BACKGROUND:Hypoxia is a critical physiological and pathological condition known to influence various cellular processes, including steroidogenesis. While previous studies, including our own, have highlighted the regulatory effects of Hypoxia-Inducible Factor 1α (HIF1α) on steroid production, the specific molecular mechanisms remain poorly understood. This study investigates the role of hypoxia and HIF1α in steroid biosynthesis across multiple experimental models during acute exposure to low oxygen levels. METHODS:To assess the extent to which acute hypoxia modulates steroidogenesis, we employed several approaches, including the Y1 adrenocortical cell line, and a conditional HIF1α-deficient mouse line in the adrenal cortex. We focused on various regulatory patterns that may critically suppress steroidogenesis. RESULTS:In Y1 cells, hypoxia upregulated specific microRNAs in a HIF1α-dependent manner, resulting in the suppression of mRNA levels of critical steroidogenic enzymes and a subsequent reduction in steroid hormone production. The hypoxia/HIF1α-dependent induction of these microRNAs and the consequent modulation of steroid production were confirmed in vivo. Notably, using our adrenocortical-specific HIF1α-deficient mouse line, we demonstrated that the increase in miRNA expression in vivo is also directly HIF1α-dependent, while the regulation of steroidogenic enzymes (e.g., StAR and Cyp11a1) and steroid production occurs at the level of protein translation, revealing an unexpected layer of control under hypoxic/HIF1 α conditions in vivo. CONCLUSIONS:These findings elucidate the molecular mechanisms underlying acute hypoxia/HIF1α-induced changes in steroid biosynthesis and may also be useful in developing new strategies for various steroid hormone pathologies.

Endoglin (Eng) is an accessory receptor for transforming growth factor-β (TGF-β) that is critical for maintaining vascular integrity. Mutations in Eng cause hereditary hemorrhagic telangiectasia (HHT), resulting in arteriovenous malformations (AVMs) and blood abnormalities. Despite the known association between Eng deficiency and AVMs, the underlying mechanisms are unclear. In addition, the role of the bone marrow (BM), a major source of immune and blood cells, in endothelial Eng (EC-Eng) deficiency is unexplored. We show that BM blood vessels conditionally deficient in Eng (cKO) undergo a structured remodeling process over four weeks, with distinct proliferative and resolution phases. These phases involve angiogenic set points, the involvement of integrins, and the modulation of vascular integrity. In addition, we observe changes in hematopoietic stem and progenitor cells (HSPC) and circulating granulocytes, along with reduced red blood cells and platelets due to splenic sequestration. Using a conditional heterozygous EC-Eng deficient mouse model, reflecting the genetics of HHT patients, we identify vascular changes similar to those in the cKO model. Taken together, using multiple in vivo approaches, we suggest that reduced Eng expression in the endothelium drives significant BM vascular remodeling, sharing mechanisms with early vascular processes associated with AVM formation. Explanation of Novelty Our findings reveal that BM blood vessels deficient in endoglin undergo an orchestrated remodeling process with distinct proliferative and resolution phases over several weeks. We identify specific angiogenic set points and profound alterations in vascular integrity, along with hematopoietic changes starting at the level of hematopoietic stem and progenitor cells. These findings advance our understanding of the role of Eng in vascular remodeling and may provide novel therapeutic targets for HHT. Key Points 1. Conditional EC-Eng deficiency leads to vascular remodeling in the BM of mice in a temporally orchestrated manner. 2. EC-Eng facilitates vascular integrity, hematopoietic homeostasis, and immune cell mobilization. ### Competing Interest Statement The authors have declared no competing interest.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Orchestrated recruitment of neutrophils to inflamed tissue is essential during the initiation of inflammation. Inflamed areas are usually hypoxic, and adaptation to reduced oxygen pressure is typically mediated by hypoxia pathway proteins. However, it remains unclear how these factors influence the migration of neutrophils to and at the site of inflammation during their transmigration through the blood-endothelial cell barrier, as well as their motility in the interstitial space. Here, we reveal that activation of hypoxia-inducible factor 2 (HIF2α) as a result of a deficiency in HIF prolyl hydroxylase domain protein 2 (PHD2) boosts neutrophil migration specifically through highly confined microenvironments. In vivo, the increased migratory capacity of PHD2-deficient neutrophils resulted in massive tissue accumulation in models of acute local inflammation. Using systematic RNA sequencing analyses and mechanistic approaches, we identified RhoA, a cytoskeleton organizer, as the central downstream factor that mediates HIF2α-dependent neutrophil motility. Thus, we propose that the novel PHD2-HIF2α-RhoA axis is vital to the initial stages of inflammation because it promotes neutrophil movement through highly confined tissue landscapes.

