Inhibition of transforming growth factor-beta-induced liver fibrosis by a retinoic acid derivative via the suppression of Col 1A2 promoter activity

Transforming growth factor-beta1 (TGF-beta1) mediates expression of collagen 1A2 (Col 1A2) gene via a synergistic cooperation between Smad2/Smad3 and Sp1, both act on the Col 1A2 gene promoter. In our previous study, we reported that a retinoic acid derivative obtained from Phellinus linteus (designated PL) antagonizes TGF-beta-induced liver fibrosis through regulation of ROS and calcium influx. In this continuing study we seek further the effect of PL on the Smad signaling pathway. We used a Col 1A2 promoter-luciferase construct to study the action of PL on Smad through TGF-beta. We found that PL decreases the promoter activity of Col 1A2, hinders the translocalization of phosphorylated Smad2/3-Smad 4 complex from cytosol into nucleus and inhibits Sp1 binding activity. These results suggest that PL inhibits TGF-beta1-induced Col 1A2 promoter activity through blocking ROS and calcium influx as well as impeding Sp1 binding and translocalization of pSmad 2/3-Smad4 complex into nucleus.     

Sp1 and Sp3 transcription factors mediate malondialdehyde-induced collagen alpha 1(I) gene expression in cultured hepatic stellate cells

Malondialdehyde, the end product of lipid peroxidation, has been shown to stimulate collagen alpha1(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 microm 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.     

B-Myb represses trans-activation of the Col5A2 collagen promoter indirectly via inhibition of binding of factors interacting with positive elements within the first exon.

B-myb, a member of the myb gene family, was originally isolated based on its high homology with c-myb in the DNA-binding domain. Previously we showed that B-myb is expressed in bovine vascular smooth muscle cells (SMCs) in a cell cycle-dependent fashion, and inhibits type I collagen gene promoter activity. Here, we have explored its role in regulation of another fibrillar collagen gene, Col5A2, encoding the (alpha2 chain of type V collagen. Ectopic expression of B-Myb decreased alpha 2(V) promoter activity and endogenous alpha 2(V) collagen mRNA levels. The responsive region of the alpha 2(V) collagen gene was localized to a fragment including 100 bp of basal promoter and 150 bp of exon 1 sequences, which contained two CRE-like elements. Binding to these elements increased upon deprivation of serum-growth factors, when expression of the Col5A2 gene is elevated, leading us to test their role despite the failure of excess unlabelled CRE oligonucleotide from the somatostatin gene to successfully compete for binding. Mutation of the elements significantly decreased the basal level of alpha2(V) collagen promoter activity and ablated inhibition by B-Myb. Furthermore, addition of B-Myb-glutathionine S-transferase fusion protein inhibited complex formation. Thus, these results confirm a major role for B-Myb in mediating intracellular signals controlling collagen gene expression in vascular SMCs. A model of indirect repression of the Col5A2 gene by B-Myb, via interaction with a positively-acting matrix regulatory factor, termed MRF-V, is discussed.