Multiple factors affecting cellular redox status and energy metabolism modulate hypoxia-inducible factor prolyl hydroxylase activity in vivo and in vitro

Prolyl hydroxylation of hypoxible-inducible factor alpha (HIF-alpha) proteins is essential for their recognition by pVHL containing ubiquitin ligase complexes and subsequent degradation in oxygen (O(2))-replete cells. Therefore, HIF prolyl hydroxylase (PHD) enzymatic activity is critical for the regulation of cellular responses to O(2) deprivation (hypoxia). Using a fusion protein containing the human HIF-1alpha O(2)-dependent degradation domain (ODD), we monitored PHD activity both in vivo and in cell-free systems. This novel assay allows the simultaneous detection of both hydroxylated and nonhydroxylated PHD substrates in cells and during in vitro reactions. Importantly, the ODD fusion protein is regulated with kinetics identical to endogenous HIF-1alpha during cellular hypoxia and reoxygenation. Using in vitro assays, we demonstrated that the levels of iron (Fe), ascorbate, and various tricarboxylic acid (TCA) cycle intermediates affect PHD activity. The intracellular levels of these factors also modulate PHD function and HIF-1alpha accumulation in vivo. Furthermore, cells treated with mitochondrial inhibitors, such as rotenone and myxothiazol, provided direct evidence that PHDs remain active in hypoxic cells lacking functional mitochondria. Our results suggest that multiple mitochondrial products, including TCA cycle intermediates and reactive oxygen species, can coordinate PHD activity, HIF stabilization, and cellular responses to O(2) depletion.     

USP13 enzyme regulates Siah2 ligase stability and activity via noncatalytic ubiquitin-binding domains

The RING finger E3 ubiquitin ligase Siah2 is implicated in control of diverse cellular biological events, including MAPK signaling and hypoxia. Here we demonstrate that Siah2 is subject to regulation by the deubiquitinating enzyme USP13. Overexpression of USP13 increases Siah2 stability by attenuating its autodegradation. Consequently, the ability of Siah2 to target its substrates prolyl hydroxylase 3 and Spry2 (Sprouty2) for ubiquitin-mediated proteasomal degradation is attenuated. Conversely, inhibition of USP13 expression with corresponding shRNA decreases the stability of both Siah2 and its substrate Spry2. Thus, USP13 limits Siah2 autodegradation and its ubiquitin ligase activity against its target substrates. Strikingly, the effect of USP13 on Siah2 is not mediated by its isopeptidase activity: mutations in its ubiquitin-binding sequences positioned within the ubiquitin-specific processing protease and ubiquitin-binding domains, but not within putative catalytic sites, abolish USP13 binding to and effect on Siah2 autodegradation and targeted ubiquitination. Notably, USP13 expression is attenuated in melanoma cells maintained under hypoxia, thereby relieving Siah2 inhibition and increasing its activity under low oxygen levels. Significantly, on melanoma tissue microarray, high nuclear expression of USP13 coincided with high nuclear expression of Siah2. Overall, this study identifies a new layer of Siah2 regulation mediated by USP13 binding to ubiquitinated Siah2 protein with a concomitant inhibitory effect on its activity under normoxia.     

Activation of the prolyl hydroxylase oxygen-sensor results in induction of GLUT1, heme oxygenase-1, and nitric-oxide synthase proteins and confers protection from metabolic inhibition to cardiomyocytes

Recently an oxygen-sensing/transducing mechanism has been identified as a family of O2-dependent prolyl hydroxylase domain-containing enzymes (PHD). In normoxia, PHD hydroxylates a specific proline residue that directs the degradation of constitutively synthesized hypoxia-inducible factor-1alpha. During hypoxia, the cessation of hydroxylation of this proline results in less degradation and thus increases hypoxia-inducible factor-1alpha protein levels. In this study we have examined the consequences of activating the PHD oxygen-sensing pathway in cultured neonatal myocytes using ethyl-3,4 dihydroxybenzoate and dimethyloxalylglycine, inhibitors that, similar to hypoxia, inhibit this family of O2-dependent PHD enzymes. Increased glucose uptake and enhanced glycolytic metabolism are classical cellular responses to hypoxia. Ethyl-3,4 dihydroxybenzoate treatment of cardiomyocyte cultures for 24 h increased [3H]deoxy-4-glucose uptake concurrent with an induction of GLUT1 protein. In addition, ethyl-3,4 dihydroxybenzoate, dimethyloxalylglycine, and hypoxia treatments were found to induce protein levels of nitricoxide synthase-2 and heme oxygenase-1, two important cardioregulatory proteins whose expression in response to hypoxic conditions is poorly understood. In conjunction with these changes in gene expression, activation of the PHD oxygen-sensing mechanism was found to preserve myocyte viability in the face of metabolic inhibition with cyanide and 2-deoxyglucose. These results point to a key role for the PHD pathway in the phenotypic changes that are observed in a hypoxic myocyte and may suggest a strategy to pharmacologically induce protection in heart.     

