Large chemokine binding spectrum of human and mouse atypical chemokine receptor GPR182 (ACKR5).

Atypical chemokine receptors (ACKRs) play pivotal roles in immune regulation by binding chemokines and regulating their spatial distribution without inducing G-protein activation. Recently, GPR182, provisionally named ACKR5, was identified as a novel ACKR expressed in microvascular and lymphatic endothelial cells, with functions in hematopoietic stem cell homeostasis. Here, we comprehensively investigated the chemokine binding profile of human and mouse GPR182. Competitive binding assays using flow cytometry revealed that besides CXCL10, CXCL12 and CXCL13, also human and mouse CXCL11, CXCL14 and CCL25, as well as human CCL1, CCL11, CCL19, CCL26, XCL1 and mouse CCL22, CCL24, CCL27 and CCL28 bind with an affinity of less than 100 nM to GPR182. In line with the binding affinity observed in vitro, elevated serum levels of CCL22, CCL24, CCL25, and CCL27 were observed in GPR182-deficient mice, underscoring the role of GPR182 in chemokine scavenging. These data show a broader chemokine binding repertoire of GPR182 than previously reported and they will be important for future work exploring the physiological and pathophysiological roles of GPR182, which we propose to be renamed atypical chemokine receptor 5 (ACKR5).

Endothelial FAT1 inhibits angiogenesis by controlling YAP/TAZ protein degradation via E3 ligase MIB2.

Activation of endothelial YAP/TAZ signaling is crucial for physiological and pathological angiogenesis. The mechanisms of endothelial YAP/TAZ regulation are, however, incompletely understood. Here we report that the protocadherin FAT1 acts as a critical upstream regulator of endothelial YAP/TAZ which limits the activity of these transcriptional cofactors during developmental and tumor angiogenesis by promoting their degradation. We show that loss of endothelial FAT1 results in increased endothelial cell proliferation in vitro and in various angiogenesis models in vivo. This effect is due to perturbed YAP/TAZ protein degradation, leading to increased YAP/TAZ protein levels and expression of canonical YAP/TAZ target genes. We identify the E3 ubiquitin ligase Mind Bomb-2 (MIB2) as a FAT1-interacting protein mediating FAT1-induced YAP/TAZ ubiquitination and degradation. Loss of MIB2 expression in endothelial cells in vitro and in vivo recapitulates the effects of FAT1 depletion and causes decreased YAP/TAZ degradation and increased YAP/TAZ signaling. Our data identify a pivotal mechanism of YAP/TAZ regulation involving FAT1 and its associated E3 ligase MIB2, which is essential for YAP/TAZ-dependent angiogenesis.

Nanobody inhibitors of Plexin-B1 identify allostery in plexin-semaphorin interactions and signaling.

Plexin-B1 is a receptor for the cell surface semaphorin, Sema4D. This signaling system has been implicated in a variety of human diseases, including cancer, multiple sclerosis and osteoporosis. While inhibitors of the Plexin-B1:Sema4D interaction have been previously reported, understanding their mechanism has been hindered by an incomplete structural view of Plexin-B1. In this study, we have raised and characterized a pair of nanobodies that are specific for mouse Plexin-B1 and which inhibit the binding of Sema4D to mouse Plexin-B1 and its biological activity. Structural studies of these nanobodies reveal that they inhibit the binding of Sema4D in an allosteric manner, binding to epitopes not previously reported. In addition, we report the first unbound structure of human Plexin-B1, which reveals that Plexin-B1 undergoes a conformational change on Sema4D binding. These changes mirror those seen upon binding of allosteric peptide modulators, which suggests a new model for understanding Plexin-B1 signaling and provides a potential innovative route for therapeutic modulation of Plexin-B1.

Plexin-B1 Mutation Drives Metastasis in Prostate Cancer Mouse Models.

Metastatic prostate cancer is essentially incurable and is a leading cause of cancer-related morbidity and mortality in men, yet the underlying molecular mechanisms are poorly understood. Plexins are transmembrane receptors for semaphorins with divergent roles in many forms of cancer. We show here that prostate epithelial cell-specific expression of a mutant form of Plexin-B1 (P1597L) which was identified in metastatic deposits in patients with prostate cancer, significantly increases metastasis, in particular metastasis to distant sites, in two transgenic mouse models of prostate cancer (PbCre+Ptenfl /flKrasG12V and PbCre+Ptenfl /flp53fl/ fl ). In contrast, prostate epithelial cell-specific expression of wild-type (WT) Plexin-B1 in PbCre+Ptenfl /flKrasG12V mice significantly decreases metastasis, showing that a single clinically relevant Pro1597Leu amino-acid change converts Plexin-B1 from a metastasis-suppressor to a metastasis-promoter. Furthermore, PLXNB1P1597L significantly increased invasion of tumor cells into the prostate stroma, while PLXNB1WT reduced invasion, suggesting that Plexin-B1 has a role in the initial stages of metastasis. Deletion of RhoA/C or PDZRhoGEF in Ptenfl /flKrasG12VPLXNB1P1597L mice suppressed metastasis, implicating the Rho/ROCK pathway in this phenotypic switch. Germline deletion of Plexin-B1, to model anti-Plexin-B1 therapy, significantly decreased invasion and metastasis in both models. Our results demonstrate that Plexin-B1 plays a complex yet significant role in metastasis in mouse models of prostate cancer and is a potential therapeutic target to block the lethal spread of the disease. Significance: Few therapeutic targets have been identified specifically for preventing locally invasive/oligometastatic prostate cancer from becoming more widely disseminated. Our findings suggest Plexin-B1 signaling, particularly from the clinically relevant P1597L mutant, is such a target.

