Erythrocyte ENT1-AMPD3 Axis is an Essential Purinergic Hypoxia Sensor and Energy Regulator Combating CKD in a Mouse Model.

SIGNIFICANCE STATEMENT: Hypoxia drives kidney damage and progression of CKD. Although erythrocytes respond rapidly to hypoxia, their role and the specific molecules sensing and responding to hypoxia in CKD remain unclear. In this study, we demonstrated in a mouse model that erythrocyte ENT1-AMPD3 is a master energy regulator of the intracellular purinergic hypoxic compensatory response that promotes rapid energy supply from extracellular adenosine, eAMPK-dependent metabolic reprogramming, and O 2 delivery, which combat renal hypoxia and progression of CKD. ENT1-AMPD3-AMPK-BPGM comprise a group of circulating erythroid-specific biomarkers, providing early diagnostic and novel therapeutic targets for CKD. BACKGROUND: Hypoxia drives kidney damage and progression of CKD. Although erythrocytes respond rapidly to hypoxia, their role and the specific molecules sensing and responding to hypoxia in CKD remain unclear. METHODS: Mice with an erythrocyte-specific deficiency in equilibrative nucleoside transporter 1 ( eEnt1-/- ) and a global deficiency in AMP deaminase 3 ( Ampd3-/- ) were generated to define their function in two independent CKD models, including angiotensin II (Ang II) infusion and unilateral ureteral obstruction (UUO). Unbiased metabolomics, isotopic adenosine flux, and various biochemical and cell culture analyses coupled with genetic studies were performed. Translational studies in patients with CKD and cultured human erythrocytes examined the role of ENT1 and AMPD3 in erythrocyte function and metabolism. RESULTS: eEnt1-/- mice display severe renal hypoxia, kidney damage, and fibrosis in both CKD models. The loss of eENT1-mediated adenosine uptake reduces intracellular AMP and thus abolishes the activation of AMPK α and bisphosphoglycerate mutase (BPGM). This results in reduced 2,3-bisphosphoglycerate and glutathione, leading to overwhelming oxidative stress in eEnt1-/- mice. Excess reactive oxygen species (ROS) activates AMPD3, resulting in metabolic reprogramming and reduced O 2 delivery, leading to severe renal hypoxia in eEnt1-/- mice. By contrast, genetic ablation of AMPD3 preserves the erythrocyte adenine nucleotide pool, inducing AMPK-BPGM activation, O 2 delivery, and antioxidative stress capacity, which protect against Ang II-induced renal hypoxia, damage, and CKD progression. Translational studies recapitulated the findings in mice. CONCLUSION: eENT1-AMPD3, two highly enriched erythrocyte purinergic components that sense hypoxia, promote eAMPK-BPGM-dependent metabolic reprogramming, O 2 delivery, energy supply, and antioxidative stress capacity, which mitigates renal hypoxia and CKD progression.

p97 dysfunction underlies a loss of quality control of damaged membrane proteins and promotes oxidative stress and sickling in sickle cell disease.

Sickling is the central pathogenic process of sickle cell disease (SCD), one of the most prevalent inherited hemolytic disorders. Having no easy access to antioxidants in the cytosol, elevated levels of reactive oxygen species (ROS) residing at the plasma membrane in sickle red blood cells (sRBCs) easily oxidize membrane proteins and thus contribute to sickling. Although the ubiquitin-proteasome system (UPS) is essential to rapidly clear ROS-damaged membrane proteins and maintain cellular homeostasis, the function and regulatory mechanism of the UPS for their clearance in sRBCs remains unidentified. Elevated levels of polyubiquitinated membrane-associated proteins in human sRBCs are reported here. High throughput and untargeted proteomic analyses of membrane proteins immunoprecipitated by ubiquitin antibodies detected elevated levels of ubiquitination of a series of proteins including cytoskeletal proteins, transporters, ROS-related proteins, and UPS machinery components in sRBCs. Polyubiquitination of membrane-associated catalase was increased in sRBCs, associated with decreased catalase activity and elevated ROS. Surprisingly, shuttling of p97 (ATP-dependent valosin-containing chaperone protein), a key component of the UPS to shuttle polyubiquitinated proteins from the membrane to cytosol for proteasomal degradation, was significantly impaired, resulting in significant accumulation of p97 along with polyubiquitinated proteins in the membrane of human sRBCs. Functionally, inhibition of p97 directly promoted accumulation of polyubiquitinated membrane-associated proteins, excessive ROS levels, and sickling in response to hypoxia. Overall, we revealed that p97 dysfunction underlies impaired UPS and contributes to oxidative stress in sRBCs.

