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

Abstract

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 (DeltaRUNX2) 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+/-9.5 U/g protein, P<0.05) and osteocalcin expression (172+/-17 vs 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+/-33 pg/ml, normal serum=117+/-15 pg/ml, P<0.05). The incubation of BVSMCs with noggin, an inhibitor of BMP, decreased RUNX2 expression. In addition, BMP-2 secretion progressively increased during calcification and uremic serum enhanced its secretion compared to normal serum. In conclusion, this study demonstrates that RUNX2 transcriptional activity is critical in uremic serum-induced bone matrix protein expression in BVSMCs and that the cAMP/PKA pathway is involved. BMP-2 is also increased in uremic serum and can upregulate RUNX2 and calcification in vitro in VSMCs.

BMP-2 promotes phosphate uptake, phenotypic modulation, and calcification of human vascular smooth muscle cells

“…BMP-2 has been previously observed to enhance phosphate-induced calcification of bovine SMC [13]. Therefore, we first confirmed that human SMC contained BMP receptors, and that BMP-2 could enhance phosphate-induced calcification in these cells.…”

“…BMP-2 was also expressed in calcified arteries of LDLR −/− mice fed a high-fat diet [11]. In addition, treatment of calcifying vascular cells or bovine smooth muscle cells in vitro with BMP-2 resulted in enhanced calcification [12,13]. Finally, mice with targeteddeletion of Smad6, an inhibitory Smad in the BMP2 signaling cascade, present with vascular calcification [14].…”

“…The concentrations of BMP-2 used were chosen based on previous study [13] and the potency of commercial preparations as indicated by the manufacturers. As shown in Fig 2A, calcification analysis indicated that BMP-2 increased matrix mineralization in the presence of various elevated phosphate concentrations (2.0, 2.3 and 2.6 mM) compared with vehicle.…”

Oxidative Stress Induces Vascular Calcification through Modulation of the Osteogenic Transcription Factor Runx2 by AKT Signaling

“…The DNA binding sites of Runx2 have been identified in the genes of major bone matrix proteins including Col I and OC, and Runx2 induces the expression of these genes or activates their promoters in osteoblasts (21,63). Blocking Runx2 transcriptional activity by a dominant-negative Runx2 has been shown to decrease uremic serum-induced ALP activity and OC expression (28). Consistently, we found that increased expression of ALP, Col I, and OC was associated with increased Runx2 during VSMC calcification (Figs.…”

“…Runx2 expression has been identified in atherosclerotic calcified human vascular tissue specimens (24 -26) and in calcifying aortic smooth muscle cells in mice (16) but not in normal vessels. Furthermore, increased expression of Runx2 is associated with VSMC calcification in vitro (16,27,28), supporting a role for Runx2 in vascular calcification. However, the potential link between Runx2 regulation and oxidative stress-induced vascular calcification has not been examined.…”