title: RANKL is a downstream mediator for insulin-induced osteoblastic differentiation of vascular smooth muscle cells.
abstract: Several reports have shown that circulating insulin level is positively correlated with arterial calcification; however, the relationship between insulin and arterial calcification remains controversial and the mechanism involved is still unclear. We used calcifying vascular smooth muscle cells (CVSMCs), a specific subpopulation of vascular smooth muscle cells that could spontaneously express osteoblastic phenotype genes and form calcification nodules, to investigate the effect of insulin on osteoblastic differentiation of CVSMCs and the cell signals involved. Our experiments demonstrated that insulin could promote alkaline phosphatase (ALP) activity, osteocalcin expression and the formation of mineralized nodules in CVSMCs. Suppression of receptor activator of nuclear factor κB ligand (RANKL) with small interfering RNA (siRNA) abolished the insulin-induced ALP activity. Insulin induced the activation of extracellular signal-regulated kinase (ERK)1/2, mitogen-activated protein kinase (MAPK) and RAC-alpha serine/threonine-protein kinase (Akt). Furthermore, pretreatment of human osteoblasts with the ERK1/2 inhibitor PD98059, but not the phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, or the Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO), abolished the insulin-induced RANKL secretion and blocked the promoting effect of insulin on ALP activities of CVSMCs. Recombinant RANKL protein recovered the ALP activities decreased by RANKL siRNA in insulin-stimulated CVSMCs. These data demonstrated that insulin could promote osteoblastic differentiation of CVSMCs by increased RANKL expression through ERK1/2 activation, but not PI3K/Akt activation.
authors.affiliation: Institute of Metabolism and Endocrinology, Second Xiang-Ya Hospital, Central South University, Changsha, Hunan, People's Republic of China.
authors.forename: Ling-Qing
authors.initials: LQ
authors.lastname: Yuan
authors.forename: Jia-Hua
authors.initials: JH
authors.lastname: Zhu
authors.forename: Hua-Wen
authors.initials: HW
authors.lastname: Wang
authors.forename: Qiu-Hua
authors.initials: QH
authors.lastname: Liang
authors.forename: Hui
authors.initials: H
authors.lastname: Xie
authors.forename: Xian-Ping
authors.initials: XP
authors.lastname: Wu
authors.forename: Hua
authors.initials: H
authors.lastname: Zhou
authors.forename: Rong-Rong
authors.initials: RR
authors.lastname: Cui
authors.forename: Zhi-Feng
authors.initials: ZF
authors.lastname: Sheng
authors.forename: Hou-De
authors.initials: HD
authors.lastname: Zhou
authors.forename: Xiao
authors.initials: X
authors.lastname: Zhu
authors.forename: Guan-Ying
authors.initials: GY
authors.lastname: Liu
authors.forename: You-Shuo
authors.initials: YS
authors.lastname: Liu
authors.forename: Er-Yuan
authors.initials: EY
authors.lastname: Liao
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008297:Male; D008903:Minerals / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D053245:RANK Ligand / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D007328:Insulin; D008903:Minerals; D053245:RANK Ligand; D012333:RNA, Messenger; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases