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.     

慢性阻塞性肺疾病患者肺小血管低氧诱导因子1α与其羟化酶表达

目的:通过观察慢性阻塞性肺疾病(COPD)患者肺小动脉内低氧诱导因子α亚基(HIF-1α)及HIF脯氨酸羟化酶(PHD)、HIF抑制因子的表达,探讨其在肺血管重塑中的可能作用。方法:选因肺肿瘤行肺叶切除者,COPD组(12例),对照组(14例)。取2组患者的肺组织,原位杂交和免疫组化检测HIF-1α、PHD1、PHD2、PHD3、FIH的mRNA及蛋白表达水平。观察并计算肺小动脉管壁厚度(PAMT)及肺小动脉管壁面积与血管总面积的比值(WA%)。结果:①COPD组PAMT(40μm±5μm)、WA%(50%±9%)均较对照组(分别为(31μm±4μm,39%±6%)高(均P<0.01);②COPD组肺小血管HIF-1αmRNA和蛋白表达(吸光度(A)值)(0.230±0.036,0.275±0.039)较对照组(0.174±0.029,0.102±0.015)增强(均P<0.01),蛋白质表达增高更明显。COPD组肺小血管PHD1 mRNA表达(0.180±0.030)与对照组(0.191±0.029)比无明显改变(P>0.05)。COPD组PHD2、PHD3 mRNA表达(0.245±0.044,0.252±0.023)较对照组(0.182±0.028,0.127±0.017)明显增高(均P<0.01)。PHD1蛋白质表达(0.104±0.015)较对照组(0.209±0.023)降低(P<0.01)。PHD2蛋白质表达(0.274±0.044)较对照组(0.219±0.043)增高(P<0.01)。PHD3蛋白质表达(0.161±0.023)较对照组(0.146±0.021)略增高,但差异无统计学意义(P>0.05)。两组间FIHmRNA和蛋白质表达差异均无显著性(P>0.05);③相关分析表明HIF-1α蛋白质水平与WA%,PAMT及PHD2、PHD3 mRNA及PHD2蛋白质呈正相关,与PHD1蛋白质呈负相关。结论:PHDs可能通过调节HIF-1α表达参与COPD患者的肺血管重塑。     

Overexpression of HIF Prolyl‐Hydoxylase‐2 transgene in the renal medulla induced a salt sensitive hypertension

Renal medullary hypoxia‐inducible factor (HIF)‐1α and its target genes, such as haem oxygenase and nitric oxide synthase, have been indicated to play an important role in the regulation of sodium excretion and blood pressure. HIF prolyl hydroxylase domain‐containing proteins (PHDs) are major enzymes to promote the degradation of HIF‐1α. We recently reported that high salt intake suppressed the renal medullary PHD2 expression and thereby activated HIF‐1α‐mediated gene regulation in the renal medulla in response to high salt. To further define the functional role of renal medullary PHD2 in the regulation of renal adaptation to high salt intake and the longer term control of blood pressure, we transfected PHD2 expression plasmids into the renal medulla in uninephrectomized rats and determined its effects on pressure natriuresis, sodium excretion after salt overloading and the long‐term control of arterial pressure after high salt challenge. It was shown that overexpression of PHD2 transgene increased PHD2 levels and decreased HIF‐1α levels in the renal medulla, which blunted pressure natriuresis, attenuated sodium excretion, promoted sodium retention and produced salt sensitive hypertension after high salt challenge compared with rats treated with control plasmids. There was no blood pressure change in PHD2‐treated rats that were maintained in low salt diet. These results suggested that renal medullary PHD2 is an important regulator in renal adaptation to high salt intake and a deficiency in PHD2‐mediated molecular adaptation in response to high salt intake in the renal medulla may represent a pathogenic mechanism producing salt sensitive hypertension.     

