Hif-2α programs oxygen chemosensitivity in chromaffin cells.

The study of transcription factors that determine specialized neuronal functions has provided invaluable insights into the physiology of the nervous system. Peripheral chemoreceptors are neurone-like electrophysiologically excitable cells that link the oxygen concentration of arterial blood to the neuronal control of breathing. In the adult, this oxygen chemosensitivity is exemplified by type I cells of the carotid body, and recent work has revealed one isoform of the hypoxia-inducible transcription factor (HIF), HIF-2α, as having a nonredundant role in the development and function of that organ. Here, we show that activation of HIF-2α, including isolated overexpression of HIF-2α but not HIF-1α, is sufficient to induce oxygen chemosensitivity in adult adrenal medulla. This phenotypic change in the adrenal medulla was associated with retention of extra-adrenal paraganglioma-like tissues resembling the fetal organ of Zuckerkandl, which also manifests oxygen chemosensitivity. Acquisition of chemosensitivity was associated with changes in the adrenal medullary expression of gene classes that are ordinarily characteristic of the carotid body, including G protein regulators and atypical subunits of mitochondrial cytochrome oxidase. Overall, the findings suggest that, at least in certain tissues, HIF-2α acts as a phenotypic driver for cells that display oxygen chemosensitivity, thus linking 2 major oxygen-sensing systems.

N-terminal cysteine acetylation and oxidation patterns may define protein stability.

Oxygen homeostasis is maintained in plants and animals by O2-sensing enzymes initiating adaptive responses to low O2 (hypoxia). Recently, the O2-sensitive enzyme ADO was shown to initiate degradation of target proteins RGS4/5 and IL32 via the Cysteine/Arginine N-degron pathway. ADO functions by catalysing oxidation of N-terminal cysteine residues, but despite multiple proteins in the human proteome having an N-terminal cysteine, other endogenous ADO substrates have not yet been identified. This could be because alternative modifications of N-terminal cysteine residues, including acetylation, prevent ADO-catalysed oxidation. Here we investigate the relationship between ADO-catalysed oxidation and NatA-catalysed acetylation of a broad range of protein sequences with N-terminal cysteines. We present evidence that human NatA catalyses N-terminal cysteine acetylation in vitro and in vivo. We then show that sequences downstream of the N-terminal cysteine dictate whether this residue is oxidised or acetylated, with ADO preferring basic and aromatic amino acids and NatA preferring acidic or polar residues. In vitro, the two modifications appear to be mutually exclusive, suggesting that distinct pools of N-terminal cysteine proteins may be acetylated or oxidised. These results reveal the sequence determinants that contribute to N-terminal cysteine protein modifications, with implications for O2-dependent protein stability and the hypoxic response.

Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type-Specific and Time-Resolved Responses to Vhl Inactivation in the Kidney.

Defining the initial events in oncogenesis and the cellular responses they entrain, even in advance of morphologic abnormality, is a fundamental challenge in understanding cancer initiation. As a paradigm to address this, we longitudinally studied the changes induced by loss of the tumor suppressor gene von Hippel Lindau (VHL), which ultimately drives clear cell renal cell carcinoma. Vhl inactivation was directly coupled to expression of a tdTomato reporter within a single allele, allowing accurate visualization of affected cells in their native context and retrieval from the kidney for single-cell RNA sequencing. This strategy uncovered cell type-specific responses to Vhl inactivation, defined a proximal tubular cell class with oncogenic potential, and revealed longer term adaptive changes in the renal epithelium and the interstitium. Oncogenic cell tagging also revealed markedly heterogeneous cellular effects including time-limited proliferation and elimination of specific cell types. Overall, this study reports an experimental strategy for understanding oncogenic processes in which cells bearing genetic alterations can be generated in their native context, marked, and analyzed over time. The observed effects of loss of Vhl in kidney cells provide insights into VHL tumor suppressor action and development of renal cell carcinoma. SIGNIFICANCE: Single-cell analysis of heterogeneous and dynamic responses to Vhl inactivation in the kidney suggests that early events shape the cell type specificity of oncogenesis, providing a focus for mechanistic understanding and therapeutic targeting.

Deficiency of factor-inhibiting HIF creates a tumor-promoting immune microenvironment.

