Dexamethasone alters messenger RNA levels but not synthesis of collagens, fibronectin, or laminin by cultured rat fat-storing cells

Abstract

Glucocorticoids have been shown to suppress collagen synthesis and gene expression by fibroblasts. However, little is known about their effects on fat-storing cells, the major matrix-producing cells in liver fibrosis. In this study we investigated the effect of dexamethasone on the extracellular matrix expression by cultured rat fat-storing cells. Fat-storing cells were isolated from male Wistar rats by collagenase/pronase digestion and purified by density gradient centrifugation. Fat-storing cells in early primary culture (3-day-old, representing a relatively quiescent phenotype) and in subculture (one passage, about 2-week-old, representing an activated phenotype) were treated with 10(-6) mol/L dexamethasone for messenger RNA (mRNA) study or with 10(-8) to 10(-6) mol/L dexamethasone for protein study. Expression of collagen type I, III, IV, fibronectin, and laminin was analyzed at the mRNA level by Northern hybridization, and at the protein level by metabolic labeling and immunoprecipitation. Dexamethasone had a variable effect on the expression of collagen alpha1(I) mRNA level. While a tendency for modest suppression was observed (5%-50%) in primary cells, the difference was not statistically significant. Variable response was observed in subcultured cells. Collagen alpha1(III) mRNA level showed a tendency for stimulation. Dexamethasone stimulated the expression of collagen alpha1 (IV), fibronectin, and laminin B1 mRNA levels by 1.4-, 2.4-, and 1.6-fold respectively, in primary fat-storing cells. Subcultured cells showed a similar response, but the magnitude of stimulation was more variable than that of primary cells. Unexpectedly, at the protein level dexamethasone had no effect on the expression of these proteins. Our results indicate that glucocorticoids do not possess a net suppressive effect on extracellular matrix synthesis by fat-storing cells. Beneficial effects of glucocorticoids may be attributable to other mechanisms of action, such as their anti-inflammatory effect.

Antifibrogenic effects of canrenone, an antialdosteronic drug, on human hepatic stellate cells

“…14,31 Briefly, subconfluent cultures were incubated 24 hours with SFIF medium before treatment with canrenone, TGF-␤1, or both. One group of HSC was treated with 25 mol/L of canrenone, and another group was exposed to 1 ng/mL of TGF-␤1.…”

“…32 For collagen type I and IV, incubation with the primary antibody was followed by incubation with affinity-purified rabbit anti-goat immunoglobulin G (Jackson Immunochemicals, West Grove, PA) as a second antibody for 1 hour at 4°C. 14,31 After immunoprecipitation, proteins were separated by SDS-PAGE, followed by enhancement with Amplify (Amersham), dried, and exposed to preflashed Hyperfilm-MP (Amersham). Band intensities were quantified by Phosphor Imaging (Molecular Imager, GS-525; Bio-Rad, Hercules, CA).…”

A histone deacetylase inhibitor, trichostatin A, suppresses myofibroblastic differentiation of rat hepatic stellate cells in primary culture

“…Cells at day 3 after isolation (primary cells) or passaged cells at day 14 after isolation (subcultured cells) were exposed to 0.01 to 1 mmol/L sodium butyrate or 1 to 100 nmol/L TSA for 24 hours. Subsequently, cells were metabolically labeled for 24 hours using 25 µCi/mL of Trans 35 S-label (specific activity of 35 S-methionine Ͼ1,000 Ci/mmol; ICN Biomedicals, Costa Mesa, CA) while exposure to test compounds continued. Media and cell layers were harvested separately as described previously.…”

“…Media and cell layers were harvested separately as described previously. 35 Total incorporation of Trans 35 S-label into protein was determined by the hot trichloroacetic acid (TCA) precipitation method. 35,36 Total incorporation in media and cell layers was expressed per 9.6-cm 2 culture dish.…”

“…Labeled medium or cell layer was then subjected to immunoprecipitation as described, 35 using antibodies against collagen type I (Southern Biotechnology, Birmingham, AL), type III, 37,38 or smooth muscle ␣-actin (Sigma, Little Chalfort, UK). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).…”