Targeting EGLN2/PHD1 protects motor neurons and normalizes the astrocytic interferon response.

Neuroinflammation and dysregulated energy metabolism are linked to motor neuron degeneration in amyotrophic lateral sclerosis (ALS). The egl-9 family hypoxia-inducible factor (EGLN) enzymes, also known as prolyl hydroxylase domain (PHD) enzymes, are metabolic sensors regulating cellular inflammation and metabolism. Using an oligonucleotide-based and a genetic approach, we showed that the downregulation of Egln2 protected motor neurons and mitigated the ALS phenotype in two zebrafish models and a mouse model of ALS. Single-nucleus RNA sequencing of the murine spinal cord revealed that the loss of EGLN2 induced an astrocyte-specific downregulation of interferon-stimulated genes, mediated via the stimulator of interferon genes (STING) protein. In addition, we found that the genetic deletion of EGLN2 restored this interferon response in patient induced pluripotent stem cell (iPSC)-derived astrocytes, confirming the link between EGLN2 and astrocytic interferon signaling. In conclusion, we identified EGLN2 as a motor neuron protective target normalizing the astrocytic interferon-dependent inflammatory axis in vivo, as well as in patient-derived cells.

Zebrafish phd1 enhances mavs-mediated antiviral responses in a hydroxylation-independent manner.

PHD1 is a member of the prolyl hydroxylase domain protein (PHD1-4) family, which plays a prominent role in the post-translational modification of its target proteins by hydroxylating proline residues. The best-characterized targets of PHD1 are hypoxia-inducible factor α (HIF-1α and HIF-2α), two master regulators of the hypoxia signaling pathway. In this study, we show that zebrafish phd1 positively regulates mavs-mediated antiviral innate immunity. Overexpression of phd1 enhances the cellular antiviral response. Consistently, zebrafish lacking phd1 are more susceptible to spring viremia of carp virus infection. Further assays indicate that phd1 interacts with mavs through the C-terminal transmembrane domain of mavs and promotes mavs aggregation. In addition, zebrafish phd1 attenuates K48-linked polyubiquitination of mavs, leading to stabilization of mavs. However, the enzymatic activity of phd1 is not required for phd1 to activate mavs. In conclusion, this study reveals a novel function of phd1 in the regulation of antiviral innate immunity.IMPORTANCEPHD1 is a key regulator of the hypoxia signaling pathway, but its role in antiviral innate immunity is largely unknown. In this study, we found that zebrafish phd1 enhances cellular antiviral responses in a hydroxylation-independent manner. Phd1 interacts with mavs through the C-terminal transmembrane domain of mavs and promotes mavs aggregation. In addition, phd1 attenuates K48-linked polyubiquitination of mavs, leading to stabilization of mavs. Zebrafish lacking phd1 are more susceptible to spring viremia of carp virus infection. These findings reveal a novel role for phd1 in the regulation of mavs-mediated antiviral innate immunity.

Loss of prolyl hydroxylase 1 and 2 in SM22α-expressing cells prevents Hypoxia-Induced pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a disease characterized by increased vasoconstriction and vascular remodeling. Pulmonary artery smooth muscle cells (PASMCs) highly express the transcription factor hypoxia-inducible factor-1α (HIF-1α), yet the role of PASMC HIF-1α in the development of PAH remains controversial. To study the role of SMC HIF-1α in the pulmonary vascular response to acute and chronic hypoxia, we used a gain-of-function strategy to stabilize HIF-1α in PASMC by generating mice lacking prolyl hydroxylase domain (PHD) 1 and 2 in SM22α-expressing cells. This strategy increased HIF-1α expression and transcriptional activity under conditions of normoxia and hypoxia. Acute hypoxia increased right ventricular systolic pressure (RVSP) in control, but not in SM22α-PHD1/2-/- mice. Chronic hypoxia increased RVSP and vascular remodeling more in control SM22α-PHD1/2+/+ than in SM22α-PHD1/2-/- mice. In vitro studies demonstrated increased contractility and myosin light chain phosphorylation in isolated PHD1/2+/+ compared with PHD1/2-/- PASMC under both normoxic and hypoxic conditions. After chronic hypoxia, there was more p27 and less vascular remodeling in SM22α-PHD1/2-/- compared with SM22α-PHD1/2+/+ mice. Hypoxia increased p27 in PASMC isolated from control patients, but not in cells from patients with idiopathic pulmonary arterial hypertension (IPAH). These findings highlight an SM22α-expressing cell-specific role for HIF-1α in the inhibition of pulmonary vasoconstriction and vascular remodeling. Modulating HIF-1α expression in PASMC may represent a promising preventative and therapeutic strategy for patients with PAH.NEW & NOTEWORTHY In a mouse model wherein hypoxia-inducible factor 1 alpha (HIF-1α) is stabilized in vascular smooth muscle cells, we found that HIF-1α regulates vasoconstriction by limiting phosphorylation of myosin light chain and regulates vascular remodeling through p27 induction. These findings highlight a cell-specific role for HIF-1α in the inhibition of pulmonary vasoconstriction and vascular remodeling.

