ABSTRACT
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.
Subject(s)
Myoblasts/metabolism , rho GTP-Binding Proteins/metabolism , rho-Associated Kinases/metabolism , rhoA GTP-Binding Protein/metabolism , Animals , Cell Differentiation , Cell Fusion , Cell Line , Cell Shape , Down-Regulation , GTPase-Activating Proteins/metabolism , Male , Mice , Mice, Inbred C57BL , Microscopy, Electron, Scanning , Muscle Fibers, Skeletal/cytology , Myoblasts/cytology , Myoblasts/ultrastructure , Signal Transduction , Up-RegulationABSTRACT
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.
Subject(s)
B-Lymphocytes/cytology , Proteasome Endopeptidase Complex/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Amino Acid Sequence , Animals , Apoptosis , Calpain/metabolism , Cell Death , Cell Line , Cycloheximide/pharmacology , Flow Cytometry , Green Fluorescent Proteins/metabolism , Humans , Immunoprecipitation , Lysine/chemistry , Mice , Minor Histocompatibility Antigens , Models, Biological , Molecular Sequence Data , Mutagenesis , Mutation , Phenotype , Plasmids/metabolism , Protein Binding , Protein Biosynthesis , Protein Structure, Tertiary , Proto-Oncogene Proteins c-bcl-2/chemistry , Sequence Homology, Amino Acid , Staurosporine/pharmacology , Transfection , Ubiquitin/metabolismABSTRACT
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.
Subject(s)
Brain Neoplasms/pathology , Cell Cycle Proteins , Gastrins/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Glioblastoma/pathology , Neoplasm Proteins/biosynthesis , Actins/metabolism , Amino Acid Sequence , Biopolymers , Cell Movement/drug effects , Cell Movement/genetics , DNA, Complementary/genetics , Gene Expression Profiling , Genes, Reporter , Humans , Molecular Sequence Data , Myosin-Light-Chain Kinase/biosynthesis , Myosin-Light-Chain Kinase/genetics , Myosin-Light-Chain Kinase/physiology , Neoplasm Invasiveness/genetics , Neoplasm Proteins/genetics , Neoplasm Proteins/physiology , Promoter Regions, Genetic/drug effects , Protein Biosynthesis , Proteins/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Neoplasm/biosynthesis , RNA, Neoplasm/genetics , Reverse Transcriptase Polymerase Chain Reaction , S100 Calcium Binding Protein A6 , S100 Proteins/biosynthesis , S100 Proteins/genetics , S100 Proteins/physiology , Stress Fibers/metabolism , Subtraction Technique , Tenascin/biosynthesis , Tenascin/genetics , Tenascin/physiology , Transfection , Tumor Cells, Cultured/cytology , Tumor Cells, Cultured/drug effects , Wiskott-Aldrich Syndrome Protein Family , rhoA GTP-Binding Protein/physiologyABSTRACT
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.
