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

Kidney Int. 2006 Sep;70(6):1046-53. doi: 10.1038/sj.ki.5001663. Epub 2006 Jul 12.

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Alkaline Phosphatase / metabolism
  • Animals
  • Aorta, Thoracic / cytology
  • Blood Proteins / pharmacology*
  • Bone Morphogenetic Protein 2
  • Bone Morphogenetic Proteins / blood
  • Bone Morphogenetic Proteins / metabolism*
  • Calcification, Physiologic
  • Carrier Proteins / metabolism
  • Carrier Proteins / pharmacology
  • Cattle
  • Cell Culture Techniques
  • Cells, Cultured
  • Core Binding Factor Alpha 1 Subunit / genetics
  • Core Binding Factor Alpha 1 Subunit / metabolism*
  • Cyclic AMP / metabolism
  • Muscle, Smooth, Vascular / cytology
  • Muscle, Smooth, Vascular / metabolism*
  • Osteocalcin / metabolism
  • Transforming Growth Factor beta / blood
  • Transforming Growth Factor beta / metabolism*
  • Up-Regulation
  • Uremia / blood*

Substances

  • BMP2 protein, human
  • Blood Proteins
  • Bone Morphogenetic Protein 2
  • Bone Morphogenetic Proteins
  • Carrier Proteins
  • Core Binding Factor Alpha 1 Subunit
  • Transforming Growth Factor beta
  • Osteocalcin
  • noggin protein
  • Cyclic AMP
  • Alkaline Phosphatase