title: Runx2-upregulated receptor activator of nuclear factor κB ligand in calcifying smooth muscle cells promotes migration and osteoclastic differentiation of macrophages.
abstract: OBJECTIVE
  Clinical and experimental studies demonstrate the important roles of vascular smooth muscle cells (VSMC) in the pathogenesis of atherosclerosis. We have previously determined that the osteogenic transcription factor Runx2 is essential for VSMC calcification. The present study characterized Runx2-regulated signals and their potential roles in vascular calcification.
  
  
  RESULTS
  In vivo studies with atherogenic apolipoprotein E(-/-) mice demonstrated that increased oxidative stress was associated with upregulation of Runx2 and receptor activator of nuclear factor κB ligand (RANKL), which colocalized in the calcified atherosclerotic lesions and were juxtaposed to infiltrated macrophages and osteoclast-like cells that are positively stained for an osteoclast marker, tartrate-resistant acid phosphatase. Mechanistic studies using RNA interference, a luciferase reporter system, chromatin immunoprecipitation, and electrophoretic mobility shift assays indicated that Runx2 regulated the expression of RANKL via a direct binding to the 5'-flanking region of the RANKL. Functional characterization revealed that RANKL did not induce VSMC calcification, nor was RANKL required for oxidative stress-induced VSMC calcification. Using a coculture system, we demonstrated that VSMC-expressed RANKL induced migration as well as differentiation of bone marrow-derived macrophages into multinucleated, tartrate-resistant acid phosphatase-positive osteoclast-like cells. These effects were inhibited by the RANKL antagonist osteoprotegerin and with VSMC deficient in Runx2 or RANKL.
  
  
  CONCLUSIONS
  We demonstrate that Runx2 directly binds to the promoter and controls the expression of RANKL, which mediates the crosstalk between calcifying VSMC and migration and differentiation of macrophages into osteoclast-like cells in the atherosclerotic lesions. Our studies provide novel mechanistic insights into the regulation and function of VSMC-derived RANKL in the pathogenesis of atherosclerosis and vascular calcification.
authors: Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL, USA.
authors: Chang Hyun
authors: CH
authors: Byon
authors: Yong
authors: Y
authors: Sun
authors: Jianfeng
authors: J
authors: Chen
authors: Kaiyu
authors: K
authors: Yuan
authors: Xia
authors: X
authors: Mao
authors: Jack M
authors: JM
authors: Heath
authors: Peter G
authors: PG
authors: Anderson
authors: Yin
authors: Y
authors: Tintut
authors: Linda L
authors: LL
authors: Demer
authors: Deli
authors: D
authors: Wang
authors: Yabing
authors: Y
authors: Chen
mesh_terms: D000135:Acid Phosphatase / Q000032:analysis; D000818:Animals; D050197:Atherosclerosis / Q000209:etiology; D002114:Calcinosis / Q000209:etiology* / Q000378:metabolism; D002454:Cell Differentiation; D002465:Cell Movement; D050659:Core Binding Factor Alpha 1 Subunit / Q000502:physiology*; D005786:Gene Expression Regulation; D007527:Isoenzymes / Q000032:analysis; D008264:Macrophages / Q000166:cytology / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000502:physiology; D032389:Myocytes, Smooth Muscle / Q000502:physiology*; D010010:Osteoclasts / Q000166:cytology*; D018384:Oxidative Stress; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D053245:RANK Ligand / Q000235:genetics / Q000502:physiology*; D000071681:Tartrate-Resistant Acid Phosphatase; D014652:Vascular Diseases / Q000209:etiology* / Q000378:metabolism
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D007527:Isoenzymes; D053245:RANK Ligand; C493716:Runx2 protein, mouse; C505689:Tnfsf11 protein, mouse; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase