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Endocrinology Vol. 143, No. 6 2376-2384
Copyright © 2002 by The Endocrine Society

RECEPTORS

Divergent Effects of Selective Peroxisome Proliferator-Activated Receptor-{gamma}2 Ligands on Adipocyte Versus Osteoblast Differentiation

Beata Lecka-Czernik, Elena J. Moerman, David F. Grant, Jürgen M. Lehmann, Stavros C. Manolagas and Robert L. Jilka

Department of Geriatrics, Reynolds Center on Aging (B.L.-C., E.J.M.), Division of Endocrinology and Metabolism, Center for Osteoporosis and Metabolic Bone Diseases (B.L.-C., S.C.M., R.L.J.), Central Arkansas Veterans Healthcare System, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; Department of Pharmaceutical Sciences, University of Connecticut (D.F.G.), Storrs, Connecticut 06269-2092; and Tularik, Inc. (J.M.L.), South San Francisco, California 94080

Address all correspondence and requests for reprints to: Dr. Beata Lecka-Czernik, Department of Geriatrics, Reynolds Center on Aging, University of Arkansas for Medical Sciences, Slot 807, 629 South Elm Street, Little Rock, Arkansas 72205. E-mail: . leckaczernikbeata{at}uams.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PPAR{gamma} is activated by diverse ligands and regulates the differentiation of many cell types. Based on evidence that activation of PPAR{gamma}2 by rosiglitazone stimulates adipogenesis and inhibits osteoblastogenesis in U-33/{gamma}2 cells, a model mesenchymal progenitor of adipocytes and osteoblasts, we postulated that the increase in marrow fat and the decrease in osteoblast number that occur during aging are due to increased PPAR{gamma}2 activation. Here, we show that the naturally occurring PPAR{gamma} ligands 9,10-dihydroxyoctadecenoic acid, and 15-deoxy-{Delta}12,14-PGJ2, also stimulate adipocytes and inhibit osteoblast differentiation of U-33/{gamma}2 cells. Strikingly, 9,10-epoxyoctadecenoic acid and the thiazolidine acetamide ligand GW0072 [(±)-(2S,5S)-4-(4-(4-carboxyphenyl)butyl)-2-heptyl-4-oxo-5-thaizolidineN,N-dibenzyl-acetamide] prevent osteoblast differentiation, but do not stimulate adipogenesis, whereas 9-hydroxyoctadecadienoic acid stimulates adipogenesis but does not affect osteoblast differentiation. The divergent effects of PPAR{gamma}2 ligands on osteoblast and adipocyte differentiation were confirmed in primary murine bone marrow cultures using rosiglitazone and GW0072. These findings indicate that the proadipogenic and antiosteoblastogenic effects of PPAR{gamma}2 are mediated by distinct regulatory pathways that can be differentially modulated depending on the nature of the ligand, and they support the idea that increased fatty acid oxidation during aging may inhibit osteoblast differentiation. Moreover, there may be selective PPAR{gamma}2 modulators that block the adverse effects of fatty acid oxidation products while retaining beneficial activities such as insulin sensitization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PPAR{gamma} is a member of the nuclear receptor family of transcription factors, a large and diverse group of proteins that mediate ligand-dependent transcriptional activation or repression (1). PPAR{gamma} exists in two isoforms, PPAR{gamma}1 and PRAR{gamma}2, as a result of alternative promoter usage and alternative splicing. PPAR{gamma} plays an important role in adipogenesis and glucose homeostasis, and has also been implicated in inflammatory responses, atherosclerosis, and cancer (1, 2, 3). This diverse spectrum of activities appears to be due to the cell specificity of PPAR{gamma} function and the nature of the ligand (4, 5, 6). Indeed, the ligand-binding domain of PPAR{gamma} is quite promiscuous and interacts with a wide variety of substances, including thiazolidinediones, modified tyrosine and leucine derivatives, PGJ2 metabolites, polyunsaturated fatty acids and their oxidation products, and certain alkyl phospholipids (7, 8, 9, 10, 11, 12, 13).

The ability of PPAR{gamma}2 to stimulate adipocyte differentiation is of relevance to skeletal metabolism because of evidence that marrow fat increases with age in both animals and humans concomitant with a fall in osteoblast production (14, 15, 16). Because marrow adipocytes and osteoblasts are derived from a common mesenchymal progenitor (17), we have proposed that the bone loss commonly seen during aging in both males and females is due in part to a reciprocal increase in the development of adipocytes and a decrease in osteoblast differentiation. In support of this contention, we have previously demonstrated that activation of PPAR{gamma}2 with rosiglitazone in the murine marrow-derived mesenchymal progenitor cell line U-33/{gamma}2 stimulated their differentiation to adipocytes and irreversibly blocked their ability to differentiate into osteoblasts (18). The latter response appeared to be due to the suppression of runt-related transcription factor 2/core-binding factor-{alpha}1 (Runx2/Cbfa1; also known as AML3 and PEBP2{alpha}A), a transcription factor required for osteoblast differentiation (19), and the synthesis of osteoblast-specific proteins such as alkaline phosphatase, osteocalcin, osteopontin, and {alpha}1(I)-procollagen (20). Thus, PPAR{gamma}2 is a potent suppressor of the osteoblast phenotype and may be critically involved in the differentiation of bone marrow mesenchymal progenitors toward adipocytes. Based on this, it is likely that the age-related increase in adipocyte and the decrease in osteoblast differentiation may be due to increased activation of PPAR{gamma}2. The fact that fatty acid oxidation products can bind and activate PPAR{gamma}2, together with evidence for their increased production with advancing age (21), suggests that they could contribute to the increased fat and bone loss that characterize the aging skeleton.

Here we compared a variety of PPAR{gamma} ligands for their ability to influence differentiation of U-33/{gamma}2 cells to adipocytes or osteoblasts. We found that the linoleic acid (LA) peroxidation products 9,10-epoxyoctadecenoic acid (9,10-EOA) and 9,10-dihydroxyoctadecenoic acid (9,10-DHOA) are PPAR{gamma}2 ligands, and that 9,10-DHOA is both proadipocytic and antiosteoblastic. Strikingly, however, 9,10-EOA and the thiazolidine acetamide ligand GW0072 [(±)-(2S,5S)-4-(4-(4-carboxyphenyl)butyl)-2-heptyl-4-oxo-5-thaizolidine N,N-dibenzyl-acetamide] are antiosteogenic without stimulating adipocyte differentiation. On the other hand, 9-hydroxyoctadecadienoic acid (9-HODE) is proadipogenic without affecting osteoblast differentiation. These findings indicate that PPAR{gamma}2 stimulates multiple pathways that promote adipocyte differentiation, inhibit osteoblast differentiation, or both depending on the nature of the ligand, and that fatty acid oxidation metabolites could be involved in age-related bone loss.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Rosiglitazone (BRL 49653) was obtained from Tularik, Inc. (South San Francisco CA); GW0072 was obtained from GlaxoSmithKline Pharmaceuticals (Research Triangle Park, NC); 15-deoxy-{Delta}12,14-PGJ2 (15dPGJ2) and 9(S)HODE were purchased from Cayman Chemical (Ann Arbor, MI), and LA was from Sigma (St. Louis, MO). 9,10-EOA and 9,10-DHOA were synthesized as previously described (22). At the concentrations used in the experiments none of the compounds tested for their effect on cell differentiation had an effect on cell viability as measured by trypan blue exclusion; however, oxidized fatty acids caused cell toxicity at higher than tested concentrations.

