Antiapoptotic and antioxidative protein ALR in Cholestatic Liver Diseases – Do bile acids regulate ALR expression via Egr1?

Cholestatic liver diseases result when the excretion of bile acids from the liver is interrupted. It has been shown that inflammation contributes to liver injury during cholestasis and previous studies indicated that bile acids up- regulate early growth response fator-1 (Egr1) through the activation of MAPK signaling pathway. Egr1 is responsible for the development of the inflammation through up-regulation of several pro-inflammatory genes. Augmenter of liver regeneration (ALR) is a hepatotrophic factor with anti- oxidative and anti-apoptotic properties which has been found to be highly expressed in regenerating livers after tissue loss or insults through toxic substances. Nevertheless, little is known about the impact of ALR in cholestasis. Therefore, the aim of our study is to investigate the potential role of bile acids in regulating ALR expression as a protective protein in cholestasis, and to determine which transcription factors might regulate the tissue-specific expression of ALR.

Promoter studies of ALR gene (-733/+527) revealed several potential regulatory elements of Egr1. Luciferase assay was performed using two different constructs of ALR promoter cloned into pGL2 vector (-733/+240, -733/+527). The assay revealed that co-transfection with Egr1 significantly induced the activity of only one of the two ALR promoter constructs (-733/+527) in both HepG2 and Huh7 cells. In addition, co-transfection with a dominant negative EGR1 (dnEGR1) expression plasmid revealed no activity of either promoter constructs. Furthermore, different bile acids (known to be associated with cholestasis) were used to analyze their potential for ALR promoter activation. To further verify the results of the luciferase assay, an in-vitro model was used to imitate cholestasis. Expression of ALR in HepG2 and Huh7 cells after bile acid treatment was analyzed on mRNA and protein levels using qPCR and western blot techniques respectively. Additionally we investigate the role of Egr1 in regulating ALR expression by preforming EMSA analysis.

Corresponding author: Weiss, Thomas

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