protein interactions or compete for the same coregulatory proteins (52). There is accumulating evidence that the association of ligand-bound receptors with coregulators can be promoter specific (53). Thus, it is plausible that DEX and TZD could have different effects (enhancing or opposing) on gene transcription depending on the promoter context and the composite array of coregulators that associate with the receptors.

Summary and conclusions
The interaction between TZD and GR demonstrated in our studies may have direct relevance to the action of thiazolidinediones on insulin resistance. Although glucocorticoids are usually thought to increase insulin resistance, some studies have found that glucocorticoids can modulate the actions of insulin and insulin-like growth factor I (54) and up-regulate insulin receptors, insulin receptor substrate-1, and phosphoinositide 3-kinase (55). As osteoblasts and adipocytes are derived from the same progenitor cells in the bone marrow, the interaction between TZD and GR observed in osteoblasts raises the possibility that in addition to PPARs, GR might be involved in the actions of these antidiabetic agents. Although in vitro studies on gene transcription, gene expression, and mineralization cannot predict the effect of TZD on bone formation in vivo, our results suggest the need for further studies in bone.

In conclusion, we have demonstrated a complex interaction between PPAR and GR signaling pathways that modulates the response to TZD and glucocorticoids in ligand-dependent transcription assays and on gene expression and differentiation in osteoblastic cells. The mechanism of this interaction remains to be elucidated, but might involve other signaling pathways and factors in addition to PPARs.


   Footnotes
 
1 Current address: Department of Genetic and Cellular Toxicology, Merck Research Laboratories, West Point, Pennsylvania 19486. Back

Received August 20, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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