Ligand binding to PPAR{gamma}
The binding affinities of 9,10-EOA, 9,10-DHOA, and LA were determined using a previously described scintillation proximity assay (SPA; Amersham Pharmacia Biotech, Piscataway, NJ) (23). Briefly, the PPAR{gamma} ligand-binding domain was expressed in Escherichia coli as a glutathione-S-transferase-tagged fusion protein. The protein was purified and immobilized on poly-L-lysine-coated yttrium silicate SPA beads. The labeled PPAR{gamma} ligand [3H]T0900393 [N-ethyl 2-(3-chloro-5-pyridyloxy) 5-(2,4-dichloro-5-methylbenzenesulfonamido)benzamide; Tularik, Inc.] was used for determination of binding to PPAR{gamma} in the absence or presence of the compounds tested.

Differentiation of U-33/{gamma}2 cells
Murine marrow-derived UAMS-33 cells stably transfected with a vector expressing mRNA for PPAR{gamma}2, referred to as U-33/{gamma}2 cells, and UAMS-33 cells transfected with an empty vector control, referred to as U-33/c cells, have been previously described (18). In the former, PPAR{gamma}2 transcription is under the control of a promoter fragment of human EF-1{alpha} translation elongation factor. The level of PPAR{gamma}2 protein is comparable to that seen in a marrow-derived adipocyte cell line (18). To avoid artifacts due to potential differences among PPAR{gamma}2 stable transfectants, all experiments were performed using two independently derived clones, {gamma}2.28 and {gamma}2.45 (18). Representative data from clone {gamma}2.28 are shown in this report. Cells were maintained in {alpha}MEM supplemented with 10% FBS (HyClone Laboratories, Inc., Logan, UT), 0.5 mg/ml G418, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin (Sigma) at 37 C in a humidified atmosphere containing 5% CO2. Media and additives were purchased from Life Technologies, Inc. (Gaithersburg, MD).

To measure adipogenesis, cells were cultivated on 24-well plates, and after achieving about 70% confluence, media were supplemented with the ligand to be tested or with dimethylsulfoxide (DMSO) as a vehicle control. Medium containing ligand was changed every 2 d for 6 d. Cells were fixed with 10% formalin in PBS, rinsed, and stained for 30 min with 0.15% Oil Red O in a 55:45 mix of isopropanol and water (Sigma). After rinsing, cells were counterstained with 0.5% methyl green (Fisher Scientific, Fairlawn, NJ) in 0.1 M sodium acetate (pH 4.0). For quantification of adipocytes, cells in each replicate well (n = 3) were evaluated microscopically at x400 magnification. Each field contained approximately 200 cells. For enumeration, three or four randomly selected fields were evaluated for the presence of Oil Red O droplets, and the percent fraction of cells that contained fat droplets was calculated. Photomicrographs of Oil Red O-stained cells were obtained after culture of cells as described above on Lab-Tek glass slides (Nalge Nunc International, Naperville, IL).

For analysis of osteoblast differentiation, the cells were initially cultured for 6 d without or with ligands as described above and then maintained for an additional 13 d in medium without ligands but containing 0.2 mM ascorbic acid (Sigma) and 10 mM ß-glycerophosphate (Sigma) to stimulate osteoblast differentiation (18). The culture medium was replaced every other day. Calcium deposited into the extracellular matrix, a marker of differentiated osteoblasts, was extracted with 0.1% acetic acid for 5 h at room temperature and quantified colorimetrically using calcium-binding reagent (Sigma).

Differentiation of bone marrow-derived mesenchymal progenitors
Bone marrow cells were obtained from adult Swiss-Webster mice and maintained in the presence of {alpha}MEM with 15% FBS and 1 mM ascorbate-2-phosphate at 1.5 x 106/10-cm2 culture dish as previously described (24). Test ligands or vehicle (DMSO) were added after 7 d of culture. Half of the medium was changed at 10, 15, and 20 d of culture with replacement of ligand. After 25 d of culture, adipogenesis was quantified by enumerating colonies containing at least 5% Oil Red O-positive cells. Osteoblastogenesis was quantified in a parallel set of cultures by enumerating colonies containing extracellular calcium, as determined by von Kossa staining.

Northern and RT-PCR analysis of gene expression
Total RNA was isolated using an RNAeasy kit (QIAGEN, Chatsworth, CA). The following cDNA probes were used for detection of transcripts on Northern blots: a 1.8-kb fragment of human CAAT enhancer-binding protein-{alpha} (C/EBP{alpha}), a 1.4-kb fragment of murine lipoprotein lipase (LPL), a 0.6-kb fragment of murine fatty acid-binding protein aP2, a 1.5-kb fragment of murine osteopontin, and a 1.5-kb fragment of human {alpha}1(I)-procollagen. The human probes were more than 80% homologous to the murine sequences. Transcripts were visualized with a phosphorimager. The equivalence of loading and transfer of RNA onto membranes was assessed by detection of 18S rRNA using human cDNA as a probe. All hybridizations were performed under high stringency conditions as previously described (25).

RT-PCR was performed using Advantage RT-for-PCR and Advantage cDNA PCR kits (CLONTECH Laboratories, Inc., Palo Alto, CA). The RT reaction was incubated at 42 C for 1 h. The amount of cDNA used for each PCR corresponded to the 0.014 µg total RNA used originally for the RT reaction. The amplification reactions were performed using the following primers and protocols: Runx2/Cbfa1: forward, 5'-CTACAACCTTGAAGGCCACG-3'; reverse, 5'-ATGCTTCATTCGCCTCACAAAC-3' (annealing at 60 C; 35 cycles; product, 655 bp) (26); Wnt-10b: forward, 5'-CTGCCACTGTCGTTTCCACTG-3'; reverse, 5'-AGACCCTTTCAACAACTGAACG-3' (annealing at 60 C; 32 cycles; product, 660 bp); glyceraldehyde-3-phosphate dehydrogenase: forward, 5'-ATTGGGAAGCTTGTCATCAACG-3'; reverse, 5'-CACCCTGTTGCTGTAGCCGT-3' (annealing at 60 C; 23 cycles; product, 781 bp). Reactions were carried out using a Perkin-Elmer Corp./Cetus DNA Thermal Cycler (Norwalk, CT). PCR products were resolved on 2% ultraPURE agarose (Life Technologies, Inc.). Preliminary experiments varying the amount of template between 0.05 and 0.005 µg RNA and the number of cycles showed that the reaction products generated under the conditions described above for each transcript were within the linear range of the amplification.

