RESUMO
Peroxisome proliferator-activated receptor (PPAR) gamma was shown to be required for adipocyte formation both in vivo and in vitro. However, the role of PPARgamma in the initial steps of adipose cell development was not distinguished from its role in the terminal steps. We now show that PPARgamma is expressed early in embryoid bodies (EBs) derived from embryonic stem cells and in E.8.5 mouse embryos. Addition of a specific ligand for PPARgamma in developing EBs over-expressing PPARgamma did not commit stem cells towards the adipose lineage. In differentiated PPARgamma(-/-) EBs, only markers characteristic of preadipocytes were found to be expressed. PPARdelta is present in EBs but did not compensate for the lack of PPARgamma in terminal differentiation. Taken together, these results favor a critical PPARgamma-independent phase culminating in preadipocyte formation that precedes a PPARgamma-dependent phase in the development of adipose cells from pluripotent stem cells.
Assuntos
Adipócitos/citologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Células-Tronco/citologia , Tiazolidinedionas , Fatores de Transcrição/metabolismo , Adipócitos/metabolismo , Animais , Biomarcadores , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Expressão Gênica , Proteína HMGA2/genética , Lipase Lipoproteica/genética , Camundongos , Camundongos Endogâmicos C57BL , Receptores Citoplasmáticos e Nucleares/genética , Rosiglitazona , Células-Tronco/metabolismo , Tiazóis/farmacologia , Fatores de Transcrição/genética , Tretinoína/metabolismo , Tretinoína/farmacologiaRESUMO
The essential role of CCAAT/enhancer binding proteins (C/EBPs) beta and delta for adipocyte differentiation has been clearly established. In preadipocytes, their expression is up-regulated by the activation of leukemia inhibitory factor receptor (LIF-R) and prostacyclin receptor (IP-R) via the extracellular signal-regulated kinase (ERK) pathway and cAMP production, respectively. However, the molecular mechanisms by which LIF and prostacyclin-induced signals are propagated to the nucleus and the transcription factors mediating ERK and cAMP-induced C/EBP gene expression were unknown. Here we report that both pathways share cAMP responsive element binding protein/activation transcription factor 1 (CREB/ATF-1) as common downstream effectors. LIF-R and IP-R activation induced binding of CREB and/or ATF-1 to C/EBP promoters and CREB-dependent transcription. Expression of dominant negative forms of CREB dramatically reduced the LIF- and prostacyclin-stimulated C/EBP beta and C/EBP delta expression. Upon stimulation of the IP-R, the ERK pathway was activated in a PKA-dependent manner. ERK activation by the PKA pathway was not required for CREB/ATF-1 phosphorylation but rather was necessary for CREB-dependent up-regulation of C/EBPs expression. Our findings suggest that ERK activation is required for CREB transcriptional activity, possibly by recruitment of a coactivator.
