Figure 2. Insulin increased RANKL mRNA expression through ERK1/2, but not Akt signal pathway in calcifying vascular smooth muscle cells (CVSMCs). Cell lysates were subjected to western blot and incubated with anti-p-ERK1/2, ERK1/2, p-Akt, and Akt antibodies. (A) Effects of insulin on ERK1/2 and Akt activation in CVSMCs. Representative results are shown for cells exposed to 10 nM insulin for 5–60 min. (B) Cells incubated with PD98059 (10 mM) for 2 h prior to treatment with 10 nM insulin for 30 min. (C) Cells incubated with LY294002 (10 mM) or HIMO (10 mM) for 2 h prior to treatment with 10 nM insulin for 15 min. (D) Cells were incubated with PD98059 (10 mM), LY294002 (10 mM) or HIMO (10 mM) for 2 h before treatment with 10 nM insulin for 48 h. Cells were treated with 1027 M 1a,25- Insulin Induces Calcification of VSMC via RANKL dihydroxyvitamin D3 (1,25 vitD), or 1,25 vitD+10 mM PD98059 as another control. RANKL mRNA expression was determined by real-time quantitative PCR. Results are expressed as percent of control. Bars represent mean 6 standard deviation (SD) (n = 3; **p,0.01 vs. insulin- treated control). doi:10.1371/journal.pone.0029037.g002 omentin-attenuated arterial calcification in osteoprotegerin-defi- cient mice [24]. Our present data showed that insulin could increase the expression of RANKL and promote ALP activities in CVSMCs, concurrently. Furthermore, inhibition of RANKL expression by addition of siRNA abrogated the induction by insulin of CVSMC ALP activity. Then, recombinant RANKL protein recovered the ALP activity that was reduced by RANKL- siRNA addition in insulin-stimulated CVSMCs. These data suggest RANKL is the key regulator in insulin induced osteoclastic differentiation of VSMCs. In contrast, Tseng et al.’s result showed that upregulated RANKL induced by PKA in aortic smooth muscle cells could not mediate matrix calcification [30]. We think that these differences may result from use of a different cell model, as our previous study confirmed adenovirus-mediated over- expression of omentin-1 attenuated arterial calcification by decreased in RANKL expression in OPG knockout mice [24]. Thus we have shown that insulin regulated osteoblastic differen- tiation of CVSMCs through upregulation of RANKL in CVSMCs. To gain further insight into the mechanisms by which insulin promoted RANKL expression in CVSMCs, we evaluated the signaling events. Recent studies have demonstrated the existence of two major signaling pathways, namely Akt and ERK1/2, which mediate insulin action [22,23]. Signal transduction along both pathways is initiated by insulin binding to the insulin receptor. Transduction of insulin signal via the insulin receptor substrate (IRS) proteins PI3K and Akt is responsible for most, if not all, of the metabolic aspects of insulin action. The second signaling pathway, which involves Ras, Raf, and ERK1/2, is responsible for the mitogenic aspects of insulin action and is not involved in the mechanisms of metabolic insulin action [31,32]. There are several independent signaling pathways documented that regulate RANKL expression in osteoblasts/stromal cells. These pathways include the ERK1/2 pathway (annexin II), the vitamin D receptor pathway [1,25 vitD], the protein kinase A pathway (parathyroid hormone (PTH) and prostaglandin (PGE)2), the gp130 pathway (interleukin (IL)-6 and IL-11), and the calcium/protein kinase C pathway (high extracellular Ca2+) [33–37]. Here, we reported that insulin induced activation of Akt and ERK1/2 in CVSMCs. Furthermore, we demonstrated that pretreatment of cells with ERK1/2 inhibitor, but not PI3K inhibitor or Akt inhibitor, abolished the insulin-promoted RANKL expression in CVSMCs. These data indicated that insulin-induced RANKL expression was mediated by the ERK1/2 pathway. In conclusion, the present data has provided evidence that insulin promoted osteoblastic differentiation of CVSMCs, and this effect is mediated by the ERK1/2/RANKL pathway. These findings suggest that insulin directly facilitates arterial calcification. Materials and Methods Reagents Anti-rat extracellular signal-regulated kinase1/2 (ERK1/2), p- ERK, Akt, p-Akt antibody, anti a-actin polyclonal antibody, anti- RANKL, and rabbit IgG peroxidase conjugate antibodies were purchased from Santa Cruz Biotechnology (Waltham, MA, USA). The ERK 1/2 inhibitor PD98059, PI3K inhibitor LY294002 and PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e29037