Transient transfection and trans-activation assay
Transfections were performed using Lipofectamine Plus reagent (Life Technologies, Inc.). Typically, cells were plated in triplicate at the density of 3 x 104 cells/1.8-cm2 well and transfected for 6 h with 0.4 µg DNA of firefly luciferase gene reporter constructs (Promega Corp., Madison WI) under the transcriptional control of either peroxisome proliferator response element (PPRE) sequences in a 2AOx construct (27) or osteoblast-specific element 2 (OSE2) sequences in a 1.3luc construct (28). The 2AOx construct has two copies of PPRE from the acetyl-coenzyme A oxidase gene promoter introduced into the minimal promoter from the rat liver carbamoyl-phosphate synthetase gene. Cells were cotransfected with 0.2 µg DNA of pRL-TK vector encoding Renilla luciferase enzyme (Promega Corp.). Then, the medium was changed, and the cells were cultured with vehicle or PPAR{gamma} ligand for 24 h. The firefly and Renilla luciferase activities were detected using the Dual Luciferase Reporter Assay (Promega Corp.). Firefly luciferase activity was normalized with Renilla luciferase activity. Each experiment was repeated three times.

Statistics
Statistically significant differences between mean values were detected by t test using SigmaStat (SPSS, Inc., Chicago, IL) after establishing the homogeneity of variances and normal distribution of the data. SPA ligand binding assays were analyzed with curve-fitting software SigmaPlot (SPSS, Inc.) to calculate IC50 values using logistic regression. ANOVA and post hoc testing for significant differences by the Bonferroni method were used to demonstrate dose-dependent effects of ligands on adipocyte and osteoblast differentiation. In all cases, P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Binding affinity of tested compounds
Several PPAR{gamma} ligands were tested for their ability to influence adipocyte and osteoblast differentiation (see Table 1Go). They included the established PPAR{gamma} ligands rosiglitazone and 15dPGJ2, and GW0072, a PPAR{gamma} modulator that blocks the stimulatory effect of rosiglitazone on adipogenesis (29). In addition, the effects of LA and its oxidation products, 9-HODE, 9,10-DHOA, and 9,10-EOA, were studied. The latter two compounds are found in vivo (21, 30) and are potential PPAR{gamma} ligands based on their structural similarity to LA. Both 9,10-DHOA and 9,10-EOA displaced the thiazolidinedione radioligand [3H]T0900393 from PPAR{gamma}, as determined by SPA (Fig. 1Go). The IC50 values for 9,10-DHOA and 9,10-EOA were 2.8 ± 1.0 and 0.8 ± 0.6 µM, respectively. The LA IC50 value of 2.9 ± 0.2 µM (±SEM) was similar to that previously reported (23).


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  		Table 1. Compounds tested for their effect on adipocyte and osteoblast differentiation

 


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  		Figure 1. Binding of LA and LA oxidation products to PPAR{gamma}. The abilities of LA, 9,10-DHOA, and 9,10-EOA to bind to PPAR{gamma} were determined by displacement of the thiazolidinedione ligand [3H]T0900393 using SPA. The displacement curves shown were calculated by logistic regression, and gave IC50 values of 2.9 ± 0.2 µM (±SEM) for LA, 2.8 ± 1.0 µM for 9,10-DHOA, and 0.8 ± 0.6 µM for 9,10-EOA.

 
Effects of ligands on adipocyte and osteoblast development
The effects of various PPAR{gamma} ligands on adipocyte and osteoblast differentiation were studied in the model bipotential mesenchymal progenitor cell line U-33/{gamma}2 stably transfected with a PPAR{gamma}2 expression construct (18). U-33/c cells that do not express PPAR{gamma}2 served as a negative control. In the presence of ascorbic acid and ß-glycerophosphate, both cell lines express an osteogenic phenotype, whereas addition of the PPAR{gamma} ligand rosiglitazone induces fat accumulation and irreversibly suppresses the osteoblast phenotype only in U-33/{gamma}2 cells (18).

Figure 2AGo shows that of those ligands that stimulated lipid accumulation, they did so in rank order of their affinity for PPAR{gamma} (rosiglitazone > 15dPGJ2 > 9(S)-HODE ~ 9,10-DHOA). LA, 9,10-EOA, and GW0072 did not stimulate lipid accumulation. In experiments not presented here, GW0072 antagonized rosiglitazone-stimulated adipogenesis, consistent with a previous report (29). Practically all of the cells were converted to adipocytes by rosiglitazone at concentrations of 1 µM or more. Maximal concentrations of rosiglitazone and 15dPGJ2 induced accumulation of large fat-containing droplets within the cells, and the cells changed from a fibroblastic to a rhomboid morphology (Fig. 3Go). Doses of rosiglitazone as low as 0.01 µM, which is below its Kd for PPAR{gamma} (Table 1Go), caused the appearance of fat in approximately 50% of treated cells; however, the size of fat droplets was smaller than after the same time of treatment with intermediate concentrations of the ligand (not shown). As we did not attempt to quantify the amount of fat per cell, low and intermediate levels of rosiglitazone thus appeared to be of equivalent potency in this assay.



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  		Figure 2. Effects of PPAR{gamma}2 ligands on differentiation of U-33/{gamma}2 cells. A, Adipogenesis. U-33/{gamma}2 cells were cultured on 24-well plates for 6 d in vehicle (DMSO) or in the presence of ligands at the indicated concentrations. The percentage of cells containing fat was then determined after staining with Oil Red O as described in Materials and Methods. B, Osteoblastogenesis. Cells were cultured and exposed to compounds as described in A. The cells were then rinsed and medium was replaced with proosteoblastogenic medium for an additional 13 d. Osteoblast differentiation was quantified by determining the calcium content in each well. ANOVA and post hoc testing for significant differences using the Bonferroni method demonstrated dose-dependent effects of specific ligands on lipid accumulation (rosiglitazone, 15dPGJ2, 9-HODE, and 9,10-DHOA) and on mineral deposition (rosiglitazone, GW0072, 15dPGJ2, 9,10-DHOA, and 9,10-EOA).

