Abstract 5890: Runx2 Inhibits TGFβ-induced Connective Tissue Growth Factor Gene Expression Through Physical Association With Smad3 in Vascular Smooth Muscle Cells
[Background]Balance between the synthesis and degradation of extracellular matrix (ECM) by vascular smooth muscle cells (VSMC) is a critical determinant of the plaque stability. We recently reported that Runx2, a key transcription factor for osteoblastic differentiation, represses 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 ECM. In this study, we investigated the regulatory mechanism of CTGF gene expression by Runx2 in VSMC.
[Methods and Results] 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. Co-immunoprecipitation showed that Runx2 formed a complex with Smad3. Immunohistochemistry of human athersclerotic plaque revealed that Runx2-positive cells stained negative for CTGF.
[Conclusion]Results of this study indicate that Runx2 inhibits TGFβ-induced CTGF gene expression via physical association with Smad3 in VSMC. These findings imply that besides the potential for inducing vascular calcification, Runx2 may promote plaque instability by reducing TGFβ-dependent ECM synthesis.