Disruption of an SP2/KLF6 Repression Complex by SHP Is Required for Farnesoid X Receptor-induced Endothelial Cell Migration

Amitava Das,Martin E Fernandez-Zapico,Sheng Cao,Janet Yao,Stefano Fiorucci,Robert P Hebbel,Raul Urrutia,Vijay H Shah

doi:10.1074/jbc.m607720200

Abstract

The farnesoid X receptor (FXR) signaling pathway regulates bile acid and cholesterol homeostasis. Here, we demonstrate, using a variety of gain- and loss-of-function approaches, a role of FXR in the process of cell motility, which involves the small heterodimeric partner (SHP)-dependent up-regulation of matrix metalloproteinase-9. We use this observation to reveal a transcriptional regulatory mechanism involving the SP/KLF transcription factors, SP2 and KLF6. Small interference RNA-based silencing studies in combination with promoter, gel shift, and chromatin immunoprecipitation assays indicate that SP2 and KLF6 bind to the matrix metalloproteinase-9 promoter and together function to maintain this gene in a silenced state. However, upon activation of FXR, SHP interacts with SP2 and KLF6, disrupting the SP2/KLF6 repressor complex. Thus, together, these studies identify a mechanism for antagonizing Sp/KLF protein repression function via SHP, with this process regulating endothelial cell motility.

Highlights

Bile acid, cholesterol, and triglyceride metabolism [1,2,3,4,5,6,7,8,9]
farnesoid X receptor (FXR) Regulates Cell Motility via the Up-regulation of matrix metalloproteinase-9 (MMP-9)—Our work initiated from the observation that FXR is expressed in BOECs and regulates cell motility via the up-regulation of MMP-9, a key celincubated with vehicle or CDCA (50 ␮M)
Samples were immunoprecipitated using aga- approach (Fig. 1A). These results were confirmed by qRT-PCR, rose-conjugated antibodies to SP2 or KLF6 (Santa Cruz Bio- which showed that the FXR ligand, CDCA, induced a technology, Santa, Cruz, CA), control (IgG) antibody, or concentration-dependent up-regulation of MMP-9 mRNA levagarose beads alone

Summary

Introduction

Bile acid, cholesterol, and triglyceride metabolism [1,2,3,4,5,6,7,8,9]. FXR heterodimerizes with the 9-cis-retinoic acid receptor ␣, which allows binding to a specific DNA sequence composed of two inverted hexamer repeats separated by one nucleotide (IR-1), thereby regulating target gene transcription (10 –12). Another indition was not observed in cells transfected with MMP-9 siRNA rect pathway occurs through transcriptional activation of the indicating that this molecule is required to trigger FXR- atypical non-DNA binding nuclear receptor SHP [13].

Results

Conclusion