Fig 1.
Gene expression profiling of oligodendrocyte progenitors treated with the p38 MAPK inhibitor, PD169316, reveals gene targets with diverse cellular functions.
(A) Schematic diagram of the microarray analysis performed on rat oligodendrocyte (OL) progenitors treated for 1d with the p38 inhibitor, PD169316. Groups of genes were classified by biological function using UniProt and Gene Ontology functional analysis. (B) Functional classes of up-regulated genes included those involved in cytokinesis, spindle formation, replication, cytoskeleton and vesicular transport, p38 target genes, extracellular matrix (ECM), transcription and oxidative stress. (C) down-regulated gene transcript classes included receptors, ligands and transporters, cholesterol and lipid biosynthesis, extracellular matrix (ECM), cytoskeletal remodeling and vesicle trafficking, kinases, phosphatases, cell cycle, bone morphogenetic (BMP) signaling, DNA damage and apoptosis, ubiquitination, transcriptional regulators, and p38 signaling targets.
Table 1.
Myelin-specific and other pro-myelin gene transcripts upregulated and/or downregulated during OLG differentiation.
OLGs were differentiated by removal of PDGF-AA and bFGF for 2d.
Fig 2.
p38 MAPK regulates the expression of myelin gene transcripts and myelin gene activators and repressors that control oligodendrocyte identity.
Gene transcript expression levels of (A) myelin specific (Mag), (B) transcriptional activators (Fyn, Hdac11) and (C) transcriptional repressors (Tcf4, Hes5, Id2) are altered after PD169316 treatment as determined by qRT-PCR. OLPs were treated with 5 μM PD169316 for 1d, 2d or 4d and RNA was harvested, reverse transcribed and analyzed by qRT-PCR. All gene transcripts were normalized to ctrl at 1d all relative to 28S rRNA. Statistical differences were determined using independent t-tests with Bonferroni’s correction (*p < 0.05, **p< 0.01, ***p< 0.001 vs same day ctrl).
Table 2.
Myelin genes and transcriptional activators are decreased by a 24h treatment of OLPs with 5 mM PD169316.
Fig 3.
p38 MAPK regulates expression of oligodendrocyte specification genes, vesicular transport regulators and cell cycle regulators.
Gene transcript expression levels of early (A) specification markers (Nkx2.2), (B) vesicular transport (Rab33a), and (C) cell cycle regulators (p57kip2, p21Cip1, Cyclin A1, Cyclin D1) are altered after PD169316 treatment as determined by qRT-PCR. OLPs were treated with 5 μM PD169316 for 1d, 2d or 4d. All gene transcripts were normalized to ctrl at 1d all relative to 28S rRNA. Statistical differences were determined using independent t-tests with Bonferroni correction (*p< 0.05, **p< 0.01, ***p< 0.001 vs. same day ctrl).
Table 3.
Transcriptional repressors and early OLP markers are upregulated following 24h treatment with 5 mM PD169316.
Table 4.
PD169316 treatment elevates mRNA levels of p38 upstream activators.
Table 5.
Transcripts encoding cell cycle regulators, kinesins, centromere, spindle and kinetochore associated proteins are upregulated by 5 mM PD169316 treatment.
Fig 4.
p38 inhibitors maintain oligodendrocyte progenitor cells in a non-resting state of the cell cycle.
(A, B) Oligodendrocyte progenitors were maintained in serum-free medium in the presence or absence of PD169316 for 2d or 4d and the A2B5 and O4-positive population analyzed for the presence of nuclear Ki67. (A) Representative fluorescent images for the 2d time point, scale bar represents 50 μm. (B) Plot of the relative distribution of Ki67+ or Ki67- A2B5+O4-positive cells. (C) Oligodendrocyte progenitors were treated with 5 μM PD169316 for 1d or 2d, followed by 3H-thymidine incorporation overnight in the presence of absence of growth factors (PDGF-AA and bFGF). Statistical differences in (C) were determined by one-way ANOVA followed by Dunnett’s correction (***p< 0.001 vs ctrl).