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Keywords:

  • oxysterol;
  • mesenchymal stem cells;
  • peroxisome proliferator-activated receptor γ;
  • Hedgehog;
  • CCAAT/enhancer-binding protein α

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Specific oxysterols have been shown to be pro-osteogenic and anti-adipogenic. However, the molecular mechanism(s) by which oxysterols inhibit adipogenic differentiation is unknown. We show that the anti-adipogenic effects of osteogenic oxysterol, 20(S)-hydroxycholesterol, are mediated through a hedgehog-dependent mechanism(s) and are associated with inhibition of PPARγ expression.

Introduction: Multipotent bone marrow stromal cells (MSCs) are common progenitors of osteoblasts and adipocytes. A reciprocal relationship between osteogenic and adipogenic differentiation may explain the increased adipocyte and decreased osteoblast formation in aging and osteoporosis. We have previously reported that specific oxysterols stimulate osteogenic differentiation of MSCs while inhibiting their adipogenic differentiation.

Materials and Methods: The M2–10B4 (M2) murine pluripotent bone MSC line was used to assess the inhibitory effects of 20(S)-hydroxycholesterol (20S) and sonic hedgehog (Shh) on peroxisome proliferator-activated receptor γ (PPARγ) and adipogenic differentiation. All results were analyzed for statistical significance using ANOVA.

Results and Conclusions: Treatment of M2 cells with the osteogenic oxysterol 20S completely inhibited adipocyte formation induced by troglitazone after 10 days. PPARγ mRNA expression assessed by RT-qPCR was significantly induced by Tro after 48 (5-fold) and 96 h (130-fold), and this induction was completely inhibited by 20S. In contrast, 20S did not inhibit PPARγ transcriptional activity in M2 cells overexpressing PPARγ and retinoid X receptor (RXR). To elucidate the molecular mechanism(s) by which 20S inhibits PPARγ expression and adipogenic differentiation, we focused on the hedgehog signaling pathway, which we previously showed to be the mediator of osteogenic responses to oxysterols. The hedgehog signaling inhibitor, cyclopamine, reversed the inhibitory effects of 20S and Shh on troglitazone-induced adipocyte formation in 10-day cultures of M2 cells by 70% and 100%, respectively, and the inhibitory effect of 20S and Shh on troglitazone-induced PPARγ expression was fully reversed at 48 h by cyclopamine. Furthermore, 20S and Shh greatly inhibited PPARγ2 promoter activity induced by CCAAT/enhancer-binding protein α overexpression. These studies show that, similar to the induction of osteogenesis, the inhibition of adipogenesis in murine MSCs by the osteogenic oxysterol, 20S, is mediated through a hedgehog-dependent mechanism(s).


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Age-related bone loss is associated with a progressive decrease in bone formation and an increase in adipogenesis in the bone marrow, increasing the risk of bone fractures.(1,2) Multipotent bone marrow stromal cells (MSCs) are common progenitors of osteoblasts and adipocytes,(3,4) and a potential reciprocal relationship between osteogenic and adipogenic differentiation of MSC has been suggested.(5–8) Furthermore, an increase in adipose tissue volume and a decrease in trabecular bone volume in bone marrow has been observed with aging and in patients with osteoporosis.(2) However, the molecular mechanisms underlying the reciprocal relationship between osteogenic and adipogenic differentiation during aging and pathological states are not well understood.

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear hormone receptor superfamily and a key regulator of adipogenic differentiation.(9,10) In early adipogenic differentiation, CCAAT/enhancer-binding protein β (C/EBPβ) and C/EBPδ induce the expression of PPARγ and C/EBPα.(11,12) PPARγ and C/EBPα regulate each other's expression through a positive feedback mechanism and induce other adipogenic genes that establish terminal adipogenic differentiation.(13) PPARγ consists of two protein isoforms produced by alternative promoter use and splicing.(14) PPARγ1 is expressed at low levels in many tissues, whereas PPARγ2 is expressed at high levels in adipose tissue.(9) The introduction of PPARγ2 into fibroblastic cells using retroviral infection stimulates adipocyte differentiation,(15) whereas PPARγ null embryonic stem (ES) cells fail to differentiate into adipocytes.(16)

