RESUMO
Valproic acid (VPA) has been shown to increase the reprogramming efficiency of induced pluripotent stem cells (iPSC) from somatic cells, but the mechanism by which VPA enhances iPSC induction has not been defined. Here we demonstrated that VPA directly activated Oct4 promoter activity through activation of the PI3K/Akt/mTOR signaling pathway that targeted the proximal hormone response element (HRE, -41â¼-22) in this promoter. The activating effect of VPA is highly specific as similar compounds or constitutional isomers failed to instigate Oct4 promoter activity. We further demonstrated that the upstream 2 half-sites in this HRE were essential to the activating effect of VPA and they were targeted by a subset of nuclear receptors, such as COUP-TFII and TR2. These findings show the first time that NRs are implicated in the VPA stimulated expression of stem cell-specific factors and should invite more investigation on the cooperation between VPA and NRs on iPSC induction.
Assuntos
Fator II de Transcrição COUP/genética , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células Musculares/efeitos dos fármacos , Membro 1 do Grupo C da Subfamília 2 de Receptores Nucleares/genética , Fator 3 de Transcrição de Octâmero/genética , Ácido Valproico/farmacologia , Animais , Sequência de Bases , Fator II de Transcrição COUP/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Reprogramação Celular , Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Dados de Sequência Molecular , Células Musculares/citologia , Células Musculares/metabolismo , Membro 1 do Grupo C da Subfamília 2 de Receptores Nucleares/metabolismo , Fator 3 de Transcrição de Octâmero/agonistas , Fator 3 de Transcrição de Octâmero/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Ácido Valproico/análogos & derivadosRESUMO
Induced pluripotent stem (iPS) cells are reprogrammed from somatic cells through ectopic expression of stem cell-specific transcription factors, including Oct4, Nanog, Sox2, Lin28, Klf4, and c-Myc. Although iPS cells are similar to embryonic stem (ES) cells in their pluripotency, their inherited defects, such as insertion mutagenesis, employment of oncogenes, and low efficiency, associated with the reprogramming procedure have hindered their clinical application. A study has shown that valproic acid (VPA) treatment can significantly enhance the reprogramming efficiency and avoid the usage of oncogenes. To understand how VPA can enhance pluripotency, we stably transfected an Oct4 promoter driven luciferase reporter (Oct4-1.9k-Luc) into P19 embryonic carcinoma (EC) cells and C2C12 myoblasts and examined their response to VPA. We found that VPA could both activate Oct4 promoter and rescue its inhibition by retinoic acid (RA). In C2C12 myoblasts, VPA treatment also enhanced endogenous Oct4 expression but repressed that of MyoD. Furthermore, both RARalpha over-expression and mutation of a proximal hormone response element (HRE) blocked the activation effect of VPA on Oct4 promoter, implying that VPA may exert its activation effect through factors targeting this HRE. Taken together, these observations identify a molecular mechanism by which VPA directly regulate Oct4 expression to ensure the acquirement and maintenance of pluripotency.
Assuntos
Músculos/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Regiões Promotoras Genéticas , Ácido Valproico/farmacologia , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Imuno-Histoquímica , Fator 4 Semelhante a Kruppel , Camundongos , Músculos/citologia , Reação em Cadeia da Polimerase/métodosRESUMO
Oct4 and Nanog are two embryonic stem (ES) cell-specific transcription factors that play critical roles in the maintenance of ES cell pluripotency. In this study, we investigated the effects of Oct4 and Nanog expression on the differentiation state of myogenic cells, which is sustained by a strong positive feedback loop. Oct4 and Nanog, either independently or simultaneously, were overexpressed in C2C12 myoblasts, and the expression of myogenic lineage-specific genes and terminal differentiation was observed by RT-PCR. Overexpression of Oct4 in C2C12 cultures repressed, while exogenous Nanog did not significantly alter C2C12 terminal differentiation. The expression of Pax7 was reduced in all Oct4-overexpressing myoblasts, and we identified a major Oct4-binding site in the Pax7 promoter. Simultaneous expression of Oct4 and Nanog in myoblasts inhibited the formation of myotubes, concomitant with a reduction in the endogenous levels of hallmark myogenic markers. Furthermore, overexpression of Oct4 and Nanog induced the expression of their endogenous counterparts along with the expression of Sox2. Using mammalian two-hybrid assays, we confirmed that Oct4 functions as a transcriptional repressor whereas Nanog functions as a transcriptional activator during muscle terminal differentiation. Importantly, in nonobese diabetic (NOD) severe combined immunodeficiency (SCID) mice, the pluripotency of C2C12 cells was conferred by overexpression of Oct4 and Nanog. These results suggest that Oct4 in cooperation with Nanog strongly suppresses the myogenic differentiation program and promotes pluripotency in myoblasts.
