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.     

ATF3 represses PDX-1 expression in pancreatic β-cells

The downregulation of PDX-1 expression plays an important role in development of type 2 diabetes. However, the negative regulator of PDX-1 expression is not well known. In this study, we analyzed the mouse PDX-1 promoter to characterize the effects of ATF3 on PDX-1 expression in pancreatic β-cells. Both thapsigargin treatment, an inducer of ER stress, and ATF3 expression decreased PDX-1 expression in pancreatic β-cells, MIN6N8. Furthermore, they also repressed the activity of −4.5 Kb promoter of mouse PDX-1 gene. Transfection studies with 5′ deleted-reporters showed that ATF3 repressed the activity of 0.9 Kb PDX-1 promoter, whereas it did not affect the activity of 0.7 Kb PDX-1 promoter, suggesting that ATF3 responsive element is located between the −903 and −702. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 binds directly to the promoter region spanning from −759 to −738. Moreover, mutation of the putative ATF/CRE site between −752 and −745 abrogated ATF3-mediated transrepression of the PDX-1 promoter. PDX-1 was decreased in MIN6N8 cells treated with high glucose or high palmitate, whereas ATF3 was increased, indicating that ATF3 plays a role in hyperglycemia or hyperlipidemia-mediated downregulation of PDX-1 expression. Collectively, these results demonstrate that ATF3 represses PDX-1 expression via binding to an ATF3-responsive element in its promoter, which plays an important role in suppression of pancreatic β-cells function.     

Hypoxia-induced assembly of prolyl hydroxylase PHD3 into complexes: implications for its activity and susceptibility for degradation by the E3 ligase Siah2

PHD1-3 (prolyl hydroxylases 1-3) catalyse the hydroxylation of HIF (hypoxia-inducible factor)-alpha subunit that triggers the substrate ubiquitination and subsequent degradation. The RING (really interesting new gene) finger E3 ligase Siah2 preferentially targets PHD3 for degradation. Here, we identify the requirements for such selective targeting. Firstly, PHD3 lacks an N-terminal extension found in PHD1 and PHD2; deletion of this domain from PHD1 and PHD2 renders them susceptible to degradation by Siah2. Secondly, PHD3 can homo- and hetero-multimerize with other PHDs. Consequently, PHD3 is found in high-molecular-mass fractions that were enriched in hypoxia. Interestingly, within the lower-molecular-mass complex, PHD3 exhibits higher specific activity towards hydroxylation of HIF-1alpha and co-localizes with Siah2, suggesting that Siah2 limits the availability of the more active form of PHD3. These findings provide new insight into the mechanism underlying the regulation of PHD3 availability and activity in hypoxia by the E3 ligase Siah2.     

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 hydroxylation- and glycosylation-dependent functions of Skp1 in O2-regulated development of Dictyostelium

O₂ regulates multicellular development of the social amoeba Dictyostelium, suggesting it may serve as an important cue in its native soil environment. Dictyostelium expresses an HIFα-type prolyl 4-hydroxylase (P4H1) whose levels affect the O₂-threshold for culmination implicating it as a direct O₂-sensor, as in animals. But Dictyostelium lacks HIFα, a mediator of animal prolyl 4-hydroxylase signaling, and P4H1 can hydroxylate Pro143 of Skp1, a subunit of E3^SCFubiquitin-ligases. Skp1 hydroxyproline then becomes the target of five sequential glycosyltransferase reactions that modulate the O₂-signal. Here we show that genetically induced changes in Skp1 levels also affect the O₂-threshold, in opposite direction to that of the modification enzymes suggesting that the latter reduce Skp1 activity. Consistent with this, overexpressed Skp1 is poorly hydroxylated and Skp1 is the only P4H1 substrate detectable in extracts. Effects of Pro143 mutations, and of combinations of Skp1 and enzyme level perturbations, are consistent with pathway modulation of Skp1 activity. However, some effects were not mirrored by changes in modification of the bulk Skp1 pool, implicating a Skp1 subpopulation and possibly additional unknown factors. Altered Skp1 levels also affected other developmental transitions in a modification-dependent fashion. Whereas hydroxylation of animal HIFα results in its polyubiquitination and proteasomal degradation, Dictyostelium Skp1 levels were little affected by its modification status. These data indicate that Skp1 and possibly E3^SCFubiquitin-ligase activity modulate O₂-dependent culmination and other developmental processes, and at least partially mediate the action of the hydroxylation/glycosylation pathway in O₂-sensing.     

Loss of the Oxygen Sensor PHD3 Enhances the Innate Immune Response to Abdominal Sepsis

Hypoxia and HIFs (HIF-1α and HIF-2α) modulate innate immune responses in the setting of systemic inflammatory responses and sepsis. The HIF prolyl hydroxylase enzymes PHD1, PHD2 and PHD3 regulate the mammalian adaptive response to hypoxia; however, their significance in the innate immune response has not been elucidated. We demonstrate in this study that deficiency of PHD3 (PHD3(-/-)) specifically shortens the survival of mice subjected to various models of abdominal sepsis because of an overwhelming innate immune response, leading to premature organ dysfunction. By contrast, this phenotype was absent in mice deficient for PHD1 (PHD1(-/-)) or PHD2 (PHD2(+/-)). In vivo, plasma levels of proinflammatory cytokines were enhanced, and recruitment of macrophages to internal organs was increased in septic PHD3-deficient mice. Reciprocal bone marrow transplantation in sublethally irradiated mice revealed that enhanced susceptibility of PHD3-deficient mice to sepsis-related lethality was specifically caused by loss of PHD3 in myeloid cells. Several in vitro assays revealed enhanced cytokine production, migration, phagocytic capacity, and proinflammatory activation of PHD3-deficient macrophages. Increased proinflammatory activity of PHD3-deficient macrophages occurred concomitantly with enhanced HIF-1α protein stabilization and increased NF-κB activity, and interference with the expression of HIF-1α or the canonical NF-κB pathway blunted their proinflammatory phenotype. It is concluded that impairment of PHD3 enzyme function aggravates the clinical course of abdominal sepsis via HIF-1α- and NF-κB-mediated enhancement of the innate immune response.     

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.