The mechanisms of uremic serum-induced expression of bone matrix proteins in bovine vascular smooth muscle cells
The mechanisms of uremic serum-induced expression of bone matrix proteins in bovine vascular smooth muscle cells
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DOI：
10.1038/sj.ki.5001663
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发表时间：
2006-09-01
影响因子：
19.6
通讯作者：
Moe, S. M.
中科院分区：
文献类型：
--
作者：
Chen, N. X.;Duan, D.;Moe, S. M.
我们先前发现尿毒症人血清上调血管平滑肌细胞（VSMC）中的RUNX 2，并且RUNX 2在体内血管钙化区域中上调。为了证实RUNX 2的作用，我们在牛血管平滑肌细胞（BVSMC）中瞬时转染显性负性RUNX 2（Delta RUNX 2）构建体。阻断RUNX 2转录活性显著降低尿毒症血清诱导的碱性磷酸酶（ALP）活性（268 +/- 34 vs 188 +/- 9.5 U/ g蛋白，P < 0.05）和骨钙素表达（172 +/- 17 vs 125 +/- 9 ODU，P < 0.05）。为了确定尿毒症血清上调RUNX 2的机制，我们检查了细胞信号通路。在存在或不存在抑制剂的情况下孵育BVSMC，并测定RUNX 2表达和ALP活性。结果表明，环AMP（cAMP）/蛋白激酶A（PKA），而不是蛋白激酶C，信号通路参与尿毒症血清诱导的BVSMCs的RUNX 2表达和ALP活性。为了检测潜在的尿毒症“毒素”，我们测量了骨形态发生蛋白（BMP）-2浓度，发现尿毒症血清含有增加的BMP-2（尿毒症血清= 169 +/-33 pg/ml，正常血清117 +/-15 pg/ml，P < 0.05）。BVSMC与BMP抑制剂noggin孵育，降低RUNX 2表达。此外，BMP-2分泌进行性增加，在钙化和尿毒症血清增强其分泌相比，正常血清。总之，这项研究表明，RUNX 2的转录活性是尿毒症血清诱导的骨基质蛋白在BVSMCs的表达和cAMP/PKA途径参与的关键。BMP-2在尿毒症血清中也增加，并且可以在体外VSMC中上调RUNX 2和钙化。
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 (Delta RUNX2) 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.