Purchase this article with an account.
George Hoppe, John Au, Julieta Zutel, Jonathan E. Sears; Capillary Rescue in Oxygen Induced Retinopathy by Inhibition of Prolyl Hydroxylation is Associated with Transcriptional Induction of HIF-1α through Non-inflammatory Activation of NF-B. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2535.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Systemic inhibition of prolyl hydroxylase (PHD) activity prevents oxygen-induced loss of retinal capillaries in the mouse model of retinopathy of prematurity. PHD inhibition was not only associated with the canonical post-translational stabilization of hepatic hypoxia-inducible factor (HIF), but also with an unanticipated increase in retinal HIF mRNA. Since HIF expression requires transcriptional activity of NF-ΚB, we asked whether hypoxia-mimetics are capable of NF-ΚB activation.
Three structurally distinct PHD inhibitors, i.e., α-ketoglutarate analog dimethyloxalylglycine (DMOG), triazinoindole hydrazone derivative CB1, and phthalazine derivative hydralazine (HZ), were tested for their ability upregulate NF-ΚB pathway in cultured Muller cells and hepatocytes. Phosphorylation at Ser-536 and nuclear translocation of p65 subunit, phosphorylation of IKK, protein levels of A20, COX-2 and HIF-1α were investigated by Western blotting following subcellular fractionation. Gene products of HIF and NF-kB were assessed by measuring erythropoietin and IL-6 in cell culture media using ELISA. Finally the transcriptional activity of NF-ΚB was measured by binding of p50 to ΚB consensus sequence and the activity of ΚB promoter was evaluated by ΚB reporter gene assay using HEK293-ΚB-Luc stable cells. The effect of intraperitoneal (i.p.) administration of DMOG on the levels of HIF-1α transcript in the liver, kidney, brain and retina was assessed by quantitative RT-PCR.
PHD inhibitors demonstrated wide range of potency to induce p65 phosphorylation and nuclear translocation from 3 mM (DMOG) to 1 μM (CB1). All compounds caused rapid accumulation of HIF-1α, while NF-ΚB activation was delayed by 24 hrs. Based on the ability of CB1 and HZ, but not of DMOG, to stimulate sustained secretion of erythropoietin, it is likely that DMOG is relatively unstable. In fact, in vivo DMOG was undetectable 3 hrs after i.p. injection in mice. Despite robust mobilization of p65 subunit, neither activation of IKK nor induction of inflammatory mediators IL-6 and COX-2 was induced by PHD inhibition. In contrast, TNFα treatment activated multiple steps of the NF-ΚB pathway, e.g., p65 phosphorylation and nuclear accumulation, IKK phosphorylation, DNA-binding of p50 to ΚB site, ΚB-driven transcription, and secretion of IL-6.
In addition to blocking oxygen-dependent HIF degradation and transcriptional upregulation of HIF, PHD inhibitors induce p65 nuclear translocation in Muller cells. Non-inflammatory activation of retinal NF-ΚB may regulate HIF expression and vascular protection.
This PDF is available to Subscribers Only