HECT Domain-containing E3 Ubiquitin Ligase Nedd4 Interacts with and Ubiquitinates Sprouty2

Sprouty (Spry) proteins are important regulators of receptor tyrosine kinase signaling in development and disease. Alterations in cellular Spry content have been associated with certain forms of cancers and also in cardiovascular diseases. Thus, understanding the mechanisms that regulate cellular Spry levels are important. Herein, we demonstrate that Spry1 and Spry2, but not Spry3 or Spry4, associate with the HECT domain family E3 ubiquitin ligase, Nedd4. The Spry2/Nedd4 association involves the WW domains of Nedd4 and requires phosphorylation of the Mnk2 kinase sites, Ser¹¹² and Ser¹²¹, on Spry2. The phospho-Ser^112/121 region on Spry2 that binds WW domains of Nedd4 is a novel non-canonical WW domain binding region that does not contain Pro residues after phospho-Ser. Endogenous and overexpressed Nedd4 polyubiquitinate Spry2 via Lys⁴⁸ on ubiquitin and decrease its stability. Silencing of endogenous Nedd4 increased the cellular Spry2 content and attenuated fibroblast growth factor-elicited ERK1/2 activation that was reversed when elevations in Spry2 levels were prevented by Spry2-specific small interfering RNA. Mnk2 silencing decreased Spry2-Nedd4 interactions and also augmented the ability of Spry2 to inhibit fibroblast growth factor signaling. This is the first report demonstrating the regulation of cellular Spry content and its ability to modulate receptor tyrosine kinase signaling by a HECT domain-containing E3 ubiquitin ligase.     

Intersectin 1 enhances Cbl ubiquitylation of epidermal growth factor receptor through regulation of Sprouty2-Cbl interaction

Ubiquitylation of receptor tyrosine kinases plays a critical role in regulating the trafficking and lysosomal degradation of these important signaling molecules. We identified the multidomain scaffolding protein intersectin 1 (ITSN1) as an important regulator of this process (N. P. Martin et al., Mol. Pharmacol. 70:1463-1653, 2006) ITSN1 stimulates ubiquitylation of the epidermal growth factor receptor (EGFR) through enhancing the activity of the Cbl E3 ubiquitin ligase. However, the precise mechanism through which ITSN1 enhances Cbl activity was unclear. In this study, we found that ITSN1 enhances Cbl activity through disrupting the interaction of Cbl with the Sprouty2 (Spry2) inhibitory protein. We demonstrate that ITSN1 binds Pro-rich regions in both Cbl and Spry2 and that interaction of ITSN1 with Spry2 disrupts Spry2-Cbl interaction, resulting in enhanced ubiquitylation of the EGFR. Disruption of ITSN1 binding to Spry2 through point mutation of the Pro-rich ITSN1 binding site in Spry2 results in enhanced Cbl-Spry2 interaction and inhibition of receptor ubiquitylation. This study demonstrates that ITSN1 enhances Cbl activity by modulating the interaction of Cbl with Spry2. In addition, our results reveal a new level of complexity in the regulation of Cbl through the interaction with ITSN1 and Spry2.     

