MicroRNA-194 reciprocally stimulates osteogenesis and inhibits adipogenesis via regulating COUP-TFII expression.

B-C Jeong,Y-C Hwang,I-H Kang,J-T Koh,S-H Kim

doi:10.1038/cddis.2014.485

Abstract

Osteoblasts and adipocytes are differentiated from common mesenchymal stem cells (MSCs) in processes which are tightly controlled by various growth factors, signaling molecules, transcriptional factors and microRNAs. Recently, chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) was identified as a critical regulator of MSC fate. In the present study, we aimed to identify some microRNAs (miR), which target COUP-TFII, and to determine the effects on MSCs fate. During osteoblastic or adipocytic differentiation from MSCs lineage cells, miR-194 expression was found to be reversal. In the cultures of mesenchymal C3H10T1/2 and primary bone marrow stromal cells, osteogenic stimuli increased miR-194 expression with accompanying decreases in COUP-TFII expression, whereas adipogenic stimuli reduced miR-194 expression with accompanying increases in COUP-TFII expression. A luciferase assay with COUP-TFII 3′-untranslated region (UTR) reporter plasmid, including the miR-194 binding sequences, showed that the introduction of miR-194 reduced the luciferase activity. However, it did not affect the activity of mutated COUP-TFII 3′-UTR reporter. Enforced expression of miR-194 significantly enhanced osteoblast differentiation, but inhibited adipocyte differentiation by decreasing COUP-TFII mRNA and protein levels. In contrast, inhibition of the endogenous miR-194 reduced matrix mineralization in the MSCs cultures, promoting the formation of lipid droplets by rescuing COUP-TFII expression. Furthermore, overexpression of COUP-TFII reversed the effects of miR-194 on the cell fates. Taken together, our results showed that miR-194 acts as a critical regulator of COUP-TFII, and can determinate the fate of MSCs to differentiate into osteoblasts and adipocytes. This suggests that miR-194 and COUP-TFII may be good target molecules for controlling bone and metabolic diseases.

Highlights

General bone homeostasis relies on the inverse relation between osteogenesis and adipogenesis of mesenchymal stem cell (MSC)
Expression of miR-194 was differentially regulated during osteoblast and adipocyte differentiation
To determine if the miRNAs expression is modulated with COUP-TFII expression during osteogenesis and adipogenesis, mesenchymal C3H10T1/2 cells were cultured in osteogenic medium (OM) or adipogenic medium (AM) for 3 days, after which qRT-PCR and Western blotting were performed

Summary

Introduction

General bone homeostasis relies on the inverse relation between osteogenesis and adipogenesis of MSCs. Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII; NR2F2), a member of the orphan nuclear receptor superfamily, modulates mesenchymal cell commitment and differentiation to decide whether cells take part in osteogenesis, adipogenesis and chondrogenesis.[10,11] COUP-TFII activates PPARγ expression and simultaneously inhibits Runx[2] activity, promoting the entry of mesenchymal progenitors to the adipocyte lineage, while impeding progression to alternative lineage pathways.[10] ablation of COUP-TFII was found to increase bone formation and osteoblast differentiation, as well as reduce fat tissue and insulin sensitivity.[10] Haploinsufficiency of COUP-TFII displayed impairment of white adipose tissue development and reduced body fat mass.[11] We previously demonstrated that COUP-TFII inhibited bone morphogenetic protein 2 (BMP2)-mediated osteoblast differentiation in mesenchymal C3H10T1/2 cells through the suppression of Runx[2] activity.[12] In addition, we observed that BMP2 reduced the cellular levels of COUP-TFII protein with osteoblast differentiation. The specific miRNAs for targeting COUP-TFII and subsequently controlling the differentiation of pluripotent mesenchymal cells have not yet been fully clarified

Objectives

Methods

Results

Conclusion