Background: CCL2 is a cytokine of the CC chemokine family primarily secreted by monocytes, macrophages and dendritic cells. CCL2 has been shown to be elevated in patients with hepatitis C virus (HCV) induced liver diseases including fibrosis and cirrhosis. The promoter region of CCL2 gene in chromosome 17 contains regulatory sequences that bind to transcription factors implicated in chronic liver diseases. In this study, by sequence analysis of the CCL2 promoter using computational methods followed by biochemical assays we determined the transcription factors that regulate CCL2 expression and function.

Methods: Computational sequence analyses and reporter assays using the CCL2 promoter region were done to analyze transcription factors. Protein overexpression and gene knockdowns in human primary hepatocytes were carried out using cDNA plasmids and siRNA transfections respectively. Chromatin (ChIP) and protein (CoIP) immunoprecipitations were done to establish interactions between transcription factors and DNA.

Results: Reporter analysis using -2000 bp of the CCL2 promoter region demonstrated that its expression is regulated by the cytokine IL6 in human hepatocytes. Further, computational analysis of the CCL2 promoter identified putative binding sites for STAT3, PU.1 and CEBPΑ. Mutation in the STAT3 binding site abolished IL6 mediated activation of the CCL2 promoter (>3 fold). Overexpression of STAT3, PU.1 and CEBPΑ resulted in activation of the CCL2 promoter in hepatocytes (>10 fold) and their knockdown reduced its expression (>2 fold). Co-expression of STAT3, PU.1 and CEBPΑ demonstrated significantly higher level of activation (10-12 fold) compared to each alone (3-7 fold) indicating cooperation between these three factors in regulating CCL2 expression. CoIP experiments established a positive interaction between STAT3, PU.1 and CEBPΑ and ChIP showed sequence specific binding of these factors to the CCL2 promoter in hepatocytes.

Conclusions: Our results demonstrated that expression of the inflammatory biomarker CCL2 in human hepatocytes is regulated at the transcriptional level through a cooperative action by STAT3, PU.1 and CEBPΑ. Therefore, defining transcriptional regulatory mechanisms which control inflammation following HCV infection will be important in developing strategies towards preventing hepatic fibrosis following liver transplantation of HCV recipients.

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