Hydralazine differentially increases mRNAs for the α and β subunits of prolyl 4-hydroxylase whereas it decreases proα1(I) collagen mRNAs in human skin fibroblasts

Abstract

We have used specific oligonucleotide probes to measure the effect of hydralazine on mRNA levels of the α and β subunits of prolyl 4-hydroxylase (PH), a key post-translational modifying enzyme in collagen biosynthesis. Hydralazine exerts a paradoxical effect on collagen biosynthesis in cultured fibroblasts. Cells exposed to hydralazine synthesize substantially reduced amounts of collagen, which is severely deficient in hydroxyproline. Surprisingly, however, the level of prolyl hydroxylase activity assayed in extracts of treated cells is markedly increased, suggesting overproduction of the enzyme. Hybridization analysis indicated that in untreated cells the concentration of the αPH subunit mRNA was about 20–25% of the βPH subunit mRNA concentration. Hydralazine treatment increased the mRNAs for both α and β subunits of PH by three- to fourfold. A differential induction of these mRNAs was observed, however. The α subunit mRNA was maximally increased within 24 h, whereas the β subunit mRNA was increased more slowly, reaching a maximum at 72 h. In contrast, the 5.8 and 4.8-kb mRNAs for proα1(I) collagen were virtually eliminated by 72 h. This study demonstrates that the increased prolyl hydroxylase activity is a direct result of hydralazine-mediated increases in steady state mRNA content for the α and β subunits of this enzyme. Moreover, the earlier induction of αPH mRNA may provide the first evidence at the mRNA level that regulation of PH activity occurs mainly through regulation of the α subunit of PH. In addition, the decrease in collagen synthesis by hydralazine appears to result directly from suppression of both species of mRNA for proα1(I) collagen.