Erythrocyte transglutaminase-2 combats hypoxia and chronic kidney disease by promoting oxygen delivery and carnitine homeostasis.

Due to lack of nuclei and de novo protein synthesis, post-translational modification (PTM) is imperative for erythrocytes to regulate oxygen (O2) delivery and combat tissue hypoxia. Here, we report that erythrocyte transglutminase-2 (eTG2)-mediated PTM is essential to trigger O2 delivery by promoting bisphosphoglycerate mutase proteostasis and the Rapoport-Luebering glycolytic shunt for adaptation to hypoxia, in healthy humans ascending to high altitude and in two distinct murine models of hypoxia. In a pathological hypoxia model with chronic kidney disease (CKD), eTG2 is critical to combat renal hypoxia-induced reduction of Slc22a5 transcription and OCNT2 protein levels via HIF-1α-PPARα signaling to maintain carnitine homeostasis. Carnitine supplementation is an effective and safe therapeutic approach to counteract hypertension and progression of CKD by enhancing erythrocyte O2 delivery. Altogether, we reveal eTG2 as an erythrocyte protein stabilizer orchestrating O2 delivery and tissue adaptive metabolic reprogramming and identify carnitine-based therapy to mitigate hypoxia and CKD progression.

Erythrocyte adenosine A2B receptor prevents cognitive and auditory dysfunction by promoting hypoxic and metabolic reprogramming.

Hypoxia drives aging and promotes age-related cognition and hearing functional decline. Despite the role of erythrocytes in oxygen (O2) transport, their role in the onset of aging and age-related cognitive decline and hearing loss (HL) remains undetermined. Recent studies revealed that signaling through the erythrocyte adenosine A2B receptor (ADORA2B) promotes O2 release to counteract hypoxia at high altitude. However, nothing is known about a role for erythrocyte ADORA2B in age-related functional decline. Here, we report that loss of murine erythrocyte-specific ADORA2B (eAdora2b-/-) accelerates early onset of age-related impairments in spatial learning, memory, and hearing ability. eAdora2b-/- mice display the early aging-like cellular and molecular features including the proliferation and activation of microglia and macrophages, elevation of pro-inflammatory cytokines, and attenuation of hypoxia-induced glycolytic gene expression to counteract hypoxia in the hippocampus (HIP), cortex, or cochlea. Hypoxia sufficiently accelerates early onset of cognitive and cochlear functional decline and inflammatory response in eAdora2b-/- mice. Mechanistically, erythrocyte ADORA2B-mediated activation of AMP-activated protein kinase (AMPK) and bisphosphoglycerate mutase (BPGM) promotes hypoxic and metabolic reprogramming to enhance production of 2,3-bisphosphoglycerate (2,3-BPG), an erythrocyte-specific metabolite triggering O2 delivery. Significantly, this finding led us to further discover that murine erythroblast ADORA2B and BPGM mRNA levels and erythrocyte BPGM activity are reduced during normal aging. Overall, we determined that erythrocyte ADORA2B-BPGM axis is a key component for anti-aging and anti-age-related functional decline.

Adenosine A2B receptor: A pathogenic factor and a therapeutic target for sensorineural hearing loss.

Over 466 million people worldwide are diagnosed with hearing loss (HL). About 90% of HL cases are sensorineural HL (SNHL) with treatments limited to hearing aids and cochlear implants with no FDA-approved drugs. Intriguingly, ADA-deficient patients have been reported to have bilateral SNHL, however, its underlying cellular and molecular basis remain unknown. We report that Ada-/- mice, phenocopying ADA-deficient humans, displayed SNHL. Ada-/- mice cochlea with elevated adenosine caused substantial nerve fiber demyelination and mild hair cell loss. ADA enzyme therapy in these mice normalized cochlear adenosine levels, attenuated SNHL, and prevented demyelination. Additionally, ADA enzyme therapy rescued SNHL by restoring nerve fiber structure in Ada-/- mice post two-week drug withdrawal. Moreover, elevated cochlear adenosine in untreated mice was associated with enhanced Adora2b gene expression. Preclinically, ADORA2B-specific antagonist treatment in Ada-/- mice significantly improved HL, nerve fiber density, and myelin compaction. We also provided genetic evidence that ADORA2B is detrimental for age-related SNHL by impairing cochlear myelination in WT aged mice. Overall, understanding purinergic molecular signaling in SNHL in Ada-/- mice allows us to further discover that ADORA2B is also a pathogenic factor underlying aged-related SNHL by impairing cochlear myelination and lowering cochlear adenosine levels or blocking ADORA2B signaling are effective therapies for SNHL.

