Paracrine Induction of HIF by Glutamate in Breast Cancer: EglN1 Senses Cysteine.

The HIF transcription factor promotes adaptation to hypoxia and stimulates the growth of certain cancers, including triple-negative breast cancer (TNBC). The HIFα subunit is usually prolyl-hydroxylated by EglN family members under normoxic conditions, causing its rapid degradation. We confirmed that TNBC cells secrete glutamate, which we found is both necessary and sufficient for the paracrine induction of HIF1α in such cells under normoxic conditions. Glutamate inhibits the xCT glutamate-cystine antiporter, leading to intracellular cysteine depletion. EglN1, the main HIFα prolyl-hydroxylase, undergoes oxidative self-inactivation in the absence of cysteine both in biochemical assays and in cells, resulting in HIF1α accumulation. Therefore, EglN1 senses both oxygen and cysteine.

Activation of the hypoxia-inducible factor pathway by roxadustat improves glucose metabolism in human primary myotubes from men.

AIMS/HYPOTHESIS: Hypoxia-inducible factor prolyl 4-hydroxylase (HIF-P4H) enzymes regulate adaptive cellular responses to low oxygen concentrations. Inhibition of HIF-P4Hs leads to stabilisation of hypoxia-inducible factors (HIFs) and activation of the HIF pathway affecting multiple biological processes to rescue cells from hypoxia. As evidence from animal models suggests that HIF-P4H inhibitors could be used to treat metabolic disorders associated with insulin resistance, we examined whether roxadustat, an HIF-P4H inhibitor approved for the treatment of renal anaemia, would have an effect on glucose metabolism in primary human myotubes. METHODS: Primary skeletal muscle cell cultures, established from biopsies of vastus lateralis muscle from men with normal glucose tolerance (NGT) (n=5) or type 2 diabetes (n=8), were treated with roxadustat. Induction of HIF target gene expression was detected with quantitative real-time PCR. Glucose uptake and glycogen synthesis were investigated with radioactive tracers. Glycolysis and mitochondrial respiration rates were measured with a Seahorse analyser. RESULTS: Exposure to roxadustat stabilised nuclear HIF1α protein expression in human myotubes. Treatment with roxadustat led to induction of HIF target gene mRNAs for GLUT1 (also known as SLC2A1), HK2, MCT4 (also known as SLC16A4) and HIF-P4H-2 (also known as PHD2 or EGLN1) in myotubes from donors with NGT, with a blunted response in myotubes from donors with type 2 diabetes. mRNAs for LDHA, PDK1 and GBE1 were induced to a similar degree in myotubes from donors with NGT or type 2 diabetes. Exposure of myotubes to roxadustat led to a 1.4-fold increase in glycolytic rate in myotubes from men with NGT (p=0.0370) and a 1.7-fold increase in myotubes from donors with type 2 diabetes (p=0.0044), with no difference between the groups (p=0.1391). Exposure to roxadustat led to a reduction in basal mitochondrial respiration in both groups (p<0.01). Basal glucose uptake rates were similar in myotubes from donors with NGT (20.2 ± 2.7 pmol mg-1 min-1) and type 2 diabetes (25.3 ± 4.4 pmol mg-1 min-1, p=0.4205). Treatment with roxadustat enhanced insulin-stimulated glucose uptake in myotubes from donors with NGT (1.4-fold vs insulin-only condition, p=0.0023). The basal rate of glucose incorporation into glycogen was lower in myotubes from donors with NGT (233 ± 12.4 nmol g-1 h-1) than in myotubes from donors with type 2 diabetes (360 ± 40.3 nmol g-1 h-1, p=0.0344). Insulin increased glycogen synthesis by 1.9-fold (p=0.0025) in myotubes from donors with NGT, whereas roxadustat did not affect their basal or insulin-stimulated glycogen synthesis. Insulin increased glycogen synthesis by 1.7-fold (p=0.0031) in myotubes from donors with type 2 diabetes. While basal glycogen synthesis was unaffected by roxadustat, pretreatment with roxadustat enhanced insulin-stimulated glycogen synthesis in myotubes from donors with type 2 diabetes (p=0.0345). CONCLUSIONS/INTERPRETATION: Roxadustat increases glycolysis and inhibits mitochondrial respiration in primary human myotubes regardless of diabetes status. Roxadustat may also improve insulin action on glycogen synthesis in myotubes from donors with type 2 diabetes.

