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.     

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.     

细胞氧感受器：天冬酰胺酰羟化酶

缺氧诱导因子（hypoxia-inducible factor, HIF）是异二聚体的转录因子,由氧敏感的α亚基和在细胞内稳定表达的β亚基组成,在细胞缺氧应答反应中起核心作用.缺氧诱导因子脯氨酰羟化酶（prolyl hydroxylase domain-containing proteins, PHDs）和天冬酰胺酰羟化酶,即缺氧诱导因子抑制因子(factor-inhibiting HIF, FIH）是调节缺氧诱导因子蛋白质水平和活性的2类关键酶,它们自身的催化活性受细胞内氧张力的调节,因而被称为细胞氧感受器.目前,大多数的研究都集中于PHDs,而对FIH的研究相对较少.本文主要就FIH的发现、晶体结构、生物学特征以及表达水平和活性调节等方面作一综述.     

Hypoxia-inducible factor 1α under rapid enzymatic hypoxia: Cells sense decrements of oxygen but not hypoxia per se

HIF1 (hypoxia-inducible factor 1α) is considered a central oxygen-threshold sensor in mammalian cells. In the presence of oxygen, HIF1 is marked by prolyl hydroxylases (PHDs) at the oxygen-dependent degradation (ODD) domain for ubiquitination followed by rapid proteasomal degradation. However, the actual mechanisms of oxygen sensing by HIF1 are still controversial. Thus, HIF1 expression correlates poorly with tissue oxygen levels, and PHDs are themselves target genes of HIF1 considered to readjust to new oxygen thresholds. In contrast to hypoxia chambers, we here establish an enzymatic model that allows both the rapid induction of stable hypoxia and independent control of H₂O₂. Rapid enzymatic hypoxia only transiently induced HIF1 in various cell types and the HIF1 was completely degraded within 8–12 h despite sustained hypoxia. HIF1 degradation under sustained hypoxia could be blocked by a competitive ODD–GFP construct and PHD siRNA, but also by cobalt chloride and micromolar H₂O₂ levels. Concomitant induction of PHDs further confirmed their role in degrading HIF1 under enzymatic hypoxia. The rapid and complete degradation of HIF1 under enzymatic hypoxia suggests that, in addition to hypoxia sensing, the HIF1/PHD loop may rather compensate for fluctuations of tissue oxygen staying tuned to other, e.g., metabolic, signals. In addition to hypoxia chambers, enzymatic hypoxia provides a valuable tool for independently studying the regulatory functions of hypoxia and oxidative stress in vitro.     

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.     

Hypoxia inducible factor(HIF) in the tumor microenvironment:friend or foe?

Hypoxia acts as an important regulator of physiological and pathological processes. Hypoxia inducible factors (HIFs) are the central players involved in the cellular adaptation to hypoxia and are regulated by oxygen sensing EGLN prolyl hydroxylases. Hypoxia affects many aspects of cellular growth through both redox effects and through the stabilization of HIFs. The HIF isoforms likely have differential effects on tumor growth via alteration of metabolism, growth, and self-renewal and are likely highly context-dependent. In some tumors such as renal cell carcinoma, the EGLN/HIF axis appears to drive tumorigenesis, while in many others HIF1 and HIF2 may actually have a tumor suppressive role. An emerging role of HIF biology is its effects on the tumor microenvironment. The EGLN/HIF axis plays a key role in regulating the function of the various components of the tumor microenvironment, which include cancer-associated fibroblasts, endothelial cells, immune cells, and the extracellular matrix (ECM). Here, we discuss hypoxia and the diverse roles of HIFs in the setting of tumorigenesis and the maintenance of the tumor microenvironment as well as possible future directions of the field.