Maternal erythrocyte ENT1-mediated AMPK activation counteracts placental hypoxia and supports fetal growth.

Insufficient O2 supply is frequently associated with fetal growth restriction (FGR), a leading cause of perinatal mortality and morbidity. Although the erythrocyte is the most abundant and only cell type to deliver O2 in our body, its function and regulatory mechanism in FGR remain unknown. Here, we report that genetic ablation of mouse erythrocyte equilibrative nucleoside transporter 1 (eENT1) in dams, but not placentas or fetuses, results in FGR. Unbiased high-throughput metabolic profiling coupled with in vitro and in vivo flux analyses with isotopically labeled tracers led us to discover that maternal eENT1-dependent adenosine uptake is critical in activating AMPK by controlling the AMP/ATP ratio and its downstream target, bisphosphoglycerate mutase (BPGM); in turn, BPGM mediates 2,3-BPG production, which enhances O2 delivery to maintain placental oxygenation. Mechanistically and functionally, we revealed that genetic ablation of maternal eENT1 increases placental HIF-1α; preferentially reduces placental large neutral aa transporter 1 (LAT1) expression, activity, and aa supply; and induces FGR. Translationally, we revealed that elevated HIF-1α directly reduces LAT1 gene expression in cultured human trophoblasts. We demonstrate the importance and molecular insight of maternal eENT1 in fetal growth and open up potentially new diagnostic and therapeutic possibilities for FGR.

Erythrocyte Metabolic Reprogramming by Sphingosine 1-Phosphate in Chronic Kidney Disease and Therapies.

RATIONALE: Hypoxia promotes renal damage and progression of chronic kidney disease (CKD). The erythrocyte is the only cell type for oxygen (O2) delivery. Sphingosine 1-phosphate (S1P)-a highly enriched biolipid in erythrocytes-is recently reported to be induced under high altitude in normal humans to enhance O2 delivery. However, nothing is known about erythrocyte S1P in CKD. OBJECTIVE: To investigate the function and metabolic basis of erythrocyte S1P in CKD with a goal to explore potential therapeutics. METHODS AND RESULTS: Using erythrocyte-specific SphK1 (sphingosine kinase 1; the only enzyme to produce S1P in erythrocytes) knockout mice (eSphK1-/-) in an experimental model of hypertensive CKD with Ang II (angiotensin II) infusion, we found severe renal hypoxia, hypertension, proteinuria, and fibrosis in Ang II-infused eSphk1-/- mice compared with controls. Untargeted metabolomics profiling and in vivo U-13C6 isotopically labeled glucose flux analysis revealed that SphK1 is required for channeling glucose metabolism toward glycolysis versus pentose phosphate pathway, resulting in enhanced erythroid-specific Rapoport-Luebering shunt in Ang II-infused mice. Mechanistically, increased erythrocyte S1P functioning intracellularly activates AMPK (AMP-activated protein kinase) 1α and BPGM (bisphosphoglycerate mutase) by reducing ceramide/S1P ratio and inhibiting PP2A (protein phosphatase 2A), leading to increased 2,3-bisphosphoglycerate (an erythrocyte-specific metabolite negatively regulating Hb [hemoglobin]-O2-binding affinity) production and thus more O2 delivery to counteract kidney hypoxia and progression to CKD. Preclinical studies revealed that an AMPK agonist or a PP2A inhibitor rescued the severe CKD phenotype in Ang II-infused eSphK1-/- mice and prevented development of CKD in the control mice by inducing 2,3-bisphosphoglycerate production and thus enhancing renal oxygenation. Translational research validated mouse findings in erythrocytes of hypertensive CKD patients and cultured human erythrocytes. CONCLUSIONS: Our study elucidates the beneficial role of eSphk1-S1P in hypertensive CKD by channeling glucose metabolism toward Rapoport-Luebering shunt and inducing 2,3-bisphosphoglycerate production and O2 delivery via a PP2A-AMPK1α signaling pathway. These findings reveal the metabolic and molecular basis of erythrocyte S1P in CKD and new therapeutic avenues.

Reciprocal upregulation of hypoxia-inducible factor-1α and persistently enhanced placental adenosine signaling contribute to the pathogenesis of preeclampsia.

