Effect of retinoic acid on the enhancing effect of acetaldehyde on mouse type I collagen expression

Acetaldehyde alone and retinoic acid alone have been shown to increase and decrease, respectively, collagen production by stellate cells in culture. In this study the effects of retinoic acid on alpha(1)(I) and alpha(2)(I) collagen expression and its influence on the enhancing effects of acetaldehyde were determined. Retinoic acid decreased the activation of the alpha(2)(I) collagen promoter and decreased the message of alpha(2)(I) collagen in cultured stellate cells, but had no effect on either the activation of the alpha(1)(I) collagen promoter or on the alpha(1)(I) collagen message. This depressant effect of retinoic acid was also evident in the transfected alpha(2)(I) collagen promoter mutated at the retinoic acid response element (RARE). The activation of the alpha(2)(I) collagen promoter by acetaldehyde was not decreased significantly by retinoic acid, but was suppressed by the retinoic acid receptor (RAR) selective retinoid SRI-6751-84. Retinoic acid, however, decreased the acetaldehyde-induced enhancement of the alpha(1)(I) and alpha(2)(I) collagen messages. Acetaldehyde also resulted in a decrease in RAR beta message and RARbeta protein. This study shows that retinoic acid depresses alpha(2)(I) collagen gene expression but that this effect is less pronounced when the expression of this collagen is enhanced by acetaldehyde, which also decreases RARbeta message and protein. Furthermore, the action of retinoic acid in inhibiting alpha(2)(I) collagen gene expression occurs at sites other than the RARE site.     

Two regulatory proteins that bind to the basic transcription element (BTE), a GC box sequence in the promoter region of the rat P-4501A1 gene.

The cDNAs for two DNA binding proteins of BTE, a GC box sequence in the promoter region of the P-450IA1(CYP1A1) gene, have been isolated from a rat liver cDNA library by using the BTE sequence as a binding probe. While one is for the rat equivalent to human Sp1, the other encodes a primary structure of 244 amino acids, a novel DNA binding protein designated BTEB. Both proteins contain a zinc finger domain of Cys-Cys/His-His motif that is repeated three times with sequence similarity of 72% to each other, otherwise they share little or no similarity. The function of BTEB was analysed by transfection of plasmids expressing BTEB and/or Sp1 with appropriate reporter plasmids into a monkey cell line CV-1 and compared with Sp1. BTEB and Sp1 activated the expression of genes with repeated GC box sequences in promoters such as the simian virus 40 early promoter and the human immunodeficiency virus-1 long terminal repeat promoter. In contrast, BTEB repressed the activity of a promoter containing BTE, a single GC box of the CYP1A1 gene that is stimulated by Sp1. When the BTE sequence was repeated five times, however, BTEB turned out to be an activator of the promoter. RNA blot analysis showed that mRNAs for BTEB and Sp1 were expressed in all tissues tested, but their concentrations varied independently in tissues. The former mRNA was rich in the brain, kidney, lung and testis, while the latter was relatively abundant in the thymus and spleen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Curcumin suppresses the expression of extracellular matrix genes in activated hepatic stellate cells by inhibiting gene expression of connective tissue growth factor

Upon liver injury, quiescent hepatic stellate cells (HSCs), the most relevant cell type for hepatic fibrogenesis, become active and overproduce extracellular matrix (ECM). Connective tissue growth factor (CTGF) promotes ECM production. Overexpression of CTGF during hepatic fibrogenesis is induced by transforming growth factor (TGF)-beta. We recently demonstrated that curcumin reduced cell growth and inhibited ECM gene expression in activated HSCs. Curcumin induced gene expression of peroxisome proliferator-activated receptor (PPAR)-gamma and stimulated its activity in activated HSCs, which was required for curcumin to suppress ECM gene expression, including alphaI(I)-collagen. The underlying mechanisms remain largely unknown. The aim of this study was to elucidate the mechanisms by which curcumin suppresses alphaI(I)-collagen gene expression in activated HSCs. We hypothesize that inhibition of alphaI(I)-collagen gene expression in HSCs by curcumin is mediated by suppressing CTGF gene expression through attenuating oxidative stress and interrupting TGF-beta signaling. The present report demonstrated that curcumin significantly reduced the abundance of CTGF in passaged HSCs and suppressed its gene expression. Exogenous CTGF dose dependently abrogated the inhibitory effect of curcumin. Activation of PPAR-gamma by curcumin resulted in the interruption of TGF-beta signaling by suppressing gene expression of TGF-beta receptors, leading to inhibition of CTGF gene expression. The phytochemical showed its potent antioxidant property by significantly increasing the level of total glutathione (GSH) and the ratio of GSH to GSSG in activated HSCs. De novo synthesis of cellular GSH was a prerequisite for curcumin to interrupt TGF-beta signaling and inhibited gene expression of CTGF and alphaI(I)-collagen in activated HSCs. Taken together, our results demonstrate that inhibition of alphaI(I)-collagen gene expression by curcumin in activated HSCs results from suppression of CTGF gene expression through increasing cellular GSH contents and interruption of TGF-beta signaling. These results provide novel insights into the mechanisms underlying inhibition of HSC activation by curcumin.     

ERK is involved in EGF-mediated protection of tight junctions, but not adherens junctions, in acetaldehyde-treated Caco-2 cell monolayers

The role of mitogen-activated protein kinases (MAPK) in the mechanism of EGF-mediated prevention of acetaldehyde-induced tight junction disruption was evaluated in Caco-2 cell monolayers. Pretreatment of cell monolayers with EGF attenuated acetaldehyde-induced decrease in resistance and increase in inulin permeability and redistribution of occludin, zona occludens-1 (ZO-1), E-cadherin, and β-catenin from the intercellular junctions. EGF rapidly increased the levels of phospho-ERK1/2, phospho-p38 MAPK, and phospho-JNK1. Pretreatment of cell monolayers with U-0126 (inhibitor of ERK activation), but not SB-202190 and SP-600125 (p38 MAPK and JNK inhibitors), significantly attenuated EGF-mediated prevention of acetaldehyde-induced changes in resistance, inulin permeability, and redistribution of occludin and ZO-1. U-0126, but not SB-202190 and SP-600125, also attenuated EGF-mediated prevention of acetaldehyde effect on the midregion F-actin ring. However, EGF-mediated preservation of junctional distribution of E-cadherin and β-catenin was unaffected by all three inhibitors. Expression of wild-type or constitutively active MEK1 attenuated acetaldehyde-induced redistribution of occludin and ZO-1, whereas dominant-negative MEK1 prevented EGF-mediated preservation of occludin and ZO-1 in acetaldehyde-treated cells. MEK1 expression did not alter E-cadherin distribution in acetaldehyde-treated cells in the presence or absence of EGF. Furthermore, EGF attenuated acetaldehyde-induced tyrosine-phosphorylation of occludin, ZO-1, claudin-3, and E-cadherin. U-0126, but not SB-202190 and SP-600125, prevented EGF effect on tyrosine-phosphorylation of occludin and ZO-1, but not claudin-3, E-cadherin, or β-catenin. These results indicate that EGF-mediated protection of tight junctions from acetaldehyde requires the activity of ERK1/2, but not p38 MAPK or JNK1/2, and that EGF-mediated protection of adherens junctions is independent of MAPK activities.