Protein kinase C activity modulates myelin gene expression in enriched oligodendrocytes

Abstract

Protein kinase C (PKC) and its potential role in myelin gene expression were investigated in primary cultured rat oligodendrocytes. The major myelin genes were expressed in a developmentally coordinated manner in cultured oligodendrocytes. PKC activity in these cells was similarly regulated with differential expression of PKC isozyme mRNAs. PKC-gamma mRNA was expressed transiently and was most abundant in 9-day cells in vitro. PKC-alpha and PKC-beta mRNAs were present at low levels throughout development in these cells, and their expression increased in 18-25 day cells. Immunocytochemical colocalization of PKC with oligodendrocyte-specific markers--O4, galactosyl cerebroside, MBP, and PLP--in enriched oligodendrocyte cultures suggested that the PKC enzyme activities assayed in these cultures were predominantly contributed by oligodendrocytes. PKC inhibition resulting from long-term exposure to 4 beta-phorbol-12,13-dibutyrate (4 beta-PDB) reduced steady-state levels of MBP, PLP, MAG, CNP, and PKC-alpha mRNAs, as detected by slot blots or in situ hybridization, and downregulated the oligodendrocyte-specific markers O4, galactosyl cerebroside, and the major constituent proteins MBP and PLP, as detected by immunocytochemistry. PKC-mediated downmodulation of myelin gene expression was most profound in normally differentiating oligodendrocytes at or before the onset of myelin protein synthesis. Six-day oligodendrocytes were most susceptible to such modulation. To elucidate the mechanism of reduction in various myelin gene messages upon modulation of PKC, we analyzed mRNA levels in oligodendrocytes, which were pretreated with either the transcriptional inhibitor actinomycin D or the protein synthesis blocker cycloheximide before exposure to 4 beta-PDB. Our results demonstrate that the PKC inhibition-mediated loss in myelin mRNA levels did not require the transcription of any genes, but appeared to be at least partially dependent on continuous protein synthesis.

Very large G protein-coupled receptor 1 regulates myelin-associated glycoprotein via Gαs/Gαq-mediated protein kinases A/C

“…PKCmediated posttranscriptional suppression of myelin gene expression is most profound in differentiating OLs at or before the onset of myelin protein synthesis (19). This correlates well with our data showing that MASS1 regulates MAG protein stability.…”

“…PKC was previously shown to play a role in regulating myelin proteins levels (19). To further reveal the mechanism and assess MASS1 regulation of PKC in vivo, we used primary OL cultures and found that phosphorylated PKCδ/θ levels were dramatically decreased in Frings OLs vs. control (Fig.…”

Disruption of the murine nuclear factor I-A gene ( Nfia ) results in perinatal lethality, hydrocephalus, and agenesis of the corpus callosum

“…Some embryos and fetuses were fixed in 4% paraformaldehyde, Histochoice (AMRESCO), or snap frozen in OCT and cryosectioned or embedded in paraffin and sectioned. Immunostaining was performed as described previously (35) by using monoclonal antibodies to glial fibrillary acidic protein (GFAP) (G-A-5; Sigma) and myelin phospholipid protein (PLP) (M2; ref. 35).…”

“…Immunostaining was performed as described previously (35) by using monoclonal antibodies to glial fibrillary acidic protein (GFAP) (G-A-5; Sigma) and myelin phospholipid protein (PLP) (M2; ref. 35).…”

Expression of a Myelin Proteolipid Protein (Plp)-lacZ Transgene is Reduced in both the CNS and PNS of Plp jp Mice

“…Unfortunately the only antibody available to detect DM20 protein by Western blot analysis recognizes a Cterminal epitope, which is altered in Plp jp PLP/DM20 proteins. Thus, only the mutant PLP protein in Plp jp mice could be examined due to the existence of PLP-specific antibodies [21]. Although the amounts, if any, of mutant DM20 in Plp jp sciatic nerve could not be determined, the PCR data (Fig.…”