AMPK-Dependent Angiogenesis by Nitrated Lipids Figure 4. OA-NO2-induced HIF-1a is partially dependent on ERK1/2. A) BAECs were incubated with the indicated concentrations of OA-NO2 for 16 h. Protein expression and phosphorylation of p38 MAPK and ERK1/2 were assayed as described in Materials and Methods. The blot is representative of three blots obtained from three independent experiments. B) BAECs were treated with ZnBG (1 mM) for 30 min followed by incubation with OA-NO2 for 16 h. ERK1/2 phosphorylation levels were monitored by immunoblot analysis, and band density was normalized to total ERK1/2 levels (lower panel). The blot is representative of three blots obtained from three independent experiments. *p,0.05 vs. control; #p,0.05 vs. OA-NO2 group. C) BAECs were treated with OA-NO2 alone or with the ERK1/2 signaling inhibitors, UO126 (10 mM) or PD98059 (50 mM), 1 h before the addition of OA-NO2. HIF-1a protein levels were monitored by immunoblot analysis, and band density was normalized to b-actin levels (lower panel). The blot is representative of three blots obtained from three independent experiments. *p,0.05 vs. control; #p,0.05 vs. OA-NO2 group. doi:10.1371/journal.pone.0031056.g004 further investigated whether a Ca2+ signal is involved in OA-NO2- induced AMPK activation using BAPTA-AM, a cell-permeable Ca2+ chelator that is widely used as an intracellular Ca2+ ‘‘sponge’’. Our results indicate that chelation of intracellular Ca2+ by BAPTA-AM (25 mM, 30 min) significantly inhibited OA-NO2-stimulated AMPK phosphorylation (,60%, p,0.05; Figure 5E). Together, these data show that intracellular Ca2+ is necessary for OA-NO2-induced AMPK activation. CaMKKb mediates OA-NO2-induced AMPK activation Both the tumor suppressor LKB1 [14] and CaMKK [15] are important AMPK kinases as each activates AMPK by directly phosphorylating the AMPK a subunit on Thr172. As shown above (Figure 2A–C), LKB1 is unlikely involved in the activation of AMPK by OA-NO2. Because treatment with OA-NO2 elevated [Ca2+]i and OA-NO2-stimulated AMPK activation was Ca2+ sensitive, we speculate that the AMPK kinase, CaMKKb, which is activated by Ca2+/calmodulin binding, may be involved. To test this hypothesis, we used the relatively selective CaMKKa and CaMKKb inhibitor, STO-609 (1 mM) [25] and the competitive CaM inhibitor, KN-93 (3 mM). Either STO-609 or KN-93 was sufficient to prevent activation of AMPK by OA-NO2 (Figure 6A), suggesting the involvement of CaMKK in this activation. A specific role for CaMKKb was indicated by the results of siRNA experiments, which showed that downregulation of CaMKKb caused a significant reduction in OA-NO2-stimulated AMPK phosphorylation (Figure 6B). Downregulation of CaMKKb protein was verified by western blot analysis. These results suggest that CaMKKb is the major AMPK kinase under these conditions. To further validate this notion, we determined whether treatment with OA-NO2 enhanced the association between CaMKK and AMPK as this interaction is a prerequisite for AMPK phosphorylation/ activation. Indeed, following OA-NO2 treatment, an enhanced CaMKK and AMPK association was detected by immunopre- cipitation with either AMPK or CaMKK antibodies (Figure 6C). Moreover, pre-treatment with YC-1 dramatically inhibited the OA-NO2-induced increased CaMKK-AMPK association, fur- ther substantiating the indispensable role of HIF-1a in OA-NO2- induced AMPK activation. OA-NO2-induced eNOS phosphorylation is dependent on AMPK We previously demonstrated that AMPK phosphorylates and activates eNOS in cultured endothelial cells [26]. Similarly, Zhang et al. demonstrated that infection of endothelial cells with a recombinant adenovirus expressing constitutively active AMPK resulted in eNOS activation and increased NO production [27]. Here, we used phosphorylation of Ser1177 in eNOS, which is a reported substrate of AMPK, as an indicator of AMPK activity in this system. As shown in Figure 7A, incubation of BAECs with OA-NO2 increased eNOS Ser1177 phosphorylation in a dose-dependent manner that was very similar to that for AMPK phosphorylation. The time course of OA-NO2–induced eNOS phosphorylation was also similar to that for AMPK phosphorylation (Figure 7B). To confirm that OA-NO2-stimulated eNOS phosphorylation involved AMPK, we infected BAECs with adenovirus encoding a dominant- negative form of AMPK (Ad-DN-AMPK). As expected, phosphorylation of both AMPK and eNOS was increased after treatment of control BAECs (Ad-GFP-infected or non-infected BAECs) with 2.5 mM OA-NO2 for 16 h (Figure 7C). In contrast, overexpression of Ad-DN-AMPK completely abolished OA- NO2-induced eNOS phosphorylation. Consistent with a role for AMPK in phosphorylating eNOS, treatment with STO-609 or KN-93 to inhibit the AMPK kinase CaMKK or downregulation of CaMKKb using siRNA prevented OA-NO2-induced eNOS phosphorylation (Figure 6A and B). Both Akt and AMPK are capable of phosphorylating eNOS at Ser1179 [28]. Thus, we determined whether Akt also contributes to OA-NO2-induced eNOS phosphorylation. OA-NO2 did not increase basal Akt phosphorylation of Ser473 (data not shown), suggesting that OA-NO2-stimulated eNOS phosphorylation does not require Akt but depends on activation of AMPK. PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e31056