Hypoxia signaling influences tumor development through both cell-intrinsic and -extrinsic pathways. Inhibiting hypoxia-inducible factor (HIF) function has recently been approved as a cancer treatment strategy. Hence, it is important to understand how regulators of HIF may affect tumor growth under physiological conditions. Here we report that in aging mice factor-inhibiting HIF (FIH), one of the most studied negative regulators of HIF, is a haploinsufficient suppressor of spontaneous B cell lymphomas, particular pulmonary B cell lymphomas. FIH deficiency alters immune composition in aged mice and creates a tumor-supportive immune environment demonstrated in syngeneic mouse tumor models. Mechanistically, FIH-defective myeloid cells acquire tumor-supportive properties in response to signals secreted by cancer cells or produced in the tumor microenvironment with enhanced arginase expression and cytokine-directed migration. Together, these data demonstrate that under physiological conditions, FIH plays a key role in maintaining immune homeostasis and can suppress tumorigenesis through a cell-extrinsic pathway.

Comparative analysis of N-terminal cysteine dioxygenation and prolyl-hydroxylation as oxygen-sensing pathways in mammalian cells.

In animals, adaptation to changes in cellular oxygen levels is coordinated largely by 2-oxoglutarate-dependent prolyl-hydroxylase domain (PHD) dioxygenase family members, which regulate the stability of their hypoxia-inducible factor (HIF) substrates to promote expression of genes that adapt cells to hypoxia. Recently, 2-aminoethanethiol dioxygenase (ADO) was identified as a novel O2-sensing enzyme in animals. Through N-terminal cysteine dioxygenation and the N-degron pathway, ADO regulates the stability of a set of non-transcription factor substrates; the regulators of G-protein signaling 4, 5. and 16 and interleukin-32. Here, we set out to compare and contrast the in cellulo characteristics of ADO and PHD enzymes in an attempt to better understand their co-evolution in animals. We find that ADO operates to regulate the stability of its substrates rapidly and with similar O2-sensitivity to the PHD/HIF pathway. ADO appeared less sensitive to iron chelating agents or transition metal exposure than the PHD enzymes, possibly due to tighter catalytic-site Fe2+ coordination. Unlike the PHD/HIF pathway, the ADO/N-degron pathway was not subject to feedback by hypoxic induction of ADO, and induction of ADO substrates was well sustained in response to prolonged hypoxia. The data also reveal strong interactions between proteolytic regulation of targets by ADO and transcriptional induction of those targets, that shape integrated cellular responses to hypoxia. Collectively, our comparative analysis provides further insight into ADO/N-degron-mediated oxygen sensing and its integration into established mechanisms of oxygen homeostasis.

Multi-level interaction between HIF and AHR transcriptional pathways in kidney carcinoma.

Hypoxia-inducible factor (HIF) and aryl hydrocarbon receptor (AHR) are members of the bHLH-PAS family of transcription factors that underpin cellular responses to oxygen and to endogenous and exogenous ligands, respectively, and have central roles in the pathogenesis of renal cancer. Composed of heterodimers, they share a common HIF-1ß/ARNT subunit and similar DNA-binding motifs, raising the possibility of crosstalk between the two transcriptional pathways. Here, we identify both general and locus-specific mechanisms of interaction between HIF and AHR that act both antagonistically and cooperatively. Specifically, we observe competition for the common HIF-1ß/ARNT subunit, in cis synergy for chromatin binding, and overlap in their transcriptional targets. Recently, both HIF and AHR inhibitors have been developed for the treatment of solid tumours. However, inhibition of one pathway may promote the oncogenic effects of the other. Therefore, our work raises important questions as to whether combination therapy targeting both of these pro-tumourigenic pathways might show greater efficacy than targeting each system independently.

Pan-cancer analysis of tissue and single-cell HIF-pathway activation using a conserved gene signature.

Activation of cellular hypoxia pathways, orchestrated by HIF (hypoxia-inducible factor) transcription factors, is a common feature of multiple tumor types, resulting from microenvironment factors and oncogenic mutation. Although they help drive many of the "hallmarks" of cancer and are associated with poor outcome and resistance to therapy, the transcriptional targets of HIF vary considerably depending on the cell type. By integrating 72 genome-wide assays of HIF binding and transcriptional regulation from multiple cancer types, we define a consensus set of 48 HIF target genes that is highly conserved across cancer types and cell lineages. These genes provide an effective marker of HIF activation in bulk and single-cell transcriptomic analyses across a wide range of cancer types and in malignant and stromal cell types. This allows the tissue-orchestrated responses to the hypoxic tumor microenvironment and to oncogenic HIF activation to be deconvoluted at the tumor and single-cell level.

Isoform-resolved mRNA profiling of ribosome load defines interplay of HIF and mTOR dysregulation in kidney cancer.

