Hydralazine differentially increases mRNAs for the alpha and beta subunits of prolyl 4-hydroxylase whereas it decreases pro alpha 1(I) collagen mRNAs in human skin fibroblasts.
We have used specific oligonucleotide probes to measure the effect of hydralazine on mRNA levels of the alpha and beta 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 alpha PH subunit mRNA was about 20-25% of the beta PH subunit mRNA concentration. Hydralazine treatment increased the mRNAs for both alpha and beta subunits of PH by three- to fourfold. A differential induction of these mRNAs was observed, however. The alpha subunit mRNA was maximally increased within 24 h, whereas the beta subunit mRNA was increased more slowly, reaching a maximum at 72 h. In contrast, the 5.8 and 4.8-kb mRNAs for pro alpha 1(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 alpha and beta subunits of this enzyme. Moreover, the earlier induction of alpha PH mRNA may provide the first evidence at the mRNA level that regulation of PH activity occurs mainly through regulation of the alpha subunit of PH. In addition, the decrease in collagen synthesis by hydralazine appears to result directly from suppression of both species of mRNA for pro alpha 1(I) collagen.
Yeowell
H N
HN
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Murad
S
S
Pinnell
S R
SR
D001483:Base Sequence; D002478:Cells, Cultured; D015342:DNA Probes; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005353:Fibronectins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D006830:Hydralazine / Q000494:pharmacology*; D008969:Molecular Sequence Data; D011347:Procollagen / Q000096:biosynthesis* / Q000235:genetics; D011393:Procollagen-Proline Dioxygenase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012867:Skin / Q000187:drug effects / Q000378:metabolism
D015342:DNA Probes; D005353:Fibronectins; D011347:Procollagen; D012333:RNA, Messenger; D006830:Hydralazine; D011393:Procollagen-Proline Dioxygenase