The mechanisms of uremic serum-induced expression of bone matrix proteins in bovine vascular smooth muscle cells

We have previously found that uremic human serum upregulates RUNX2 in vascular smooth muscle cells (VSMCs), and that RUNX2 is upregulated in areas of vascular calcification in vivo. To confirm the role of RUNX2, we transiently transfected a dominant-negative RUNX2 (ΔRUNX2) construct in bovine vascular smooth muscle cells (BVSMCs). Blocking RUNX2 transcriptional activity significantly decreased uremic serum induced alkaline phosphatase (ALP) activity (268±34 vs 188±9.5U/g protein, P<0.05) and osteocalcin expression (172±17vs 125±9 ODU, P<0.05). To determine the mechanism by which uremic serum upregulates RUNX2, we examined cell signaling pathways. BVSMCs were incubated in the presence or absence of inhibitors and RUNX2 expression and ALP activity were determined. The results demonstrate that the cyclic AMP (cAMP)/protein kinase A (PKA), but not protein kinase C, signaling pathway is involved in uremic serum-induced RUNX2 expression and ALP activity in BVSMCs. To examine potential uremic ‘toxins’, we measured bone morphogenetic protein (BMP)-2 concentration and found that uremic serum contained increased BMP-2 (uremic serum=169±33pg/ml, normal serum=117±15pg/ml, P