Decreased expression of prolyl hydroxylase 1 is associated with poor prognosis in colorectal cancers.

BACKGROUND: Prolyl hydroxylase 1 (PHD1) is a prognostic marker in several cancers. AIMS AND SCOPES: This study was undertaken to elucidate the clinical relevance of PHD1 in colorectal cancer (CRC) prognosis. MATERIALS AND METHODS: We compared PHD1 expression on a tissue microarray (TMA) containing samples from 1800 CRCs with corresponding clinicopathological tumor variables and patient survival. RESULTS: While PHD1 staining was always high in benign colorectal epithelium, high PHD1 staining was detectable in only 71.8% of CRCs. Low PHD1 staining was associated with advanced tumor stage (p = 0.0101) and shortened overall survival in CRC patients (p = 0.0011). In a multivariable analysis including tumor stage, histological type and PHD1 staining revealed tumor stage and histological type (p < 0.0001 each), but also PHD1 staining (p = 0.0202) to be independent prognostic markers for CRC. CONCLUSIONS: In our cohort, loss of PHD1 expression independently identified a subset of CRC patients with poor overall survival and might, thus, be a promising prognostic marker. PHD1 targeting may even allow for specific therapeutic approaches for these patients.

Elevation of Intracellular Alpha-Ketoglutarate Levels Inhibits Osteoclastogenesis by Suppressing the NF-κB Signaling Pathway in a PHD1-Dependent Manner.

Age-related osteoporosis, a high-prevalence disease in the aged population, is generally attributed to the excessive activity of osteoclasts. Most approved drugs treat osteoporosis by inhibition of osteoclasts. Although in vivo studies have shown that alpha-ketoglutarate (AKG), an intermediate in the TCA cycle, can ameliorate age-related osteoporosis, the effects of AKG on osteoclastogenesis and the underlying mechanism of its action have not been studied yet. Here, we showed that the elevation of intracellular AKG levels by supplementing dimethyl AKG (DM-AKG, a cell-permeable derivative of AKG) inhibits the receptor activator of NF-κB ligand (RANKL)-induced osteoclasts differentiation from primary bone marrow-derived macrophages (BMMs) and RAW264.7 cells in vitro. We further found that DM-AKG treatment suppresses NF-κB signaling and oxidative phosphorylation (OXPHOS) during RANKL-induced osteoclastogenesis in RAW264.7 cells. Interestingly, dimethyl oxalylglycine (DMOG), an AKG competitive inhibitor of AKG-dependent prolyl hydroxylases (PHDs), antagonizes the suppression of the RANKL-activated NF-κB signaling pathway caused by DM-AKG treatment. Furthermore, blocked PHD1 expression (also known as EglN2), instead of PHD2 or PHD3, was confirmed to reverse the DM-AKG treatment-induced suppression of the RANKL-activated NF-κB signaling pathway. Accordingly, blocked PHD1 expression antagonized the inhibitory effects of DM-AKG on osteoclastogenesis. Together, our finding suggests that the elevation of intracellular AKG levels inhibits osteoclastogenesis by suppressing RANKL-activated NF-κB signaling in a PHD1-dependent manner, which may provide a novel nutritional strategy for osteoporosis treatment.

