RESUMO
Evidence indicates that the balance between osteoblastogenesis and adipogenesis of mesenchymal stem cells (MSCs) is regulated by several hormones, growth factors, and their downstream signaling cascades. Previous studies suggest that retinoic acid (RA) plays a role in osteoblastogenesis and adipogenesis. However, it is unknown whether RA regulates commitment of MSCs into osteoblasts and adipocytes. In this study, we investigated the role of RA in differentiation of MSCs using the C3H10T1/2 cell line. RA stimulated activity and expression of alkaline phosphatase (ALP) and upregulated activity of the ALP gene promoter. The effects of RA were further enhanced by bone morphogenetic protein 2 (BMP2) and resultant Smad signaling. Furthermore, overexpression of Runx2 and Msx2, critical transcription factors for bone formation and BMP2-dependent osteoblastogenesis, enhanced RA-dependent ALP activity. In view of these findings, RA likely stimulates osteoblast differentiation through the BMP2-Smad-Runx2/Msx2 pathway. In contrast, RA markedly inhibited BMP2-induced adipocyte differentiation, suppressing expression of peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer-binding protein (C/EBP)α and C/EBPδ, and inhibiting adipogenic function of C/EBPß, C/EBPδ, and PPARγ. In conclusion, our data suggest that RA regulates commitment of MSCs into osteoblasts and adipocytes by controlling transcriptional regulators.
Assuntos
Adipócitos/metabolismo , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Tretinoína/farmacologia , Adipócitos/citologia , Fosfatase Alcalina/biossíntese , Animais , Proteína Morfogenética Óssea 2/biossíntese , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Proteínas de Homeodomínio/biossíntese , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Osteoblastos/citologia , PPAR gama/metabolismo , Proteínas Smad/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Although both osteoblasts and adipocytes have a common origin, i.e., mesenchymal cells, the molecular mechanisms that define the direction of two different lineages are presently unknown. In this study, we investigated the role of a transcription factor, CCAAT/enhancer binding protein beta (C/EBPbeta), and its isoform in the regulation of balance between osteoblast and adipocyte differentiation. We found that C/EBPbeta, which is induced along with osteoblast differentiation, promotes the differentiation of mesenchymal cells into an osteoblast lineage in cooperation with Runx2, an essential transcription factor for osteogenesis. Surprisingly, an isoform of C/EBPbeta, liver-enriched inhibitory protein (LIP), which lacks the transcriptional activation domain, stimulates transcriptional activity and the osteogenic action of Runx2, although LIP inhibits adipogenesis in a dominant-negative fashion. Furthermore, LIP physically associates with Runx2 and binds to the C/EBP binding element present in the osteocalcin gene promoter. These data indicate that LIP functions as a coactivator for Runx2 and preferentially promotes the osteoblast differentiation of mesenchymal cells. Thus, identification of a novel role of the C/EBPbeta isoform provides insight into the molecular basis of the regulation of osteoblast and adipocyte commitment.
Assuntos
Adipócitos/citologia , Proteína beta Intensificadora de Ligação a CCAAT/fisiologia , Células-Tronco Mesenquimais/fisiologia , Proteínas de Neoplasias/fisiologia , Osteoblastos/citologia , Fatores de Transcrição/fisiologia , Adipócitos/fisiologia , Animais , Proteína beta Intensificadora de Ligação a CCAAT/análise , Proteína beta Intensificadora de Ligação a CCAAT/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core , Expressão Gênica , Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/metabolismo , Camundongos , Proteínas de Neoplasias/metabolismo , Osteoblastos/química , Osteoblastos/fisiologia , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Deleção de Sequência/genética , Fatores de Transcrição/metabolismoRESUMO
Mice deficient in the Msx2 gene manifest defects in skull ossification and a marked reduction in bone formation associated with decreases in osteoblast numbers, thus suggesting that Msx2 is involved in bone formation. However, the precise role of Msx2 during osteoblast differentiation is not fully understood. In the present study, we investigated the role of Msx2 in the regulation of osteoblast differentiation in the multipotent mesenchymal cell lines C3H10T1/2 and C2C12 and in murine primary osteoblasts. Introduction of Msx2 induced alkaline phosphatase activity in C3H10T1/2 and C2C12 cells and promoted the calcification of murine primary osteoblasts. This effect of Msx2 was also observed in mesenchymal cells isolated from Runx2-deficient mice. Interestingly the expression of Msx2 was induced by bone morphogenetic protein 2 treatment in Runx2-deficient mesenchymal cells. In contrast, Msx2 diminished peroxisome proliferator-activated receptor gamma (PPARgamma) expression and adipogenesis of the preadipocytic cell line 3T3-F442A. Moreover Msx2 inhibited the transcriptional activity of PPARgamma, CCAAT/enhancer-binding protein beta (C/EBPbeta), and C/EBPdelta and blocked adipocyte differentiation of mesenchymal cells induced by overexpression of PPARgamma, C/EBPalpha, C/EBPbeta, or C/EBPdelta. These data indicate that Msx2 promotes osteoblast differentiation independently of Runx2 and negatively regulates adipocyte differentiation through inhibition of PPARgamma and the C/EBP family.