Metabolic characteristics of transmembrane prolyl 4-hydroxylase (P4H-TM) deficient mice.

Transmembrane prolyl 4-hydroxylase (P4H-TM) is an enigmatic enzyme whose cellular function and primary substrate remain to be identified. Its loss-of-function mutations cause a severe neurological HIDEA syndrome with hypotonia, intellectual disability, dysautonomia and hypoventilation. Previously, P4H-TM deficiency in mice was associated with reduced atherogenesis and lower serum triglyceride levels. Here, we characterized the glucose and lipid metabolism of P4h-tm-/- mice in physiological and tissue analyses. P4h-tm-/- mice showed variations in 24-h oscillations of energy expenditure, VO2 and VCO2 and locomotor activity compared to wild-type (WT) mice. Their rearing activity was reduced, and they showed significant muscle weakness and compromised coordination. Sedated P4h-tm-/- mice had better glucose tolerance, lower fasting insulin levels, higher fasting lactate levels and lower fasting free fatty acid levels compared to WT. These alterations were not present in conscious P4h-tm-/- mice. Fasted P4h-tm-/- mice presented with faster hepatic glycogenolysis. The respiratory rate of conscious P4h-tm-/- mice was significantly lower compared to the WT, the decrease being further exacerbated by sedation and associated with acidosis and a reduced ventilatory response to both hypoxia and hypercapnia. P4H-TM deficiency in mice is associated with alterations in whole-body energy metabolism, day-night rhythm of activity, glucose homeostasis and neuromuscular and respiratory functions. Although the underlying mechanism(s) are not yet fully understood, the phenotype appears to have neurological origins, controlled by brain and central nervous system circuits. The phenotype of P4h-tm-/- mice recapitulates some of the symptoms of HIDEA patients, making this mouse model a valuable tool to study and develop tailored therapies.

Higher haemoglobin levels are associated with impaired periodontal status.

AIM: Cellular oxygen sensing mechanisms have been linked to periodontal condition, and levels of haemoglobin (Hb) (the main carrier of oxygen) can be used as a surrogate measure for hypoxia. We aimed to examine relations between Hb levels and key periodontal health parameters in a general population. MATERIALS AND METHODS: The population comprised 1711 (47% male) subjects from the Northern Finland Birth Cohort 1966, for whom an oral health examination was carried out at 46 years of age and whose Hb levels were within the Finnish reference values. Relative risks (RRs) were estimated using Poisson regression models. RESULTS: The low-Hb tertile (mean Hb 133 g/L) had healthier anthropometric, metabolic and periodontal health parameters than the high-Hb tertile (mean Hb 151 g/L). Multivariable regression models adjusted for risk factors showed Hb levels to be positively associated with alveolar bone loss (ABL) and periodontal pocket depth (PPD), although the associations were weaker after adjustment for key metabolic parameters and were strongly influenced by smoking status. CONCLUSIONS: Hb levels within the normal variation are positively associated with PPD and ABL. The association between Hb levels and periodontal condition appeared to be more complex than had previously been anticipated.

Contribution of HIF-P4H isoenzyme inhibition to metabolism indicates major beneficial effects being conveyed by HIF-P4H-2 antagonism.

Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases (HIF-P4Hs 1-3) are druggable targets in renal anemia, where pan-HIF-P4H inhibitors induce an erythropoietic response. Preclinical data suggest that HIF-P4Hs could also be therapeutic targets for treating metabolic dysfunction, although the contributions of HIF-P4H isoenzymes in various tissues to the metabolic phenotype are inadequately understood. Here, we used mouse lines that were gene-deficient for HIF-P4Hs 1 to 3 and two preclinical pan-HIF-P4H inhibitors to study the contributions of these isoenzymes to the anthropometric and metabolic outcome and HIF response. We show both inhibitors induced a HIF response in wildtype white adipose tissue (WAT), liver, and skeletal muscle and alleviated metabolic dysfunction during a 6-week treatment period, but they did not alter healthy metabolism. Our data indicate that HIF-P4H-1 contributed especially to skeletal muscle and WAT metabolism and that its loss lowered body weight and serum cholesterol levels upon aging. In addition, we found HIF-P4H-3 had effects on the liver and WAT and its loss increased body weight, adiposity, liver weight and triglyceride levels, WAT inflammation, and cholesterol levels and resulted in hyperglycemia and insulin resistance, especially during aging. Finally, we demonstrate HIF-P4H-2 affected all tissues studied; its inhibition lowered body and liver weight and serum cholesterol levels and improved glucose tolerance. We found very few HIF target metabolic mRNAs were regulated by the inhibition of three isoenzymes, thus suggesting a potential for selective therapeutic tractability. Altogether, these data provide specifications for the future development of HIF-P4H inhibitors for the treatment of metabolic diseases.