 


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  		Figure 3. The histological appearance of U-33/{gamma}2 cells treated with different compounds. U-33/{gamma}2 cells were cultured on Lab-Tek glass slides and treated as described in Materials and Methods with ligands at the following concentrations: 5 µM 15dPGJ2, 5 µM rosiglitazone, 60 µM 9-HODE, 120 µM 9,10-DHOA, 120 µM 9,10-EOA, and vehicle. Cells were stained with Oil Red O (red) and counterstained with methyl green (blue). Magnification, x400.

 
Compared with rosiglitazone, the degree of maximal adipogenesis stimulation achieved by 15dPGJ2, 9-HODE, and 9,10-DHOA was significantly lower. This may be due to the comparatively lower stability of these ligands in aqueous media compared with rosiglitazone and binding to the serum albumin (Grant, D., unpublished observations). Fat droplets in cells treated with 9-HODE or 9,10-DHOA were small, and the cells retained their fibroblastic appearance (Fig. 3Go). Figure 2BGo shows that rosiglitazone and 15dPGJ2, which induced adipogenesis, also inhibited mineral deposition. However, GW0072 and 9,10-EOA, which did not influence adipogenesis, did inhibit mineral deposition, whereas 9-HODE, which stimulated fat accumulation, failed to suppress mineral deposition. As in the case of adipogenesis, LA had no effect on osteoblast differentiation. In data not shown here, we found that none of the tested compounds stimulated fat accumulation or inhibited mineralization in cultures of U-33/c cells, indicating that the effects on adipocyte and osteoblast differentiation are PPAR{gamma}2 dependent.

To confirm divergent effects of different PPAR{gamma}2 ligands on osteoblast and adipocyte differentiation, we compared the effects of rosiglitazone and GW0072 on murine marrow-derived mesenchymal progenitors (Table 2Go). In these cultures, early mesenchymal progenitors replicate to form a colony of cells that can differentiate into osteoblasts and/or adipocytes upon exposure to appropriate prodifferentiating agents (17, 24). We found that both ligands modestly reduced the total number of colonies that developed in the cultures. Nevertheless, and consistent with the findings in the U-33/{gamma}2 cell model, only rosiglitazone stimulated adipocyte differentiation in these cultures, whereas both ligands strongly suppressed osteoblast differentiation.


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  		Table 2. Effect of PPAR{gamma}2 ligands on adipocyte and osteoblast differentiation from murine marrow-derived progenitors

 
Effects of ligands on the expression of phenotype-specific genes and trans-activation of PPRE and OSE2 regulatory elements
We next examined the effect of each ligand on the expression of genes associated with the development and/or expression of the adipocyte or osteoblast phenotype. Northern blot analysis showed that all ligands, except LA, stimulated the expression of C/EBP{alpha} and LPL, early markers of adipogenesis (Fig. 4AGo). However, only rosiglitazone and 15dPGJ2 stimulated aP2 mRNA expression, a marker of fully differentiated adipocytes. Consistent with these findings, only these two ligands were able to activate PPRE of the acetyl-CoA oxidase promoter in a reporter gene assay (Fig. 5AGo) (27). Activation of PPRE was PPAR{gamma}2 dependent, as evidenced by the lack of effect of any ligand when the reporter gene was transfected into PPAR{gamma}2-negative U-33/c cells.



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  		Figure 4. Effects of PPAR{gamma}2 ligands on gene expression. A, Northern analysis. U-33/{gamma}2 cells were cultured as described in Fig. 2AGo with vehicle (veh), 5 µM rosiglitazone (rosigl), 5 µM 15dPGJ2, 60 µM 9-HODE, 120 µM LA, 120 µM 9,10-DHOA, 120 µM 9,10-EOA, or 5 µM GW0072. Total RNA was isolated and subjected to Northern blot analysis using 15 µg/lane. The same blot was probed subsequently with the indicated 32P-labeled cDNAs. B, RT-PCR analysis. Semiquantitative RT-PCR analysis for Runx2/Cbfa1, Wnt-10b, and glyceraldehyde-3-phosphate dehydrogenase was performed using total RNA from U-33/{gamma}2 or U-33/c cells, cultured as described in A.

 


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  		Figure 5. Effects of PPAR{gamma} ligands on 2AOx (PPRE) and osteocalcin (OSE2) promoter activities. U-33/{gamma}2 and U-33/c cells were transiently transfected with a luciferase construct driven by the PPRE-containing 2AOx promoter (A) or the OSE2-containing osteocalcin promoter (B), as described in Materials and Methods. Replicate cultures were established, and 24 h later, cells were treated with vehicle (veh), 5 µM rosiglitazone (rosigl), 5 µM GW0072, 5 µM 15dPGJ2, 60 µM 9-HODE, 120 µM LA, 120 µM 9,10-DHOA, or 120 µM 9,10-EOA for an additional 24 h. Luciferase expression was then determined in cell lysates. {blacksquare}, U-33/{gamma}2; , U-33/c cells.

 
Semiquantitative RT-PCR analysis showed that rosiglitazone, 15dPGJ2, and GW0072 strongly suppressed the expression of Wnt-10b, a negative regulator of adipocyte formation (Fig. 4BGo) (31). Marginal inhibitory effects of 9-HODE and LA were also noted, but 9,10-DHOA and 9,10-EOA failed to inhibit Wnt-10b expression. These effects were PPAR{gamma}2 dependent, as they were observed in U-33/{gamma}2 cells, but not in U-33/c control cells.

Every ligand that inhibited calcium deposition by U-33/{gamma}2 cells also suppressed expression of {alpha}1(I)-procollagen mRNA; however, osteopontin mRNA levels were significantly affected only by rosiglitazone and 15dPGJ2 (Fig. 4AGo). Interestingly, 9-HODE, which did not significantly inhibit calcium deposition, also decreased {alpha}1(I)-procollagen mRNA. Expression of Runx2/Cbfa1 was inhibited by rosiglitazone and 15dPGJ2, but GW0072, 9,10-EOA, and 9,10-DHOA, which inhibited mineralization, did not affect Runx2/Cbfa1 expression (Fig. 4BGo). LA and 9-HODE, which did not exert antiosteoblastic activity, also did not affect Runx2/Cbfa1 expression.