Differential roles for the oxygen sensing enzymes PHD1 and PHD3 in the regulation of neutrophil metabolism and function.

Background: Neutrophils are essential in the early innate immune response to pathogens. Harnessing their antimicrobial powers, without driving excessive and damaging inflammatory responses, represents an attractive therapeutic possibility. The neutrophil population is increasingly recognised to be more diverse and malleable than was previously appreciated. Hypoxic signalling pathways are known to regulate important neutrophil behaviours and, as such, are potential therapeutic targets for regulating neutrophil antimicrobial and inflammatory responses. Methods: We used a combination of in vivo and ex vivo models, utilising neutrophil and myeloid specific PHD1 or PHD3 deficient mouse lines to investigate the roles of oxygen sensing prolyl hydroxylase enzymes in the regulation of neutrophilic inflammation and immunity. Mass spectrometry and Seahorse metabolic flux assays were used to analyse the role of metabolic shifts in driving the downstream phenotypes. Results: We found that PHD1 deficiency drives alterations in neutrophil metabolism and recruitment, in an oxygen dependent fashion. Despite this, PHD1 deficiency did not significantly alter ex vivo neutrophil phenotypes or in vivo outcomes in mouse models of inflammation. Conversely, PHD3 deficiency was found to enhance neutrophil antibacterial properties without excessive inflammatory responses. This was not linked to changes in the abundance of core metabolites but was associated with increased oxygen consumption and increased mitochondrial reactive oxygen species (mROS) production. Conclusions: PHD3 deficiency drives a favourable neutrophil phenotype in infection and, as such, is an important potential therapeutic target.

Lack of Skeletal Effects in Mice with Targeted Disruptionof Prolyl Hydroxylase Domain 1 (Phd1) Gene Expressed in Chondrocytes.

The critical importance of hypoxia-inducible factor (HIF)s in the regulation of endochondral bone formation is now well established. HIF protein levels are closely regulated by the prolyl hydroxylase domain-containing protein (PHD) mediated ubiquitin-proteasomal degradation pathway. Of the three PHD family members expressed in bone, we previously showed that mice with conditional disruption of the Phd2 gene in chondrocytes led to a massive increase in the trabecular bone mass of the long bones. By contrast, loss of Phd3 expression in chondrocytes had no skeletal effects. To investigate the role of Phd1 expressed in chondrocytes on skeletal development, we conditionally disrupted the Phd1 gene in chondrocytes by crossing Phd1 floxed mice with Collagen 2α1-Cre mice for evaluation of a skeletal phenotype. At 12 weeks of age, neither body weight nor body length was significantly different in the Cre+; Phd1flox/flox conditional knockout (cKO) mice compared to Cre−; Phd1flox/flox wild-type (WT) control mice. Micro-CT measurements revealed significant gender differences in the trabecular bone volume adjusted for tissue volume at the secondary spongiosa of the femur and the tibia for both genotypes, but no genotype differences were found for any of the trabecular bone measurements of either femur or tibia. Similarly, cortical bone parameters were not affected in the Phd1 cKO mice compared to control mice. Histomorphometric analyses revealed no significant differences in bone area, bone formation rate or mineral apposition rate in the secondary spongiosa of femurs between cKO and WT control mice. Loss of Phd1 expression in chondrocytes did not affect the expression of markers of chondrocytes (collage 2, collagen 10) or osteoblasts (alkaline phosphatase, bone sialoprotein) in the bones of cKO mice. Based on these and our published data, we conclude that of the three PHD family members, only Phd2 expressed in chondrocytes regulates endochondral bone formation and development of peak bone mass in mice.

Mucosal inflammation downregulates PHD1 expression promoting a barrier-protective HIF-1α response in ulcerative colitis patients.

