Abstract
The prostaglandin (PG) receptors EP4 and FP have the potential to exert negative effects on adipogenesis, but the exact contribution of endogenous PG-driven receptor signaling to this process is not fully understood. In this study, we employed an adipocyte differentiation system from mouse embryonic fibroblasts (MEF) and compared the effects of each PG receptor-deficiency on adipocyte differentiation. In wild-type (WT) MEF cells, inhibition of endogenous PG synthesis by indomethacin augmented the differentiation, whereas exogenous PGE₂, as well as an FP agonist, reversed the effect of indomethacin. In EP4-deficient cells, basal differentiation was upregulated to the levels in indomethacin-treated WT cells, and indomethacin did not further enhance differentiation. Differentiation in FP-deficient cells was equivalent to WT and was still sensitive to indomethacin. PGE₂ or indomethacin treatment of WT MEF cells for the first two days was enough to suppress or enhance transcription of the Pparg2 gene as well as the subsequent differentiation, respectively. Differentiation stimuli induced COX-2 gene and protein expression, as well as PGE₂ production, in WT MEF cells. These results suggest that PGE₂-EP4 signaling suppresses adipocyte differentiation by affecting Pparg2 expression in an autocrine manner and that FP-mediated inhibition is not directly involved in adipocyte differentiation in the MEF system.
Highlights
The prostaglandin (PG) receptors EP4 and prostaglandin F receptor (FP) have the potential to exert negative effects on adipogenesis, but the exact contribution of endogenous PG-driven receptor signaling to this process is not fully understood
When we examined gene expression of PPAR␥, a transcription factor playing a central role in adipocyte differentiation, its induction was observed upon differentiation-inducing cocktail (DIC) treatment, and such gene expression was augmented by indomethacin (Fig. 1E)
Both PGI2 and PGE2, the two PGs predominantly synthesized by fat cells, appear to have opposing effects on early adipogenesis
Summary
Specific-pathogen-free C57BL/6 mice were obtained from Japan SLC (Hamamatsu, Japan). Mice were maintained on a 12-h light, 12-h dark cycle under specific-pathogen-free conditions. MEF cell culture, adipocyte differentiation, and triglyceride content measurement. Total RNA was isolated from MEF cells on the indicated days of the differentiation program with the RNeasy mini kit (QIAGEN, Venlo, Netherlands), subjected to the RT reaction with a Superscript II First-strand Synthesis Kit, and subjected to real time PCR with a LightCycler (Roche Applied Science, Penzberg, Germany) using Fast Start DNA Master SYBR Green I as reported previously [19]. Immunoblot analysis MEF cells grown in a 100 mm dish were harvested at the indicated h of the differentiation program in SDS sample buffer and sonicated. In both cell groups, TG was undetected on day 2, slight but significant levels of TG were detected on day 4, and levels drastically increased on days 6 and 8.
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