Finally, we examined the effects of the ligands on the activity of the osteoblast-specific osteocalcin promoter. Cells were transiently transfected with a construct comprising the OSE2 of the rat osteocalcin promoter driving the expression of luciferase (28). As shown in Fig. 5BGo, rosiglitazone, 15dPGJ2, and GW0072 suppressed osteocalcin promoter activity in U-33/{gamma}2, but not in U-33/c, cells, whereas LA and its oxidized metabolites had no effect.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PPAR{gamma}2 modulates numerous physiological and pathological processes. The findings presented herein show that, depending on the activating ligand, PPAR{gamma}2 may stimulate adipocyte differentiation, suppress osteoblast differentiation, or exhibit both activities in U-33/{gamma}2 cells, a model bipotential mesenchymal progenitor cell line. These effects were mediated by PPAR{gamma}2, as evidenced by the fact that U-33/c cells lacking PPAR{gamma}2 failed to respond to any of the ligands tested. The same divergent effects of two of the ligands, namely rosiglitazone and GW0072, were confirmed in primary cultures of murine marrow-derived mesenchymal progenitors. Reminiscent of our findings in marrow stromal cells, previous studies have demonstrated that different PPAR{gamma} ligands exert disparate effects on macrophage differentiation (32, 33, 34).

Osteoblasts and marrow adipocytes originate from a common mesenchymal progenitor. Lineage commitment probably depends on specific transcription factors that simultaneously suppress factors that are required for expression of the alternate phenotype. Previous experiments with cell lines, primary marrow-derived stromal cell cultures, as well as in vivo studies in rodents and humans have provided evidence indicating that proadipogenic agents suppress osteoblast differentiation (18, 35, 36, 37, 38, 39). However, we found that whereas rosiglitazone, 15dPGJ2, and 9,10-DHOA activate both proadipogenic and antiosteoblastogenic pathways, GW0072 and 9,10-EOA inhibit osteoblast differentiation without activating adipocyte differentiation, and 9-HODE stimulates lipid accumulation, but has no effect on osteogenesis. Therefore, reciprocal changes in adipogenesis and osteoblastogenesis are not an inevitable consequence of PPAR{gamma}2 activation.

Rosiglitazone and 15dPGJ2 activated pathways that are functionally distinct from those of LA metabolites to stimulate adipocyte differentiation. Thus, rosiglitazone and 15dPGJ2 induced the complete spectrum of adipogenic responses, including activation of a PPRE and expression of early (C/EBP{alpha} and LPL) and late (aP2) markers of adipocyte differentiation. As shown here for the first time, both ligands also down-regulated Wnt-10b expression, as would be expected from evidence that Wnt-10b prevents expression of the adipocyte phenotype (31). Interestingly, however, GW0072 also suppressed Wnt-10b, but had no effect on adipogenesis, indicating only partial activation of PPAR{gamma}2 by this ligand. In contrast, none of the LA oxidation products activated PPRE or induced aP2 mRNA, but they did stimulate C/EBP{alpha} and LPL synthesis, which are necessary for adipocyte development and function (40). Recent evidence indicates that expression of aP2 mRNA is governed by a PPRE and controls certain adipocyte functions, but it is not required for adipocyte differentiation (41, 42). Therefore, thiazolidinediones and 15dPGJ2 may promote the development of marrow adipocytes that are functionally distinct from those induced by oxidized fatty acids.

Distinct pathways are also involved in suppression of the osteoblastic phenotype by various PPAR{gamma}2 ligands. This is in keeping with the fact that full expression of the osteoblast phenotype depends not only on Runx2/Cbfa1 and other transcriptional regulators (43, 44, 45), but also on integrin signaling generated by interaction with collagen in the extracellular matrix (46). Thus, the loss of such signaling by down-regulation of {alpha}1(I)-procollagen expression in response to PPAR{gamma}2 activation may contribute to the suppression of osteogenesis. Inhibition of osteoblast differentiation by rosiglitazone and 15dPGJ2 was associated with the reduction of Runx2/Cbfa1, {alpha}1(I)-procollagen, and reduced OSE2 promoter activity. GW0072 strongly inhibited both OSE2 activity and {alpha}1(I)-procollagen expression, responses that were not apparently mediated by down-regulation of Runx2/Cbfa1 expression. It is possible that GW0072 exerts its effect by interfering with, or down-regulating, other transcriptional regulators, such as Msx2, Dlx5, or osterix, which are also required for OSE2 activity (44, 47, 48). Further, the most potent antiosteogenic ligands (rosiglitazone, 15dPGJ2, and GW0072) almost completely abrogated the expression of Wnt-10b, an activator of frizzled/low density lipoprotein (LDL) receptor-related protein receptors leading to ß-catenin-mediated changes in gene expression (49). It was recently shown that mutations in the LDL receptor-related protein-5 component of the receptor affect bone accrual in human and rodents (50, 51), and that Wnt-induced ß-catenin signaling can stimulate osteoblast differentiation in vitro (52, 53, 54). Thus, the ability of these PPAR{gamma}2 ligands to inhibit Wnt-10b synthesis may also contribute to their ability to inhibit osteogenesis.

What could explain the ligand-dependent effects of PPAR{gamma}2 activation reported herein? As in the case of other members of the nuclear receptor family of transcription factors, ligand binding to PPAR{gamma} induces allosteric alterations of the activating function-2 domain, resulting in dissociation of transcriptional corepressors and concomitant association of transcriptional coactivators (55). However, different coactivators may be recruited depending on the ligand (4, 5, 6, 56). For example, it has been reported that rosiglitazone and 15dPGJ2 recruited similar coactivators to the complex, including SRC-1, CBP/p300, and TRAP220/DRIP205, whereas 9-HODE did not (5, 6) (55). On the other hand, GW0072 exhibited limited capacity to recruit SRC-1 and CBP/p300 compared with rosiglitazone (29). It is also important to consider activation of nongenotropic intracellular signaling, as has been observed for several members of the nuclear receptor family, including ER, AR, VDR, and PR (57). Of relevance to the present discussion, ligand-specific activation of the genomic vs. nongenomic actions of the ER has been demonstrated with structurally different ligands, an estren and a pyrazole, respectively (58). In view of this, it is tempting to speculate that PPAR{gamma}2-mediated activation of nongenomic pathways can partially account for the divergent effects of PPAR{gamma}2 ligands reported in our studies.

Our results demonstrate that two LA oxidation products, 9,10-EOA and 9,10-DHOA, are ligands for PPAR{gamma}2, and they both inhibit osteoblast differentiation. These effects required high concentrations of the free acids in our culture system, perhaps due to their binding to serum proteins and/or instability in aqueous media. However, these metabolites are components of oxidized LDL, the level of which increases with aging (59), perhaps due to an increase in 15-lipoxygenase and free oxygen radicals (21, 60). Oxidized LDL inhibits osteoblast differentiation in vitro, and administration of a high fat atherogenic diet suppresses bone formation in mice (61, 62). Based on these lines of evidence, we propose that an increase in the level of LA oxidation products, such as 9-EOA and 9,10-DHOA, might contribute to decreased bone formation during aging. Interestingly, the parent ligand, LA, had no effect on osteoblast or adipocyte differentiation, but modestly decreased Wnt-10b. Thus, the response of our stromal cell model to LA differs from that of the NIH-3T3 cell model (63), consistent with the idea that responses to PPAR{gamma}2 activation are cell specific.