Inactivation of mouse transmembrane prolyl 4-hydroxylase increases blood brain barrier permeability and ischemia-induced cerebral neuroinflammation.

Hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) regulate the hypoxic induction of >300 genes required for survival and adaptation under oxygen deprivation. Inhibition of HIF-P4H-2 has been shown to be protective in focal cerebral ischemia rodent models, while that of HIF-P4H-1 has no effects and inactivation of HIF-P4H-3 has adverse effects. A transmembrane prolyl 4-hydroxylase (P4H-TM) is highly expressed in the brain and contributes to the regulation of HIF, but the outcome of its inhibition on stroke is yet unknown. To study this, we subjected WT and P4htm-/- mice to permanent middle cerebral artery occlusion (pMCAO). Lack of P4H-TM had no effect on lesion size following pMCAO, but increased inflammatory microgliosis and neutrophil infiltration was observed in the P4htm-/- cortex. Furthermore, both the permeability of blood brain barrier and ultrastructure of cerebral tight junctions were compromised in P4htm-/- mice. At the molecular level, P4H-TM deficiency led to increased expression of proinflammatory genes and robust activation of protein kinases in the cortex, while expression of tight junction proteins and the neuroprotective growth factors erythropoietin and vascular endothelial growth factor was reduced. Our data provide the first evidence that P4H-TM inactivation has no protective effect on infarct size and increases inflammatory microgliosis and neutrophil infiltration in the cortex at early stage after pMCAO. When considering HIF-P4H inhibitors as potential therapeutics in stroke, the current data support that isoenzyme-selective inhibitors that do not target P4H-TM or HIF-P4H-3 would be preferred.

Lower hemoglobin levels associate with higher baroreflex sensitivity and heart rate variability.

The aim of this study was to cross-sectionally examine whether hemoglobin (Hb) levels within the normal variation associate with heart rate variability (HRV) measures and baroreflex sensitivity (BRS). The study population included 733 Finnish subjects of the OPERA cohort (aged 41-59 yr, 53% males, 51.7% treated for hypertension) of whom HRV was measured from a standardized 45-min period and whose Hb levels were within the Finnish reference intervals. The low Hb tertile (mean Hb, 135 g/L) had an overall healthier metabolic profile compared with the high Hb tertile (mean Hb, 152 g/L). BRS was higher in the low Hb tertile compared with the high Hb tertile (P < 0.05). R-R interval (RRi) and standard deviation (SD) of the RRi (SDNN)index were the longest in the low Hb tertile regardless of posture. Of the spectral components of HRV, HF power was the highest in the low Hb tertile regardless of posture (P < 0.05). In a stepwise logistic regression model, BRS associated negatively with Hb levels after adjusting for covariates (B = -0.160 [-0.285; -0.035]). Similar associations were observed for SDNNindex when lying down (B = -0.105 [-0.207; -0.003]) and walking (B = -0.154 [-0.224; -0.083]). For HF power negative associations with Hb levels were observed when lying down (B = -0.110 [-0.180; -0.040]), sitting (B = -0.150 [-0.221; -0.079]), and in total analysis (B = -0.124 [-0.196; -0.053]). Overall, lower Hb levels associated independently with healthier cardiac autonomic function.NEW & NOTEWORTHY Heart rate variability (HRV) and baroreflex sensitivity (BRS), which can be measured noninvasively, can predict cardiac and metabolic diseases. Our findings show that within normal variation subjects with lower hemoglobin (Hb) levels have an overall healthier HRV profile and increased cardiac parasympathetic activity in middle age, independent of age, sex, smoking status, and key metabolic covariates. These findings support our previous findings that Hb levels can be used in assessing long-term risks for cardiometabolic diseases.

Characterization of genetic variants in the EGLN1/PHD2 gene identified in a European collection of patients with erythrocytosis.

