Signal transduction pathways responsible for serotonin-mediated prostaglandin G/H synthase expression in rat mesangial cells.
Prostaglandin G/H synthase (PGHS) is one of the key enzymes in prostaglandin synthesis. Regulation of the mRNA expression of the two isozymes PGHS-1 and PGHS-2 was investigated in mesangial cells. PGHS-1 was constitutively expressed and not modulated by any of the stimuli used. PGHS-2 was induced by the platelet products serotonin (5-HT) and thromboxane A2 (used as its analogue U46619), but not by ATP. Expression of PGHS protein was regulated correspondingly; whereas PGHS-1 protein was constitutively expressed, PGHS-2 protein was virtually absent in unstimulated cells, but could increasingly be induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), 5-HT, or fetal calf serum. Induction of PGHS-2 mRNA was transient with a peak after 2-3 h. Stimulated mRNA levels persisted for more than 6 h when transcription was inhibited by actinomycin D or when translation was inhibited by cycloheximide. As shown by specific inhibitors, 5-HT signal transduction was mediated by 5-HT2 receptors, which couple to phospholipase C via pertussis toxin-sensitive G-proteins. Induction of PGHS-2 mRNA by 5-HT was dependent on protein kinase C. Down-regulation of the enzyme by prolonged incubation with TPA abolished 5-HT-induced PGHS-2 mRNA expression. Short time activation of protein kinase C by TPA induced PGHS-2 mRNA expression. On the other hand, TPA given immediately before 5-HT decreased the 5-HT-induced PGHS-2 mRNA expression, indicating a negative feedback. The immunosuppressive drug cyclosporin A reduced induction of PGHS-2 mRNA expression by 5-HT, indicating interference with the signaling cascade, most likely with the Ser/Thr phosphatase calcineurin. Involvement of Tyr phosphorylation in 5-HT signaling was shown by the Tyr kinase inhibitor genistein, which inhibited the induction, while the Tyr phosphatase inhibitor vanadate by itself was able to induce PGHS-2 mRNA expression, which was further augmented when vanadate was combined with 5-HT. PGHS-2 mRNA expression is thus tightly regulated in mesangial cells and therefore allows modulation at various levels by physiological and pharmacological stimuli.
Stroebel
M
M
Department of Medicine IV, University of Erlangen-Nuernberg, Federal Republic of Germany.
Goppelt-Struebe
M
M
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D003513:Cycloheximide / Q000494:pharmacology; D017931:DNA Primers; D003609:Dactinomycin / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D005920:Glomerular Mesangium / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011450:Prostaglandin Endoperoxides, Synthetic / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis* / Q000235:genetics; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D051381:Rats; D012701:Serotonin / Q000502:physiology*; D015398:Signal Transduction; D013928:Thromboxane A2 / Q000031:analogs & derivatives / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism
D017931:DNA Primers; D007527:Isoenzymes; D011450:Prostaglandin Endoperoxides, Synthetic; D012333:RNA, Messenger; D003609:Dactinomycin; D012701:Serotonin; D013928:Thromboxane A2; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D000255:Adenosine Triphosphate; D003513:Cycloheximide; D011451:Prostaglandin-Endoperoxide Synthases; D011493:Protein Kinase C; D013936:Thymidine