RESUMO
We have previously shown that low concentrations of a specific proteasome inhibitor accelerate exit from the cell cycle and enhance oligodendroglial cell (OLGc) differentiation. To elucidate the mechanisms involved in this process, OLGcs of the N20.1 cell line, transfected with a reporter gene driven by the MBP promoter, were treated with proteasome inhibitors and/or inhibitors of different signaling pathways. Partial proteasome inhibition resulted in enhanced activation of the MBP promoter which involved the tyrosine kinase, PI3-Akt and PKC pathways, accompanied by an increase in the levels of p21(Cip1), p27(Kip1) and Sp1 and by a decrease in Nkx2.2. Binding of Sp1 to DNA was also increased. These results were not observed when the Sp1 binding site was mutated. We conclude that the enhanced activation of the MBP promoter induced by partial inhibition of the proteasome could be due, at least in part, to the stabilization of p27(Kip1) and Sp1.
Assuntos
Diferenciação Celular/genética , Proteína Básica da Mielina/genética , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Transdução de Sinais/fisiologia , Animais , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Proteína Homeobox Nkx-2.2 , Imunoprecipitação , Camundongos , Proteína Básica da Mielina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição Sp1/metabolismo , TransfecçãoRESUMO
In the CNS, transferrin (Tf) is expressed by the oligodendroglial cells (OLGcs) and is essential for their development. We have previously shown that apotransferrin (aTf) accelerates maturation of OLGcs in vivo as well as in vitro. The mechanisms involved in this action appear to be complex and have not been completely elucidated. The aim of this study was to investigate if Tf participates in the regulation of the cell cycle of oligodendroglial progenitor cells (OPcs). Primary cultures of OPcs were treated with aTf and/or with different combinations of mitogenic factors. Cell cycle progression was studied by BrdU incorporation, flow cytometry and by the expression of cell cycle regulatory proteins. Apotransferrin decreased the number of BrdU+ cells, increasing the cell cycle time and decreasing the number of cells in S phase. The cell cycle inhibitors p27kip1, p21cip1 and p53 were increased, and in agreement with these results, the activity of the complexes involved in G1-S progression (cyclin D/CDK4, cyclin E/CDK2), was dramatically decreased. Apotransferrin also inhibited the mitogenic effects of PDGF and PDGF/IGF on OPcs, but did not affect their proliferation rate in the presence of bFGF, bFGF/PDGF or bFGF/IGF. Our results indicate that inhibition of the progression of the cell cycle of OPcs by aTf, even in the presence of PDGF, leads to an early beginning of the differentiation program, evaluated by different maturation markers (O4, GC and MBP) and by morphological criteria. The modulation by aTf of the response of OPcs to PDGF supports the idea that this glycoprotein might act as a key regulator of the OLGc lineage progression.
Assuntos
Apoproteínas/farmacologia , Ciclo Celular/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Fator de Crescimento Derivado de Plaquetas/farmacologia , Células-Tronco/efeitos dos fármacos , Transferrina/farmacologia , Animais , Antimetabólitos , Western Blotting , Bromodesoxiuridina , Diferenciação Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/metabolismo , DNA/biossíntese , Depressão Química , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Fase G1 , Imuno-Histoquímica , Oligodendroglia/ultraestrutura , Ratos , Fase S , Células-Tronco/ultraestrutura , Sais de Tetrazólio , TiazóisRESUMO
Twenty-one-day-old Wistar rats were fed a diet containing 0.6% cuprizone for 2 weeks. Studies carried out after withdrawal of cuprizone showed histological evidences of marked demyelination in the corpus callosum. Biochemical studies of isolated myelin showed a marked decrease in myelin proteins, phospholipids, and galactocerebrosides as well as a marked decrease in myelin yield. Treatment of these animals with a single intracranial injection of 350 ng of apotransferrin at the time of withdrawal of cuprizone induced a marked increase in myelin deposition resulting in a significantly improved remyelination, evaluated by histological, immunocytochemical, and biochemical parameters, in comparison to what was observed in spontaneous recovery. Immunocytochemical studies of cryotome sections to analyze developmental parameters of the oligodendroglial cell population at the time of termination of cuprizone and at different times thereafter showed that in the untreated animals, there was a marked increase in the number of NG2-BrdU-positive precursor cells together with a marked decrease in MBP expression at the peak of cuprizone-induced demyelination. As expected, the amount of precursor cells decreased markedly during spontaneous remyelination and was accompanied by an increase in MBP reactivity. In the apotransferrin-treated animals, these phenomena occurred much faster, and remyelination was much more efficient than in the untreated controls. The results of this study suggest that apotransferrin is a very active promyelinating agent which could be important for the treatment of certain demyelinating conditions.