Hereditary erythrocytosis is a rare hematologic disorder characterized by an excess of red blood cell production. Here we describe a European collaborative study involving a collection of 2,160 patients with erythrocytosis sequenced in ten different laboratories. We focused our study on the EGLN1 gene and identified 39 germline missense variants including one gene deletion in 47 probands. EGLN1 encodes the PHD2 prolyl 4-hydroxylase, a major inhibitor of hypoxia-inducible factor. We performed a comprehensive study to evaluate the causal role of the identified PHD2 variants: (i) in silico studies of localization, conservation, and deleterious effects; (ii) analysis of hematologic parameters of carriers identified in the UK Biobank; (iii) functional studies of the protein activity and stability; and (iv) a comprehensive study of PHD2 splicing. Altogether, these studies allowed the classification of 16 pathogenic or likely pathogenic mutants in a total of 48 patients and relatives. The in silico studies extended to the variants described in the literature showed that a minority of PHD2 variants can be classified as pathogenic (36/96), without any differences from the variants of unknown significance regarding the severity of the developed disease (hematologic parameters and complications). Here, we demonstrated the great value of federating laboratories working on such rare disorders in order to implement the criteria required for genetic classification, a strategy that should be extended to all hereditary hematologic diseases.

Remote ischemic preconditioning and hypoxia-induced biomarkers in acute myocardial infarction: study on a porcine model.

Objectives. Remote ischemic preconditioning (RIPC) mitigates acute myocardial infarction (AMI). We hypothesized that RIPC reduces the size and severity of AMI and explored molecular mechanisms behind this phenomenon. Design. In two series of experiments, piglets underwent 60 min of the circumflex coronary artery occlusion, resulting in AMI. Piglets were randomly assigned into the RIPC groups (n = 7 + 7) and the control groups (n = 7 + 7). The RIPC groups underwent four 5-min hind limb ischemia-reperfusion cycles before AMI. In series I, the protective efficacy of RIPC was investigated by using biomarkers and echocardiography with a follow-up of 24 h. In series II, the heart of each piglet was harvested for TTC-staining to measure infarct size. Muscle biopsies were collected from the hind limb to explore molecular mechanisms of RIPC using qPCR and Western blot analysis. Results. The levels of CK-MBm (p = 0.032) and TnI (p = 0.007) were lower in the RIPC group. Left ventricular ejection fraction in the RIPC group was greater at the end of the follow-up. The myocardial infarct size in the RIPC group was smaller (p = 0.033). Western blot indicated HIF1α stabilization in the skeletal muscle of the RIPC group. PCR analyses showed upregulation of the HIF target mRNAs for glucose transporter (GLUT1), glucose transporter 4 (GLUT4), phosphofructokinase 1 (PFK1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), enolase 1 (ENO1), lactate dehydrogenase (LDHA) and endothelial nitric oxidate synthase (eNOS). Conclusions. Biochemical, physiologic, and histologic evidence confirms that RIPC decreases the size of AMI. The HIF pathway is likely involved in the mechanism of the RIPC.

Activation of the hypoxia response protects mice from amyloid-ß accumulation.

Alzheimer's disease (AD) is the most common cause of dementia with limited treatment options affecting millions of people and the prevalence increasing with the aging population. The current knowledge on the role of the hypoxia/hypoxia-inducible factor (HIF) in the AD pathology is restricted and controversial. We hypothesized based on benefits of the genetic long-term inactivation of HIF prolyl 4-hydroxylase-2 (HIF-P4H-2) on metabolism, vasculature and inflammatory response that prolonged moderate activation of the hypoxia response could hinder AD pathology. We used an aging model to study potential spontaneous accumulation of amyloid-ß (Aß) in HIF-P4H-2-deficient mice and a transgenic APP/PSEN1 mouse model subjected to prolonged sustained environmental hypoxia (15% O2 for 6 weeks) at two different time points of the disease; at age of 4 and 10 months. In both settings, activation of the hypoxia response reduced brain protein aggregate levels and this associated with higher vascularity. In the senescent HIF-P4H-2-deficient mice metabolic reprogramming also contributed to less protein aggregates while in APP/PSEN1 mice lesser Aß associated additionally with hypoxia-mediated favorable responses to neuroinflammation and amyloid precursor protein processing. In conclusion, continuous, non-full-scale activation of the HIF pathway appears to mediate protection against neurodegeneration via several mechanisms and should be studied as a treatment option for AD.