Recent evidence indicates that elevated placental adenosine signaling contributes to preeclampsia (PE). However, the molecular basis for the chronically enhanced placental adenosine signaling in PE remains unclear. Here, we report that hypoxia-inducible factor-1α (HIF-1α) is crucial for the enhancement of placental adenosine signaling. Utilizing a pharmacologic approach to reduce placental adenosine levels, we found that enhanced adenosine underlies increased placental HIF-1α in an angiotensin receptor type 1 receptor agonistic autoantibody (AT1 -AA)-induced mouse model of PE. Knockdown of placental HIF-1α in vivo suppressed the accumulation of adenosine and increased ecto-5'-nucleotidase (CD73) and adenosine A2B receptor (ADORA2B) in the placentas of PE mouse models induced by AT1 -AA or LIGHT, a TNF superfamily cytokine (TNFSF14). Human in vitro studies using placental villous explants demonstrated that increased HIF-1α resulting from ADORA2B activation facilitates the induction of CD73, ADORA2B, and FLT-1 expression. Overall, we demonstrated that (a) elevated placental HIF-1α by AT1 -AA or LIGHT upregulates CD73 and ADORA2B expression and (b) enhanced adenosine signaling through upregulated ADORA2B induces placental HIF-1α expression, which creates a positive feedback loop that promotes FLT-1 expression leading to disease development. Our results suggest that adenosine-based therapy targeting the malicious cycle of placental adenosine signaling may elicit therapeutic effects on PE.

Differential role of adenosine signaling cascade in acute and chronic pain.

Adenosine is a signaling molecule induced under stress such as energy insufficiency and ischemic/hypoxic conditions. Adenosine controls multiple physiological and pathological cellular and tissue function by activation of four G protein-coupled receptors (GPCR). Functional role of adenosine signaling in acute pain has been widely studied. However, the role of adenosine signaling in chronic pain is poorly understood. At acute levels, adenosine can be beneficial to anti-pain whereas a sustained elevation of adenosine can be detrimental to promote chronic pain. In recent years, extensive progress has been made to define the role of adenosine signaling in chronic pain and to dissect molecular new insight underlying the development of chronic pain. In this review, we summarize the differential role of adenosine signaling cascade in acute and chronic pain with a major focus on recent studies revealing adenosine ADORA2B receptor activation in the pathology of chronic pain. We further provide a therapeutic outlook of how multiple adenosine signaling components can be useful to treat chronic pain.

Increased LIGHT leading to sFlt-1 elevation underlies the pathogenic link between hydatidiform mole and preeclampsia.

Hydatidiform moles are known to pose an extremely high risk of severe early-onset preeclampsia if left untreated. TNF superfamily cytokine, LIGHT has recently been reported to contribute to pathophysiology of preeclampsia. The present study aimed to investigate the involvement of LIGHT in hydatidiform moles. We measured the serum levels of LIGHT and sFlt-1 by ELISA in 17 women with complete hydatidiform mole (HM) and 20 gestational-age-matched normal pregnant women (control). As a result, the serum LIGHT levels were significantly higher in HM as compared with those in control (69.9 ± 9.6 pg/ml vs 25.4 ± 5.3 pg/ml, p = 0.0001) and the serum levels of LIGHT were significantly positively correlated with those of sFlt-1 in HM (r = 0.68, p = 0.0029). Immunohistochemical analysis revealed that the expression levels of LIGHT were increased in HM placentas as compared with controls, and LIGHT and sFlt-1 were co-localized in the trophoblast cells of HM. In vitro studies using primary syncytiotrophoblast cells demonstrated that LIGHT directly induced sFlt-1 expression in trophoblast cells. Our results indicated that elevated LIGHT in the trophoblast cells of hydatidiform mole induces sFlt-1, which might underlie the pathogenic mechanism of early-onset preeclampsia developing secondary to molar pregnancies.

Circadian period 2: a missing beneficial factor in sickle cell disease by lowering pulmonary inflammation, iron overload, and mortality.

The circadian clock is important for cellular and organ function. However, its function in sickle cell disease (SCD), a life-threatening hemolytic disorder, remains unknown. Here, we performed an unbiased microarray screen, which revealed significantly altered expression of circadian rhythmic genes, inflammatory response genes, and iron metabolic genes in SCD Berkeley transgenic mouse lungs compared with controls. Given the vital role of period 2 (Per2) in the core clock and the unrecognized role of Per2 in SCD, we transplanted the bone marrow (BM) of SCD mice to Per2Luciferase mice, which revealed that Per2 expression was up-regulated in SCD mouse lung. Next, we transplanted the BM of SCD mice to period 1 (Per1)/Per2 double deficient [Per1/Per2 double knockout (dKO)] and wild-type mice, respectively. We discovered that Per1/Per2 dKO mice transplanted with SCD BM (SCD → Per1/Per2 dKO) displayed severe irradiation sensitivity and were more susceptible to an early death. Although we observed an increase of peripheral inflammatory cells, we did not detect differences in erythrocyte sickling. However, there was further lung damage due to elevated pulmonary congestion, inflammatory cell infiltration, iron overload, and secretion of IL-6 in lavage fluid. Overall, we demonstrate that Per1/Per2 is beneficial to counteract elevated systemic inflammation, lung tissue inflammation, and iron overload in SCD.-Adebiyi, M. G., Zhao, Z., Ye, Y., Manalo, J., Hong, Y., Lee, C. C., Xian, W., McKeon, F., Culp-Hill, R., D' Alessandro, A., Kellems, R. E., Yoo, S.-H., Han, L., Xia, Y. Circadian period 2: a missing beneficial factor in sickle cell disease by lowering pulmonary inflammation, iron overload, and mortality.

