AMPK-Dependent Angiogenesis by Nitrated Lipids Figure 2. Activation of AMPK by OA-NO2 does not require LKB1. A) Phosphorylation of LKB1 Ser428 was not affected by OA-NO2 in BAECs. Confluent BAECs were exposed to 2.5 mM OA-NO2 for 16 h, and phosphorylated LKB1-Ser428 was detected by a phospho-specific antibody in western blots. The blot is a representative of three blots obtained from three independent experiments. Lower panels: summary data (n = 3). B) LKB1 is not required for AMPK activation by OA-NO2. Confluent LKB1-deficient Hela-S3 cells were exposed to 2.5 mM OA-NO2 for 16 h, and then AMPK and ACC phosphorylation were assayed as described in Materials and Methods. The blot is representative of three blots obtained from three independent experiments. Lower panels: summary data (*p,0.05 vs. control; n = 3). C) LKB1 siRNA did not abolish OA-NO2-stimulated AMPK activation in HUVECs. HUVECs were incubated with LKB1-specific siRNA or control siRNA for 48 h and then treated with OA-NO2 or vehicle for 16 h. After treatment, cell lysates were analyzed for LKB1 protein levels and AMPK phosphorylation at Thr172. Lower panels: summary data (*p,0.05 vs. control; n = 3). doi:10.1371/journal.pone.0031056.g002 Figure 3. OA-NO2-induced HIF-1a is dependent on HO-1. A) HUVECs were treated with 1 mM ZnBG for 30 min followed by incubation with OA-NO2 for the indicated times. AMPK protein and phosphorylation levels and HIF-1a protein expression were assayed as described in Materials and Methods. The blot is representative of three blots obtained from three independent experiments. B) HUVECs were incubated with HO-1-specific siRNA or control siRNA for 48 h and then treated with OA-NO2 or vehicle for 16 h. After treatment, cell lysates were analyzed for HIF-1a and HO-1 protein levels and AMPK phosphorylation at Thr172. Lower panels: summary data (*p,0.05 vs. control; #p,0.05 vs. OA-NO2 group; n = 3). doi:10.1371/journal.pone.0031056.g003 alone (Figure 4C), suggesting that OA-NO2-mediated HIF-1a induction is due, at least in part, to MEK/ERK1/2 activity. UO126 and PD98059 did not affect basal HIF-1a expression. In addition to increasing HIF-1a expression, ERK1/2 has previously been reported to phosphorylate HIF-1a in vitro, and this post- translational modification can increase HIF-1a activity, presum- ably by impeding proteasome/von-Hippel-Lindau (VHL) recog- nition [20]. Although very little is known about the phosphory- lation of HIF-1a, this phosphorylation event may be important for optimal activity of the HIF-1 pathway. HIF-1a induction by OA-NO2 contributes to intracellular Ca2+ accumulation and AMPK activation To examine the involvement of the HIF-1a pathway in OA- NO2-mediated AMPK activation, we employed the widely used HIF-1a inhibitor, 3-(59-hydroxymethyl-29-furyl)-1-benzylindazole (YC-1) and HIF-1a siRNA. Pre-treatment of BAECs with YC-1 or HIF-1a siRNA significantly inhibited the OA-NO2-induced increase in AMPK Thr172 phosphorylation levels relative to OA-NO2 treatment alone (Figure 5A and B). Since OA-NO2- induced AMPK activation is LKB1-independent, we postulated that the Ca2+/CaMKK pathway plays a role in the activation of AMPK by OA-NO2/HIF-1a. In addition, because HIF-1a has been reported to regulate Ca2+ homeostasis in pulmonary arterial smooth muscle cells via upregulation of store-operated Ca2+ channels and enhanced Ca2+ influx [24], we examined a possible role for HIF-1a in this process. To determine whether OA-NO2 affected endothelial cell [Ca2+]i, we measured intracellular fluorescence in Fluo-4–loaded BAECs. We found that OA-NO2 treatment produced an approximately 1.4- to 2.2-fold increase in [Ca2+]i at 6 and 16 h after treatment, respectively (Figure 5C). To confirm a role for HIF-1a in this OA-NO2-mediated [Ca2+]i increase, we used pharmaceutical- and genetic-inhibition strategies to downregulate HIF-1a. Pre-treatment of BAECs with YC-1 or transfection of these cells with HIF-1a siRNA partially suppressed the OA-NO2-induced increases in [Ca2+]i (Figure 5D), indicating that this process is at least partially dependent on HIF-1a. We PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e31056