Kim Woo-Kyun, Meliton Vicente, Amantea Christopher M, Hahn Theodore J, Parhami Farhad
Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
J Bone Miner Res. 2007 Nov;22(11):1711-9. doi: 10.1359/jbmr.070710.
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 PPARgamma expression.
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.
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 gamma (PPARgamma) and adipogenic differentiation. All results were analyzed for statistical significance using ANOVA.
Treatment of M2 cells with the osteogenic oxysterol 20S completely inhibited adipocyte formation induced by troglitazone after 10 days. PPARgamma 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 PPARgamma transcriptional activity in M2 cells overexpressing PPARgamma and retinoid X receptor (RXR). To elucidate the molecular mechanism(s) by which 20S inhibits PPARgamma 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 PPARgamma expression was fully reversed at 48 h by cyclopamine. Furthermore, 20S and Shh greatly inhibited PPARgamma2 promoter activity induced by CCAAT/enhancer-binding protein alpha 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).
特定的氧化甾醇已被证明具有促骨生成和抗脂肪生成作用。然而，氧化甾醇抑制脂肪生成分化的分子机制尚不清楚。我们发现，成骨氧化甾醇20(S)-羟基胆固醇的抗脂肪生成作用是通过一种依赖于刺猬信号通路的机制介导的，并且与PPARγ表达的抑制有关。
多能骨髓基质细胞(MSCs)是成骨细胞和脂肪细胞的共同祖细胞。成骨分化和脂肪生成分化之间的相互关系可能解释了衰老和骨质疏松症中脂肪细胞增加和成骨细胞减少的现象。我们之前报道过，特定的氧化甾醇可刺激MSCs的成骨分化，同时抑制其脂肪生成分化。
使用M2-10B4 (M2)小鼠多能骨髓MSC系评估20(S)-羟基胆固醇(20S)和音猬因子(Shh)对过氧化物酶体增殖物激活受体γ (PPARγ)和脂肪生成分化的抑制作用。所有结果均采用方差分析进行统计学显著性分析。
用成骨氧化甾醇20S处理M2细胞10天后，完全抑制了曲格列酮诱导的脂肪细胞形成。RT-qPCR检测显示，曲格列酮在48小时(5倍)和96小时(130倍)后显著诱导PPARγ mRNA表达，而20S完全抑制了这种诱导。相反，20S并未抑制过表达PPARγ和视黄酸X受体(RXR)的M2细胞中PPARγ的转录活性。为了阐明20S抑制PPARγ表达和脂肪生成分化的分子机制，我们聚焦于刺猬信号通路，我们之前证明该通路是氧化甾醇成骨反应的介导者。刺猬信号通路抑制剂环杷明分别逆转了20S和Shh对M2细胞10天培养中曲格列酮诱导的脂肪细胞形成的抑制作用，分别为70%和100%，并且环杷明在48小时时完全逆转了20S和Shh对曲格列酮诱导的PPARγ表达的抑制作用。此外，20S和Shh极大地抑制了CCAAT/增强子结合蛋白α过表达诱导的PPARγ2启动子活性。这些研究表明，与成骨诱导相似，成骨氧化甾醇20S对小鼠MSCs脂肪生成的抑制作用是通过一种依赖于刺猬信号通路的机制介导的。
