Hepatic stellate cells are the major cellular sources of extracellular matrix in chronic liver diseases leading to fibrosis. We explored the antifibrogenic effect of two histone deacetylase inhibitors, sodium butyrate and trichostatin A (TSA), on this cell type in vitro. Primary hepatic stellate cells as well as culture activated cells were exposed to butyrate (0.01-1 mmol/L) or TSA (1-100 nmol/L); their effect on collagen types I and III and smooth muscle β£-actin was examined by quantitative immunoprecipitation and by Northern analysis. Their antiproliferative effect was examined by 3 H-thymidine incorporation and cell counting. Hyperacetylation of histones was demonstrated by acid urea/Triton-X-100 (AUT) polyacrylamide gel electrophoresis. Possible cytotoxic effects were judged on stellate cells by evaluating de novo total protein synthesis, and on hepatocytes by measuring lactate dehydrogenase (LDH) leakage, albumin secretion, and epoxide hydrolase and ethoxycoumarin O-deethylase activity. TSA at 100 nmol/L and butyrate at 1 mmol/L retarded the morphological changes characteristic for activation of primary stellate cells. TSA at 100 nmol/L inhibited synthesis of collagen types I and III and smooth muscle β£-actin by 62%, 70%, and 88%. Butyrate at 1 mmol/L showed a modest inhibitory effect on collagen type III and smooth muscle β£-actin, but had no effect on collagen type I. Northern analysis suggested that these inhibitory effects on collagen type III and smooth muscle β£-actin were transcriptional, while the effect on collagen type I was largely posttranscriptional. At 100 nmol/L, TSA strongly suppressed proliferation of primary hepatic stellate cells. Inhibition of activation of stellate cells was preceded by hyperacetylation of histone H4. When tested on cells at day 14 in culture, butyrate had no inhibitory effects on the synthesis of collagens or smooth muscle β£-actin. One hundred or 10 nmol/L TSA modestly inhibited the synthesis of collagens type I (Οͺ24%,Οͺ22%) and III (Οͺ34%,Οͺ22%), and smooth muscle β£-actin (Οͺ27%,Οͺ12%). We conclude that TSA inhibits transdifferentiation of stellate cells into myofibroblasts by interfering with the level of acetylation of histone H4. (HEPATOLOGY 1999;29:858-867.)
Eukaryotic gene expression has been mainly studied in the context of trans-acting transcription factors and their interaction with regulatory cis-elements. Evidence is accumulating, however, that the high-order structure of chromatin plays an essential role in eukaryotic gene regulation. [1][2][3] One major process by which chromatin structure is modulated is histone acetylation. Lysine residues in the N-terminal domain of histone molecules are reversibly acetylated, the level of which is determined by an equilibrium between histone acetyltransferases and deacetylases. 4 It is thought that acetylation of lysine reduces the positive charge of the N-terminal tail domain of histones to weaken their interaction with DNA in the nucleosome. 2,4 Genetic, biochemical, and immunological studies have shown that actively transcribed genes are generally associated with hyperacetylated histones. 4 Use of specific pharmacological inhibitors of histone deacetylases may provide further insight. Currently known inhibitors include butyrate, 5 trichostatin A (TSA), 6 cyclic tetrapeptides such as trapoxin and apicidin, 7,8 trichostatin/peptide hybrid molecules, 9,10 and depudecin. 11 Sodium butyrate is a natural 4-carbon fatty acid that inhibits histone deacetylase in a noncompetitive manner, and requires millimolar concentrations for its biological activities. 5 TSA and the cyclic tetrapeptides are specific inhibitors of histone deacetylase, and are much more potent than sodium butyrate, being effective in the submicromolar range. 6,7 TSA is a reversible inhibitor, 6 whereas trapoxin inhibits histone deacetylase in an irreversible manner. 7 Experiments from many laboratories have shown several beneficial effects of these histone deacetylase inhibitors. Butyrate inhibits cell proliferation and causes differentiation in many tumorous and nontumorous cells. 5,12 Butyrate stimulates gene expression of albumin while inhibiting that of Abbreviations: TSA, trichostatin A; PBS, phosphate-buffered saline; TCA, trichloroacetic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AUT, acid urea/Triton-X-100; LDH, lactate dehydrogenase; ECOD, 7-ethoxycoumarin O-deethylase; mEH, microsomal epoxide hydrolase.