Down-regulation Of Monocyte Chemoattractant Protein-1 Expression By Farnesoid X Receptor

The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily. Bile acid is the physiological ligand of FXR(hence the name bile acid receptor). FXR is highly expressed in liver, intestine, kidney and adrenal glands and also can be detected in heart and fat tissues. It regulates target gene expression by binding to FXR response elements (FXREs), after heterodimerization with the retinoid X receptor (RXR). FXR plays an essential role in feedback regulation of bile acid biosynthesis and maintaining cholesterol, glucose homeostasis. However, more recent work has expanded the scope of this nuclear receptor system into a diversity of cell types and functions including vascular wall cells and immune cells. FXR-based therapy may also benefit from its direct effect on vasculature via regulating the expression of vasoactive mediators.Monocyte chemoattractant protein–1 (MCP-1) is produced predominantly by macrophages and endothelial cells and is a potent chemotactic factor for monocytes. Expression of this proinflammatory chemokine is increased in atherosclerotic lesions, and inhibition of its expression or that of its receptor, CC chemokine receptor 2 (CCR2), reduces the extent of atheroma formation in hypercholesterolemic mice. These observations indicate that MCP-1 plays an important role in atherogenesis. Recent studies have revealed important insight into regulation of the mouse MCP-1 expression. Regions essential for high basal level of MCP-1 promoter activity in cells include binding sites for transcription factors such as activator protein (AP)-1, nuclear factor (NF)-кB and SP/KLF family proteins. And these transcription factors have also been shown to be involved in MCP-1 regulation in macrophages that are stimulated with either lipopolysaccharide(LPS) or cytokines. Interestingly, MCP-1 expression has been shown to be negatively regulated by several members of the nuclear receptor superfamily of ligand-activated transcription factors including Liver X receptors (LXRs), retinoic acid receptor (RAR) and peroxisome proliferator-activated receptors (PPARs).With more and more recent work expanding the scope of FXR into a diversity of cell types and functions, and especially the important role of FXR in inflammation and AS, we investigate whether FXR regulates MCP-1 expression. We carried out this study, and the major results are summarized below:1. Using RT-PCR and Western blotting analysis, it was investigated that FXR was constitutively expressed in murine macrophage cell line ANA-1. Immunostaining showed that FXR localized in the nucleus and cytoplasm of ANA-1 cells. Western blot revealed that activated by its agonist CDCA, FXR acted locally in ANA-1 cells in a positive auto-regulatory fashion.2. To study the transcriptional regulation of MCP-1 by FXR, RT-PCR and real-time PCR revealed that treatment with FXR agonist CDCA for 12h and 24h of ANA-1 cells decreased transcription level of MCP-1. And MCP-1 mRNA expression decreased in a CDCA(0, 25, 50, 100μM) dose-dependent manner in ANA-1 cells. Results from Western blot and ELISA showed that CDCA(0, 25, 50, 100μM) treatment also caused a dose-dependent decrease of protein level of MCP-1 in ANA-1 cells.3. The 2.7 kb 5′flanking region of MCP-1 promoter was amplified from genomic DNA of ANA-1 cells and used as template to create two different 5′deletion constructs of the MCP-1 promoter which were cloned into pGL3-Basic luciferase vector. They were transiently transfected into HEK293 cells with or without FXR and RXR expression vectors by Liposome. Functional assay showed that activity of the two deleted MCP-1 promoters were equivalently inhibited by CDCA(50μM) activated FXR in HEK293 cells.In summary, we find that FXR is expressed in murine macrophage cell line ANA-1. Activated by its agonist CDCA, FXR act locally in ANA-1 cells in an autoregulatory fashion. Meanwhile CDCA treatment decreases MCP-1 mRNA and protein level in ANA-1 cells. And promoter analysis indicates that FXR may down-regulate transcriptional activation of MCP-1 gene in a direct or indirect way.