Increasing evidence indicates that PPARγ plays an important role in the regulation of bone metabolism. Heterozygous PPARγ-deficient mice exhibit increased osteogenesis with enhanced osteoblastogenesis and decreased adipogenesis from bone marrow progenitors.(17) The ES cells from homozygous PPARγ-deficient mice do not form adipocytes in the presence of troglitazone, whereas the formation of von Kossa-positive bone nodules and the expression of osteogenic marker genes such as Runx2, osteocalcin, and type I collagen are greatly increased.(17) However, introduction and activation of PPARγ in PPARγ-deficient ES cells stimulates adipogenesis and inhibits osteoblastogenesis.(17) Transfection with a PPARγ2 expression construct and activation with thiazolidinediones in a murine bone marrow-derived cell line stimulates the terminal differentiation of these cells to adipocytes and simultaneously suppresses osteogenic markers, such as Runx2, α1(1) procollagen, and osteocalcin synthesis.(18) Moreover, rosiglitazone, a PPARγ ligand, causes bone loss associated with an increase in marrow adipocytes and a decrease in bone formation rate in mice.(19) A potential strategy to reduce bone loss in age-related osteoporosis may be to increase bone formation by enhancing osteogenic and inhibiting adipogenic differentiation of progenitor MSC. Thus, PPARγ could be an important target for inducing a shift in MSC differentiation from the adipogenic to osteogenic pathway.

Oxysterols are potential candidates for shifting MSC differentiation pathways. Oxysterols, a large family of 27-carbon oxygenated products of cholesterol, are present in the circulation and in human and animal tissues,(20–22) and can be formed from cholesterol by either enzymatic or nonenzymatic oxidation.(22) Oxysterols have been identified as bioactive compounds involved in various biological and pathological processes, such as cholesterol efflux, lipoprotein metabolism, calcium uptake, cell differentiation, atherosclerosis, and apoptosis.(23–27) We previously reported that specific oxysterols including 20(S)-hydroxycholesterol (20S) induce osteoblastic differentiation markers, such as alkaline phosphatase activity, osteocalcin expression, and matrix mineralization in murine M2-10B4 (M2) MSCs.(28,29) Furthermore, the osteogenic oxysterols inhibit adipocyte formation and the expression of adipogenic differentiation marker genes, such as lipoprotein lipase (LPL) and adipocyte-specific fatty acid binding protein 2 (aP2).(28) Recently, we reported that oxysterols are novel activators of the hedgehog signaling pathway,(30) which is pro-osteogenic and anti-adipogenic.(31,32) Therefore, we hypothesized that inhibitory effects of oxysterols on adipogenic differentiation of MSCs would be mediated by hedgehog signaling. Because 20S is the most potent naturally occurring osteogenic oxysterol that we have identified to date, in this study, we further investigated the molecular mechanisms by which it inhibits adipogenic differentiation of MSCs. We found that similar to sonic hedgehog (Shh), 20S inhibited PPARγ mRNA expression induced by the thiazolidinedione, troglitazone (Tro), which stimulates adipogenesis by activating PPARγ. The inhibitory effects of 20S and Shh on PPARγ expression were completely blocked by the hedgehog signaling inhibitor, cyclopamine. Furthermore, 20S and Shh significantly inhibited PPARγ promoter activity induced by C/EBPα overexpression. However, 20S did not inhibit the transcriptional activity of PPARγ. In conclusion, we showed that the inhibition of adipogenesis by 20S is mediated predominantly through a hedgehog pathway-dependent mechanism(s).

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Materials

20S was purchased from Sigma-Aldrich (St Louis, MO, USA), recombinant mouse sonic hedgehog, amino-terminal peptide from R&D Systems (Minneapolis, MN, USA), troglitazone from BioMol Research Laboratories (Plymouth Meeting, PA, USA), cyclopamine and PD98059 from Calbiochem (La Jolla, CA, USA), RPMI 1640 from Irvine Scientific (Santa Ana, CA, USA), and FBS from Hyclone (Logan, UT, USA).

