RESUMO
Differentiation of skeletal myoblasts into multinucleated myotubes is a multi-step process orchestrated by several signaling pathways. The Rho small G protein family plays critical roles both during myogenesis induction and myoblast fusion. We report here that in C2C12 myoblasts, expression of RhoE, an atypical member of this family, increases until the onset of myoblast fusion before resuming its basal level once fusion has occurred. We show that RhoE accumulates in elongated, aligned myoblasts prior to fusion and that its expression is also increased during injury-induced skeletal muscle regeneration. Moreover, although RhoE is not required for myogenesis induction, it is essential for myoblast elongation and alignment before fusion and for M-cadherin expression and accumulation at the cell-cell contact sites. Myoblasts lacking RhoE present with defective p190RhoGAP activation and RhoA inhibition at the onset of myoblast fusion. RhoE interacts also with the RhoA effector Rho-associated kinase (ROCK)I whose activity must be downregulated to allow myoblast fusion. Consistently, we show that pharmacological inactivation of RhoA or ROCK restores myoblast fusion in RhoE-deficient myoblasts. RhoE physiological upregulation before myoblast fusion is responsible for the decrease in RhoA and ROCKI activities, which are required for the fusion process. Therefore, we conclude that RhoE is an essential regulator of myoblast fusion.
Assuntos
Mioblastos/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Diferenciação Celular , Fusão Celular , Linhagem Celular , Forma Celular , Regulação para Baixo , Proteínas Ativadoras de GTPase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Fibras Musculares Esqueléticas/citologia , Mioblastos/citologia , Mioblastos/ultraestrutura , Transdução de Sinais , Regulação para CimaRESUMO
Bfl-1/A1 is generally recognized as a Bcl-2-related inhibitor of apoptosis. We show that Bfl-1 undergoes constitutive ubiquitin/proteasome-mediated turnover. Moreover, while Bfl-1 suppresses apoptosis induced by staurosporine or cytokine withdrawal, it is proapoptotic in response to tumor necrosis factor (TNF) receptor activation in FL5.12 pro-B cells. Its anti- versus proapoptotic effect is regulated by two proteolytic events: (1) its constitutive proteasome-mediated turnover and (2) its TNF/cycloheximide (CHX)-induced cleavage by mu-calpain, or a calpain-like activity, coincident with acquisition of a proapoptotic phenotype. In vitro studies suggest that calpain-mediated cleavage of Bfl-1 occurs between its Bcl-2 homology (BH)4 and BH3 domains. This would be consistent with the generation of a proapoptotic Bax-like BH1-3 molecule. Overall, our studies uncovered two new regulatory mechanisms that play a decisive role in determining Bfl-1's prosurvival versus prodeath activities. These findings might provide important clues to counteract chemoresistance in tumor cells that highly express Bfl-1.
Assuntos
Linfócitos B/citologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Calpaína/metabolismo , Morte Celular , Linhagem Celular , Cicloeximida/farmacologia , Citometria de Fluxo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunoprecipitação , Lisina/química , Camundongos , Antígenos de Histocompatibilidade Menor , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese , Mutação , Fenótipo , Plasmídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/química , Homologia de Sequência de Aminoácidos , Estaurosporina/farmacologia , Transfecção , Ubiquitina/metabolismoRESUMO
Astrocytic tumors are the most common and the most malignant primary tumors of the central nervous system. We had previously observed that gastrin could significantly modulate both cell proliferation and migration of astrocytoma cells. We have investigated in the present study which genes could be targeted by gastrin in tumor astrocyte migration. Using a subtractive hybridization PCR technique we have cloned genes differentially over-expressed in human astrocytoma U373 cells treated or not with gastrin. We found about 70 genes over-expressed by gastrin. Among the genes overexpressed by gastrin, we paid particular attention to tenascin-C, S100A6 and MLCK genes because their direct involvement in cell migration features. Their gastrin-induced overexpression was quantitatively determined by competitive RT-PCR technique. We also showed by means of a reporter gene system that S100A6 and tenascin-C respective promoters were upregulated after gastrin treatment. These data show that gastrin-mediated effects in glioblastoma cells occur through activation of a number of genes involved in cell migration and suggest that gastrin could be a target in new therapeutic strategies against malignant gliomas.
Assuntos
Neoplasias Encefálicas/patologia , Proteínas de Ciclo Celular , Gastrinas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/patologia , Proteínas de Neoplasias/biossíntese , Actinas/metabolismo , Sequência de Aminoácidos , Biopolímeros , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , DNA Complementar/genética , Perfilação da Expressão Gênica , Genes Reporter , Humanos , Dados de Sequência Molecular , Quinase de Cadeia Leve de Miosina/biossíntese , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/fisiologia , Invasividade Neoplásica/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Biossíntese de Proteínas , Proteínas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína A6 Ligante de Cálcio S100 , Proteínas S100/biossíntese , Proteínas S100/genética , Proteínas S100/fisiologia , Fibras de Estresse/metabolismo , Técnica de Subtração , Tenascina/biossíntese , Tenascina/genética , Tenascina/fisiologia , Transfecção , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacos , Família de Proteínas da Síndrome de Wiskott-Aldrich , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
The RPE65 protein appears late during the retinal development. To study the basis for this regulation, the rat RPE65 cDNA was sequenced and the mRNA subsequently quantitated at various stages by competitive RT-PCR. RPE65 mRNA was detected as early as E18 (36 copies/ng of whole eye total RNA). It gradually accumulates up to P12 (27000 copies/ng) at which point it reaches a steady state level. This increase is interrupted for 3 days (P2-P4) during which the levels of mRNA remain stable. This timing and rate of accumulation parallels that of rat and mouse opsin mRNA and suggests that common factors may control the activation of genes in photoreceptors and retinal pigment epithelium cells.
