Transforming growth factor-β/Smads signaling induces transcription of the cell type-restricted ankyrin repeat protein CARP gene through CAGA motif in vascular smooth muscle cells

Transforming growth factor (TGF)-beta plays a major role in the development of vascular diseases. Despite the pleiotropic effects of TGF-beta on vascular smooth muscle cells (VSMCs), only a few genes have been characterized as direct targets of TGF-beta in VSMCs. Cardiac ankyrin repeat protein (CARP) has been thought to be expressed exclusively in the heart. In the present study, we showed that CARP is expressed in the vasculature after balloon injury and in cultured VSMCs in response to TGF-beta. Analysis of a half-life of the cytoplasmic CARP mRNA levels and the transient transfection of the CARP promoter/luciferase gene indicates that the regulation of CARP expression is increased by TGF-beta at the transcriptional level. Transfection of expression vectors encoding Smads significantly activated the CARP promoter/luciferase activity. Deletion analysis and site-specific mutagenesis of the CARP promoter indicate that TGF-beta response element is localized to CAGA motif at -108 bp relative to the transcription start site. Electrophoretic mobility shift assays showed that the binding activity to the CAGA motif was increased in nuclear extracts of cultured VSMCs by TGF-beta. Cells transfected with adenovirus vector expressing CARP showed a significant decrease in DNA synthesis. Overexpression of CARP enhanced the TGF-beta -mediated inhibition of the DNA synthesis. These data indicate that CARP is a downstream target of TGF-beta /Smad signaling in VSMCs and suggest a role of CARP in mediation of the inhibitory effects of TGF-beta on the proliferation of VSMCs.