In summary, we have shown that diverse ligands of PPAR{gamma}2 act as selective PPAR{gamma}2 modulators designated SPPARMs (64) to regulate distinct pathways, the sum of which may lead to full or partial expression of the adipocyte phenotype, suppression of osteoblast differentiation, or both. Future studies are needed to elucidate the pathways that mediate PPAR{gamma}2-induced inhibition of osteoblast differentiation and to identify SPPARMs that can block the adverse effects of PPAR{gamma}2 activation on osteoblast differentiation while retaining other beneficial activities, such as insulin sensitization.


   Acknowledgments
 
We thank Mitchell V. Hull (Tularik, Inc.) for performing SPA assays, David C. Morris (GlaxoSmithKline Pharmaceuticals) for providing the PPAR{gamma} modulator GW0072, and Rebecca Wynne for technical assistance.


   Footnotes
 
This work was supported by NIH Grants R03-AG-15605 and R01-AG-17482 (to B.L.C.), R01-GM-56708 (to D.F.G.), P01-AG-13918 (to S.C.M.), and R01-AR-46823 (to R.L.J.); the Department of Veterans Affairs (REAP and Merit Review support to S.C.M. and R.L.J.); and the Department of Defense (N00014-99-1-0905 and N00014-99-1-0606 to D.F.G.).

Abbreviations: C/EBP{alpha}, CAAT enhancer-binding protein-{alpha}; 9,10-DHOA, 9,10-dihydroxyoctadecenoic acid; DMSO, dimethylsulfoxide; 15dPGJ2, 15-deoxy-{Delta}12,14-PGJ2; 9,10-EOA, 9,10-epoxyoctadecenoic acid; GW0072, (±)-(2S,5S)-4-(4-(4-carboxyphenyl)butyl)-2-heptyl-4-oxo-5-thaizolidine N,N-dibenzyl-acetamide; 9-HODE, 9-hydroxyoctadecadienoic acid; LA, linoleic acid; LDL, low density lipoprotein; LPL, lipoprotein lipase; OSE2, osteoblast-specific element 2; PPRE, peroxisome proliferator response element; Runx2/Cbfa1, runt-related transcription factor 2/core-binding factor-{alpha}1; SPA, scintillation proximity assay.

Received October 25, 2001.

