Abstract 5554: Runx2 Represses TGFβ/Smad-Mediated Connective Tissue Growth Factor Gene Expression in Vascular Smooth Muscle Cells

Abstract

[Background] Runx2, a key transcription factor for osteoblastic differentiation, is expressed in calcified atherosclerotic plaques. We recently reported that Runx2 represses vascular smooth muscle cells (VSMC) differentiation and promotes its osteogenic differentiation. Connective tissue growth factor (CTGF) has been implicated in the progression to vulnerable plaque by inducing mononuclear cell chemotaxis and VSMC apoptosis despite of its potent stimulatory effect on synthesis of extracellular matrix. In this study, we investigated the regulatory mechanism of CTGF gene expression by Runx2 in VSMC. [Methods and Results] RT-PCR analyses showed that adenovirally overexpressed Runx2 significantly repressed the basal expression of the CTGF gene in human aortic SMCs (HASMCs). Consistent with this, knockdown of the Runx2 expression in HASMCs by small interfering RNA (siRNA) increased CTGF mRNA levels. Luciferase assays showed that Runx2 reduced the transcriptional activity of the CTGF promoter. Transfection of a series of 5′-deletion constructs revealed that Runx2 inhibited CTGF expression through the sequence element located at 5′ untranslated region of CTGF mRNA. We next examined the effects of Runx2 on the TGFβ-induced CTGF expression. Runx2 overexpression significantly attenuated the TGFβ-mediated induction of CTGF expression in HASMCs, and knockdown of Runx2 by siRNA enhanced the induction of CTGF expression in response to TGFβ. Runx2 repressed TGFβ-induced CTGF promoter activity through the sequence containing Smad binding element (SBE), and luciferase assay using SBE-specific mutation construct showed that Runx2 repressed CTGF promoter activity in an SBE-dependent manner. Overexpression of Runx2 significantly reduced TGFβ and Smad3-mediated luciferase activity of 4xSBE-tkLuc, which contains four copies of SBE. Co-immunoprecipitation showed that Runx2 formed a complex with Smad4. [Conclusion] These data demonstrate that Runx2 represses basal and TGFβ-induced CTGF gene expression in VSMC, and thus suggest that besides the potential for inducing vascular calcification, Runx2 may affect plaque stability by modulating extracellular matrix synthesis through inhibiting CTGF gene expression and TGFβ signaling.