Interferon-Gamma Induces Prolyl Hydroxylase (PHD)3 Through a STAT1-Dependent Mechanism in Human Endothelial Cells

Abstract

Objective-We previously reported that interferons (IFNs) regulate transcription of HIF-1␣ in human endothelial cells (ECs), linking immunity and hypoxia. Prolyl hydroxylases (PHDs) regulate expression of HIF-1␣ in response to hypoxia. We examined whether IFNs affect PHD expression and whether PHDs regulate the EC response to IFNs. Methods and Results-Human cell cultures were treated with various cytokines, and PHD expression was examined using qRT-PCR and immunoblotting. IFN␥ and, to a lesser extent, IFN␣ significantly induced PHD3, but not PHD1 or 2, mRNA, and protein expression selectively in ECs directly via a JAK/STAT1 pathway as demonstrated by pharmacological inhibition, siRNA knockdown, and chromatin immunoprecipitation. Inhibition of PHD activity with dimethyloxallyl glycine or desferroxamine reduced IFNg-dependent responses in these same cells. H ypoxia activates transcription of genes necessary for adaptation to low oxygen. 1,2 The best described response system uses hypoxia-inducible factors (HIFs) composed of the constitutively expressed subunit HIF-1␤ bound to labile subunits HIF-1␣ or HIF-2␣, forming HIF-1 or HIF-2, respectively. 3 HIF expression is regulated by a family of prolyl hydroxylases, PHD1, PHD2, and PHD3, that sense oxygen tension through its binding to an associated iron atom. 4,5 When the PHD iron is occupied by O 2 , these enzymes catalyze a reaction in which one oxygen atom reacts with 2-oxoglutarate to form succinate and CO 2 while the other is transferred to a proline residue in a protein substrate, such as HIF-1␣, to form a hydroxyproline side chain. Hydroxylation of proline in HIF-1␣ recruits the von HippelLindau (pVHL) complex, targeting HIF-1␣ for ubiquitination and proteasomal degradation. 6 -8 Molecular oxygen is normally rate limiting, and hypoxia causes HIF-1␣ protein stabilization and accumulation by inhibiting PHD-mediated proline hydroxylation. As its levels increase, HIF-1␣ enters the nucleus and dimerizes with HIF-1␤ to form active HIF-1, initiating transcription of genes that aid in adaptation to hypoxic conditions including enzymes that favor anaerobic glycolysis and factors that stimulate both angiogenesis and erythropoiesis. 5,9 PHD3 transcription is induced by HIF-1 under hypoxic conditions through a functional hypoxic response element (HRE) located in the first intron of PHD3. 10 HIF-1-dependent induction of PHD3 most likely serves as a negative feedback mechanism during hypoxia, and may promote the rapid degradation of HIF-1␣ or HIF-2␣ on reoxygenation. 6,11,12 Although most closely identified with the hypoxic response, PHDs may also regulate other molecular systems. PHD1 negatively regulates the NF-B pathway by repressing the activity of the positive regulator, IKK␤, through hydroxylation in an oxygen-sensitive reaction. 13 In rodents, PHD3 is required for normal neurological development through induction of neuron apoptosis. 14,15 Loss of PHD3 impairs development of sympathetic neurons and may lead to the formation of pheochromocytomas. 16 PHD3 interacts with ...

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