Role and regulation of prolyl hydroxylase domain proteins

Oxygen-dependent hydroxylation of hypoxia-inducible factor (HIF)-alpha subunits by prolyl hydroxylase domain (PHD) proteins signals their polyubiquitination and proteasomal degradation, and plays a critical role in regulating HIF abundance and oxygen homeostasis. While oxygen concentration plays a major role in determining the efficiency of PHD-catalyzed hydroxylation reactions, many other environmental and intracellular factors also significantly modulate PHD activities. In addition, PHDs may also employ hydroxylase-independent mechanisms to modify HIF activity. Interestingly, while PHDs regulate HIF-alpha protein stability, PHD2 and PHD3 themselves are subject to feedback upregulation by HIFs. Functionally, different PHD isoforms may differentially contribute to specific pathophysiological processes, including angiogenesis, erythropoiesis, tumorigenesis, and cell growth, differentiation and survival. Because of diverse roles of PHDs in many different processes, loss of PHD expression or function triggers multi-faceted pathophysiological changes as has been shown in mice lacking different PHD isoforms. Future investigations are needed to explore in vivo specificity of PHDs over different HIF-alpha subunits and differential roles of PHD isoforms in different biological processes.     

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.     

Prolyl Hydroxylase 3 (PHD3) Modulates Catabolic Effects of Tumor Necrosis Factor-α (TNF-α) on Cells of the Nucleus Pulposus through Co-activation of Nuclear Factor κB (NF-κB)/p65 Signaling

Recent studies suggest a differential role of prolyl hydroxylase (PHD) isoforms in controlling hypoxia-inducible factor (HIF)-α degradation and activity in nucleus pulposus (NP) cells. However, the regulation and function of PHDs under inflammatory conditions that characterize disc disease are not yet known. Here, we show that in NP cells, TNF-α and IL-1β induce PHD3 expression through NF-κB. Lentiviral delivery of Sh-p65 and Sh-IKKβ confirms that cytokine-mediated PHD3 expression is NF-κB-dependent. It is noteworthy that although both cytokines induce HIF activity, mechanistic studies using Sh-HIF-1α and PHD3 promoter/enhancer constructs harboring well characterized hypoxia response element (HRE) show lack of HIF involvement in cytokine-mediated PHD3 expression. Loss-of-function studies clearly indicate that PHD3 serves as a co-activator of NF-κB signaling activity in NP cells; PHD3 interacts with, and co-localizes with, p65. We observed that when PHD3 is silenced, there is a significant decrease in TNF-α-induced expression of catabolic markers that include ADAMTS5, syndecan4, MMP13, and COX2, and at the same time, there is restoration of aggrecan and collagen type II expression. It is noteworthy that hydroxylase function of PHDs is not required for mediating cytokine-dependent gene expression. These findings show that by enhancing the activity of inflammatory cytokines, PHD3 may serve a critical role in degenerative disc disease.     

Retention of prolyl hydroxylase PHD2 in the cytoplasm prevents PHD2-induced anchorage-independent carcinoma cell growth

Cellular oxygen tension is sensed by a family of prolyl hydroxylases (PHD1-3) that regulate the degradation of hypoxia-inducible factors (HIF-1α and -2α). The PHD2 isoform is considered as the main downregulator of HIF in normoxia. Our previous results have shown that nuclear translocation of PHD2 associates with poorly differentiated tumor phenotype implying that nuclear PHD2 expression is advantageous for tumor growth. Here we show that a pool of PHD2 is shuttled between the nucleus and the cytoplasm. In line with this, accumulation of wild type PHD2 in the nucleus was detected in human colon adenocarcinomas and in cultured carcinoma cells. The PHD2 isoforms showing high nuclear expression increased anchorage-independent carcinoma cell growth. However, retention of PHD2 in the cytoplasm inhibited the anchorage-independent cell growth. A region that inhibits the nuclear localization of PHD2 was identified and the deletion of the region promoted anchorage-independent growth of carcinoma cells. Finally, the cytoplasmic PHD2, as compared with the nuclear PHD2, less efficiently downregulated HIF expression. Forced HIF-1α or -2α expression decreased and attenuation of HIF expression increased the anchorage-independent cell growth. However, hydroxylase-inactivating mutations in PHD2 had no effect on cell growth. The data imply that nuclear PHD2 localization promotes malignant cancer phenotype.     