The HIF hydroxylase enzymes (PHD1-3 and FIH) are cellular oxygen-sensors which confer hypoxic-sensitivity upon the hypoxia-inducible factors HIF-1α and HIF-2α. Microenvironmental hypoxia has a strong influence on the epithelial and immune cell function through HIF-dependent gene expression and consequently impacts upon the course of disease progression in ulcerative colitis (UC), with HIF-1α being protective while HIF-2α promotes disease. However, little is known about how inflammation regulates hypoxia-responsive pathways in UC patients. Here we demonstrate that hypoxia is a prominent microenvironmental feature of the mucosa in UC patients with active inflammatory disease. Furthermore, we found that inflammation drives transcriptional programming of the HIF pathway including downregulation of PHD1 thereby increasing the tissue responsiveness to hypoxia and skewing this response toward protective HIF-1 over detrimental HIF-2 activation. We identified CEBPα as a transcriptional regulator of PHD1 mRNA expression which is downregulated in both inflamed tissue derived from patients and in cultured intestinal epithelial cells treated with inflammatory cytokines. In summary, we propose that PHD1 downregulation skews the hypoxic response toward enhanced protective HIF-1α stabilization in the inflamed mucosa of UC patients.

The molecular characterization, expression pattern and alternative initiation of Megalobrama amblycephala Hif prolyl hydroxylase Phd1.

HIF prolyl hydroxylase 1 (PHD1) functions in prolyl hydroxylation on mammal hypoxia-inducible factors (HIF), important transcription factors involved in hypoxia, however the roles of Phd1 in fish remain unclear. In this study, the full-length cDNA and promoter sequences of blunt snout bream (Megalobrama amblycephala) phd1 gene were isolated by a modified RACE strategy. The phd1 cDNA was 2672 bp for encoding 481 amino acid residues. In silico assays indicated that phd1 had 5 exons, and a 348 bp CpG island in the exon1, and several transcription factor binding sites (CAAT box, HRE, ARNT, FOX, etc) were also found on the promoter. The quantitative real-time PCR results suggested that phd1 mRNA was constitutively expressed in all detected tissues, and higher in the blood, brain and heart in normoxia, but significantly decreased after hypoxia in all detected tissues except for gill. Western blot assays indicated that two Phd1 isoforms were generated by alternative translation initiation. Moreover, these two isoforms were both localized in the nucleus, therein only the senior isoform promoted cell proliferation. Taken together, the present study firstly describes the functions of M. amblycephala two Phd1 isoforms in hypoxia and cell proliferation.

Effects of Simultaneous Downregulation of PHD1 and Keap1 on Prevention and Reversal of Liver Fibrosis in Mice.

Background and Aim: To investigate whether double-knockdown of PHD1 and Keap1 in mice could enhance the resolution of carbon tetrachloride (CCl4)-induced liver fibrosis. Methods: The liver fibrosis model of mice was established by intraperitoneal injection of 25% CCl4 in olive oil (4 ul/g) twice a week for 8 weeks. PHD1shRNA and Keap1shRNA eukaryotic expression plasmids were simultaneously administered from the beginning of the first to fourth week (preventive group) or from the fifth to eighth week of CCl4 injection (therapeutic group) via hydrodynamic-based tail vein injection. Successful transfection was confirmed with the expression of red fluorescent protein and green fluorescent protein in hepatocytes. Western blot was used for determining the expression of PHD1 and Keap1, HE, Sirius red, and Masson staining for evaluating the histopathological stages of fibrosis. Immunohistochemical techniques were applied to evaluate the expression of a-SMA. Results: The fluorescence of red and green were observed mainly in hepatocytes, and downregulation of PHD1 and Keap1 expression in liver was detected by western blot. Meanwhile, double-knockdown of PHD1 and Keap1 in mice alleviated liver fibrosis, and the effect was further enhanced especially in the preventive group. Immunocytochemical staining showed decreased expression of a-SMA when both PHD1 and Keap1 were knockdown. Conclusion: Downregulation of PHD1 and Keap1 expression in the liver could be achieved via hydrodynamic injection of PHD1shRNA and Keap1shRNA, thereby, preventing liver fibrosis.

The PHD1 oxygen sensor in health and disease.

