Abstract
The proteolipid protein (PLP) gene codes for the major central nervous system myelin protein. We have studied the effects of different agents on the expression of the PLP gene in C6 glioma cells. Retinoic acid (RA), but not dexamethasone, estradiol, insulin, growth hormone, or vitamin D3, had a drastic effect, increasing 10-20-fold the level of PLP mRNA. Concomitantly, RA also induced the appearance of the corresponding immunoreactive protein. The increase in PLP RNA level showed a slow kinetics and was blocked by cycloheximide, suggesting a posttranscriptional regulation by RA. Nuclear run-on assays confirmed that the rate of PLP gene transcription was unchanged by RA. In contrast, we found that retinoic acid augmented PLP mRNA stability, causing a substantial increase in its half-life. RA action was independent of cell density, serum, or PDGF but was partially inhibited by bFGF. On the other hand, thyroid hormone caused a moderate increase in PLP mRNA levels in C6 cells but only when the low numbers of thyroid receptors in these cells were increased by retrovirally mediated expression of an exogenous c-erbA/TR alpha-1 gene. Our results indicate that RA specifically up-regulates PLP expression in glioma C6 cells at a posttranscriptional level by increasing PLP RNA half-life.
Highlights
Monica Lopez-BarahonaSO, MartMa iiianol, Emilia MiraSII, Teresa IglesiasSQ, Hendrik G
Thyroid hormone caused a modeprraecteursor cells obtained in primary cultures, the ratC6 glioma increase in PLmP RNA levels in C6 cells but only when cell line was chosen because it retains certain characteristics the low numbers of thyroid receptors in these cells of glial precursors, expresses some astrocyte and oligodendrowere increased byretrovirally mediated expression of cyte genes including some myelin genes, and is able at least an exogenous c-erbAITRa1-gene
We report an increase of steady-state Proteolipid protein (PLP) mRNA levels byRA mirrored by an increase in the amount of immunoreactive protein
Summary
Blotted onto Genescreen membranes (DuPont NEN) according to standardprotocols (Sambrook et al, 1989).All probes were labeled by the random priming method (Feinberg and Vogelstein, 1983)using [CT-~~P]~CTP. sTouhrece of probes were as follows. Rat PLP and Retinoic Acid Increases PLP mRNA Levels in Glioma C6 Cells-To search for physiological regulators of PLP gene expression and to test whether thyroid hormone could cyclophilin were from Dr J. Hybridizations were carried out overnight at the developing rat brain (Muiioz et al, 1991),rat glioma C6 cells were treated with these or other hormones and agents for 48 h (Fig. I).No increase in the steady-state PLP mRNA level was observed in T3-treated cells. 65 "C in 7% SDS, 500 mM sodium phosphate buffer, pH 7.2, and 1 the level of PLP mRNA after 48 h which was not detected in mM EDTA according to Church and Gilbert (1984). Insulin failed to increase PLP mRNA expression, and forskolin, a CAMP-inducing agent, had only a minor effect (not shown). Additional minor bands of 5.5 and 4.5 kb correspondingprobably to precursor PLP mRNAs were usually detected (see Campagnoni and Macklin, 1988)
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