Activation of the hypoxia response pathway protects against age-induced cardiac hypertrophy.

AIMS: We have previously demonstrated protection against obesity, metabolic dysfunction, atherosclerosis and cardiac ischemia in a hypoxia-inducible factor (HIF) prolyl 4-hydroxylase-2 (Hif-p4h-2) deficient mouse line, attributing these protective effects to activation of the hypoxia response pathway in a normoxic environment. We intended here to find out whether the Hif-p4h-2 deficiency affects the cardiac health of these mice upon aging. METHODS AND RESULTS: When the Hif-p4h-2 deficient mice and their wild-type littermates were monitored during normal aging, the Hif-p4h-2 deficient mice had better preserved diastolic function than the wild type at one year of age and less cardiomyocyte hypertrophy at two years. On the mRNA level, downregulation of hypertrophy-associated genes was detected and shown to be associated with upregulation of Notch signaling, and especially of the Notch target gene and transcriptional repressor Hairy and enhancer-of-split-related basic helix-loop-helix (Hey2). Blocking of Notch signaling in cardiomyocytes isolated from Hif-p4h-2 deficient mice with a gamma-secretase inhibitor led to upregulation of the hypertrophy-associated genes. Also, targeting Hey2 in isolated wild-type rat neonatal cardiomyocytes with siRNA led to upregulation of hypertrophic genes and increased leucine incorporation indicative of increased protein synthesis and hypertrophy. Finally, oral treatment of wild-type mice with a small molecule inhibitor of HIF-P4Hs phenocopied the effects of Hif-p4h-2 deficiency with less cardiomyocyte hypertrophy, upregulation of Hey2 and downregulation of the hypertrophy-associated genes. CONCLUSIONS: These results indicate that activation of the hypoxia response pathway upregulates Notch signaling and its target Hey2 resulting in transcriptional repression of hypertrophy-associated genes and less cardiomyocyte hypertrophy. This is eventually associated with better preserved cardiac function upon aging. Activation of the hypoxia response pathway thus has therapeutic potential for combating age-induced cardiac hypertrophy.

Structure of transmembrane prolyl 4-hydroxylase reveals unique organization of EF and dioxygenase domains.

Prolyl 4-hydroxylases (P4Hs) catalyze post-translational hydroxylation of peptidyl proline residues. In addition to collagen P4Hs and hypoxia-inducible factor P4Hs, a third P4H-the poorly characterized endoplasmic reticulum-localized transmembrane prolyl 4-hydroxylase (P4H-TM)-is found in animals. P4H-TM variants are associated with the familiar neurological HIDEA syndrome, but how these variants might contribute to disease is unknown. Here, we explored this question in a structural and functional analysis of soluble human P4H-TM. The crystal structure revealed an EF domain with two Ca2+-binding motifs inserted within the catalytic domain. A substrate-binding groove was formed between the EF domain and the conserved core of the catalytic domain. The proximity of the EF domain to the active site suggests that Ca2+ binding is relevant to the catalytic activity. Functional analysis demonstrated that Ca2+-binding affinity of P4H-TM is within the range of physiological Ca2+ concentration in the endoplasmic reticulum. P4H-TM was found both as a monomer and a dimer in the solution, but the monomer-dimer equilibrium was not regulated by Ca2+. The catalytic site contained bound Fe2+ and N-oxalylglycine, which is an analogue of the cosubstrate 2-oxoglutarate. Comparison with homologous P4H structures complexed with peptide substrates showed that the substrate-interacting residues and the lid structure that folds over the substrate are conserved in P4H-TM, whereas the extensive loop structures that surround the substrate-binding groove, generating a negative surface potential, are different. Analysis of the structure suggests that the HIDEA variants cause loss of P4H-TM function. In conclusion, P4H-TM shares key structural elements with other P4Hs while having a unique EF domain.

HIDEA syndrome is caused by biallelic, pathogenic, rare or founder P4HTM variants impacting the active site or the overall stability of the P4H-TM protein.

