RESUMO
LIM homeobox transcription factor Lhx2 is known to be an important regulator of neuronal development, homeostasis of hair follicle stem cells, and self-renewal of hematopoietic stem cells; however, its function in skeletal muscle development is poorly understood. In this study, we found that overexpression of Lhx2 completely inhibits the myotube-forming capacity of C2C12 cells and primary myoblasts. The muscle dedifferentiation factors Msx1 and Msx2 were strongly induced by the Lhx2 overexpression. Short interfering RNA-mediated knockdown of Lhx2 in the developing limb buds of mouse embryos resulted in a reduction in Msx1 and Msx2 mRNA levels, suggesting that they are downstream target genes of Lhx2. We found two Lhx2 consensus-binding sites in the -2097 to -1189 genomic region of Msx1 and two additional sites in the -536 to +73 genomic region of Msx2. These sequences were shown by luciferase reporter assay to be essential for Lhx2-mediated transcriptional activation. Moreover, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that Lhx2 is present in chromatin DNA complexes bound to the enhancer regions of the Msx1 and Msx2 genes. These data demonstrate that Msx1 and Msx2 are direct transcriptional targets of Lhx2. In addition, overexpression of Lhx2 significantly enhanced the mRNA levels of bone morphogenetic protein 4 and transforming growth factor beta family genes. We propose that Lhx2 is involved in the early stage of skeletal muscle development by inducing multiple differentiation inhibitory factors.
Assuntos
Proteínas de Homeodomínio/genética , Proteínas com Homeodomínio LIM/metabolismo , Fator de Transcrição MSX1/genética , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/citologia , Mioblastos/citologia , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Sítios de Ligação/genética , Proteína Morfogenética Óssea 4/biossíntese , Proteína Morfogenética Óssea 4/genética , Diferenciação Celular/genética , Linhagem Celular , Ensaio de Desvio de Mobilidade Eletroforética , Elementos Facilitadores Genéticos/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Proteínas de Homeodomínio/biossíntese , Humanos , Proteínas com Homeodomínio LIM/genética , Fator de Transcrição MSX1/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno , Fatores de Transcrição/genética , Transcrição Gênica , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genética , Regulação para CimaRESUMO
Adult skeletal muscle maintenance and regeneration depend on efficient muscle stem cell (MuSC) functions. The mechanisms coordinating cell cycle with activation, renewal, and differentiation of MuSCs remain poorly understood. Here, we investigated how adult MuSCs are regulated by CDKN1c (p57kip2), a cyclin-dependent kinase inhibitor, using mouse molecular genetics. In the absence of CDKN1c, skeletal muscle repair is severely impaired after injury. We show that CDKN1c is not expressed in quiescent MuSCs, while being induced in activated and proliferating myoblasts and maintained in differentiating myogenic cells. In agreement, isolated Cdkn1c-deficient primary myoblasts display differentiation defects and increased proliferation. We further show that the subcellular localization of CDKN1c is dynamic; while CDKN1c is initially localized to the cytoplasm of activated/proliferating myoblasts, progressive nuclear translocation leads to growth arrest during differentiation. We propose that CDKN1c activity is restricted to differentiating myoblasts by regulated cyto-nuclear relocalization, coordinating the balance between proliferation and growth arrest.
Assuntos
Células-Tronco Adultas/citologia , Diferenciação Celular/genética , Inibidor de Quinase Dependente de Ciclina p57/genética , Desenvolvimento Muscular/genética , Animais , Pontos de Checagem do Ciclo Celular/genética , Proliferação de Células/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/crescimento & desenvolvimento , Mioblastos/citologia , Mioblastos/metabolismo , Regeneração/genética , Células Satélites de Músculo Esquelético/citologiaRESUMO
Viral vector-mediated foreign gene expression in cultured cells has been extensively used in stem cell studies to explore gene function. However, it is difficult to obtain high-quality stem cells and primary cells after viral vector infection. Here, we describe a new protocol for high-efficiency retroviral infection of primary muscle stem cell (satellite cell) cultures. We compared multiple commercially available transfection reagents to determine which was optimal for retroviral infections of primary myoblasts. Centrifugation force was also tested, and a spin infection protocol with centrifugation at 2800 × g for 90 min had the highest infection efficiency for primary myoblasts. We confirmed that infected muscle stem cells maintain cell proliferation and the capacity for in vitro and in vivo myogenic differentiation. Our new, efficient retroviral infection protocol for muscle stem cells can be applied to molecular biology experiments as well as translational studies.
Assuntos
Vetores Genéticos/genética , Desenvolvimento Muscular , Mioblastos/citologia , Retroviridae/genética , Células-Tronco/citologia , Transfecção/métodos , Animais , Linhagem Celular , Proliferação de Células , Células Cultivadas , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Mioblastos/metabolismo , Células-Tronco/metabolismoRESUMO
Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the potential to differentiate into various types of cells including skeletal muscle cells. The approach of converting ESCs/iPSCs into skeletal muscle cells offers hope for patients afflicted with the skeletal muscle diseases such as the Duchenne muscular dystrophy (DMD). Patient-derived iPSCs are an especially ideal cell source to obtain an unlimited number of myogenic cells that escape immune rejection after engraftment. Currently, there are several approaches to induce differentiation of ESCs and iPSCs to skeletal muscle. A key to the generation of skeletal muscle cells from ESCs/iPSCs is the mimicking of embryonic mesodermal induction followed by myogenic induction. Thus, current approaches of skeletal muscle cell induction of ESCs/iPSCs utilize techniques including overexpression of myogenic transcription factors such as MyoD or Pax3, using small molecules to induce mesodermal cells followed by myogenic progenitor cells, and utilizing epigenetic myogenic memory existing in muscle cell-derived iPSCs. This review summarizes the current methods used in myogenic differentiation and highlights areas of recent improvement.