Cell culture

M2 mouse MSCs were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). These cells were maintained in growth medium consisting of RPMI 1640 with 10% heat-inactivated FBS and supplemented with 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 U/ml streptomycin. Cell culture was performed in 24- and 6-well plates for adipogenic differentiation and gene expression studies, respectively, and treatment with test agents was done in growth medium.

Oil red O staining

Oil red O staining for detection of adipocytes was performed as previously described.(33) The number of adipocytes was quantitated by counting Oil red O-positive cells in five separate fields per well, in three wells per experimental condition. The results are reported as the mean of triplicate determination ± SD.

Quantitative RT-PCR

Total RNA was extracted with the RNA isolation kit from Stratagene (La Jolla, CA, USA) according to the manufacturer's instructions. RNA was DNase-treated using DNA-free kit from Ambion (Austin, TX, USA). Three micrograms of RNA was reverse-transcribed using reverse transcriptase from Stratagene to make single-stranded cDNA. The cDNA was mixed with Qi SYBR Green Supermix (Bio-Rad) for quantitative RT-PCR assay using a Bio-Rad I-cycler IQ quantitative thermocycler. All PCR samples were prepared in triplicate wells of a 96-well plate. After 40 cycles of PCR, melt curves were examined to ensure primer specificity. Fold changes in gene expression were calculated using the ΔΔCt method.(34) Primers used are as follows: PPARγ2 (5′-TGAAACTCTGGGAGATTCTCCTG-3′ and 5′-CCATGGTAATTTCTTGTGAAGTGC-3′),(31)C/EBPα (5′-GGACAAGAACAGCAACGAGTACC-3′ and 5′-GGCGGTCATTGTCACTGGTC-3′),(31)aP2 (5′-GRCACCATCCGGTCAGAGAGTAC-3′ and 5′-TCGTCTGCGGTGATTTCATC-3′),(35)LPL (5′-GTGGCCGAGAGCGAGAAC-3′ and 5′-AAGAAGGAGTAGGTTTTATTTGTGGAA-5′),(35)GATA2 (5′-ATCCACCCTTCCTCCAGTCT-3′ and 5′-CTCTCCAAGTGCATGCAAGA-3′),(32)GATA3 (5′-AGAAGGCATCCAGACCCGAAAC-3′ and 5′-ACTTGGAGACTCCTCACGCATGTG-3′),(32)GILZ (5′-GCTGCACAATTTCTCCACCT-3′ and 5′-GCTCACGAATCTGCTCCTTT-3′),(36) and pref-1 (5′-CTGTGTCAATGGAGTCTGCAAG-3′ and 5′-CTACGATCTCACAGAAGTTGC-3′).(37)

Reporter assays

M2 cells at 70% confluency in a 24-well plate were transiently transfected with: a plasmid containing three tandem repeats of the PPAR response element (PPRE) upstream of the basic thymidine kinase promoter (p3xPPRE-TK-Luciferase), a control pTK-Luciferase plasmid devoid of PPRE, a CMX-PPARγ expression plasmid (all kind gifts of Dr Peter Tontonoz), a CMX-RXRα expression plasmid (kind gift of Dr Sotirios Tetradis), and a pTK-Renilla-Luciferase plasmid (Promega, Madison, WI, USA) using Fugene 6 Transfection Reagents from Roche (Indianapolis, IN, USA). Luciferase activity assay was performed using Dual-Luciferase Reporter 1000 Assay System (Promega, Madison, WI, USA). Luciferase reporter activity was normalized to Renilla luciferase activity. Transfection efficiency was monitored by co-transfecting with a plasmid expressing green fluorescent protein and found to be >30%. For GATA reporter assays, M2 cells were transfected with a GATA luciferase reporter vector or a control reporter vector (both from Panomics, Fremont, CA, USA), and pTK-Renilla-Luciferase plasmid.