Endogenous steroid hormones, especially glucocorticoids and mineralocorticoids, derive from the adrenal cortex, and drastic or sustained changes in their circulatory levels affect multiple organ systems. Although hypoxia signaling in steroidogenesis has been suggested, knowledge on the true impact of the HIFs (Hypoxia-Inducible Factors) in the adrenocortical cells of vertebrates is scant. By creating a unique set of transgenic mouse lines, we reveal a prominent role for HIF1α in the synthesis of virtually all steroids in vivo. Specifically, mice deficient in HIF1α in adrenocortical cells displayed enhanced levels of enzymes responsible for steroidogenesis and a cognate increase in circulatory steroid levels. These changes resulted in cytokine alterations and changes in the profile of circulatory mature hematopoietic cells. Conversely, HIF1α overexpression resulted in the opposite phenotype of insufficient steroid production due to impaired transcription of necessary enzymes. Based on these results, we propose HIF1α to be a vital regulator of steroidogenesis as its modulation in adrenocortical cells dramatically impacts hormone synthesis with systemic consequences. In addition, these mice can have potential clinical significances as they may serve as essential tools to understand the pathophysiology of hormone modulations in a number of diseases associated with metabolic syndrome, auto-immunity or even cancer.

The adrenal gland and its hormones regulate numerous fundamental biological processes; however, the impact of hypoxia signaling on adrenal function remains poorly understood. Here, we reveal that deficiency of HIF (hypoxia inducible factors) prolyl hydroxylase domain protein-2 (PHD2) in the adrenal medulla of mice results in HIF2α-mediated reduction in phenylethanolamine N-methyltransferase (PNMT) expression, and consequent reduction in epinephrine synthesis. Simultaneous loss of PHD2 in renal erythropoietin (EPO)-producing cells (REPCs) stimulated HIF2α-driven EPO overproduction, excessive RBC formation (erythrocytosis), and systemic hypoglycemia, which is necessary and sufficient to enhance exocytosis of epinephrine from the adrenal medulla. Based on these results, we propose that the PHD2-HIF2α axis in the adrenal medulla regulates the synthesis of epinephrine, whereas in REPCs, it indirectly induces the release of this hormone. Our findings are also highly relevant to the testing of small molecule PHD inhibitors in phase III clinical trials for patients with renal anemia. KEY MESSAGES: HIF2α and not HIF1α modulates PNMT during epinephrine synthesis in chromaffin cells. The PHD2-HIF2α-EPO axis induces erythrocytosis and hypoglycemia. Reduced systemic glucose facilitates exocytosis of epinephrine from adrenal gland.

Endogenous steroid hormones, especially glucocorticoids and mineralocorticoids, are essential for life regulating numerous physiological and pathological processes. These hormones derive from the adrenal cortex, and drastic or sustained changes in their circulatory levels affect multiple organ systems. Although a role for hypoxia pathway proteins (HPP) in steroidogenesis has been suggested, knowledge on the true impact of the HIFs (Hypoxia Inducible Factors) and oxygen sensors (HIF-prolyl hydroxylase domain-containing enzymes; PHDs) in the adrenocortical cells of vertebrates is scant. By creating a unique set of transgenic mouse lines, we reveal a prominent role for HIF1α in the synthesis of virtually all steroids under steady state conditions. Specifically, mice deficient in HIF1α in a part of the adrenocortical cells displayed enhanced levels of enzymes responsible for steroidogenesis and a cognate increase in circulatory steroid levels. These changes resulted in cytokine alterations and changes in the profile of circulatory mature hematopoietic cells. Conversely, HIF1α overexpression due to combined PHD2 and PHD3 deficiency in the adrenal cortex resulted in the opposite phenotype of insufficient steroid production due to impaired transcription of necessary enzymes. Based on these results, we propose HIF1α to be a central and vital regulator of steroidogenesis as its modulation in adrenocortical cells dramatically impacts hormone synthesis with systemic consequences. Additionally, these mice can have potential clinical significances as they may serve as essential tools to understand the pathophysiology of hormone modulations in a number of diseases associated with metabolic syndrome, auto-immunity or even cancer.