Differential and Reciprocal Regulation between Hypoxia-inducible Factor-α Subunits and Their Prolyl Hydroxylases in Pulmonary Arteries of Rat with Hypoxia-induced Hypertension

Hypoxia-inducible factor (HIF)-α subunits (HIF-1α,HIF-2α and HIF-3α),which play a pivotalrole during the development of hypoxia-induced pulmonary hypertension (HPH),are regulated through post-U'anslational hydroxylation by their three prolyl hydroxylase domain-containing proteins (PHD 1,PHD2 and PHD3).PHDs could also be regulated by HIF.But differential and reciprocal regulation between HIF-α and PHDs duringthe development of HPH remains unclear.To investigate this problem,a rat HPH model was established.Meanpulmonary arterial pressure increased significantly after 7 d of hypoxia.Pulmonary artery remodeling indexand right ventricular hypertrophy became evident after 14 d of hypoxia.HIF-1α and HIF-2α mRNA increasedslightly after 7 d of hypoxia,but HIF-3α increased significantly after 3 d of hypoxia.The protein expressionlevels of all three HIF-α were markedly upregulated after exposure to hypoxia.PHD2 mRNA and proteinexpression levels were upregulated after 3 d of hypoxia;PHD 1 protein declined after 14 d of hypoxia withoutsignificant mRNA changes.PHD3 mRNA and protein were markedly upregulated after 3 d of hypoxia,then themRNA remained at a high level,but the protein declined after 14 d of hypoxia.In hypoxic animals,HIF-lotproteins negatively correlated with PHD2 proteins,whereas HIF-2α and HIF-3α proteins showed negativecorrelations with PHD3 and PHD 1 proteins,respectively.All three HIF-α proteins were positively correlatedwith PHD2 and PHD3 mRNA.In the present study,HIF-α subunits and PHDs showed differential andreciprocal regulation,and this might play a key pathogenesis role in hypoxia-induced pulmonary hypertension.     

Regulation of HIF prolyl hydroxylases by hypoxia-inducible factors

Hypoxia and induction of hypoxia-inducible factors (HIF-1alpha and HIF-2alpha) is a hallmark of many tumors. Under normal oxygen tension HIF-alpha subunits are rapidly degraded through prolyl hydroxylase dependent interaction with the von Hippel-Lindau (VHL) tumor suppressor protein, a component of E3 ubuiquitin ligase complex. Using microarray analysis of VHL mutated and re-introduced cells, we found that one of the prolyl hydroxylases (PHD3) is coordinately expressed with known HIF target genes, while the other two family members (PHD1 and 2) did not respond to VHL. We further tested the regulation of these genes by HIF-1 and HIF-2 and found that siRNA targeted degradation of HIF-1alpha and HIF-2alpha results in decreased hypoxia-induced PHD3 expression. Ectopic overexpression of HIF-2alpha in two different cell lines provided a much better induction of PHD3 gene than HIF-1alpha. In contrast, we demonstrate that PHD2 is not affected by overexpression or downregulation of HIF-2alpha. However, induction of PHD2 by hypoxia has HIF-1-independent and -dependent components. Short-term hypoxia (4 h) results in induction of PHD2 independent of HIF-1, while PHD2 accumulation by prolonged hypoxia (16 h) was decreased by siRNA-mediated degradation of HIF-1alpha subunit. These data further advance our understanding of the differential role of HIF factors and putative feedback loop in HIF regulation.     

Ablation of endothelial prolyl hydroxylase domain protein‐2 promotes renal vascular remodelling and fibrosis in mice

Accumulating evidence demonstrates that hypoxia‐inducible factor (HIF‐α) hydroxylase system has a critical role in vascular remodelling. Using an endothelial‐specific prolyl hydroxylase domain protein‐2 (PHD2) knockout (PHD2^ECKO) mouse model, this study investigates the regulatory role of endothelial HIF‐α hydroxylase system in the development of renal fibrosis. Knockout of PHD2 in EC up‐regulated the expression of HIF‐1α and HIF‐2α, resulting in a significant decline of renal function as evidenced by elevated levels of serum creatinine. Deletion of PHD2 increased the expression of Notch3 and transforming growth factor (TGF‐β1) in EC, thus further causing glomerular arteriolar remodelling with an increased pericyte and pericyte coverage. This was accompanied by a significant elevation of renal resistive index (RI). Moreover, knockout of PHD2 in EC up‐regulated the expression of fibroblast‐specific protein‐1 (FSP‐1) and increased interstitial fibrosis in the kidney. These alterations were strongly associated with up‐regulation of Notch3 and TGF‐β1. We concluded that the expression of PHD2 in endothelial cells plays a critical role in renal fibrosis and vascular remodelling in adult mice. Furthermore, these changes were strongly associated with up‐regulation of Notch3/TGF‐β1 signalling and excessive pericyte coverage.