Repression of myelin basic protein gene expression in developing oligodendrocytes

Regulation of oligodendrocyte (OL) differentiation occurs through specific signal transduction pathways that lead to the induction of genes required for myelin production. Protein kinase C (PKC) downregulation followed by increased levels of cyclic AMP drives high-level expression from the full-length MBP promoter. We have begun to investigate specific isozymes of the PKC family, in order to better understand their role in the regulation of MBP gene expression during primary OL development, MBP promoter activity was determined with transient transfection assays in OLs using the full-length MBP promoter (-1323 to +30) luciferase reporter. Treatment with general or isozyme-specific PKC inhibitors enhanced promoter activity. Expression of the dominant negative PKC alpha, delta, epsilon or zeta increased promoter activity, while expression of the catalytic domain of PKC alpha, epsilon or zeta had no effect and PKC delta decreased promoter activity. Phorbol esters altered interactions of a binding complex that was detected with EMSAs using distal 5' end MBP promoter sequence oligonucleotide probes and proliferating but not differentiating CG4 cell nuclear extracts (NEs). The heterogeneous nuclear ribonucleoprotein K (hnRNP-K) was identified as a component of the purified binding complex using HPLC MS/MS. Purified-recombinant GST-hnRNP-K exhibited strong binding interactions with MBP promoter probes. Expression of hnRNP-K decreased promoter activity. Total endogenous levels of hnRNP-K remained constant. However, phospho(S302)hnRNP-K levels increased during OL differentiation. Mutation of S302 to E was introduced into hnRNP-K to mimic the phosphorylated state, since this had been shown to regulate DNA binding. GST-hnRNP-K(S302E) altered binding interactions with MBP probes. Expression of hnRNP-K(S302E) increased MBP promoter activity. Mutations were introduced into MBP probes to disrupt possible hnRNP-K binding sites. GST-hnRNP-K exhibited altered interactions with the mutated MBP probes. Additional interactions and sequence specificity was detected using CG4 and OL NEs. These results suggest that the PKCalpha and delta isozymes played specific roles in the regulation of MBP gene expression during OL differentiation. In addition, changes to the phosphorylation state of S302 altered the hnRNP-K DNA interactions that mediate repression of MBP gene expression during early OL development.