For PPARγ promoter activity assays, M2 cells were transiently transfected with a murine PPARγ2 promoter construct luciferase plasmid (p19-PPARγ2; (kind gift of Dr Steven McKnight), along with MSV-C/EBPα overexpression plasmid (kind gift of Dr Sophia Tsai) and pTK-Renilla-Luciferase plasmid. Luciferase activity was measured after 24 h and normalized for transfection efficiency using the Renilla luciferase activity.

Statistical analysis

Statistical analyses were performed using the StatView 5 program. All p values were calculated using ANOVA and Fisher's projected least significant difference (PLSD) significance test. A value of p < 0.05 was considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Effects of 20S on adipogenic differentiation and PPARγ and C/EBPα mRNA expression

Consistent with our previous report,(28) treatment of M2 cells with Tro significantly increased adipocyte formation compared with control after 10 days, and this increase was significantly inhibited in the presence of 5 μM 20S (Figs. 1A and 1B).

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Figure Figure 1. 20S inhibits adipogenic differentiation of M2 bone marrow stromal cells. (A) M2 cells were treated at confluence with control vehicle, 10 μM Tro, or 5 μM 20S alone or in combination for 10 days. Adipocyte formation was examined by Oil red O staining. (B) The number of adipocytes in A was quantitated by counting Oil red O-positive cells in five separate fields per well, in three wells per experimental condition. The results are reported as the mean of triplicate determination ± SD (p < 0.0001 for control vs. Tro and Tro vs. Tro + 20S).

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To elucidate the mechanism(s) by which osteogenic oxysterols inhibit adipogenesis, we examined the effect of 20S on PPARγ expression in M2 cells. PPARγ expression was assessed by RT-qPCR after treating M2 cells with Tro in the presence or absence of 20S. Tro caused a significant increase in PPARγ mRNA expression after 24–96 h of treatment, with the level of expression increasing in a time-dependent manner (Fig. 2). Tro did not cause a detectable significant increase in PPARγ expression significantly at earlier time-points (data not shown). Tro-induced PPARγ expression was almost completely blocked by 20S at all time-points examined (Fig. 2). Furthermore, the expression of C/EBPα mRNA, a key adipogenic gene, was also significantly increased by Tro at 24, 48, and 96 h in a time-dependent manner, but 20S did not inhibit this increase in C/EBPα expression at the time-points examined (Fig. 3). The expression of aP2, a downstream target of PPARγ, was significantly increased in Tro-treated cells, and this induction was inhibited by 20S (Fig. 4).

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Figure Figure 2. Effect of 20S on PPARγ mRNA expression induced by Tro. M2 cells at confluence were treated with control vehicle, 10 μM Tro, or 5 μM 20S, alone or in combination for 24, 48, and 96 h. PPARγ mRNA expression was measured by quantitative real-time PCR. Fold changes in gene expression to the control were calculated using the ΔΔCt method and reported as the mean of triplicate determination ± SD (p < 0.0001 for Tro vs. control and p < 0.001 for Tro vs. Tro + 20S at 24 h; p < 0.0001 for Tro vs. control and Tro vs. Tro + 20S at 48 h; p < 0.0001 for Tro vs. control and Tro versus Tro + 20S at 96 h).

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Figure Figure 3. Effect of 20S on C/EBPα mRNA expression induced by Tro. M2 cells at confluence were treated with control vehicle, 10 μM Tro, or 5 μM 20S, alone or in combination for 24, 48, and 96 h. C/EBPα mRNA expression was measured by quantitative real-time PCR. Fold changes in gene expression relative to the control were calculated using the ΔΔCt method and reported as the mean of triplicate determination ± SD (p < 0.0001 for Tro vs. control at 24 h and p < 0.001 for Tro + 20S vs. control at 24 h; p < 0.0001 for Tro vs. control and p < 0.001 for Tro + 20S vs. control at 48 h; p < 0.0001 for Tro or Tro + 20S vs. control at 96 h).

