Results Effects of insulin on the expression of RANKL mRNA and protein in CVSMCs Physiologically, insulin concentrations in vivo may rise to ,1 nM [21]. In the presence of insulin resistance, serum insulin concentrations may exceed these levels. We performed insulin dose–response and time course experiments to determine the effect of physiological and supraphysiological concentrations of insulin on mRNA levels and on RANKL protein secretion in CVSMCs. After 48 h, insulin at a 1 nM concentration had no effect on the expression of RANKL mRNA. RANKL mRNA levels increased significantly at 5 nM insulin stimulation, and the maximal effect of insulin was reached at 10 nM on RANKL mRNA levels. At markedly supraphysiological insulin concentrations there was a small decrease in RANKL mRNA levels compared with the maximum effect of insulin (Fig. 1A). RANKL protein secretion followed the mRNA trend (Fig. 1B). After 48 h of culture and at a concentration of 10 nM of insulin, expression of RANKL mRNA was greater than that of the controls (p,0.01) (Fig. 1C). Insulin activated ERK1/2 and Akt signaling pathway in CVSMCs Recent studies have that demonstrated that ERK1/2 and Akt signaling pathways mediate insulin action [22,23]. We examined if insulin induced ERK1/2 and Akt signaling in CVSMCs. As shown in Figure 2A, insulin stimulated the activity of a specific ERK1/2 in the CVSMCs after 5 min of incubation, when determined by the increase in the phosphorylated ERK1/2 levels the peak activation of ERK1/2 occurred at 15–30 min. Then, we examined whether insulin induced Akt signaling in CVSMCs. Insulin treatment increased phosphorylated Akt (p-Akt) levels after 5 min of incubation; the peak activation of Akt occurred at 15 min. The activation of ERK1/2 and Akt by insulin was abolished by PD98059 (an inhibitor of ERK1/2), LY294002 (an inhibitor of PI3-K) or HIMO (an inhibitor of Akt), respectively (Fig. 2B and 2C). These data indicated that insulin stimulated ERK and PI3- K/Akt activation in CVSMCs. ERK1/2 signaling pathway mediated insulin-regulated RANKL expression in CVSMCs Figure 2D showed that pretreatment of cells with the ERK1/2 inhibitor PD98059, but not the PI3K inhibitor LY294002 or the Akt inhibitor HIMO blocked the increasing RANKL mRNA expression stimulated by 10 nM insulin. To exclude any nonspecific effect of PD98059, LY294002 or HIMO, we used 1a,25-dihydroxyvitamin D3 (1,25 vitD) and 1,25 vitD+PD98059 as the control (both at 1027 M) to induce RANKL mRNA expression. PD98059 did not block the increase in RANKL mRNA expression induced by 1,25 vitD. Insulin increased osteoblastic differentiation of CVSMCs and calcification of CVSMCs Mineralization of matrix. The calcification induced by insulin was also visualized by Alizarin Red S staining, as we show in Figure 3A. In this figure we show that incubation of CVSMCs with insulin increased the red staining that marks calcified areas. Figure 3B showed that insulin increased CVSMCs calcification as measured by CVSMC calcium levels. ALP activity, osteocalcin secretion assay. Figure 3C showed the dose–response of effects of insulin on ALP activity in cultured CVSMCs. At insulin concentrations of 5, 10, and Insulin Induces Calcification of VSMC via RANKL 100 nM, ALP activity increased markedly. Figure 3D shows the effects of insulin on osteocalcin secretion in cultured CVSMCs. Treatment with 5–100 nM insulin caused a significant increase in osteocalcin production and the maximal effect of insulin was reached after addition of 10 nM insulin. Involvement of RANKL and ERK1/2 signaling in insulin regulation of osteoblastic differentiation of CVSMCs In order to illuminate whether and how RANKL, ERK1/2, and PI3K/Akt signaling was involved in the regulation of osteoblastic differentiation of CVSMCs, we used siRNA to block RANKL, and signal inhibitor to block ERK1/2, and PI3K/Akt signal pathways, respectively. Then, we observed ALP activity, which is an important phenotypic marker of osteoblastic differentiation. Figure 4A showed that pretreatment of cells with the ERK1/2 inhibitor PD98059, but not the PI3K inhibitor LY294002 or Akt inhibitor HIMO blocked the increased ALP activity induced by 10 nM insulin. RANKL was knocked down efficiently by RNA interference in CVSMCs (Fig. 4A). Suppression of RANKL with siRNA-RANKL, but not control siRNA, also abolished the increased ALP activity indued by 10 nM insulin (Fig. 4B). Furthermore, 15 pM recombinant RANKL protein could recover ALP activities in the insulin-induced RANKL-knocked down CVSMCs (Fig. 4C). These data indicated that insulin-induced osteoblastic differentiation of CVSMCs is mediated by the ERK1/ 2/RANKL pathway. Discussion VSMCs can express RANKL and recent results have shown that RANKL plays an important role in mediating arterial calcification [8,24]. Our present data showed that insulin induced RANKL expression in CVSMCs through the ERK1/2 signal pathway, and these responses contributed to insulin-induced CVSMCs calcification. These findings suggested that RANKL induced by insulin in CVSMCs accelerated arterial calcification through an antocrine or a paracrine effect. Extensive epidemiological data have shown that diabetes is correlated with arterial calcification and higher insulin level could predicate arterial calcification independently [11,25]. The mech- anisms of insulin involved in arterial calcification in these clinical settings are still controversial. Olesen et al.’s result demonstrated that insulin enhanced in vitro-induced calcification in VSMCs [17]. However, Wang et al. suggested that insulin attenuated VSMCs calcification in high phosphate medium [18]. These contradictory results might be explained by different cell types or different experimental condition. Increasing ALP activities, osteocalcin secretion is a marker of osteoblastic differentiation, and matrix mineralization is a hallmark of osteoblast phenotype. In our present study, we demonstrated that insulin upregulated ALP activities, increased osteocalcin secretion and promoted mineral- ization nodule formation. These results show that insulin stimulated osteoblastic differentiation of CVSMCs and increased CVSMC calcification, findings that are consistent with Olesen et al.’s results [17]. RANKL is a member of the tumor necrosis factor superfamily and is expressed mainly on osteoblasts and its immature precursors. RANKL binds to RANK and is expressed on the cell surface of hematopoietic osteoclast precursor cells, inducing osteoclastogenesis and activation and prolonging osteoclast survival by suppressing apoptosis. OPG acts as a decoy receptor by binding to RANKL and blocking its interaction with RANK, thus inhibiting osteoclast development. It has been suggested that PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e29037