RESUMO
R-Ras3/M-Ras is a member of the RAS superfamily of small-molecular-weight GTP-binding proteins. Previous studies have demonstrated high levels of expression in several regions of the central nervous system, and a constitutively active form of M-Ras promotes cytoskeletal reorganization, cellular transformation, survival, and differentiation. However, the physiological functions of M-Ras during embryogenesis and postnatal development have not been elucidated. By using a specific M-Ras antibody, we demonstrated a high level of M-Ras expression in astrocytes, in addition to neurons. Endogenous M-Ras was activated by several trophic factors in astrocytes, including epidermal growth factor (EGF), basic fibroblast growth factor, and hepatocyte growth factor. Interestingly, M-Ras activation by EGF was more sustained compared to prototypic Ras. A mouse strain deficient in M-Ras was generated to investigate its role in development. M-Ras null mice appeared phenotypically normal, and there was a lack of detectable morphological and neurological defects. In addition, primary astrocytes derived from Mras(-/-) mice did not appear to display substantial alterations in the activation of both the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways in response to trophic factors.
Assuntos
Substâncias de Crescimento/farmacologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Monoméricas de Ligação ao GTP/deficiência , Proteínas Monoméricas de Ligação ao GTP/genética , Fosfotransferases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Proteínas rasRESUMO
The Kruppel-like transcription factor KLF6 is a novel tumor-suppressor gene mutated in a significant fraction of human prostate cancer. It is localized to human chromosome 10p14-15, a region that displays frequent loss of heterozygosity in glioblastoma multiforme (GBM). Indeed, mutations of the KLF6 gene have recently been reported in this tumor type. In this study, we report that the expression of KLF6 is attenuated in human GBM when compared with primary astrocytes. Expression of KLF6 in GBM cells reverts their tumorigenicity both in vitro and in vivo, which is correlated with its transactivation of the p21/CIP1/WAF1 promoter. Additionally, KLF6 inhibits cellular transformation induced by several oncogenes (c-sis/PDGF-B, v-src, H-Ras, and EGFR) that are components of signaling cascades implicated in GBM. Our results provide the first evidence of functional tumor suppression by KFL6, and its loss may contribute to glial tumor progression.
Assuntos
Genes Supressores de Tumor , Glioblastoma/genética , Proteínas Proto-Oncogênicas , Transativadores/genética , Fatores de Transcrição/metabolismo , Animais , Astrócitos/metabolismo , Neoplasias Encefálicas , Linhagem Celular , Transformação Celular Neoplásica , Glioblastoma/patologia , Glioblastoma/prevenção & controle , Humanos , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like , Camundongos , Camundongos Nus , Mutação , Transplante de Neoplasias , Ratos , Células Tumorais CultivadasRESUMO
R-Ras3/M-Ras is a novel member of the Ras subfamily of GTP-binding proteins which has a unique expression pattern highly restricted to the mammalian central nervous system. In situ hybridization using an R-Ras3 cRNA probe revealed high levels of R-Ras3 transcripts in the hippocampal region of the mouse brain as well as a pattern of expression in the cerebellum that was distinct from that of H-Ras. We found that R-Ras3 was activated by nerve growth factor (NGF) and basic fibroblast growth factor as well as by the guanine nucleotide exchange factor GRP but not by epidermal growth factor. Ectopic expression of either R-Ras3 or GRP in PC12 cells induced efficient neuronal differentiation. The ability of NGF as well as GRP to promote differentiation of PC12 cells was attenuated by an R-Ras3 dominant-negative mutant. Furthermore, the biological action of R-Ras3 in PC12 cells was dependent on the mitogen-activated protein kinase (MAPK). Interestingly, whereas R-Ras3 was unable to mediate efficient activation of MAPK activity in NIH 3T3 cells, it was able to do so in PC12 cells. This cell-type specificity is in stark contrast to that of H-Ras, which can stimulate the MAPK pathway in both cell types. Indeed, this pattern of MAPK activation could be explained by the fact that R-Ras3 was unable to activate c-Raf, while it bound and stimulated the neuronal Raf isoform, B-Raf, in PC12 cells. Thus, R-Ras3 is implicated in a novel pathway of neuronal differentiation by coupling specific trophic factors to the MAPK cascade through the activation of B-Raf.