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Figure Figure 4. Effect of 20S on aP2 mRNA expression induced by Tro. M2 cells at confluence were treated with control vehicle, 10 μM Tro, or 5 μM 20S, alone or in combination for 24, 48, and 96 h. aP2 mRNA expression was measured by quantitative real-time PCR. Fold changes in gene expression relative to the control were calculated using the ΔΔCt method and reported as the mean of triplicate determination ± SD (p < 0.0001 for Tro or Tro + 20S vs. control or 20S and p < 0.01 for Tro vs. Tro + 20S at 24 h; p < 0.0001 for Tro or Tro + 20S vs. control or 20S and for Tro vs. Tro + 20S at 48 h; p < 0.0001 for Tro vs. control, 20S, or Tro + 20S and p < 0.001 for Tro + 20S vs. control or 20S at 96 h).

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Next we examined whether, in addition to inhibiting PPARγ expression, 20S also inhibits transcriptional activity of PPARγ protein. M2 cells were transiently transfected with a reporter construct containing three tandem repeats of a PPRE (pTK-3xPPRE-Luciferase) or the control plasmid (pTK-Luciferase). Cells were treated with 10 μM Tro or control vehicle, and luciferase activity was measured after 24 and 48 h. Results showed that Tro induced a small but significant increase in reporter activity (40%; Fig. 5A). Interestingly, 20S did not inhibit Tro-induced PPRE reporter activity, but instead caused a small but significant increase in the Tro-induced response (Fig. 5A). Because Tro-induced PPRE reporter activity appeared to be low in M2 cells under baseline conditions, we used a PPARγ overexpression vector to transiently transfect the cells and assessed whether Tro-induced reporter activity was increased. Indeed, we found a more substantial reporter activity in PPARγ overexpressing cells in response to Tro after 24 (Fig. 5B) and 48 h (data not shown) of treatment. Consistent with the results obtained without PPARγ overexpression, 20S did not inhibit but rather enhanced Tro-induced PPRE reporter activity in cells overexpressing PPARγ. Because PPARγ and retinoid X receptor (RXR) form obligatory heterodimers, we studied whether 20S would have an effect when both PPARγ and RXRα were overexpressed. Co-transfection of M2 cells with PPARγ and RXRα overexpression plasmids showed similar results to transfection with only PPARγ overexpression plasmid, and 20S enhanced PPARγ-RXRα induced PPRE reported activity (Fig. 5C).

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Figure Figure 5. Effect of 20S on transcriptional activity of PPARγ. (A) M2 cells at 70% confluence in a 24-well plate were transiently transfected with a PPRE reporter construct (pTK-3xPPRE-Luciferase) plasmid (PPRE-TK-LUC) or pTK-Luciferase plasmid (pTK-LUC) and pTK-Renilla-Luciferase plasmid. Luciferase activity was measured after 24 h and normalized for transfection efficiency using the Renilla luciferase activity. Data are reported as the mean of triplicate determination ± SD (p < 0.001 for control vs. Tro and Tro vs. Tro + 20S). (B) M2 cells were transiently transfected with a PPRE reporter plasmid (pTK-3xPPRE-Luciferase) (PPRE-TK-LUC) or pTK-Luciferase plasmid (pTK-LUC), along with CMX-PPARγ expression plasmid, and pTK-Renilla-Luciferase plasmid. Luciferase activity was normalized for transfection efficiency using the Renilla luciferase activity. Data are reported as the mean of triplicate determination ± SD (p < 0.0001 for control vs. Tro and Tro vs. Tro + 20S). (C) M2 cells were transiently transfected as described in B along with CMX- RXRα expression plasmid. Luciferase activity was measured after 24 h and normalized to the Renilla luciferase activity. Data are reported as the mean of triplicate determination ± SD (p < 0.0001 for control vs. Tro and Tro vs. Tro + 20S).