Hypoxia inducible factor (HIF) and mammalian target of rapamycin (mTOR) pathways orchestrate responses to oxygen and nutrient availability. These pathways are frequently dysregulated in cancer, but their interplay is poorly understood, in part because of difficulties in simultaneous measurement of global and mRNA-specific translation. Here, we describe a workflow for measurement of ribosome load of mRNAs resolved by their transcription start sites (TSSs). Its application to kidney cancer cells reveals extensive translational reprogramming by mTOR, strongly affecting many metabolic enzymes and pathways. By contrast, global effects of HIF on translation are limited, and we do not observe reported translational activation by HIF2A. In contrast, HIF-dependent alterations in TSS usage are associated with robust changes in translational efficiency in a subset of genes. Analyses of the interplay of HIF and mTOR reveal that specific classes of HIF1A and HIF2A transcriptional target gene manifest different sensitivity to mTOR, in a manner that supports combined use of HIF2A and mTOR inhibitors in treatment of kidney cancer.

Hypoxia shapes the immune landscape in lung injury and promotes the persistence of inflammation.

Hypoxemia is a defining feature of acute respiratory distress syndrome (ARDS), an often-fatal complication of pulmonary or systemic inflammation, yet the resulting tissue hypoxia, and its impact on immune responses, is often neglected. In the present study, we have shown that ARDS patients were hypoxemic and monocytopenic within the first 48 h of ventilation. Monocytopenia was also observed in mouse models of hypoxic acute lung injury, in which hypoxemia drove the suppression of type I interferon signaling in the bone marrow. This impaired monopoiesis resulted in reduced accumulation of monocyte-derived macrophages and enhanced neutrophil-mediated inflammation in the lung. Administration of colony-stimulating factor 1 in mice with hypoxic lung injury rescued the monocytopenia, altered the phenotype of circulating monocytes, increased monocyte-derived macrophages in the lung and limited injury. Thus, tissue hypoxia altered the dynamics of the immune response to the detriment of the host and interventions to address the aberrant response offer new therapeutic strategies for ARDS.

Abnormal whole-body energy metabolism in iron-deficient humans despite preserved skeletal muscle oxidative phosphorylation.

Iron deficiency impairs skeletal muscle metabolism. The underlying mechanisms are incompletely characterised, but animal and human experiments suggest the involvement of signalling pathways co-dependent upon oxygen and iron availability, including the pathway associated with hypoxia-inducible factor (HIF). We performed a prospective, case-control, clinical physiology study to explore the effects of iron deficiency on human metabolism, using exercise as a stressor. Thirteen iron-deficient (ID) individuals and thirteen iron-replete (IR) control participants each underwent 31P-magnetic resonance spectroscopy of exercising calf muscle to investigate differences in oxidative phosphorylation, followed by whole-body cardiopulmonary exercise testing. Thereafter, individuals were given an intravenous (IV) infusion, randomised to either iron or saline, and the assessments repeated ~ 1 week later. Neither baseline iron status nor IV iron significantly influenced high-energy phosphate metabolism. During submaximal cardiopulmonary exercise, the rate of decline in blood lactate concentration was diminished in the ID group (P = 0.005). Intravenous iron corrected this abnormality. Furthermore, IV iron increased lactate threshold during maximal cardiopulmonary exercise by ~ 10%, regardless of baseline iron status. These findings demonstrate abnormal whole-body energy metabolism in iron-deficient but otherwise healthy humans. Iron deficiency promotes a more glycolytic phenotype without having a detectable effect on mitochondrial bioenergetics.

Developmental role of PHD2 in the pathogenesis of pseudohypoxic pheochromocytoma.

Despite a general role for the HIF hydroxylase system in cellular oxygen sensing and tumour hypoxia, cancer-associated mutations of genes in this pathway, including PHD2, PHD1, EPAS1 (encoding HIF-2α) are highly tissue-restricted, being observed in pseudohypoxic pheochromocytoma and paraganglioma (PPGL) but rarely, if ever, in other tumours. In an effort to understand that paradox and gain insights into the pathogenesis of pseudohypoxic PPGL, we constructed mice in which the principal HIF prolyl hydroxylase, Phd2, is inactivated in the adrenal medulla using TH-restricted Cre recombinase. Investigation of these animals revealed a gene expression pattern closely mimicking that of pseudohypoxic PPGL. Spatially resolved analyses demonstrated a binary distribution of two contrasting patterns of gene expression among adrenal medullary cells. Phd2 inactivation resulted in a marked shift in this distribution towards a Pnmt-/Hif-2α+/Rgs5+ population. This was associated with morphological abnormalities of adrenal development, including ectopic TH+ cells within the adrenal cortex and external to the adrenal gland. These changes were ablated by combined inactivation of Phd2 with Hif-2α, but not Hif-1α. However, they could not be reproduced by inactivation of Phd2 in adult life, suggesting that they arise from dysregulation of this pathway during adrenal development. Together with the clinical observation that pseudohypoxic PPGL manifests remarkably high heritability, our findings suggest that this type of tumour likely arises from dysregulation of a tissue-restricted action of the PHD2/HIF-2α pathway affecting adrenal development in early life and provides a model for the study of the relevant processes.

