title: Glucocorticoids modulate the synthesis and expression of a 72 kDa heat shock protein in cultured Graves' retroocular fibroblasts.
abstract: Glucocorticoids modulate numerous proliferative, metabolic and immunological functions in human fibroblasts, some of which appear to be mediated via glucocorticoid receptors. We studied the influence of glucocorticosteroids on the synthesis and expression of a 72 kDa heat shock protein that is thought to play a role in thyroid autoimmunity. Experiments were performed using orbital fibroblasts derived from patients with Graves' ophthalmopathy and normal individuals. Cell monolayers were exposed to various concentrations of dexamethasone, the specific glucocorticoid agonist RU 28362, the glucocorticoid antagonist RU 38486, or combinations thereof, prior to heat stress or exposure to hydrogen peroxide. Heat shock protein 72 expression was assessed using sodium dodecylsulfate polyacrylamide-gel electrophoresis of cellular extracts, followed by autoradiography or immunoblotting with a mouse monoclonal antibody against the 72 kDa heat shock protein and quantitative scanning densitometry. In addition, cellular distribution of the immunoreactivity for the 72 kDa heat shock protein was studied using indirect immunofluorescence on parallel cultures. In other experiments, aimed at studying heat shock protein synthesis, cell cultures were pulse-labeled with [35S]-methionine prior to harvesting. Treatment with dexamethasone or RU 28362 markedly attenuated the heat stress-enhanced synthesis and expression of the 72 kDa heat shock protein and several other heat shock proteins both in normal and in Graves' retroocular fibroblasts (p < 0.001). In addition, either treatment reduced baseline expression of the 72 kDa heat shock protein in Graves' retroocular fibroblasts (p < 0.01). These effects were dose-dependent and appeared to be mediated via the glucocorticoid receptor, because combined exposure to dexamethasone or RU 28362 plus RU 38486 completely restored synthesis and expression of the 72 kDa heat shock protein. Baseline or stress-enhanced expression of the 72 kDa heat shock protein was not altered by treatment of monolayers with RU 38486 alone. As demonstrated by immunofluorescence, the characteristic intracellular shifting of the 72 kDa heat shock protein in response to cellular stress was partially inhibited by glucocorticoid agonists and restored by simultaneous exposure to glucocorticoid agonists and RU 38486. These results demonstrate that dexamethasone and the specific glucocorticoid agonist RU 28362 can modulate baseline- and stress-induced synthesis and expression of the 72 kDa heat shock protein, as well as its subcellular distribution, in cultured retroocular fibroblasts. Our studies suggest that these compounds exert these effects via the glucocorticoid receptor.
authors.affiliation: Department of Internal Medicine, Mayo Clinic/Foundation, Rochester, MN 55905.
authors.forename: A E
authors.initials: AE
authors.lastname: Heufelder
authors.forename: B E
authors.initials: BE
authors.lastname: Wenzel
authors.forename: R S
authors.initials: RS
authors.lastname: Bahn
mesh_terms: D000732:Androstanols / Q000494:pharmacology; D015153:Blotting, Western; D002478:Cells, Cultured; D003238:Connective Tissue / Q000378:metabolism / Q000473:pathology; D003907:Dexamethasone / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005123:Eye / Q000378:metabolism / Q000473:pathology*; D005347:Fibroblasts / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006111:Graves Disease / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D006360:Heat-Shock Proteins / Q000096:biosynthesis*; D006358:Hot Temperature / Q000009:adverse effects; D006801:Humans; D015735:Mifepristone / Q000494:pharmacology; D011965:Receptors, Glucocorticoid / Q000502:physiology
chemical_list: D000732:Androstanols; D006360:Heat-Shock Proteins; D011965:Receptors, Glucocorticoid; D015735:Mifepristone; C049154:11,17-dihydroxy-6-methyl-17-(1-propynyl)androsta-1,4,6-triene-3-one; D003907:Dexamethasone