Accepted for publication February 14, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rosen ED, Spiegelman BM 2001 PPAR{gamma}: a nuclear regulator of metabolism, differentiation, and cell growth. J Biol Chem 276:37731–37734[Free Full Text]
  2. Kersten S, Desvergne B, Wahli W 2000 Roles of PPARs in health and disease. Nature 405:421–424[CrossRef][Medline]
  3. Olefsky JM, Saltiel AR 2000 PPAR{gamma} and the treatment of insulin resistance. Trends Endocrinol Metab 11:362–368[CrossRef][Medline]
  4. Olefsky JM 2000 Treatment of insulin resistance with peroxisome proliferator-activated receptor {gamma} agonists. J Clin Invest 106:467–472[Medline]
  5. Kodera Y, Takeyama K, Murayama A, Suzawa M, Masuhiro Y, Kato S 2000 Ligand type-specific interactions of peroxisome proliferator-activated receptor {gamma} with transcriptional coactivators. J Biol Chem 275:33201–33204[Abstract/Free Full Text]
  6. Yang W, Rachez C, Freedman LP 2000 Discrete roles for peroxisome proliferator-activated receptor gamma and retinoid X receptor in recruiting nuclear receptor coactivators. Mol Cell Biol 20:8008–8017[Abstract/Free Full Text]
  7. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA 1995 An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}). J Biol Chem 270:12953–12956[Abstract/Free Full Text]
  8. Henke BR, Blanchard SG, Brackeen MF, Brown KK, Cobb JE, Collins JL, Harrington WW, Jr., Hashim MA, Hull-Ryde EA, Kaldor I, Kliewer SA, Lake DH, Leesnitzer LM, Lehmann JM, Lenhard JM, Orband-Miller LA, Miller JF, Mook RA, Jr., Noble SA, Oliver W, Jr., Parks DJ, Plunket KD, Szewczyk JR, Willson TM 1998 N-(2-Benzoylphenyl)-L-tyrosine PPAR{gamma} agonists. I. Discovery of a novel series of potent antihyperglycemic and antihyperlipidemic agents. J Med Chem 41:5020–5036[CrossRef][Medline]
  9. Rocchi S, Picard F, Vamecq J, Gelman L, Potier N, Zeyer D, Dubuquoy L, Bac P, Champy MF, Plunket KD, Leesnitzer LM, Blanchard SG, Desreumaux P, Moras D, Renaud JP, Auwerx J 2001 A unique PPAR{gamma} ligand with potent insulin-sensitizing yet weak adipogenic activity. Mol Cell 8:737–747[CrossRef][Medline]
  10. Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, Evans RM 1995 15-Deoxy-{Delta}12,14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR{gamma}. Cell 83:803–812[CrossRef][Medline]
  11. Nagy L, Tontonoz P, Alvarez JG, Chen H, Evans RM 1998 Oxidized LDL regulates macrophage gene expression through ligand activation of PPAR{gamma}. Cell 93:229–240[CrossRef][Medline]
  12. Kliewer SA, Sundseth SS, Jones SA, Brown PJ, Wisely GB, Koble CS, Devchand P, Wahli W, Willson TM, Lenhard JM, Lehmann JM 1997 Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors {alpha} and {gamma}. Proc Natl Acad Sci USA 94:4318–4323[Abstract/Free Full Text]
  13. Davies SS, Pontsler AV, Marathe GK, Harrison KA, Murphy RC, Hinshaw JC, Prestwich GD, Hilaire AS, Prescott SM, Zimmerman GA, McIntyre TM 2001 Oxidized alkyl phospholipids are specific, high affinity peroxisome proliferator-activated receptor {gamma} ligands and agonists. J Biol Chem 276:16015–16023[Abstract/Free Full Text]
  14. Moore SG, Dawson KL 1990 Red and yellow marrow in the femur: age-related changes in appearance at MR imaging. Radiology 175:219–223[Abstract/Free Full Text]
  15. Gimble JM, Robinson CE, Wu X, Kelley KA 1996 The function of adipocytes in the bone marrow stroma: an update. Bone 19:421–428[Medline]
  16. Kajkenova O, Lecka-Czernik B, Gubrij I, Hauser SP, Takahashi K, Parfitt AM, Jilka RL, Manolagas SC, Lipschitz DA 1997 Increased adipogenesis and myelopoiesis in the bone marrow of SAMP6, a murine model of defective osteoblastogenesis and low turnover osteopenia. J Bone Miner Res 12:1772–1779[CrossRef][Medline]
  17. Robey PG, Bianco P 1999 Cellular mechanisms of age-related bone loss. In: Rosen C, Glowacki J, Bilezikian JP, eds. The aging skeleton. San Diego: Academic Press; 145–157
  18. Lecka-Czernik B, Gubrij I, Moerman EJ, Kajkenova O, Lipschitz DA, Manolagas SC, Jilka RL 1999 Inhibition of Osf2/Cbfa1 expression and terminal osteoblast differentiation by PPAR{gamma}2. J Cell Biochem 74:357–371[CrossRef][Medline]
  19. Ducy P, Zhang R, Goeffroy V, Ridall AL, Karsenty G 1997 Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89:747–754[CrossRef][Medline]
  20. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao Y-H, Inada M, Sato M, Okamoto T, Kitamura Y, Yoshiki S, Kishimoto T 1997 Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89:755–764[CrossRef][Medline]
  21. Spiteller G 2000 Peroxidation of linoleic acid and its relation to aging and age dependent diseases. Ageing Res Rev 122:617–657
  22. Jude AR, Little JM, Freeman JP, Evans JE, Radominska-Pandya A, Grant DF 2000 Linoleic acid diols are novel substrates for human UDP-glucuronosyltransferases. Arch Biochem Biophys 380:294–302[CrossRef][Medline]
  23. Xu HE, Lambert MH, Montana VG, Parks DJ, Blanchard SG, Brown PJ, Sternbach DD, Lehmann JM, Wisely GB, Willson TM, Kliewer SA, Milburn MV 1999 Molecular recognition of fatty acids by peroxisome proliferator-activated receptors. Mol Cell 3:397–403[CrossRef][Medline]
  24. Di Gregorio GB, Yamamoto M, Ali AA, Abe E, Roberson P, Manolagas SC, Jilka RL 2001 Attenuation of the self-renewal of transit-amplifying osteoblast progenitors in the murine bone marrow by 17ß-estradiol. J Clin Invest 107:803–812[CrossRef][Medline]
  25. Lecka-Czernik B, Moerman EJ, Shmookler Reis RJ, Lipschitz DA 1997 Cellular and molecular biomarkers indicate precocious in vitro senescence in fibroblasts from SAMP6 mice. Evidence supporting a murine model of premature senescence and osteopenia. J Gerontol A Biol Sci Med Sci 52:B331–B336
  26. Xiao ZS, Thomas R, Hinson TK, Quarles LD 1998 Genomic structure and isoform expression of the mouse, rat and human Cbfa1/Osf2 transcription factor. Gene 214:187–197[CrossRef][Medline]
  27. Marcus SL, Miyata KS, Zhang B, Subramani S, Rachubinski RA, Capone JP 1993 Diverse peroxisome proliferator-activated receptors bind to the peroxisome proliferator-responsive elements of the rat hydratase/dehydrogenase and fatty acyl-CoA oxidase genes but differentially induce expression. Proc Natl Acad Sci USA 90:5723–5727[Abstract/Free Full Text]
  28. Ducy P, Karsenty G 1995 Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene. Mol Cell Biol 15:1858–1869[Abstract]
  29. Oberfield JL, Collins JL, Holmes CP, Goreham DM, Cooper JP, Cobb JE, Lenhard JM, Hull-Ryde EA, Mohr CP, Blanchard SG, Parks DJ, Moore LB, Lehmann JM, Plunket K, Miller AB, Milburn MV, Kliewer SA, Willson TM 1999 A peroxisome proliferator-activated receptor {gamma} ligand inhibits adipocyte differentiation. Proc Natl Acad Sci USA 96:6102–6106[Abstract/Free Full Text]
  30. Street JM, Evans JE, Natowicz MR 1996 Glucuronic acid-conjugated dihydroxy fatty acids in the urine of patients with generalized peroxisomal disorders. J Biol Chem 271:3507–3516[Abstract/Free Full Text]
  31. Ross SE, Hemati N, Longo KA, Bennett CN, Lucas PC, Erickson RL, MacDougald OA 2000 Inhibition of adipogenesis by Wnt signaling. Science 289:950–953[Abstract/Free Full Text]
  32. Tontonoz P, Nagy L, Alvarez JG, Thomazy VA, Evans RM 1998 PPARgamma promotes monocyte/macrophage differentiation and uptake of oxidized LDL. Cell 93:241–252[CrossRef][Medline]