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Role of Hedgehog signaling in anti-adipogenic effects of 20S

We previously found that osteogenic oxysterols stimulate osteoblastic differentiation of M2 cells by inducing hedgehog pathway activity,(30,38,39) and activation of hedgehog pathway is pro-osteogenic and anti-adipogenic.(31,32) We evaluated whether the anti-adipogenic effects of 20S are mediated through the hedgehog signaling pathway by assessing the effect of hedgehog pathway inhibitor, cyclopamine, on the anti-adipogenic effects of 20S oxysterol. Consistent with previous results, Oil red O staining showed that Tro treatment greatly increased the number of adipocytes compared with control, 20S significantly inhibited adipocyte formation induced by Tro, and pretreatment with cyclopamine (4 μM) reversed the anti-adipogenic effects of 20S (Fig. 6A). Shh also significantly inhibited Tro-induced adipocyte formation, and cyclopamine pretreatment completely reversed the anti-adipogenic effects of Shh (Fig. 6B). Cyclopamine also reversed the inhibitory effects of 20S on the expression of adipogenic differentiation marker genes, LPL and aP2 (data not shown).

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Figure Figure 6. Hedgehog pathway inhibitor, cyclopamine, blocks inhibitory effects of 20S and Shh on Tro-induced adipogenic differentiation and PPARγ mRNA expression, and 20S and Shh inhibit the PPARγ promoter activity induced by C/EBPα overexpression. (A and B) M2 cells at confluence were treated with control vehicle (control), 10 μM Tro, or a combination of Tro and 5 μM 20S or 200 ng/ml Shh, with or without a 2-h pretreatment with control vehicle (VEH) or 4 μM cyclopamine (CYC). After 10 days, adipocyte formation was measured by Oil red O staining. The number of adipocytes was determined by counting Oil red O-positive cells in five separate fields per well, in three wells per experimental condition. The results are reported as the mean of triplicate determination ± SD (A: p < 0.0001 for control, Tro + 20S, or 20S vs. Tro, Tro + Cyclopamine, or Tro + 20S + Cyclopamine and Tro + 20S vs. Tro, Tro + Cyclopamine, or Tro + 20S + Cyclopamine, and Tro + Cyclopamine vs. Tro + 20S + Cyclopamine; B: p < 0.0001 for control, Tro + 20S, or 20S vs. Tro, Tro + Cyclopamine, or Tro + 20S + Cyclopamine and Tro + 20S vs. Tro, Tro + Cyclopamine, or Tro + 20S + Cyclopamine). (C and D) M2 cells at confluence were treated with control vehicle (control), 10 μM Tro, or 5 μM 20S, alone or in combination, with or without a 2-h pretreatment with control vehicle (VEH) or 4 μM cyclopamine (CYC). After 48 h, PPARγ mRNA expression was measured by quantitative real-time PCR. Fold changes in gene expression relative to the control were calculated using the ΔΔCt method and reported as the mean of triplicate determination ± SD (C: p < 0.0001 for control vs. Tro + Cyclopamine and Tro + 20S + Cyclopamine, Tro vs. Tro + 20S, and Tro + 20S vs. Tro + 20S + Cyclopamine; p < 0.001 for Tro vs. Tro + Cyclopamine; D: p < 0.0001 for control or Tro + Shh vs. Tro, Tro + Cyclopamine, or Tro + Shh + Cyclopamine; p < 0.001 for Tro vs. control or Tro + Shh). (E) M2 cells were transiently transfected with a murine PPARγ2 promoter construct luciferase plasmid (p19-PPARγ2), alone (No Vector) or with MSV-C/EBPα overexpression plasmid (C/EBP-Alpha) and pTK-Renilla-Luciferase plasmid. Luciferase activity was measured after 24 h and normalized for transfection efficiency using the Renilla luciferase activity. Data are reported as the mean of triplicate determination ± SD (p < 0.001 for control vs. control + C/EBPα and for control + C/EBPα vs. 20S + C/EBPα or Shh + C/EBPα).

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To determine if cyclopamine's effect on the anti-adipogenic actions of 20S and Shh is produced at the level of PPARγ expression, we evaluated the effects of cyclopamine on PPARγ mRNA expression by RT-qPCR after 48 h of treatment with Tro. Consistent with earlier results, Tro caused a significant increase in PPARγ expression, which was blocked by 20S and Shh, and pretreatment with cyclopamine completely abolished the inhibitory effect of 20S and Shh (Figs. 6C and 6D).

