RESUMO
Nestin, a type VI intermediate filament (IF) protein, is predominantly expressed in neurogenic and myogenic stem cells. We previously identified the first isoform of nestin, Nes-S, in rat DRG neurons. In this study, we report a previously unidentified nestin isoform, Nes-SΔ107â254, that is expressed in lower levels in DRG neurons of adult rats. The 29-kD Nes-SΔ107â254 is identical to Nes-S, except that an additional region is removed in its rod domain. Nes-SΔ107â254 is assembly compromised and forms aggregates. Unlike nestin and Nes-S, Nes-SΔ107â254 does not exert cytoprotective effect. Furthermore, elevated caspase-3 activation was observed in HEK293T cells expressing the EGFP-Nes-SΔ107â254 protein. Taken together, Nes-SΔ107â254 is a rod domain-truncated isoform of nestin that is susceptible to form cytotoxic aggregates.
Assuntos
Gânglios Espinais/metabolismo , Nestina/metabolismo , Processamento Alternativo , Animais , Apoptose , Caspase 3/metabolismo , Células Cultivadas , Humanos , Nestina/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Ratos , TransfecçãoRESUMO
Nestin is a 240-kDa intermediate filament protein expressed mainly in neural and myogenic stem cells. Although a substantial number of studies have focused on the expression of nestin during development of the central nervous system, little is known about the factors that induce and regulate its expression. Fibroblast growth factor-2 (FGF-2) is an effective mitogen and stimulates the proliferation and differentiation of a subset of nestin-expressing cells, including neural progenitor cells, glial precursor cells, and smooth muscle cells. To assess whether FGF-2 is a potent factor that induces the expression of nestin, C6 glioma cells were used. The results showed that nestin expression was up-regulated by FGF-2 via de novo RNA and protein synthesis. Our RT-PCR results showed that C6 glioma cells express FGFR1/3, and FGFRs is required for FGF-2-induced nestin expression. Further signaling analysis also revealed that FGF-2-induced nestin expression is mediated through FGFR-MAPK-ERK signaling axis and the transcriptional factor Sp1. These findings provide new insight into the regulation of nestin in glial system and enable the further studies on the function of nestin in glial cells.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/genética , Fator 2 de Crescimento de Fibroblastos/farmacologia , Proteínas de Filamentos Intermediários/genética , Proteínas do Tecido Nervoso/genética , Fator de Transcrição Sp1/genética , Quinases raf/genética , Proteínas ras/genética , Animais , Western Blotting , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Proteínas de Filamentos Intermediários/metabolismo , Microscopia de Fluorescência , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nestina , Pirróis/farmacologia , Ratos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Transcrição Sp1/metabolismo , Regulação para Cima/efeitos dos fármacos , Quinases raf/metabolismo , Proteínas ras/metabolismoRESUMO
In this study, the first nestin isoform, Nes-S, was identified in neurons of dorsal root ganglia (DRG) of adult rats. Nes-S cannot form filaments by itself in cytoplasmic intermediate filament-free SW13 cells. Instead, it co-assembles into filaments with vimentin when transfected into vimentin(+) SW13 cells, and with peripherin and neurofilament proteins when transfected into N2a cells. In primary DRG neurons, endogenous Nes-S co-assembles with peripherin and neurofilament proteins. The expression of Nes-S first appears in DRG at postnatal day 5 and persists to adulthood. Among the adult tissues we examined, the expression of Nes-S is restricted to the sensory and motor neurons. Finally, exogenous Nes-S enhances viability when transfected into N2a cells, and knockdown of endogenous Nes-S impairs the survival of DRG neurons in primary cultures. Taken together, Nes-S is a new neuronal intermediate filament protein that exerts a cytoprotective function in mature sensory and motor neurons.
Assuntos
Gânglios Espinais/citologia , Proteínas de Filamentos Intermediários/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Especificidade de Anticorpos , Sobrevivência Celular , Células Cultivadas , Citoproteção , Citoesqueleto/metabolismo , Mapeamento de Epitopos , Gânglios Espinais/crescimento & desenvolvimento , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas de Filamentos Intermediários/imunologia , Proteínas de Filamentos Intermediários/metabolismo , Masculino , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Nestina , Neurônios/fisiologia , Especificidade de Órgãos , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Análise de Célula Única , Vimentina/metabolismoRESUMO
The muscularis externa of the developing rodent esophagus is initially composed of smooth muscle, and later replaced by skeletal muscle in a craniocaudal progression. There is growing evidence of distinct developmental origins for esophageal smooth and skeletal muscles. However, the identification of skeletal muscle progenitor cells is controversial, and the detailed cell lineage of their descendants remains elusive. In the current study, we carried out multiple labeling immunofluorescence microscopy of nestin and muscle type-specific markers to characterize the dynamic process of rat esophageal myogenesis. The results showed that nestin was transiently expressed in immature esophageal smooth muscle cells in early developing stages. After nestin was downregulated in smooth muscle cells, a distinct population of nestin-positive cells emerged as skeletal muscle precursors. They were mitotically active, and subsequently co-expressed MyoD, followed by the embryonic and later the fast type of skeletal muscle myosin heavy chain. Thus, the cell lineage of esophageal skeletal muscle differentiation was established by an immunotyping approach, which revealed that skeletal myocytes arise from a distinct lineage rather than through transdifferentiation of smooth muscle cells during rat esophageal myogenesis.
