Abstract
Deletion of the Habp2 gene encoding Factor VII-activating protease (FSAP) increases liver fibrosis in mice. A single nucleotide polymorphism (G534E) in HABP2 leads to lower enzymatic activity and is associated with enhanced liver fibrosis in humans. Liver fibrosis is associated with a decrease in FSAP expression but, to date, nothing is known about how this might be regulated. Primary mouse hepatocytes or the hepatocyte cell line, AML12, were treated with different factors, and expression of FSAP was determined. Of the various regulatory factors tested, only transforming growth factor-β (TGF-β) demonstrated a concentration- and time-dependent inhibition of FSAP expression at the mRNA and protein level. The TGF-β-Type I receptor (ALK-5) antagonist SB431542 and Smad2 siRNA, but neither SIS3, which inhibits SMAD3, nor siRNA against Smad3 could block this effect. Various regions of the HABP2 promoter region were cloned into reporter constructs, and the promoter activity was determined. Accordingly, the promoter activity, which could phenocopy changes in Habp2 mRNA in response to TGF-β, was found to be located in the 177-bp region upstream of the transcription start site, and this region did not contain any SMAD binding sites. Mutation analysis of the promoter and chromatin immunoprecipitation assays were performed to identify an important role for the ATF3 binding element. Thus, TGF-β is the most likely mediator responsible for the decrease in FSAP expression in liver fibrosis.
Highlights
Liver fibrosis arises because of varying etiologies such as viral infections, alcohol abuse, fatty diet, and or ingestion/accumulation of toxins [1]
Human genetic epidemiological studies have shown that a single nucleotide polymorphism (SNP) in the FSAP-encoding gene (HABP2), rs7080536 or G534E called the Marburg I (MI) polymorphism is found in about 5% of the European population and it is associated with the severity of liver fibrosis [6]
TGF- Is an Inhibitor of Habp2 Expression in Primary Hepatocytes and AML12 Cells—To identify factors that regulate the expression of Habp2 in hepatocytes we tested a range of factors on primary mouse hepatocytes and the mouse hepatocyte cell line AML12 cells
Summary
TGF- Is an Inhibitor of Habp Expression in Primary Hepatocytes and AML12 Cells—To identify factors that regulate the expression of Habp in hepatocytes we tested a range of factors on primary mouse hepatocytes and the mouse hepatocyte cell line AML12 cells. We investigated the role of single transcription factor binding sites in basal expression using ATF3, AP-1, HNF1␣, HNF3, and C/EBP␦ deletion mutants of the TGF--responsive Ϫ1/Ϫ177 fragment (Fig. 8A). Deletion of HNF1 and HNF3 showed reduced response to TGF- treatment and a near complete loss of TGF- response was detected after deletion of the ATF3 site These results were confirmed by determining the activity of short fragments containing only the ATF3 transcription factor binding element (Ϫ120/Ϫ177, Ϫ152/Ϫ177). Both fragments showed increased activity upon SB431542 treatment and decreased activity after TGF- treatment (supplemental Fig. S6) This suggests that ATF3 is an important transcription factor binding element in the TGF--mediated down-regulation of Habp expression in hepatocytes
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