Erythrocyte Adenosine A2B Receptor-Mediated AMPK Activation: A Missing Component Counteracting CKD by Promoting Oxygen Delivery.

BACKGROUND: Oxygen deprivation or hypoxia in the kidney drives CKD and contributes to end organ damage. The erythrocyte's role in delivery of oxygen (O2) is regulated by hypoxia, but the effects of CKD are unknown. METHODS: We screened all of the metabolites in the whole blood of mice infused with angiotensin II (Ang II) at 140 ng/kg per minute up to 14 days to simulate CKD and compared their metabolites with those from untreated mice. Mice lacking a receptor on their erythrocytes called ADORA2B, which increases O2 delivery, and patients with CKD were studied to assess the role of ADORA2B-mediated O2 delivery in CKD. RESULTS: Untargeted metabolomics showed increased production of 2,3-biphosphoglycerate (2,3-BPG), an erythrocyte-specific metabolite promoting O2 delivery, in mice given Ang II to induce CKD. Genetic studies in mice revealed that erythrocyte ADORA2B signaling leads to AMPK-stimulated activation of BPG mutase, promoting 2,3-BPG production and O2 delivery to counteract kidney hypoxia, tissue damage, and disease progression in Ang II-induced CKD. Enhancing AMPK activation in mice offset kidney hypoxia by triggering 2,3-BPG production and O2 delivery. Patients with CKD had higher 2,3-BPG levels, AMPK activity, and O2 delivery in their erythrocytes compared with controls. Changes were proportional to disease severity, suggesting a protective effect. CONCLUSIONS: Mouse and human evidence reveals that ADORA2B-AMPK signaling cascade-induced 2,3-BPG production promotes O2 delivery by erythrocytes to counteract kidney hypoxia and progression of CKD. These findings pave a way to novel therapeutic avenues in CKD targeting this pathway.

Tissue Transglutaminase-Mediated AT1 Receptor Sensitization Underlies Pro-inflammatory Cytokine LIGHT-Induced Hypertension.