Non-hypoxic activation of the negative regulatory feedback loop of prolyl-hydroxylase oxygen sensors

Hypoxia inducible factors (HIF) coordinate cellular responses towards hypoxia. HIFs are mainly regulated by a group of prolyl-hydroxylases (PHDs) that in the presence of oxygen, target the HIFα subunit for degradation. Herein, we studied the role of nitric oxide (NO) in regulating PHD activities under normoxic conditions. In the present study we show that different NO-donors initially inhibited endogenous PHD2 activity which led to accumulation of HIF-1α subsequently to enhance HIF-1 dependent increased PHD2 promoter activity. Consequently PHD2 abundance and activity were strongly induced which caused downregulation of HIF-1α. Interestingly, upregulation of endogenous PHD2 activity by NO was not found in cells that lack an intact pVHL dependent degradation pathway. Recovery of PHD activity required intact cells and was not observed in cell extracts or recombinant PHD2. In conclusion induction of endogenous PHD2 activity by NO is dependent on a feedback loop initiated despite normoxic conditions.     

Prolyl-hydroxylase inhibition and HIF activation in osteoblasts promotes an adipocytic phenotype

Bone is a dynamic environment where cells sense and adapt to changes in nutrient and oxygen availability. Conditions associated with hypoxia in bone are also associated with bone loss. In vitro hypoxia (2% oxygen) alters gene expression in osteoblasts and osteocytes and induces cellular changes including the upregulation of hypoxia inducible factor (HIF) levels. Our studies show that osteoblasts respond to hypoxia (2% oxygen) by enhancing expression of genes associated with adipocyte/lipogenesis phenotype (C/EBPbeta, PPARgamma2, and aP2) and by suppressing expression of genes associated with osteoblast differentiation (alkaline phosphatase, AP). Hypoxia increased HIF protein levels, hypoxic response element (HRE) binding, and HRE-reporter activity. We also demonstrate that prolyl-hydroxylases 2 and 3 (PHD2, PHD3), one of the major factors coordinating HIF degradation under normoxic but not hypoxic conditions, are induced in osteoblasts under hypoxic conditions. To further determine the contribution of PHDs and upregulated HIF activity in modulating osteoblast phenotype, we treated osteoblasts with a PHD inhibitor, dimethyloxaloylglycine (DMOG), and maintained cells under normoxic conditions. Similar to hypoxic conditions, HRE reporter activity was increased and adipogenic gene expression was increased while osteoblastic genes were suppressed. Taken together, our findings indicate a role for PHDs and HIFs in the regulation of osteoblast phenotype.     

Prolyl hydroxylase PHD3 activates oxygen-dependent protein aggregation

The HIF prolyl hydroxylases (PHDs/EGLNs) are central regulators of the molecular responses to oxygen availability. One isoform, PHD3, is expressed in response to hypoxia and causes apoptosis in oxygenated conditions in neural cells. Here we show that PHD3 forms subcellular aggregates in an oxygen-dependent manner. The aggregation of PHD3 was seen under normoxia and was strongly reduced under hypoxia or by the inactivation of the PHD3 hydroxylase activity. The PHD3 aggregates were dependent on microtubular integrity and contained components of the 26S proteasome, chaperones, and ubiquitin, thus demonstrating features that are characteristic for aggresome-like structures. Forced expression of the active PHD3 induced the aggregation of proteasomal components and activated apoptosis under normoxia in HeLa cells. The apoptosis was seen in cells prone to PHD3 aggregation and the PHD3 aggregation preceded apoptosis. The data demonstrates the cellular oxygen sensor PHD3 as a regulator of protein aggregation in response to varying oxygen availability.