The adrenal gland and its hormones regulate numerous fundamental biological processes; however, the impact of hypoxia signalling on its function remains scarcely understood. Here, we reveal that deficiency of HIF (Hypoxia Inducible Factors) prolyl hydroxylase domain protein-2 (PHD2) in the adrenal medulla of mice results in HIF2α-mediated reduction in phenylethanolamine N-methyltransferase (PNMT) expression, and consequent reduction in epinephrine synthesis. Concomitant loss of PHD2 in renal erythropoietin (EPO) producing cells stimulated HIF2α-driven EPO overproduction, excessive RBC formation (erythrocytosis) and systemic hypoglycaemia. Using mouse lines displaying only EPO-induced erythrocytosis or anaemia, we show that hypo- or hyperglycaemia is necessary and sufficient to respectively enhance or reduce exocytosis of epinephrine from the adrenal gland. Based on these results, we propose that the PHD2-HIF2α axis in the adrenal medulla and beyond regulates both synthesis and release of catecholamines, especially epinephrine. Our findings are also of great significance in view of the small molecule PHD inhibitors being tested in phase III global clinical development trials for use in renal anaemia patients.

Although bone marrow niche cells are essential for hematopoietic stem cell (HSC) maintenance, their interaction in response to stress is not well defined. Here, we used a mouse model of acute thrombocytopenia to investigate the cross talk between HSCs and niche cells during restoration of the thrombocyte pool. This process required membrane-localized stem cell factor (m-SCF) in megakaryocytes, which was regulated, in turn, by vascular endothelial growth factor A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB). HSCs and multipotent progenitors type 2 (MPP2), but not MPP3/4, were subsequently activated by a dual-receptor tyrosine kinase (RTK)-dependent signaling event, m-SCF/c-Kit and VEGF-A/vascular endothelial growth factor receptor 2 (VEGFR-2), contributing to their selective and early proliferation. Our findings describe a dynamic network of signals in response to the acute loss of a single blood cell type and reveal the important role of 3 RTKs and their ligands in orchestrating the selective activation of hematopoietic stem and progenitor cells (HSPCs) in thrombocytopenia.

Hypoxia and inflammation are closely intertwined phenomena. Critically ill patients often suffer from systemic inflammatory conditions and concurrently experience short-lived hypoxia. We evaluated the effects of short-term hypoxia on systemic inflammation, and show that it potently attenuates pro-inflammatory cytokine responses during murine endotoxemia. These effects are independent of hypoxia-inducible factors (HIFs), but involve augmented adenosine levels, in turn resulting in an adenosine 2B receptor-mediated post-transcriptional increase of interleukin (IL)-10 production. We translated our findings to humans using the experimental endotoxemia model, where short-term hypoxia resulted in enhanced plasma concentrations of adenosine, augmentation of endotoxin-induced circulating IL-10 levels, and concurrent attenuation of the pro-inflammatory cytokine response. Again, HIFs were shown not to be involved. Taken together, we demonstrate that short-term hypoxia dampens the systemic pro-inflammatory cytokine response through enhanced purinergic signaling in mice and men. These effects may contribute to outcome and provide leads for immunomodulatory treatment strategies for critically ill patients.

Hematopoietic stem cells (HSCs) continuously replenish all blood cell types through a series of differentiation steps and repeated cell divisions that involve the generation of lineage-committed progenitors. However, whether cell division in HSCs precedes differentiation is unclear. To this end, we used an HSC cell-tracing approach and Ki67 RFP knock-in mice, in a non-conditioned transplantation model, to assess divisional history, cell cycle progression, and differentiation of adult HSCs. Our results reveal that HSCs are able to differentiate into restricted progenitors, especially common myeloid, megakaryocyte-erythroid and pre-megakaryocyte progenitors, without undergoing cell division and even before entering the S phase of the cell cycle. Additionally, the phenotype of the undivided but differentiated progenitors correlated with the expression of lineage-specific genes and loss of multipotency. Thus HSC fate decisions can be uncoupled from physical cell division. These results facilitate a better understanding of the mechanisms that control fate decisions in hematopoietic cells.

Damaged replication forks activate poly(ADP-ribose) polymerase 1 (PARP1), which catalyses poly(ADP-ribose) (PAR) formation; however, how PARP1 or poly(ADP-ribosyl)ation is involved in the S-phase checkpoint is unknown. Here we show that PAR, supplied by PARP1, interacts with Chk1 via a novel PAR-binding regulatory (PbR) motif in Chk1, independent of ATR and its activity. iPOND studies reveal that Chk1 associates readily with the unperturbed replication fork and that PAR is required for efficient retention of Chk1 and phosphorylated Chk1 at the fork. A PbR mutation, which disrupts PAR binding, but not the interaction with its partners Claspin or BRCA1, impairs Chk1 and the S-phase checkpoint activation, and mirrors Chk1 knockdown-induced hypersensitivity to fork poisoning. We find that long chains, but not short chains, of PAR stimulate Chk1 kinase activity. Collectively, we disclose a previously unrecognized mechanism of the S-phase checkpoint by PAR metabolism that modulates Chk1 activity at the replication fork.