BACKGROUND: Although numerous recent studies have shown a strong link between inflammation and hypertension, the underlying mechanisms by which inflammatory cytokines induce hypertension remain to be fully elucidated. Hypertensive disorders are also associated with elevated pressor sensitivity. Tissue transglutaminase (TG2), a potent cross-linking enzyme, is known to be transcriptionally activated by inflammatory cytokines and stabilize angiotensin II (Ang II) receptor AT1 (AT1R) via ubiquitination-preventing posttranslational modification. Here we sought to investigate the TG2-mediated AT1R stabilization in inflammation-induced hypertension and its functional consequences with a focus on receptor abundance and Ang II responsiveness. METHODS AND RESULTS: Using an experimental model of inflammation-induced hypertension established by introducing the pro-inflammatory tumor necrosis factor cytokine LIGHT, we provide pharmacologic and genetic evidence that TG2 is required for LIGHT-induced hypertension (systolic pressure on day 6: LIGHT = 152.3 ± 7.4 vs. LIGHT+ERW1041E [TG2 inhibitor] = 105.8 ± 13.1 or LIGHT+TG2-/- = 114.3 ± 4.3 mm Hg, P < 0.05, n = 4-5) and renal compromise (urine albumin/creatinine: LIGHT = 0.17 ± 0.05 vs. LIGHT+ERW1041E = 0.03 ± 0.01 or LIGHT+TG2-/- = 0.06 ± 0.01 mg/mg; plasma creatinine: LIGHT = 1.11 ± 0.04 vs. LIGHT+ERW1041E = 0.94 ± 0.04 or LIGHT+TG2-/- = 0.88 ± 0.09 mg/dl; urine volume: LIGHT = 0.23 ± 0.1 vs. LIGHT+ERW1041E = 0.84 ± 0.13 or LIGHT+TG2-/- = 1.02 ± 0.09 ml/24 hour on day 14, P < 0.05, n = 4-5). Our mechanistic studies showed that the TG2-mediated AT1R modification and accumulation (relative renal AT1R level: phosphate-buffered saline [PBS] = 1.23 ± 0.22, LIGHT = 3.49 ± 0.37, and LIGHT+ERW1041E = 1.77 ± 0.46, P < 0.05, n = 3; LIGHT+TG2+/+ = 85.28 ± 36.11 vs. LIGHT+TG2-/- = 7.01 ± 5.68, P < 0.05, n = 3) induced by LIGHT is associated with abrogated ß-arrestin binding (AT1R/associated ß-arrestin ratio: PBS = 2.62 ± 1.07, LIGHT = 38.60 ± 13.91, and LIGHT+ERW1041E = 6.97 ± 2.91, P < 0.05, n = 3; LIGHT+TG2+/+ = 66.43 ± 44.81 vs. LIGHT+TG2-/- = 2.45 ± 1.78, P < 0.01, n = 3) and could be found in renal medulla tubules of kidneys (relative tubular AT1R level: PBS = 5.91 ± 2.93, LIGHT = 92.82 ± 19.54, LIGHT+ERW1041E = 28.49 ± 11.65, and LIGHT+TG2-/- = 0.14 ± 0.10, P < 0.01, n = 5) and the blood vasculature (relative vascular AT1R level: PBS = 0.70 ± 0.30, LIGHT = 13.75 ± 2.49, and LIGHT+ERW1041E = 3.28 ± 0.87, P < 0.01, n = 3), 2 of the tissues highly related to the genesis of hypertension. Our in vitro cellular assays showed that LIGHT stimulation triggered a rapid TG2-dependent increase in the abundance of AT1Rs (relative AT1R level after 2-hour LIGHT treatment: AT1R (WT)+TG2 = 2.21 ± 0.23, AT1R (Q315A)+TG2 = 0.18 ± 0.23, P < 0.05 vs. starting point = 1, n = 2) and downstream calcium signaling (fold increase in NFAT-driven luciferase activity: Saline = 0.02 ± 0.03, Ang II = 0.17 ± 0.08, LIGHT = 0.05 ± 0.04, LIGHT+Ang II = 0.90 ± 0.04 (P < 0.01 vs. Ang II), and LIGHT+Ang II+ERW1041E = 0.15 ± 0.15 (P < 0.01 vs. LIGHT+Ang II), n = 3). CONCLUSIONS: Our data indicate an essential and systemic role for TG2 in bridging inflammation to hypertension via its posttranslational modifications stabilizing AT1 receptor and sensitizing Ang II. Our findings also suggest that TG2 inhibitors could be used as a novel group of cardiovascular agents.

Elevated ecto-5'-nucleotidase: a missing pathogenic factor and new therapeutic target for sickle cell disease.

Although excessive plasma adenosine is detrimental in sickle cell disease (SCD), the molecular mechanism underlying elevated circulating adenosine remains unclear. Here we report that the activity of soluble CD73, an ectonucleotidase producing extracellular adenosine, was significantly elevated in a murine model of SCD and correlated with increased plasma adenosine. Mouse genetic studies demonstrated that CD73 activity contributes to excessive induction of plasma adenosine and thereby promotes sickling, hemolysis, multiorgan damage, and disease progression. Mechanistically, we showed that erythrocyte adenosine 5'-monophosphate-activated protein kinase (AMPK) was activated both in SCD patients and in the murine model of SCD. AMPK functions downstream of adenosine receptor ADORA2B signaling and contributes to sickling by regulating the production of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), a negative allosteric regulator of hemoglobin-O2 binding affinity. Preclinically, we reported that treatment of α,ß-methylene adenosine 5'-diphosphate, a potent CD73 specific inhibitor, significantly decreased sickling, hemolysis, multiorgan damage, and disease progression in the murine model of SCD. Taken together, both human and mouse studies reveal a novel molecular mechanism contributing to the pathophysiology of SCD and identify potential therapeutic strategies to treat SCD.

Sphingosine-1-phosphate receptor 1 mediates elevated IL-6 signaling to promote chronic inflammation and multitissue damage in sickle cell disease.