To determine if 20S and Shh inhibit PPARγ expression by acting directly on its promoter, we focused on C/EBPα-regulated PPARγ promoter activity. PPARγ promoter activity assays using a murine PPARγ2 promoter construct luciferase plasmid (p19-PPARγ2) containing 2× C/EBPα binding sites, transfected into M2 cells along with MSV-C/EBPα overexpression plasmid, showed that C/EBPα overexpression stimulated PPARγ2 promoter activity 6-fold, which was inhibited by both 20S and Shh (Fig. 6E).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This study showed that the inhibition of adipogenesis by the osteogenic oxysterol 20S is associated with the inhibition of PPARγ mRNA expression in MSCs. Because in this study, adipogenic differentiation was induced with Tro, a ligand for PPARγ protein, and given the fact that PPARγ does not seem to induce its own expression,(13) it is likely that the positive feedback loop between C/EBPα and PPARγ regulates the induction of key adipogenic genes and adipocyte formation. 20S specifically inhibited PPARγ expression, but not C/EBPα expression, in early adipogenic differentiation induced by Tro. Given that 20S did not inhibit C/EBPα expression, we examined whether inhibition was the level of C/EBPα controlled PPARγ promoter activity. Indeed, the PPARγ2 promoter activity assays showed that 20S and Shh inhibit C/EBPα-induced PPARγ promoter activity, suggesting that inhibition of PPARγ expression may be at the level of PPARγ promoter. The molecular mechanism for this inhibition remains to be elucidated; however, it may involve 20S- and Shh-induced regulation of co-activators and/or co-repressors that mediate PPARγ promoter activity. One limitation of this study is that we did not show the effects of 20S and Shh on PPARγ protein levels directly, although their inhibitory effect on PPARγ target gene expression suggests a potentially similar inhibitory effect on PPARγ protein expression.

Our results also showed that the inhibitory effects of 20S on adipogenesis were mediated by hedgehog signaling, which is also involved in mediating the osteogenic effects of 20S.(30,38,39) This finding is consistent with previous reports that showed the role of this signaling pathway in the regulation of osteogenic and adipogenic differentiation of progenitor cells.(31,32) Cyclopamine significantly blocked the inhibition of adipocyte formation and PPARγ mRNA expression by 20S, which suggests that activation of the hedgehog signaling pathway is the prominent anti-adipogenic mechanism by which 20S regulates adipogenic, as well as osteogenic, differentiation of MSCs. Consistent with our findings, Shh has been shown to inhibit adipogenesis and the expression of PPARγ in C3H10T1/2 embryonic fibroblasts.(31) In addition, Shh was reported to inhibit adipogenic differentiation and expression of adipogenic genes in 3T3-L1 pre-adipocytes.(32) Furthermore, a dominant-negative form of Gli (the transcription factor that mediates hedgehog-regulated gene expression) and cyclopamine were shown to inhibit hedgehog signaling while stimulating adipogenic differentiation in 3T3-L1 cells.(32) One mechanism by which 20S exerts its anti-adipogenic effects through hedgehog signaling may involve the induction of anti-adipogenic transcription factors, such as Gilz, GATAs, and pref-1. Shh was shown to increase the levels of Gilz, GATA2, GATA3, or pref-1 in mouse NIH-3T3 fibroblasts, C3H10T1/2 pluripotent mesenchymal cells, and 3T3-L1 pre-adipocytes.(32,40) GATA-2 and GATA-3 inhibit PPARγ expression and adipogenic differentiation through direct binding to the PPARγ promoter, as well as by physically interacting with C/EBPα.(41,42) Gilz and pref-1 also inhibit adipogenic differentiation and keep 3T3-L1 as pre-adipocytes.(32,36) We examined the effects of 20S on GATA2, GATA3, Gilz, and pref-1 expression in M2 cells using RT-qPCR and on GATA transcriptional activity using a GATA reporter transfected into M2 cells. Results showed that 20S does not increase GATA2/GATA3, Gilz, and pref-1 gene expression nor GATA transcriptional activity assessed after 24 and 48 h of treatments (data not shown). Further study is needed to more fully define the molecular mechanism(s) by which 20S inhibits PPARγ expression in a hedgehog signaling-dependent manner. Although 4 μM cyclopamine treatment fully reversed the inhibitory effect of 20S on PPARγ mRNA expression at 48 h, it did not completely reverse 20S effects on adipocyte formation as measured by Oil red O staining at late stages of cell differentiation, and increasing the dose of cyclopamine from 4 to 8 μM did not have any additional effect on adipocyte formation (data not shown). However, the inhibitory effects of Shh on PPARγ mRNA expression as well as adipocyte formation were completely reversed by 4 μM cyclopamine. This suggests that, in addition to hedgehog signaling, other anti-adipogenic mediators may contribute to the complete inhibition of adipogenesis by 20S in MSCs.

