The JNK pathway mediates hypoxia-induced leptin expression in HCASMCs. (A and B) The c-Jun N-terminal kinase (JNK) inhibitor (SP600125), JNK small interfering RNA (siRNA), and N-acetyl-L-cysteine (NAC; a ROS scavenger) all blocked hypoxia-induced leptin expression. Exogenous addition of Dp44mT (an ROS generator; 30 nM)) under normoxia also increased leptin expression. We pretreated HCASMCs with an extracellular signal-regulated kinase (ERK) pathway inhibitor (PD98059; 50 μM), a JNK inhibitor (SP600125; 25 μM), a p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580; 3 μM), NAC (500 μM), or JNK1 siRNA prior to hypoxia for 4 hours. The HCASMCs were then harvested and analyzed by western blotting using an anti-leptin antibody. The results were normalized to α-tubulin. *P < 0.01 vs. normoxia control. #P < 0.01 vs. 4 h (n = 3). (C and D) Phosphorylation of the JNK mediated hypoxia-induced leptin expression in HCASMCs, which was blocked by SP600125, JNK1 siRNA, and NAC. HCASMCs were subjected to normoxia or hypoxia for different durations in the presence or absence of inhibitors. Cell lysates were collected for western blot analysis, using antibody for total and phospho-JNK. T-JNK = total JNK. P-JNK = JNK phosphorylation. *P < 0.01 vs. normoxia control. #P < 0.01 vs. 2 h (n = 3).