Sphingosine-1-phosphate (S1P) is a biolipid involved in chronic inflammation in several inflammatory disorders. Recent studies revealed that elevated S1P contributes to sickling in sickle cell disease (SCD), a devastating hemolytic, genetic disorder associated with severe chronic inflammation and tissue damage. We evaluated the effect of elevated S1P in chronic inflammation and tissue damage in SCD and underlying mechanisms. First, we demonstrated that interfering with S1P receptor signaling by FTY720, a U.S. Food and Drug Administration-approved drug, significantly reduced systemic, local inflammation and tissue damage without antisickling effects. These findings led us to discover that S1P receptor activation leads to substantial elevated local and systemic IL-6 levels in SCD mice. Genetic deletion of IL-6 in SCD mice significantly reduced local and systemic inflammation, tissue damage, and kidney dysfunction. At the cellular level, we determined that elevated IL-6 is a key cytokine functioning downstream of elevated S1P, which contributes to increased S1P receptor 1 ( S1pr1) gene expression in the macrophages of several tissues in SCD mice. Mechanistically, we revealed that S1P-S1PR1 signaling reciprocally up-regulated IL-6 gene expression in primary mouse macrophages in a JAK2-dependent manner. Altogether, we revealed that elevated S1P, coupled with macrophage S1PR1 reciprocally inducing IL-6 expression, is a key signaling network functioning as a malicious, positive, feed-forward loop to sustain inflammation and promote tissue damage in SCD. Our findings immediately highlight novel therapeutic possibilities.-Zhao, S., Adebiyi, M. G., Zhang, Y., Couturier, J. P., Fan, X., Zhang, H., Kellems, R. E., Lewis, D. E., Xia, Y. Sphingosine-1-phosphate receptor 1 mediates elevated IL-6 signaling to promote chronic inflammation and multitissue damage in sickle cell disease.

Comprehensive Characterization of Alternative Polyadenylation in Human Cancer.

Background: Alternative polyadenylation (APA) is emerging as a major post-transcriptional mechanism for gene regulation, and dysregulation of APA contributes to several human diseases. However, the functional consequences of APA in human cancer are not fully understood. Particularly, there is no large-scale analysis in cancer cell lines. Methods: We characterized the global APA profiles of 6398 patient samples across 17 cancer types from The Cancer Genome Atlas and 739 cancer cell lines from the Cancer Cell Line Encyclopedia. We built a linear regression model to explore the correlation between APA factors and APA events across different cancer types. We used Spearman correlation to assess the effects of APA events on drug sensitivity and the Wilcoxon rank-sum test or Cox proportional hazards model to identify clinically relevant APA events. Results: We revealed a striking global 3'UTR shortening in cancer cell lines compared with tumor samples. Our analysis further suggested PABPN1 as the master regulator in regulating APA profile across different cancer types. Furthermore, we showed that APA events could affect drug sensitivity, especially of drugs targeting chromatin modifiers. Finally, we identified 1971 clinically relevant APA events, as well as alterations of APA in clinically actionable genes, suggesting that analysis of the complexity of APA profiles could have clinical utility. Conclusions: Our study highlights important roles for APA in human cancer, including reshaping cellular pathways and regulating specific gene expression, exemplifying the complex interplay between APA and other biological processes and yielding new insights into the action mechanism of cancer drugs.

Structural and Functional Insight of Sphingosine 1-Phosphate-Mediated Pathogenic Metabolic Reprogramming in Sickle Cell Disease.

Elevated sphingosine 1-phosphate (S1P) is detrimental in Sickle Cell Disease (SCD), but the mechanistic basis remains obscure. Here, we report that increased erythrocyte S1P binds to deoxygenated sickle Hb (deoxyHbS), facilitates deoxyHbS anchoring to the membrane, induces release of membrane-bound glycolytic enzymes and in turn switches glucose flux towards glycolysis relative to the pentose phosphate pathway (PPP). Suppressed PPP causes compromised glutathione homeostasis and increased oxidative stress, while enhanced glycolysis induces production of 2,3-bisphosphoglycerate (2,3-BPG) and thus increases deoxyHbS polymerization, sickling, hemolysis and disease progression. Functional studies revealed that S1P and 2,3-BPG work synergistically to decrease both HbA and HbS oxygen binding affinity. The crystal structure at 1.9 Å resolution deciphered that S1P binds to the surface of 2,3-BPG-deoxyHbA and causes additional conformation changes to the T-state Hb. Phosphate moiety of the surface bound S1P engages in a highly positive region close to α1-heme while its aliphatic chain snakes along a shallow cavity making hydrophobic interactions in the "switch region", as well as with α2-heme like a molecular "sticky tape" with the last 3-4 carbon atoms sticking out into bulk solvent. Altogether, our findings provide functional and structural bases underlying S1P-mediated pathogenic metabolic reprogramming in SCD and novel therapeutic avenues.

Erythrocyte purinergic signaling components underlie hypoxia adaptation.

