RESUMO
When we treated rat bone marrow stromal cells (rBMSCs) with neuronal differentiation induction media, typical unfolded protein response (UPR) was observed. BIP/GRP78 protein expression was time-dependently increased, and three branches of UPR were all activated. ATF6 increased the transcription of XBP1 which was successfully spliced by IRE1. PERK was phosphorylated and it was followed by eIF2alpha phosphorylation. Transcription of two downstream targets of eIF2alpha, ATF4 and CHOP/GADD153, were transiently up-regulated with the peak level at 24 h. Immunocytochemical study showed clear coexpression of BIP and ATF4 with NeuN and Map2, respectively. UPR was also observed during the neuronal differentiation of mouse embryonic stem (mES) cells. Finally, chemical endoplasmic reticulum (ER) stress inducers, thapsigargin, tunicamycin, and brefeldin A, dose-dependently increased both mRNA and protein expressions of NF-L, and, its expression was specific to BIP-positive rBMSCs. Our results showing the induction of UPR during neuronal differentiations of rBMSCs and mES cells as well as NF-L expression by ER stress inducers strongly suggest the potential role of UPR in neuronal differentiation.
Assuntos
Animais , Camundongos , Ratos , Fator 4 Ativador da Transcrição/genética , Apoptose/efeitos dos fármacos , Células da Medula Óssea/citologia , Diferenciação Celular , Meios de Cultura/farmacologia , Células-Tronco Embrionárias/citologia , Retículo Endoplasmático/genética , Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Proteínas Associadas aos Microtúbulos/genética , Chaperonas Moleculares/genética , Proteínas do Tecido Nervoso/genética , Proteínas de Neurofilamentos/genética , Neurônios/citologia , Proteínas Nucleares/genética , Dobramento de Proteína , Células EstromaisRESUMO
Expressions of endoplasmic reticulum stress response (ERSR) genes were examined during the neuronal differentiation of rat fetal cortical precursor cells (rCPC) and rat pheochromocytoma PC12 cells. When rCPC were differentiated into neuronal cells for 7 days, early stem cell marker, nestin, expression was decreased from day 4, and neuronal markers such as neurofilament-L, -M and Tuj1 were increased after day 4. In this condition, expressions of BIP, ATF6, and phosphorylated PERK as well as their down stream signaling molecules such as CHOP, ATF4, XBP1, GADD34, Nrf2 and p58IPK were significantly increased, suggesting the induction of ERSR during neuronal differentiation of rCPC. ERSR was also induced during the differentiation of PC12 cells for 9 days with NGF. Neurofilament-L transcript was time-dependently increased. Both mRNA and protein levels of Tuj1 were increased after the induction, and the significant increase in NeuN was observed at day 9. Similar to the expression patterns of neuronal markers, BIP/GRP78 and CHOP mRNAs were highly increased at day 9, and ATF4 mRNA was also increased from day 7. These results strongly suggest the induction and possible role of ERSR in neuronal differentiation process. Further study to identify targets responsible for neuronal induction will be necessary.
Assuntos
Animais , Ratos , Estresse do Retículo Endoplasmático , Retículo Endoplasmático , Fator de Crescimento Neural , Nestina , Neurônios , Células PC12 , Feocromocitoma , Rios , RNA Mensageiro , Células-TroncoRESUMO
B/K protein is a novel protein containing double C2-like domains. We examined the specific signaling pathway that regulates the transcription of B/K in PC12 cells. When the cells were treated with forskolin (50microM), B/K mRNA and protein levels were time-dependently decreased, reaching the lowest level at 3 or 4 hr, and thereafter returning to the control level. Chemicals such as dibutyryl-cAMP, cell- permeable cyclic AMP (cAMP) analogue and CGS21680, adenosine receptor A2A agonist, also repressed the B/K transcription. However, 1, 9-dideoxyforskolin did not show inhibitory effect on B/K transcription, suggesting direct involvement of cAMP in the forskolin-induced inhibition of B/K transcription. Effect of forskolin, dibutyryl cAMP and CGS21680 was significantly reduced in PKA-deficient PC12 cell line (PC12-123.7). One cAMP-response element (CRE) -like sequence (B/K CLS) was found in the promoter region of B/K DNA, and electrophoretic mobility shift assay indicated its binding to CREM and CREB. Forskolin significantly suppressed the promoter activity in CHO-K1 cells transfected with the constructs containing B/K CLS, but not with the construct in which B/K CLS was mutated (AC: TG). Taken together, we suggest that the transcription of B/K gene in PC12 cells may be regulated by PKA-dependent mechanism.