Abstract 313: Phosphorylation of Runx2 and Osteochondrogenic Differentiation of Vascular Smooth Muscle Cells

Vascular calcification, the abnormal deposition of calcium phosphate salts in the vasculature, is associated with a heightened risk of cardiovascular disease in patients with chronic kidney disease and diabetes mellitus. Through a genetic fate mapping strategy, our laboratory has identified vascular smooth muscle cells (SMCs) as a major cell source contributing to bone- and cartilage-like cells in calcifying arteries. Runx2, a transcription factor critical for osteoblast and hypertrophic chondrocyte differentiation, is upregulated in the early stages of a wide range of calcific arterial diseases. Treating SMCs in vitro with elevated phosphate resulted in an increase in phosphorylated Runx2 at serine 319, while total Runx2 remained unaffected. We also found that ERK1/2 signaling and Runx2 were required for SMC osteogenic differentiation and calcification in vitro . To further explore whether Runx2 phosphorylation and activation are critical to Runx2 function in vascular calcification, we mutated Runx2 at serine 301 and 319, the sites phosphorylated by active ERK and shown to be crucial for Runx2 transcriptional activity in osteoblasts, to create ERK-resistant (S310A/S319A) and constitutively active (S301E/S319E) variants. We have generated Runx2 knockout SMCs from Runx2- flox/flox mice carrying SM22α-Cre recombination transgenic allele. Wild type Runx2 and its mutants were introduced into these cells retrovirally. Preliminary Runx2 functional analyses suggest a successful introduction of the constructs and a phosphate-dependent activation of Runx2. Experiments to address a role of Runx2 phosphorylation by ERK in SMC osteogenic differentiation and matrix calcification are ongoing. In conclusion, our current findings along with the ongoing experiments will provide important inside into mechanisms of SMC osteochondrogenic differentiation and vascular calcification, findings that may serve as basis for the development of appropriate therapeutic strategies for this devastating complication.