Transforming growth factor β1 induces the expression of α1(i) procollagen mRNA by a hydrogen peroxide-C/EBPβ-dependent mechanism in rat hepatic stellate cells

✍ Scribed by Elena R. García-Trevijano; María J. Iraburu; Luis Fontana; José A. Domínguez-Rosales; Anitra Auster; Amador Covarrubias-Pinedo; Marcos Rojkind

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 230 KB
• Volume: 29
• Category: Article
• ISSN: 0270-9139
• DOI: 10.1002/hep.510290346

✦ Synopsis

Oxidative stress plays a key role in liver fibrosis. Both inflammatory cells and activated Kupffer cells produce H 2 O 2 , an oxidant involved in the activation of hepatic stellate cells (HSC). Increased production of reactive oxygen intermediates (ROIs) in fibrotic livers is associated in part with the up-regulation of transforming growth factor ␤ (TGF-␤), and this cytokine enhances collagen production by cultured HSC. However, the possible link between oxidative stress and the molecular mechanisms by which TGF-␤ induces collagen gene expression in HSC remains to be elucidated. To address this question, we investigated whether H 2 O 2 is a mediator of TGF-␤-elicited ␣1(I) collagen gene (col1a1) up-regulation. We demonstrated that TGF-␤ induces the accumulation of H 2 O 2 , and that this oxidant is, in turn, directly involved in up-regulating the expression of the col1a1 gene. While the addition of H 2 O 2 to HSC induced the expression of ␣1(I) procollagen mRNA, catalase, an H 2 O 2 enzyme scavenger, abrogated TGF-␤-mediated col1a1 gene up-regulation. We transfected HSC with chimeric plasmids driven by different segments of the mouse col1a1 promoter and mapped a cis-acting element (Ϫ370 to Ϫ344) essential for TGF-␤ responsiveness. We further showed that TGF-␤ induced the activation and binding of a C/EBP␤containing transcriptional complex to this sequence, an effect that was also mimicked by the addition of H 2 O 2 . Taken together, these data demonstrate a direct connection between TGF-␤-mediated accumulation of H 2 O 2 and the up-regulation of col1a1 gene in HSC. (HEPATOLOGY 1999;29: 960-970.)

Hepatic stellate cells (HSC) are the main producers of type I collagen in normal and cirrhotic liver. 1 These cells produce and secrete transforming growth factor ␤ (TGF-␤) and respond to this cytokine with increased expression of type I collagen. 2 TGF-␤ is a pleiotropic cytokine that plays a key role in liver fibrogenesis 3 and inhibits the proliferation of various cell types. 4,5 Its expression is increased in cirrhotic livers and in human liver specimens. 6-8 TGF-␤ mRNA values correlate with the extent of liver fibrosis. 7 Moreover, it has been shown that in transgenic mice, whose hepatocytes express active TGF-␤, there is increased accumulation of collagen in liver and kidney. 9 Oxidative stress also plays an important role in the development of liver fibrosis. 10 While increased production of reactive oxygen intermediates (ROIs) occurs in cirrhotic livers, certain antioxidants ameliorate the disease. [11][12][13][14][15] In addition to the ROIs produced by injured cells, 10 inflammatory cells as well as Kupffer cells produce H 2 O 2 . 10,[16][17][18][19][20] This oxidant is involved in TGF-␤-mediated inhibition of cell proliferation. 5 Although the increased production of ROIs during active fibrogenesis is associated with increased expression of TGF-␤, the possible link between oxidative stress and the molecular mechanisms by which this cytokine induces collagen gene expression in HSC remains to be elucidated.

Over the past several years, significant progress has been made in our understanding of critical signal transduction pathways induced by TGF-␤. 4,[21][22][23] Some cis-regulatory elements have been mapped within the promoters of the ␣1(I) and ␣2(I) collage genes, and several transcription factors that bind to them have been characterized. [24][25][26][27][28][29] However, the molecular mechanisms by which TGF-␤ enhances type I collagen expression are not completely understood and remain to be fully elucidated.

In view of these data, we considered it important to investigate, at the molecular level, the mechanisms by which TGF-␤ increases mouse ␣1(I) collagen (col1a1) gene transcription in HSC. For these studies, we used an HSC line derived from a CCl 4 -cirrhotic rat liver (CFSC-2G) that has a pheno-Abbreviations: HSC, hepatic stellate cells; TGF-␤ 1 , transforming growth factor ␤ 1 ; ROI, reactive oxygen intermediate; col1a1, mouse ␣1(I) procollagen gene; C/EBP␤, CCAAT/enhancer binding protein-␤; PDTC, pyrrolidine dithiocarbamate; EMSA, electrophoretic mobility shift assay; MEM, minimum essential medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; CAT, chloramphenicol acetyltransferase; TRE, TGF-␤ 1 -responsive element.