The HIF complex recruits the histone methyltransferase SET1B to activate specific hypoxia-inducible genes.

Hypoxia-inducible transcription factors (HIFs) are fundamental to cellular adaptation to low oxygen levels, but it is unclear how they interact with chromatin and activate their target genes. Here, we use genome-wide mutagenesis to identify genes involved in HIF transcriptional activity, and define a requirement for the histone H3 lysine 4 (H3K4) methyltransferase SET1B. SET1B loss leads to a selective reduction in transcriptional activation of HIF target genes, resulting in impaired cell growth, angiogenesis and tumor establishment in SET1B-deficient xenografts. Mechanistically, we show that SET1B accumulates on chromatin in hypoxia, and is recruited to HIF target genes by the HIF complex. The selective induction of H3K4 trimethylation at HIF target loci is both HIF- and SET1B-dependent and, when impaired, correlates with decreased promoter acetylation and gene expression. Together, these findings show SET1B as a determinant of site-specific histone methylation and provide insight into how HIF target genes are differentially regulated.

Hypoxic and pharmacological activation of HIF inhibits SARS-CoV-2 infection of lung epithelial cells.

COVID-19, caused by the novel coronavirus SARS-CoV-2, is a global health issue with more than 2 million fatalities to date. Viral replication is shaped by the cellular microenvironment, and one important factor to consider is oxygen tension, in which hypoxia inducible factor (HIF) regulates transcriptional responses to hypoxia. SARS-CoV-2 primarily infects cells of the respiratory tract, entering via its spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2). We demonstrate that hypoxia and the HIF prolyl hydroxylase inhibitor Roxadustat reduce ACE2 expression and inhibit SARS-CoV-2 entry and replication in lung epithelial cells via an HIF-1α-dependent pathway. Hypoxia and Roxadustat inhibit SARS-CoV-2 RNA replication, showing that post-entry steps in the viral life cycle are oxygen sensitive. This study highlights the importance of HIF signaling in regulating multiple aspects of SARS-CoV-2 infection and raises the potential use of HIF prolyl hydroxylase inhibitors in the prevention or treatment of COVID-19.

Marked and rapid effects of pharmacological HIF-2α antagonism on hypoxic ventilatory control.

Hypoxia-inducible factor (HIF) is strikingly upregulated in many types of cancer, and there is great interest in applying inhibitors of HIF as anticancer therapeutics. The most advanced of these are small molecules that target the HIF-2 isoform through binding the PAS-B domain of HIF-2α. These molecules are undergoing clinical trials with promising results in renal and other cancers where HIF-2 is considered to be driving growth. Nevertheless, a central question remains as to whether such inhibitors affect physiological responses to hypoxia at relevant doses. Here, we show that pharmacological HIF-2α inhibition with PT2385, at doses similar to those reported to inhibit tumor growth, rapidly impaired ventilatory responses to hypoxia, abrogating both ventilatory acclimatization and carotid body cell proliferative responses to sustained hypoxia. Mice carrying a HIF-2α PAS-B S305M mutation that disrupts PT2385 binding, but not dimerization with HIF-1ß, did not respond to PT2385, indicating that these effects are on-target. Furthermore, the finding of a hypomorphic ventilatory phenotype in untreated HIF-2α S305M mutant mice suggests a function for the HIF-2α PAS-B domain beyond heterodimerization with HIF-1ß. Although PT2385 was well tolerated, the findings indicate the need for caution in patients who are dependent on hypoxic ventilatory drive.

Hypoxia drives glucose transporter 3 expression through hypoxia-inducible transcription factor (HIF)-mediated induction of the long noncoding RNA NICI.