The hypoxia-inducible factor (HIF) co-ordinates the adaptive transcriptional response to hypoxia in metazoan cells. The hypoxic sensitivity of HIF is conferred by a family of oxygen-sensing enzymes termed HIF hydroxylases. This family consists of three prolyl hydroxylases (PHD1-3) and a single asparagine hydroxylase termed factor inhibiting HIF (FIH). It has recently become clear that HIF hydroxylases are functionally non-redundant and have discrete but overlapping physiological roles. Furthermore, altered abundance or activity of these enzymes is associated with a number of pathologies. Pharmacological HIF-hydroxylase inhibitors have recently proven to be both tolerated and therapeutically effective in patients. In this review, we focus on the physiology, pathophysiology and therapeutic potential of the PHD1 isoform, which has recently been implicated in diseases including inflammatory bowel disease, ischaemia and cancer.

Double Knockdown of PHD1 and Keap1 Attenuated Hypoxia-Induced Injuries in Hepatocytes.

Background and Aims: Hypoxia and oxidative stress contribute toward liver fibrosis. In this experiment, we used small hairpin RNA (shRNA) to interfere with the intracellular oxygen sensor-prolyl hydroxylase 1 (PHD1) and the intracellular oxidative stress sensor-kelch-like ECH associated protein 1 (Keap1) in the hypoxic hepatocytes in order to investigate the function of PHD1and Keap1. Methods: We first established the CCl4-induced liver fibrosis model, subsequently, the levels of the PHD1, hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), Keap1, and nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) were detected in liver tissues. Simultaneously, AML12 cells co-transfected with PHD1 and Keap1shRNAs were constructed in vitro, then the intracellular oxidative stress, the proportion of cells undergoing apoptosis, and cell viability were measured. The expression of pro-fibrogenic molecules were analyzed via quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The level of alpha-1 type I collagen (COL1A1) was determined using an enzyme-linked immunosorbent assay (ELISA). Finally, serum-free "conditioned medium" (CM) from the supernatant of hypoxic AML12 hepatocytes was used to culture rat hepatic stellate cells (HSC-T6), and the levels of fibrosis-related molecules, apoptosis, and cell proliferation were determined. Results: The marker of hypoxia-HIF-1α and HIF-2α in the livers with fibrosis were upregulated, however, the increase in PHD1 expression was not statistically significant in comparison to the control group. Sign of oxidative stress-Keap1 was increased, while the expression of Nrf2, one of the Keap1 main downstream molecules, was reduced in the hepatocytes. And in vitro, the double-knockdown of PHD1 and Keap1 in AML12 hepatocytes presented with decreased hypoxia-induced oxidative stress and apoptosis, furthermore, these hypoxic AML12 cells showed the increased cell viability and the doweregulated expression of pro-fibrogenic molecules. In addition, HSC-T6 cells cultured in the hypoxic double-knockdown CM demonstrated the downregulation of fibrosis-related molecules, diminished cell proliferation, and enhanced apoptosis. Conclusions: Our study demonstrated that double-knockdown of PHD1 and Keap1 attenuated hypoxia and oxidative stress induced injury in the hepatocytes, and subsequently inhibited HSC activation, which offers a novel therapeutic strategy in the prophylaxis and treatment of liver fibrosis.

Over-expression of prolyl hydroxylase-1 blocks NF-κB-mediated cyclin D1 expression and proliferation in lung carcinoma cells.

Prolyl hydroxylase-1 (PHD1), a member of the hypoxia inducible factor (HIF)-PHD family, plays an important role in regulating the stability of HIFs. The nuclear factor-κB (NF-κB) pathway consists of a family of transcription factors that play critical roles in inflammation, immunity, cell proliferation, differentiation, and survival. In this study, we demonstrate that PHD1 can inhibit NF-κB activity and its target genes in lung cancer cells based on both over-expression and RNA interference-mediated knockdown of PHD1 in human A549 lung cancer cells and HEK293 T cells. Of medical importance, PHD1 could induce cell cycle arrest in lung cancer cells, resulting in the suppression of cell proliferation. Xenograft tumor growth assays indicate that PHD1 plays a critical role in suppressing lung cancer growth. These findings reveal a new role of PHD1 in lung cancer and provide new treatment perspectives for cancer therapy by characterizing PHD1 as a potential target.