  33. Jiang CY, Ting AT, Seed B 1998 PPAR-{gamma} agonists inhibit production of monocyte inflammatory cytokines. Nature 391:82–86[CrossRef][Medline]
  34. Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK 1998 The peroxisome proliferator-activated receptor-{gamma} is a negative regulator of macrophage activation. Nature 391:79–82[CrossRef][Medline]
  35. Diascro DDJ, Vogel RL, Johnson TE, Witherup KM, Pitzenberger SM, Rutledge SJ, Prescott DJ, Rodan GA, Schmidt A 1998 High fatty acid content in rabbit serum is responsible for the differentiation of osteoblasts into adipocyte-like cells. J Bone Miner Res 13:96–106[CrossRef][Medline]
  36. Nuttall ME, Patton AJ, Olivera DL, Nadeau DP, Gowen M 1998 Human trabecular bone cells are able to express both osteoblastic and adipocytic phenotype: implications for osteopenic disorders. J Bone Miner Res 13:371–382[CrossRef][Medline]
  37. Jilka RL, Lecka-Czernik B, Ali AA, O’Brien CE, Weinstein RS, Manolagas SC 2001 Activation of PPAR{gamma}2 by rosiglitazone causes bone loss associated with increased marrow adiposity and decreased osteoblast number in mice J Bone Miner Res 16:S319
  38. Jennermann C, Triantafillou J, Cowan D, Pennink BGA, Connolly KM, Morris DC 1995 Effects of thiazolidinediones on bone turnover in the rat. J Bone Miner Res 10:S241
  39. Okazaki R, Miura M, Toriumi M, Taguchi M, Hirota Y, Fukumoto S, Fujita T, Tanaka K, Takeuchi A 1999 Short-term treatment with troglitazone decreases bone turnover in patients with type 2 diabetes mellitus. Endocr J 46:795–801[Medline]
  40. Mandrup S, Lane MD 1997 Regulating adipogenesis. J Biol Chem 272:5367–5370[Free Full Text]
  41. Hotamisligil GS, Johnson RS, Distel RJ, Ellis R, Papaioannou VE, Spiegelman BM 1996 Uncoupling of obesity from insulin resistance through a targeted mutation in aP2, the adipocyte fatty acid binding protein. Science 274:1377–1379[Abstract/Free Full Text]
  42. Coe NR, Simpson MA, Bernlohr DA 1999 Targeted disruption of the adipocyte lipid-binding protein (aP2 protein) gene impairs fat cell lipolysis and increases cellular fatty acid levels. J Lipid Res 40:967–972[Abstract/Free Full Text]
  43. Wagner EF, Karsenty G 2001 Genetic control of skeletal development. Curr Opin Genet Dev 11:527–532[CrossRef][Medline]
  44. Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, de Crombrugghe B 2002 The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108:17–29[CrossRef][Medline]
  45. Miyama K, Yamada G, Yamamoto TS, Takagi C, Miyado K, Sakai M, Ueno N, Shibuya H 1999 A BMP-inducible gene, dlx5, regulates osteoblast differentiation and mesoderm induction. Dev Biol 208:123–133[CrossRef][Medline]
  46. Jikko A, Harris SE, Chen D, Mendrick DL, Damsky CH 1999 Collagen integrin receptors regulate early osteoblast differentiation induced by BMP-2. J Bone Miner Res 14:1075–1083[CrossRef][Medline]
  47. Schinke T, Karsenty G 1999 Characterization of Osf1, an osteoblast-specific transcription factor binding to a critical cis-acting element in the mouse osteocalcin promoters. J Biol Chem 274:30182–30189[Abstract/Free Full Text]
  48. Shirakabe K, Terasawa K, Miyama K, Shibuya H, Nishida E 2001 Regulation of the activity of the transcription factor Runx2 by two homeobox proteins, Msx2 and Dlx5. Genes Cells 6:851–856[Abstract]
  49. Miller JR 2002 The wnts. Genome Biol 3:3001.1–3001.15
  50. Gong Y, Slee RB, Fukai N, Rawadi G, Roman-Roman S, Reginato AM, Wang H, Cundy T, Glorieux FH, Lev D, Zacharin M, Oexle K, Marcelino J, Suwairi W, Heeger S, Sabatakos G, Apte S, Adkins WN, Allgrove J, Arslan-Kirchner M, Batch JA, Beighton P, Black GC, Boles RG, Boon LM, Borrone C, Brunner HG, Carle GF, Dallapiccola B, De Paepe A, Floege B, Halfhide ML, Hall B, Hennekam RC, Hirose T, Jans A, Juppner H, Kim CA, Keppler-Noreuil K, Kohlschuetter A, LaCombe D, Lambert M, Lemyre E, Letteboer T, Peltonen L, Ramesar RS, Romanengo M, Somer H, Steichen-Gersdorf E, Steinmann B, Sullivan B, Superti-Furga A, Swoboda W, van den Boogaard MJ, Van Hul W, Vikkula M, Votruba M, Zabel B, Garcia T, Baron R, Olsen BR, Warman ML 2001 LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development. Cell 107:513–523[CrossRef][Medline]
  51. Little RD, Carulli JP, Del Mastro RG, Dupuis J, Osborne M, Folz C, Manning SP, Swain PM, Zhao SC, Eustace B, Lappe MM, Spitzer L, Zweier S, Braunschweiger K, Benchekroun Y, Hu X, Adair R, Chee L, FitzGerald MG, Tulig C, Caruso A, Tzellas N, Bawa A, Franklin B, McGuire S, Nogues X, Gong G, Allen KM, Anisowicz A, Morales AJ, Lomedico PT, Recker SM, Van Eerdewegh P, Recker RR, Johnson ML 2002 A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait. Am J Hum Genet 70:11–19[CrossRef][Medline]
  52. Levasseur R, Kato M, Patel MS, Chan L, Karsenty G 2001 Low bone mass, low body weight and abnormal eye vascularization in mice deficient in LRP5, the gene mutated in human osteoporosis pseudoglioma syndrome (OPS). J Bone Miner Res 16:S152
  53. Rawadi G, Garcia T, Spinella-Jaegle S, Gallea S, Faucheu S, Kawai S, Baron R, Roman-Roman S 2001 Wnt1, 2, and 3 induce osteoblast commitment of mesenchymal pluripotent cells C3H10T1/2 by ß-catenin pathway dependent signaling J Bone Miner Res 16:S201
  54. Sheikh SS, Mbalaviele G, Warlow P, Civitelli R 2001 ß-Catenin signaling during osteoblast differentiation. J Bone Miner Res 16:S368
  55. Nolte RT, Wisely GB, Westin S, Cobb JE, Lambert MH, Kurokawa R, Rosenfeld MG, Willson TM, Glass CK, Milburn MV 1998 Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-{gamma}. Nature 395:137–143[CrossRef][Medline]
  56. Willson TM, Lambert MH, Kliewer SA 2001 Peroxisome proliferator-activated receptor {gamma} and metabolic disease. Annu Rev Biochem 70:341–367[CrossRef][Medline]
  57. Falkenstein E, Tillmann HC, Christ M, Feuring M, Wehling M 2000 Multiple actions of steroid hormones-A focus on rapid, nongenomic effects. Pharmacol Rev 52:513–556[Abstract/Free Full Text]
  58. Kousteni S, Bellido T, Plotkin LI, O’Brien CA, Bodenner DL, Han L, Han K, DiGregorio GB, Katzenellenbogen JA, Katzenellenbogen BS, Roberson PK, Weinstein RS, Jilka RL, Manolagas SC 2001 Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity. Cell 104:719–730[Medline]
  59. Jira W, Spiteller G 1999 Dramatic increase of linoleic acid peroxidation products by aging, atherosclerosis, and rheumatoid arthritis. Adv Exp Med Biol 469:479–483[Medline]
  60. Sohal RS, Weindruch R 1996 Oxidative stress, caloric restriction, and aging. Science 273:59–63[Abstract]
  61. Parhami F, Tintut Y, Beamer WG, Gharavi N, Goodman W, Demer LL 2001 Atherogenic high-fat diet reduces bone mineralization in mice. J Bone Miner Res 16:182–188[CrossRef][Medline]
  62. Parhami F, Jackson SM, Tintut Y, Le V, Balucan JP, Territo M, Demer LL 1999 Atherogenic diet and minimally oxidized low density lipoprotein inhibit osteogenic and promote adipogenic differentiation of marrow stromal cells. J Bone Miner Res 14:2067–2078[CrossRef][Medline]
  63. Tontonoz P, Hu E, Spiegelman BM 1994 Stimulation of adipogenesis in fibroblasts by PPAR{gamma}2, a lipid-activated transcription factor. Cell 79:1147–1156[CrossRef][Medline]
  64. Rangwala SM, Lazar MA 2002 The dawn of the SPPARMs? Sci STKE 121:PE9



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