Background-Previously we found that smooth muscle cell (SMC)-specific knockout of miR-17 similar to 92 attenuates hypoxia-induced pulmonary hypertension. However, the mechanism underlying miR-17 similar to 92-mediated pulmonary artery SMC (PASMC) proliferation remains unclear. We sought to investigate whether miR-17 similar to 92 regulates hypoxia-inducible factor (HIF) activity and PASMC proliferation via prolyl hydroxylases (PHDs). Methods and Results-We show that hypoxic sm-17 similar to 92(-/-) mice have decreased hematocrit, red blood cell counts, and hemoglobin contents. The sm-17 similar to 92 (-/-) mouse lungs express decreased mRNA levels of HIF targets and increased levels of PHD2. miR-17 similar to 92 inhibitors suppress hypoxia-induced levels of HIF1 alpha, VEGF, Glut1, HK2, and PDK1 but not HIF2 alpha in vitro in PASMC. Overexpression of miR-17 in PASMC represses PHD2 expression, whereas miR-17/20a inhibitors induce PHD2 expression. The 3'-UTR of PHD2 contains a functional miR-17/20a seed sequence. Silencing of PHD2 induces HIF1a and PCNA protein levels, whereas overexpression of PHD2 decreases HIF1 alpha and cell proliferation. SMC-specific knockout of PHD2 enhances hypoxia-induced vascular remodeling and exacerbates established pulmonary hypertension in mice. PHD2 activator R59949 reverses vessel remodeling in existing hypertensive mice. PHDs are dysregulated in PASMC isolated from pulmonary arterial hypertension patients. Conclusions-Our results suggest that PHD2 is a direct target of miR-17/20a and that miR-17 similar to 92 contributes to PASMC proliferation and polycythemia by suppression of PHD2 and induction of HIF1 alpha.

Oxygen dependent prolyl-4-hydroxylation of the alpha-subunit of the hypoxia inducible transcription factor (HIF-alpha) plays an essential role in the hypoxic response. Hydroxylation of proline residues in the N- or C-terminal oxygen dependent degradation domains (NODD or CODD) increases the affinity of HIF-alpha to the von Hippel-Lindau protein (pVHL) by approx. 1000 fold so signalling for HIF-alpha degradation. With limiting oxygen, HIF-alpha hydroxylation slows, it dimerises with HIF-beta and activates the transcription of a gene array. Prolyl-4-hydroxylation also stabilises the triple helix structure of collagen, the most abundant human protein. Both the collagen and the HIF prolyl hydroxylases (PHDs) are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. Crystal structures of PHD2 in complex with CODD were determined in the current study. Together with biochemical analyses, the results demonstrate that catalysis involves a mobile region of PHD2 that encloses the hydroxylation site and stabilises the PHD2.Fe(II).2OG complex. When bound to PHD2 the pyrrolidine ring of the non-hydroxylated proline-residue adopts a C⁴-endo conformation. Evidence is provided that 4R-hydroxylation enables a stereoelectronic effect that changes the proline conformation to the C⁴-exo state, as observed when hydroxylated HIF-alpha is bound to pVHL and in collagen. The results help to rationalise NODD/CODD selectivity data for PHD isoforms and the effects of clinically observed mutations on PHD2 catalysis. Analyses on the interaction of nitric oxide with PHD2 are described and discussed with respect to regulation of the hypoxic response by nitric oxide.