Unlike Shh, 20S activates not only the hedgehog pathway but also liver X receptor (LXR) signaling (Fig. 7).(43) Oxysterols are ligands for LXRs, which regulate cholesterol, lipid, and carbohydrate metabolism.(44–46) LXR activation increases the expression of sterol regulatory element binding protein-1c (SREBP-1c)/adipogenic differentiation of factor 1 (ADD1), which induces the expression of fatty acid synthase, glycerol-3-phosphate acyltransferase, and stearyl CoA desaturase 2 during adipogenic differentiation.(47–49) It has been shown that activation of LXRs increases lipid accumulation during adipogenic differentiation of 3T3-L1 and 3T3-F422A pre-adipocytes.(50,51) LXRs and PPARγ seem to positively regulate each other's expression. The expression of LXRα is increased directly by PPARγ activation in 3T3-L1 pre-adipocytes and in a mouse model.(50) Furthermore, PPARγ promoter contains the conserved binding site for LXR, and LXR activation increases PPARγ expression.(51) In addition, SREBP1/ADD1 regulates adipogenesis through PPARγ gene expression through E-box motifs in the PPARγ promoter(52) and through the production of an endogenous PPARγ ligand(s) to increase PPARγ transcriptional activity.(53) In this study, we examined whether 20S, in addition to inhibiting PPARγ mRNA expression, also inhibits PPARγ transcriptional activity. We found that troglitazone-induced PPARγ activity was not inhibited but rather enhanced by 20S. Given the positive interactions between LXR and PPARγ in the context of adipogenesis, one potential explanation for this enhancement is that LXR activation by 20S causes further stimulation of troglitazone-induced PPARγ transcriptional activity. Despite the activation of LXRs by 20S in M2 cells (data not shown), the activation of hedgehog signaling by 20S inhibited PPARγ mRNA expression and adipogenic differentiation of these cells, suggesting that this level of hedgehog pathway activation is capable of counteracting any LXR-mediated pro-adipogenic effects of 20S. Further study is needed to elucidate the presently unclear role of LXRs in regulating adipogenesis in bone MSCs.

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Figure Figure 7. Regulation of adipogenic differentiation of bone MSCs by 20S. 20S activates LXRs and Hh signaling pathways. LXR activation increases the expression of SREBP-1c/ADD1. LXRs and PPARγ positively regulate each other's expression. Moreover, SREBP-1c/ADD1 regulates adipogeneis through PPPAγ gene expression and through the production of a endogenouse PPARγ ligand(s). Despite activation of LXRs by 20S oxysterol, the activation of Hh signaling induced by 20S inhibits PPARγ expression and adipogenic differentiation of M2 cells.

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Altogether, this study suggests that specific oxysterols with pro-osteogenic and anti-adipogenic properties may serve as regulators of a shift in differentiation of MSCs into osteoblasts and away from adipocytes. It is intriguing to speculate that such oxysterols may be among the endogenous signals that regulate the lineage-specific differentiation of MSC under physiological and/or pathological conditions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The authors thank Jennifer Dwyer for technical assistance; Drs Sotirios Tetradis and Peter Tontonoz for helpful suggestions, RXRα and PPARγ expression vectors, and PPARγ reporter construct plasmid; and Drs Sophia Tsai and Steven McKnight for providing a murine PPARγ2 promoter construct luciferase plasmid and MSV-C/EBPα overexpression plasmid, respectively. This work was supported by NIAMS/NIH Grant RO1AR050426.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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