Erythrocytes are vital to human adaptation under hypoxic conditions because of their abundance in number and irreplaceable function of delivering oxygen (O2). However, although multiple large-scale altitude studies investigating the overall coordination of the human body for hypoxia adaptation have been conducted, detailed research with a focus on erythrocytes was missing due to lack of proper techniques. The recently maturing metabolomics profiling technology appears to be the answer to this limitation. Metabolomics profiling provides unbiased high-throughput screening data that reveal the overall metabolic status of erythrocytes. Recent studies have exploited this new technology and provided novel insight into erythrocyte physiology and pathology. In particular, a series of studies focusing on erythrocyte purinergic signaling have reported that adenosine signaling, coupled with 5' AMP-activated protein kinase (AMPK) and the production of erythrocyte-enriched bioactive signaling lipid sphingosine 1-phosphate, regulate erythrocyte glucose metabolism for more O2 delivery. Moreover, an adenosine-dependent "erythrocyte hypoxic memory" was discovered that provides an explanation for fast acclimation upon re-ascent. These findings not only shed new light on our understanding of erythrocyte function and hypoxia adaptation, but also offer a myriad of novel therapeutic possibilities to counteract various hypoxic conditions.

Elevated Adenosine Induces Placental DNA Hypomethylation Independent of A2B Receptor Signaling in Preeclampsia.

Preeclampsia is a prevalent pregnancy hypertensive disease with both maternal and fetal morbidity and mortality. Emerging evidence indicates that global placental DNA hypomethylation is observed in patients with preeclampsia and is linked to altered gene expression and disease development. However, the molecular basis underlying placental epigenetic changes in preeclampsia remains unclear. Using 2 independent experimental models of preeclampsia, adenosine deaminase-deficient mice and a pathogenic autoantibody-induced mouse model of preeclampsia, we demonstrate that elevated placental adenosine not only induces hallmark features of preeclampsia but also causes placental DNA hypomethylation. The use of genetic approaches to express an adenosine deaminase minigene specifically in placentas, or adenosine deaminase enzyme replacement therapy, restored placental adenosine to normal levels, attenuated preeclampsia features, and abolished placental DNA hypomethylation in adenosine deaminase-deficient mice. Genetic deletion of CD73 (an ectonucleotidase that converts AMP to adenosine) prevented the elevation of placental adenosine in the autoantibody-induced preeclampsia mouse model and ameliorated preeclampsia features and placental DNA hypomethylation. Immunohistochemical studies revealed that elevated placental adenosine-mediated DNA hypomethylation predominantly occurs in spongiotrophoblasts and labyrinthine trophoblasts and that this effect is independent of A2B adenosine receptor activation in both preeclampsia models. Extending our mouse findings to humans, we used cultured human trophoblasts to demonstrate that adenosine functions intracellularly and induces DNA hypomethylation without A2B adenosine receptor activation. Altogether, both mouse and human studies reveal novel mechanisms underlying placental DNA hypomethylation and potential therapeutic approaches for preeclampsia.

Inflammation, Autoimmunity, and Hypertension: The Essential Role of Tissue Transglutaminase.

Inflammatory cytokines cause hypertension when introduced into animals. Additional evidence indicates that cytokines induce the production of autoantibodies that activate the AT1 angiotensin receptor (AT1R). Extensive evidence shows that these autoantibodies, termed AT1-AA, contribute to hypertension. We review here recent studies showing that cytokine-induced hypertension and AT1-AA production require the ubiquitous enzyme, tissue transglutaminase (TG2). We consider 3 mechanisms by which TG2 may contribute to hypertension. (i) One involves the posttranslational modification (PTM) of AT1Rs at a glutamine residue that is present in the epitope sequence (AFHYESQ) recognized by AT1-AA. (ii) Another mechanism by which TG2 may contribute to hypertension is by PTM of AT1Rs at glutamine 315. Modification at this glutamine prevents ubiquitination-dependent proteasome degradation and allows AT1Rs to accumulate. Increased AT1R abundance is likely to account for increased sensitivity to Ang II activation and in this way contribute to hypertension. (iii) The increased TG2 produced as a result of elevated inflammatory cytokines is likely to contribute to vascular stiffness by modification of intracellular contractile proteins or by crosslinking vascular proteins in the extracellular matrix. This process, termed inward remodeling, results in reduced vascular lumen, vascular stiffness, and increased blood pressure. Based on the literature reviewed here, we hypothesize that TG2 is an essential participant in cytokine-induced hypertension. From this perspective, selective TG2 inhibitors have the potential to be pharmacologic weapons in the fight against hypertension.