Glucocorticoids induce a near-total suppression of hyaluronan synthase mRNA in dermal fibroblasts and in osteoblasts: a molecular mechanism contributing to organ atrophy.
Glucocorticoid (GC) administration induces atrophy of skin, bone, and other organs, partly by reducing tissue content of glycosaminoglycans, particularly hyaluronic acid (HA). We took advantage of the recent cloning of the three human hyaluronan synthase (HAS) enzymes (HAS1, HAS2 and HAS3), to explore the molecular mechanisms of this side effect. Northern and slot blots performed on RNA extracted from cultured dermal fibroblasts and the MG-63 osteoblast-like osteosarcoma cell line indicated that HAS2 is the predominant HAS mRNA in these cells. Incubation of both cell types for 24 h in the presence of 10(-6) M dexamethasone (DEX) resulted in a striking 97--98% suppression of HAS2 mRNA levels. Time-course studies in fibroblasts demonstrated suppression of HAS2 mRNA to 28% of control by 1 h, and to 1.2% of control by 2 h, after addition of DEX. Dose-response studies in fibroblasts indicated that the majority of the suppressive effect required concentrations characteristic of cell-surface GC receptors, a point confirmed by persistent DEX-induced suppression in the presence of RU486, an antagonist of classic cytosolic steroid hormone receptors. Nuclear run-off experiments showed a 70% suppression of HAS2 gene transcription in nuclei from DEX-treated fibroblasts, which is unlikely to fully explain the rapid 50--80-fold reduction in message levels. Experiments with actinomycin D (AMD) demonstrated that the message half-life was 25 min in cells without DEX, whereas the combination of AMD with DEX dramatically increased the half-life of HAS2 mRNA, suggesting that DEX acts by inducing a short-lived destabilizer of the HAS2 message. Direct assessment of HAS2 mRNA stability by wash-out of incorporated uridine label established a half-life of 31 min in cells without DEX, which substantially shortened in the presence of DEX. In conclusion, GCs induce a rapid and sustained, near-total suppression of HAS2 message levels, mediated through substantial decreases in both gene transcription and message stability. These effects may contribute to the loss of HA in GC-treated organs.
Zhang
W
W
Department of Dermatology, University of Pennsylvania, Philadelphia V.A. Hospital, 2 Rhoads Pavilion, 36th and Spruce, Philadelphia, PA 19104, USA.
Watson
C E
CE
Liu
C
C
Williams
K J
KJ
Werth
V P
VP
D001284:Atrophy / Q000201:enzymology* / Q000378:metabolism*; D015152:Blotting, Northern; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003609:Dactinomycin / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000201:enzymology* / Q000473:pathology; D005938:Glucocorticoids / Q000494:pharmacology*; D014453:Glucuronosyltransferase / Q000378:metabolism*; D016695:Glycosyltransferases; D006727:Hormone Antagonists / Q000494:pharmacology; D006801:Humans; D000076002:Hyaluronan Synthases; D008565:Membrane Proteins; D015735:Mifepristone / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010006:Osteoblasts / Q000201:enzymology* / Q000473:pathology; D012516:Osteosarcoma / Q000201:enzymology / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism*; D012867:Skin / Q000201:enzymology* / Q000473:pathology; D013997:Time Factors; D014158:Transcription, Genetic; D014166:Transferases; D014407:Tumor Cells, Cultured; D014529:Uridine / Q000378:metabolism; D029867:Xenopus Proteins
D005938:Glucocorticoids; D006727:Hormone Antagonists; D008565:Membrane Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D012333:RNA, Messenger; D029867:Xenopus Proteins; D003609:Dactinomycin; D015735:Mifepristone; D003907:Dexamethasone; D014166:Transferases; D016695:Glycosyltransferases; D014453:Glucuronosyltransferase; C056944:HAS1 protein, Xenopus; C101754:HAS2 protein, human; C105458:HAS3 protein, human; C491096:Has2 protein, Xenopus; D000076002:Hyaluronan Synthases; D014529:Uridine