HIDEA syndrome is caused by biallelic pathogenic variants in P4HTM. The phenotype is characterized by muscular and central hypotonia, hypoventilation including obstructive and central sleep apneas, intellectual disability, dysautonomia, epilepsy, eye abnormalities, and an increased tendency to develop respiratory distress during pneumonia. Here, we report six new patients with HIDEA syndrome caused by five different biallelic P4HTM variants, including three novel variants. We describe two Finnish enriched pathogenic P4HTM variants and demonstrate that these variants are embedded within founder haplotypes. We review the clinical data from all previously published patients with HIDEA and characterize all reported P4HTM pathogenic variants associated with HIDEA in silico. All known pathogenic variants in P4HTM result in either premature stop codons, an intragenic deletion, or amino acid changes that impact the active site or the overall stability of P4H-TM protein. In all cases, normal P4H-TM enzyme function is expected to be lost or severely decreased. This report expands knowledge of the genotypic and phenotypic spectrum of the disease.

Genetic Ablation of Transmembrane Prolyl 4-Hydroxylase Reduces Atherosclerotic Plaques in Mice.

[Figure: see text].

Prolyl hydroxylation by EglN2 destabilizes FOXO3a by blocking its interaction with the USP9x deubiquitinase.

The three EglN prolyl hydroxylases (EglN1, EglN2, and EglN3) regulate the stability of the HIF transcription factor. We recently showed that loss of EglN2, however, also leads to down-regulation of Cyclin D1 and decreased cell proliferation in a HIF-independent manner. Here we report that EglN2 can hydroxylate FOXO3a on two specific prolyl residues in vitro and in vivo. Hydroxylation of these sites prevents the binding of USP9x deubiquitinase, thereby promoting the proteasomal degradation of FOXO3a. FOXO transcription factors can repress Cyclin D1 transcription. Failure to hydroxylate FOXO3a promotes its accumulation in cells, which in turn suppresses Cyclin D1 expression. These findings provide new insights into post-transcriptional control of FOXO3a and provide a new avenue for pharmacologically altering Cyclin D1 activity.

Systemic long-term inactivation of hypoxia-inducible factor prolyl 4-hydroxylase 2 ameliorates aging-induced changes in mice without affecting their life span.

Hypoxia inactivates hypoxia-inducible factor (HIF) prolyl 4-hydroxylases (HIF-P4Hs), which stabilize HIF and upregulate genes to restore tissue oxygenation. HIF-P4Hs can also be inhibited by small molecules studied in clinical trials for renal anemia. Knowledge of systemic long-term inactivation of HIF-P4Hs is limited but crucial, since HIF overexpression is associated with cancers. We aimed to determine the effects of systemic genetic inhibition of the most abundant isoenzyme HIF prolyl 4-hydroxylase-2 (HIF-P4H-2)/PHD2/EglN1 on life span and tissue homeostasis in aged mice. Our data showed no difference between wild-type and HIF-P4H-2-deficient mice in the average age reached. There were several differences, however, in the primary causes of death and comorbidities, the HIF-P4H-2-deficient mice having less inflammation, liver diseases, including cancer, and myocardial infarctions, and not developing anemia. No increased cancer incidence was observed due to HIF-P4H-2-deficiency. These data suggest that chronic inactivation of HIF-P4H-2 is not harmful but rather improves the quality of life in senescence.

Expression and Roles of Individual HIF Prolyl 4-Hydroxylase Isoenzymes in the Regulation of the Hypoxia Response Pathway along the Murine Gastrointestinal Epithelium.

The HIF prolyl 4-hydroxylases (HIF-P4H) control hypoxia-inducible factor (HIF), a powerful mechanism regulating cellular adaptation to decreased oxygenation. The gastrointestinal epithelium subsists in "physiological hypoxia" and should therefore have an especially well-designed control over this adaptation. Thus, we assessed the absolute mRNA expression levels of the HIF pathway components, Hif1a, HIF2a, Hif-p4h-1, 2 and 3 and factor inhibiting HIF (Fih1) in murine jejunum, caecum and colon epithelium using droplet digital PCR. We found a higher expression of all these genes towards the distal end of the gastrointestinal tract. We detected mRNA for Hif-p4h-1, 2 and 3 in all parts of the gastrointestinal tract. Hif-p4h-2 had significantly higher expression levels compared to Hif-p4h-1 and 3 in colon and caecum epithelium. To test the roles each HIF-P4H isoform plays in the gut epithelium, we measured the gene expression of classical HIF target genes in Hif-p4h-1-/-, Hif-p4h-2 hypomorph and Hif-p4h-3-/- mice. Only Hif-p4h-2 hypomorphism led to an upregulation of HIF target genes, confirming a predominant role of HIF-P4H-2. However, the abundance of Hif-p4h-1 and 3 expression in the gastrointestinal epithelium implies that these isoforms may have specific functions as well. Thus, the development of selective inhibitors might be useful for diverging therapeutic needs.

