Selective HIF-1 Regulation under Nonhypoxic Conditions by the p42/p44 MAP Kinase Inhibitor PD184161

Hypoxia-inducible factor-1 (HIF-1) is a key gene regulator for cellular adaptation to low oxygen. In addition to hypoxia, several nonhypoxic stimuli, including hormones and growth factors, are essential for cell-specific HIF-1 regulation. Our studies have highlighted angiotensin II (AngII), a vasoactive hormone, as a potent HIF-1 activator in vascular smooth muscle cells (VSMC). AngII increases HIF-1 transcriptional activity by modulating specific signaling pathways. In VSMC, p42/p44 mitogen-activated protein kinase (MAPK) pathway activation is essential for HIF-1-mediated transcription during AngII treatment. The present study shows that PD184161, a potent MEK1/2 inhibitor, is an HIF-1 blocker in Ang II-treated VSMC. Unlike PD98059, a widely-used MEK1/2 inhibitor, we found that PD184161 blocked AngII-driven HIF-1 alpha protein induction in a dose-dependent manner. Interestingly, the effect of PD184161 was specific to nonhypoxic activators, since HIF-1 alpha induction by hypoxia (1% O-2) was unaffected under similar conditions. VSMC treatment with MG132, a proteasome inhibitor, indicated that PD184161 influenced HIF-1 alpha protein stability. PD184161 also increased HIF-1 alpha binding to von Hippel-Lindau tumor suppressor protein, an E3 ligase component and an indication of HIF-1 alpha hydroxylation. Finally, we show that PD184161 blocked mitochondrial ROS (mtROS) production and cellular ATP levels, at the same time enhancing ascorbate availability in AngII-treated VSMC. Taken together, our study indicates that, independently of p42/p44 MAPK activation, PD184161 blocks mtROS generation by AngII, leading to re-establishment of cellular ascorbate levels, increased VHL binding, and decreased HIF-1 alpha stability. Therefore, this study reveals a previously unsuspected role for PD184161 as an HIF-1 inhibitor in VSMC under nonhypoxic conditions.