Abstract 13000: Characterization of the Functional Domain of Runx2 in Regulating Vascular Calcification
Vascular calcification is a prevalent complication in patients with atherosclerosis, diabetes and end stage renal disease. We and others have reported that oxidative stress, high phosphate and uremia promotes calcification of vascular smooth muscle cells (VSMC), which is associated with increased osteogenic transcription factor Runx2. Using short hairpin RNA, we demonstrated that an essential role of Runx2 in VSMC calcification. The present studies determined the Runx2 functional domains in regulating VSMC calcification. Calcification of mouse primary VSMC was induced in osteogenic media and determined by Von Kossa staining as we previously reported. A serial of lentiviral constructs carrying Runx2 deletion mutants were generated, including Runx2-528 (full length), Runx2-495(amino acids 1–495), Runx2-432 (1–432) and Runx2-391 (1–391). In addition, VSMC isolated from Runx2 exon 8 floxed mice (provided by Javed, A at UAB) were infected with AdCre virus to generate VSMC with Runx2 exon 8 ablation (Runx2 1–370). Oxidative stress-induced calcification was inhibited in VSMC with Runx2 exon 8 ablation, demonstrating the C-terminus of the Runx2 is important for VSMC calcification. With the Runx2 deletion mutants, we demonstrated that overexpression of Runx2-528, Runx2-495 and Runx2-432 were sufficient to induce VSMC calcification in WT VSMC; and restored calcification in VSMC with Runx2 exon 8 ablation. By contrast, overexpression of Runx2-391 failed to induce calcification in WT VSMC; and did not restore calcification in VSMC with Runx2 exon 8 ablation. Taken together, our data suggested that the Runx2 domain at amino acids 391–432 was responsible for its function in regulating VSMC calcification. The present studies demonstrated an essential and sufficient role of Runx2 in regulating VSMC calcification, which is attributed to the functional domain at amino acids 391–432. This Runx2 domain contains nuclear matrix targeting signal and the Runx2 and Smad-interacting domain, suggesting the importance of such functions in mediating Runx2-regulated VSMC calcification. Our studies provide molecular insights into a definitive role of Runx2 in regulating VSMC calcification, which may lead to identifying novel target to treat vascular calcification.
- © 2010 by American Heart Association, Inc.