Biallelic loss-of-function P4HTM gene variants cause hypotonia, hypoventilation, intellectual disability, dysautonomia, epilepsy, and eye abnormalities (HIDEA syndrome).

PURPOSE: A new syndrome with hypotonia, intellectual disability, and eye abnormalities (HIDEA) was previously described in a large consanguineous family. Linkage analysis identified the recessive disease locus, and genome sequencing yielded three candidate genes with potentially pathogenic biallelic variants: transketolase (TKT), transmembrane prolyl 4-hydroxylase (P4HTM), and ubiquitin specific peptidase 4 (USP4). However, the causative gene remained elusive. METHODS: International collaboration and exome sequencing were used to identify new patients with HIDEA and biallelic, potentially pathogenic, P4HTM variants. Segregation analysis was performed using Sanger sequencing. P4H-TM wild-type and variant constructs without the transmembrane region were overexpressed in insect cells and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot. RESULTS: Five different homozygous or compound heterozygous pathogenic P4HTM gene variants were identified in six new and six previously published patients presenting with HIDEA. Hypoventilation, obstructive and central sleep apnea, and dysautonomia were identified as novel features associated with the phenotype. Characterization of three of the P4H-TM variants demonstrated yielding insoluble protein products and, thus, loss-of-function. CONCLUSIONS: Biallelic loss-of-function P4HTM variants were shown to cause HIDEA syndrome. Our findings enable diagnosis of the condition, and highlight the importance of assessing the need for noninvasive ventilatory support in patients.

The TET enzymes.

During the past decade, we have learnt that the most common DNA modification, 5-methylcytosine (5mC), playing crucial roles in development and disease, is not stable but can be actively reversed to its unmodified form via enzymatic catalysis involving the TET enzymes. These ground-breaking discoveries have been achieved thanks to technological advances in the detection of the oxidized forms of 5mC and to the boldness of individual scientists. The TET enzymes require molecular oxygen for their catalysis, making them important targets for hypoxia research. They also require special cofactors which enable additional levels of regulation. Moreover, mutations and other genetic alterations in TETs are found, especially in myeloid malignances. This review focuses on the kinetic and inhibitory properties of the TET enzymes and the role of TETs in cellular differentiation and transformation and in cancer.

Lack of P4H-TM in mice results in age-related retinal and renal alterations.

Age-related macular degeneration (AMD), affecting the retinal pigment epithelium (RPE), is the leading cause of blindness in middle-aged and older people in developed countries. Genetic and environmental risk factors have been identified, but no effective cure exists. Using a mouse model we show that a transmembrane prolyl 4-hydroxylase (P4H-TM), which participates in the oxygen-dependent regulation of the hypoxia-inducible factor (HIF), is a potential novel candidate gene for AMD. We show that P4h-tm had its highest expression levels in the mouse RPE and brain, heart, lung, skeletal muscle and kidney. P4h-tm-/- mice were fertile and had a normal life span. Lack of P4h-tm stabilized HIF-1α in cortical neurons under normoxia, while in hypoxia it increased the expression of certain HIF target genes in tissues with high endogenous P4h-tm expression levels more than in wild-type mice. Renal erythropoietin levels increased in P4h-tm-/- mice with aging, but the resulting ∼2-fold increase in erythropoietin serum levels did not lead to erythrocytosis. Instead, accumulation of lipid-containing lamellar bodies in renal tubuli was detected in P4h-tm-/- mice with aging, resulting in inflammation and fibrosis, and later glomerular sclerosis and albuminuria. Lack of P4h-tm was associated with retinal thinning, rosette-like infoldings and drusen-like structure accumulation in RPE with aging, as is characteristic of AMD. Photoreceptor recycling was compromised, and electroretinograms revealed functional impairment of the cone pathway in adult P4h-tm-/- mice and cone and rod deficiency in middle-aged mice. P4H-TM is therefore imperative for normal vision, and potentially a novel candidate for age-induced diseases, such as AMD.