Hypoxia-inducible transcription factors (HIFs) directly dictate the expression of multiple RNA species including novel and as yet uncharacterized long noncoding transcripts with unknown function. We used pan-genomic HIF-binding and transcriptomic data to identify a novel long noncoding RNA Noncoding Intergenic Co-Induced transcript (NICI) on chromosome 12p13.31 which is regulated by hypoxia via HIF-1 promoter-binding in multiple cell types. CRISPR/Cas9-mediated deletion of the hypoxia-response element revealed co-regulation of NICI and the neighboring protein-coding gene, solute carrier family 2 member 3 (SLC2A3) which encodes the high-affinity glucose transporter 3 (GLUT3). Knockdown or knockout of NICI attenuated hypoxic induction of SLC2A3, indicating a direct regulatory role of NICI in SLC2A3 expression, which was further evidenced by CRISPR/Cas9-VPR-mediated activation of NICI expression. We also demonstrate that regulation of SLC2A3 is mediated through transcriptional activation rather than posttranscriptional mechanisms because knockout of NICI leads to reduced recruitment of RNA polymerase 2 to the SLC2A3 promoter. Consistent with this we observe NICI-dependent regulation of glucose consumption and cell proliferation. Furthermore, NICI expression is regulated by the von Hippel-Lindau (VHL) tumor suppressor and is highly expressed in clear cell renal cell carcinoma (ccRCC), where SLC2A3 expression is associated with patient prognosis, implying an important role for the HIF/NICI/SLC2A3 axis in this malignancy.

Co-incidence of RCC-susceptibility polymorphisms with HIF cis-acting sequences supports a pathway tuning model of cancer.

Emerging evidence suggests that dysregulation of oncogenic pathways requires precise tuning in order for cancer to develop. To test this, we examined the overlap between cis-acting elements of the hypoxia-inducible factor (HIF) pathway and cancer-susceptibility polymorphisms as defined in genome-wide association studies (GWAS). In renal cancer, where HIF is constitutively and un-physiologically activated by mutation of the von Hippel-Lindau tumour suppressor, we observed marked excess overlap, which extended to potential susceptibility polymorphisms that are below the conventional threshold applied in GWAS. In contrast, in other cancers where HIF is upregulated by different mechanisms, including micro-environmental hypoxia, we observed no excess in overlap. Our findings support a 'pathway tuning' model of cancer, whereby precise modulation of multiple outputs of specific, activated pathways is important in oncogenesis. This implies that selective pressures to modulate such pathways operate during cancer development and should focus attempts to identify their nature and consequences.

Lack of activity of recombinant HIF prolyl hydroxylases (PHDs) on reported non-HIF substrates.

Human and other animal cells deploy three closely related dioxygenases (PHD 1, 2 and 3) to signal oxygen levels by catalysing oxygen regulated prolyl hydroxylation of the transcription factor HIF. The discovery of the HIF prolyl-hydroxylase (PHD) enzymes as oxygen sensors raises a key question as to the existence and nature of non-HIF substrates, potentially transducing other biological responses to hypoxia. Over 20 such substrates are reported. We therefore sought to characterise their reactivity with recombinant PHD enzymes. Unexpectedly, we did not detect prolyl-hydroxylase activity on any reported non-HIF protein or peptide, using conditions supporting robust HIF-α hydroxylation. We cannot exclude PHD-catalysed prolyl hydroxylation occurring under conditions other than those we have examined. However, our findings using recombinant enzymes provide no support for the wide range of non-HIF PHD substrates that have been reported.

Conserved N-terminal cysteine dioxygenases transduce responses to hypoxia in animals and plants.

Organisms must respond to hypoxia to preserve oxygen homeostasis. We identify a thiol oxidase, previously assigned as cysteamine (2-aminoethanethiol) dioxygenase (ADO), as a low oxygen affinity (high-K mO2) amino-terminal cysteine dioxygenase that transduces the oxygen-regulated stability of proteins by the N-degron pathway in human cells. ADO catalyzes the conversion of amino-terminal cysteine to cysteine sulfinic acid and is related to the plant cysteine oxidases that mediate responses to hypoxia by an identical posttranslational modification. We show in human cells that ADO regulates RGS4/5 (regulator of G protein signaling) N-degron substrates, modulates G protein-coupled calcium ion signals and mitogen-activated protein kinase activity, and that its activity extends to other N-cysteine proteins including the angiogenic cytokine interleukin-32. Identification of a conserved enzymatic oxygen sensor in multicellular eukaryotes opens routes to better understanding and therapeutic targeting of adaptive responses to hypoxia.