RANKL Is a Downstream Mediator for Insulin-Induced Osteoblastic Differentiation of Vascular Smooth Muscle Cells Ling-Qing Yuan1,3, Jia-Hua Zhu 2 , Hua-Wen Wang 2 , Qiu-Hua Liang 1 , Hui Xie1, Xian-Ping Wu 1 , Hua Zhou 1 , Rong-Rong Cui1, Zhi-Feng Sheng 1 , Hou-De Zhou1, Xiao Zhu 1 , Guan-Ying Liu1, You-Shuo Liu2,3*, Er-Yuan 1 Liao * 1 Institute of Metabolism and Endocrinology, Second Xiang-Ya Hospital, Central South University, Changsha, Hunan, People’s Republic of China, 2 Geriatric Department, Second Xiang-Ya Hospital, Central South University, Changsha, Hunan, People’s Republic of China, 3 Institute of Aging and Geratology, Second Xiang-Ya Hospital, Central South University, Changsha, Hunan, People’s Republic of China 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 kB 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. Citation: Yuan L-Q, Zhu J-H, Wang H-W, Liang Q-H, Xie H, et al. (2011) RANKL Is a Downstream Mediator for Insulin-Induced Osteoblastic Differentiation of Vascular Smooth Muscle Cells. PLoS ONE 6(12): e29037. doi:10.1371/journal.pone.0029037 Editor: Irina Agoulnik, Florida International University, United States of America Received August 4, 2011; Accepted November 18, 2011; Published December 15, 2011 Copyright: 2011 Yuan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Natural Science Foundation of China (Nos. 30801174, 30900622 and 30871191), the Ph.D. Programs Foundation of the Ministry of Education of China (No. 200805331017), Youth Scientific Funds of the Chinese Society of Endocrinology (No. 10020040226) and the Hunan Provincial Natural Science Foundation (No. 09JJ3037). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: eyliao007@yahoo.com.cn (EYL); liuyoushuo@medmail.com.cn (YSL) Introduction Arterial calcification is a significant predictor for cardiovascular events in patient with diabetes mellitus and end-stage renal disease. In these cases, the main cell type involved is the vascular smooth muscle cell (VSMCs) [1,2]; however, the mechanisms for arterial calcification still remain unclear. There is an increasing amount of evidence to suggest that medial arterial calcification is an active and tightly regulated process that resembles mineralization in bone [1,2]. Key functional aspects of bone formation and remodeling are recapitulated in vascular calcification, and many bone-related factors such as osteoprotegerin (OPG), receptor activator of NF-kB ligand (RANKL), alkaline phosphatase (ALP), osteocalcin (OC) and runt-related transcription factor 2 (RUNX2) have been found in calcified areas of the arterial wall and VSMCs [3–5]. The discovery that OPG-deficient mice develop severe osteoporosis and arterial calcification [6,7] and that RANKL directly increased VSMC calcification [8] provided the first clue that the RANKL/ OPG/RANK axis could be an important autocrine/paracrine axis for vascular calcification. Hyperinsulinemia is a clinic characteristic of type 2 diabetes as a result of insulin resistance. Several studies have demonstrated that insulin level and insulin resistance are positively correlated with arterial calcification [9–14]. Moreover, an animal study of Zucker diabetic rats, a hyperinsulinism model, increased arterial stiffness that was related to arterial calcification [15,16]. However, the relationship between insulin and VSMCs calcifi- cation remains controversial [17,18]. In the present study, we used calcifying vascular smooth muscle cells (CVSMCs), referred to as a specific subpopulation of VSMCs that could express osteoblastic phenotype gene and form calcification nodule spontaneously [19,20], to elucidate the effect of insulin on osteoblastic differentiation of CVSMCs and the mechanisms involved. PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e29037