Hypoxic upregulation of preproendothelin-1 gene expression is associated with protein tyrosine kinase-PI3K signaling in cultured lung vascular endothelial cells.

Hypoxia-increased endothelin-1 (ET-1) expression contributes to vasoconstriction and vessel wall thickening, often seen in the progression of pulmonary hypertension. We sought to investigate whether hypoxic modulation of preproET-1 transcription is associated with protein tyrosine kinase and phosphatidylinositol-3-kinase (PI3K). ET-1 is predominantly produced in and secreted from the vascular endothelium. Cultured human pulmonary artery endothelial cells (PAEC) in basic medium EBM-2 were exposed to hypoxia (1% oxygen, 5% CO(2), 37 degrees C) or normoxia (room air containing 5% CO(2)) for 0-48 hr. RNA was extracted from the treated cells and subjected to quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Hypoxia increases the relative levels of steady-state preproET-1 mRNA. The results of actinomycin D chase studies suggest that hypoxia-increased levels of preproET-1 mRNA are unlikely to be caused by increased RNA stability. A modified nuclear run-on method coupled with the sensitive qRT-PCR technique was used to assess preproET-1 gene transcription. The synthesis rate of preproET-1 mRNA in the cells exposed to hypoxia is higher than that in normoxic cells. The inhibitors of protein tyrosine kinases and PI3K, genistein and PI3Kgamma inhibitor II, were used to elucidate the role of protein tyrosine kinase and PI3K in hypoxic modulation of preproET-1 expression. Pre-incubation of human PAEC with genistein or PI3Kgamma inhibitor II abolishes hypoxia-increased levels of preproET-1 mRNA. Our observations support the notion that hypoxia increases the level of preproET-1 mRNA through upregulation of RNA synthesis, which is associated with protein tyrosine kinase- and PI3K-mediated signal transduction pathways. This implies that therapeutic interventions targeting protein tyrosine kinases and/or PI3K might be used to treat hypoxic pulmonary hypertension.