20(S)-Hydroxycholesterol Inhibits PPARγ Expression and Adipogenic Differentiation of Bone Marrow Stromal Cells Through a Hedgehog-Dependent Mechanism

Abstract

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 hedgehogdependent mechanism(s) and are associated with inhibition of PPAR␥ 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 proliferatoractivated receptor ␥ (PPAR␥) 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. PPAR␥ 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 PPAR␥ transcriptional activity in M2 cells overexpressing PPAR␥ and retinoid X receptor (RXR). To elucidate the molecular mechanism(s) by which 20S inhibits PPAR␥ 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 PPAR␥ expression was fully reversed at 48 h by cyclopamine. Furthermore, 20S and Shh greatly inhibited PPAR␥2 promoter activity induced by CCAAT/enhancer-binding protein ␣ 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).

De Novo Synthesis of Steroids and Oxysterols in Adipocytes

“…This suggests that, in addition to LXR signaling, other adipogenic mediators may contribute to the inhibition of adipogenesis by 27HC observed in our study. Indeed, the previously reported anti-adipogenic activity of the oxysterol 20(S)-hydroxycholesterol is mediated predominantly through hedgehog signaling, despite the activation of LXRs at the same time, and is associated with inhibition of peroxisome proliferatoractivated receptor ␥ levels (64). Another mechanism for 27HC action in adipocytes may involve interaction with ERs.…”

Lipokines and oxysterols: Novel adipose-derived lipid hormones linking adipose dysfunction and insulin resistance

“…Moreover, oxLDL and 7κ-C were also reported to impair (pre) adipocyte differentiation and induce IR in mature adipocytes [104,117,118]. On the other hand, as previously mentioned, oxysterols can also behave as antiadipogenic signals by diverting pluripotent mesenchymal cells′ fate away from adipogenic and in favor of the osteogenic lineage through transactivation of Wntmediated signaling pathway [90,104,119].…”

“…In human marrow stromal cells, specific products of cholesterol oxidation, namely the oxysterols (see below), have also importantly been implicated as endogenous signals modulating lineage-specific differentiation in favor of osteogenesis and against adipogenic differentiation [90,91]. Even more, the oxysterol-associated antiadipogenic actions were consistently linked with selective activation of Wnt target genes [92].…”

Frequent Deregulations in the Hedgehog Signaling Network and Cross-Talks with the Epidermal Growth Factor Receptor Pathway Involved in Cancer Progression and Targeted Therapies

“…Moreover, the SHH protein can cause changes in endosteal hematopoietic stem cell niche, including osteoblasts, and thereby alter early lymphoid differentiation (Kiuru et al, 2009). It has also been shown that SHH protein or oxysterols, certain naturally occurring oxygenated derivatives of cholesterol, including 20(S)-hydroxycholesterol, can induce the antiadipogenic and osteogenic effects on multipotent BM-stromal cells by activating the canonical Hh pathway through SMO signaling element (Kim et al, 2007a(Kim et al, , 2010Amantea et al, 2008). In fact, the osteogenic effect induced through the stimulation of the Hh cascade seems to be mediated, at CRITICAL ROLES OF HEDGEHOG AND EGFR least in part, via the enhanced expression of gene products associated with the Notch (HES-1, HEY-1, and HEY-2) and Wingless (Dkk-1) pathways (Amantea et al, 2008;Kim et al, 2010).…”