Sp1 and Sp3 Transcription Factors Mediate Malondialdehyde-induced Collagen α1(I) Gene Expression in Cultured Hepatic Stellate Cells

Abstract

Malondialdehyde, the end product of lipid peroxidation, has been shown to stimulate collagen ␣1(I) (Col1a1) gene expression. However, mechanisms of this effect are unclear. The purpose of this study was to clarify these mechanisms. Rat hepatic stellate cells were cultured in the presence of 200 M malondialdehyde, and the effects on collagen gene expression and the binding of nuclear proteins to the col1a1 promoter were analyzed. Malondialdehyde treatment induced an increase in the cellular levels of col1a1 mRNA that was abrogated by pretreating cells with cycloheximide, p-hydroxymercuribenzoate, pyridoxal 5-phosphate, and mithramycin. Transient transfections showed that malondialdehyde exerted its effect through regulatory elements located between ؊220 and ؊110 bp of the col1a1 promoter. Gel retardation assays demonstrated that malondialdehyde increased the binding of nuclear proteins to two elements located between ؊161 and ؊110 bp of the col1a1 promoter. These bindings were supershifted with Sp1 and Sp3 antibodies. Finally, malondialdehyde increased cellular levels of the Sp1 and Sp3 proteins and Sp1 mRNA. Our data indicated that treatment of hepatic stellate cells with malondialdehyde stimulated col1a1 gene expression by inducing the synthesis of Sp1 and Sp3 and their binding to two regulatory elements located between ؊161 and ؊110 bp of the col1a1 promoter.Lipid peroxidation by-products, particularly reactive aldehydes, such as malondialdehyde (MDA) 1 and 4-hydroxy-2-nonenal (4HNE), have been incriminated in the pathogenesis of liver fibrosis. Thus, Chojkier et al.(1) first showed that MDA increased significantly the collagen ␣1(I) (Col1a1) mRNA in cultured human fetal fibroblasts, and Maher et al. (2) found that collagen synthesis doubled in response to MDA in rat kidney fibroblasts. Likewise, Parola et al. (3,4) showed that 4HNE and other 4-hydroxy-2,3-alkenals, aldehydic end products of lipid peroxidations, were able to stimulate col1a1 gene expression and collagen synthesis in cultured hepatic stellate cells (HSC). Similar results have been reported by Tsukamoto and co-workers (5, 6), who also observed a significant correlation between the liver MDA and 4HNE levels and the hepatic collagen accumulation in a rat model of alcoholic liver disease.Mechanisms by which these reactive aldehydes induce col1a1 gene expression and synthesis are still unclear. However, a number of studies have provided evidence supporting the role of aldehyde-protein adducts in the regulation of col1a1 gene expression. These aldehydes are known to react with sulfhydryl or amino groups to form aldehyde-protein adducts (7). These adducts have been found in alcoholic liver disease (8, 9) and other clinical conditions of chronic liver injury associated with active fibrogenesis, as well as in animal models of lipid peroxidation (6, 10 -14). Moreover, antioxidant treatment significantly decreased the production of these adducts and prevented the fibrogenesis cascade (10, 13, 15). The purpose of this study was to clarify the mechanisms ...

Role of Protein Kinase Cδ in Endothelin-Induced Type I Collagen Expression in Cardiac Myofibroblasts Isolated from the Site of Myocardial Infarction

“…Among them, Sp1 levels were significantly increased when compared with controls in these cells. Findings from previous studies have also demonstrated the Sp1/Sp3 association with the collagen biosynthetic pathway (Chen et al, 1998;Ruiz et al, 2000;Czuwara-Ladykowska et al, 2001;Garcia-Ruiz et al, 2002). Our RT-PCR data also demonstrate ET-induced expression of Sp1, whereas rottlerin and PD98059 attenuated the Fig.…”

“…One study on osteoblastic cells suggests the involvement of PKC and ERK pathways in TGF-␤ 1 -induced type I collagen expression (Palcy and Goltzman, 1999). Several previous reports have implicated the role of Sp1 and Sp3 in collagen transcription in various models (Li et al, 1995;Chen et al, 1998;Ihn and Tamaki, 2000;Ruiz et al, 2000;Czuwara-Ladykowska et al, 2001;Garcia-Ruiz et al, 2002). In the present study, we show a role for PKC, ERK1/2, and Sp1 in ET-induced type I collagen expression in cardiac myoFb.…”

Hepatic Stellate Cells: Protean, Multifunctional, and Enigmatic Cells of the Liver

“…The best-studied component of hepatic scar is collagen type I, the expression of which is regulated both transcriptionally and posttranscriptionally in hepatic stellate cells by a growing number of stimuli and pathways. A detailed review of collagen gene regulation in stellate cells is beyond the focus of this review, but several recent references are recommended (7,188,257,258,260,354,528,(617)(618)(619)652).…”

Reactive Oxygen Species as Initiators and Mediators of Cellular Signaling Processes

“…Similar to the aforementioned net activation of a signaling cascade following inhibition of a signaling protein, the inhibition and alkylation of the lipid and protein dual phosphatase PTEN (phosphatase and tensin homolog) was demonstrated to coincide with, and postulated to mediate, stimulation of PI3K/Akt signaling by exposure of cells to HNE [ 76 ]. Further examples of such alkylations by lipid peroxidation products inducing pathophysiologically relevant signaling processes include the stimulated expression of the proinfl ammatory protein cyclooxygenase-2 (COX-2) via stress-responsive kinase p38 MAPK upon exposure to HNE [ 77 ], or the stimulation of collagen expression (a crucial step in the pathogenesis of fi brosis) in fi broblasts [ 78 ] or hepatic stellate cells [ 79 ] following exposure to MDA.…”