Suppression of CCL2 angiocrine function by adrenomedullin promotes tumor growth.

Within the tumor microenvironment, tumor cells and endothelial cells regulate each other. While tumor cells induce angiogenic responses in endothelial cells, endothelial cells release angiocrine factors, which act on tumor cells and other stromal cells. We report that tumor cell-derived adrenomedullin has a pro-angiogenic as well as a direct tumor-promoting effect, and that endothelium-derived CC chemokine ligand 2 (CCL2) suppresses adrenomedullin-induced tumor cell proliferation. Loss of the endothelial adrenomedullin receptor CALCRL or of the G-protein Gs reduced endothelial proliferation. Surprisingly, tumor cell proliferation was also reduced after endothelial deletion of CALCRL or Gs. We identified CCL2 as a critical angiocrine factor whose formation is inhibited by adrenomedullin. Furthermore, CCL2 inhibited adrenomedullin formation in tumor cells through its receptor CCR2. Consistently, loss of endothelial CCL2 or tumor cell CCR2 normalized the reduced tumor growth seen in mice lacking endothelial CALCRL or Gs. Our findings show tumor-promoting roles of adrenomedullin and identify CCL2 as an angiocrine factor controlling adrenomedullin formation by tumor cells.

Targeting of Scavenger Receptors Stabilin-1 and Stabilin-2 Ameliorates Atherosclerosis by a Plasma Proteome Switch Mediating Monocyte/Macrophage Suppression.

BACKGROUND: Scavenger receptors Stabilin-1 (Stab1) and Stabilin-2 (Stab2) are preferentially expressed by liver sinusoidal endothelial cells. They mediate the clearance of circulating plasma molecules controlling distant organ homeostasis. Studies suggest that Stab1 and Stab2 may affect atherosclerosis. Although subsets of tissue macrophages also express Stab1, hematopoietic Stab1 deficiency does not modulate atherogenesis. Here, we comprehensively studied how targeting Stab1 and Stab2 affects atherosclerosis. METHODS: ApoE-KO mice were interbred with Stab1-KO and Stab2-KO mice and fed a Western diet. For antibody targeting, Ldlr-KO mice were also used. Unbiased plasma proteomics were performed and independently confirmed. Ligand binding studies comprised glutathione-S-transferase-pulldown and endocytosis assays. Plasma proteome effects on monocytes were studied by single-cell RNA sequencing in vivo, and by gene expression analyses of Stabilin ligand-stimulated and plasma-stimulated bone marrow-derived monocytes/macrophages in vitro. RESULTS: Spontaneous and Western diet-associated atherogenesis was significantly reduced in ApoE-Stab1-KO and ApoE-Stab2-KO mice. Similarly, inhibition of Stab1 or Stab2 by monoclonal antibodies significantly reduced Western diet-associated atherosclerosis in ApoE-KO and Ldlr-KO mice. Although neither plasma lipid levels nor circulating immune cell numbers were decisively altered, plasma proteomics revealed a switch in the plasma proteome, consisting of 231 dysregulated proteins comparing wildtype with Stab1/2-single and Stab1/2-double KO, and of 41 proteins comparing ApoE-, ApoE-Stab1-, and ApoE-Stab2-KO. Among this broad spectrum of common, but also disparate scavenger receptor ligand candidates, periostin, reelin, and TGFBi (transforming growth factor, ß-induced), known to modulate atherosclerosis, were independently confirmed as novel circulating ligands of Stab1/2. Single-cell RNA sequencing of circulating myeloid cells of ApoE-, ApoE-Stab1-, and ApoE-Stab2-KO mice showed transcriptomic alterations in patrolling (Ccr2-/Cx3cr1++/Ly6Clo) and inflammatory (Ccr2+/Cx3cr1+/Ly6Chi) monocytes, including downregulation of proatherogenic transcription factor Egr1. In wildtype bone marrow-derived monocytes/macrophages, ligand exposure alone did not alter Egr1 expression in vitro. However, exposure to plasma from ApoE-Stab1-KO and ApoE-Stab2-KO mice showed a reverted proatherogenic macrophage activation compared with ApoE-KO plasma, including downregulation of Egr1 in vitro. CONCLUSIONS: Inhibition of Stab1/Stab2 mediates an anti-inflammatory switch in the plasma proteome, including direct Stabilin ligands. The altered plasma proteome suppresses both patrolling and inflammatory monocytes and, thus, systemically protects against atherogenesis. Altogether, anti-Stab1- and anti-Stab2-targeted therapies provide a novel approach for the future treatment of atherosclerosis.

Mechanosensation by endothelial PIEZO1 is required for leukocyte diapedesis.

The extravasation of leukocytes is a critical step during inflammation that requires the localized opening of the endothelial barrier. This process is initiated by the close interaction of leukocytes with various adhesion molecules such as ICAM-1 on the surface of endothelial cells. Here we reveal that mechanical forces generated by leukocyte-induced clustering of ICAM-1 synergize with fluid shear stress exerted by the flowing blood to increase endothelial plasma membrane tension and to activate the mechanosensitive cation channel PIEZO1. This leads to increases in [Ca2+]i and activation of downstream signaling events including phosphorylation of tyrosine kinases sarcoma (SRC) and protein tyrosine kinase 2 (PYK2), as well as of myosin light chain, resulting in opening of the endothelial barrier. Mice with endothelium-specific Piezo1 deficiency show decreased leukocyte extravasation in different inflammation models. Thus, leukocytes and the hemodynamic microenvironment synergize to mechanically activate endothelial PIEZO1 and subsequent downstream signaling to initiate leukocyte diapedesis.

Cysteinyl leukotrienes and acetylcholine are biliary tuft cell cotransmitters.

The gallbladder stores bile between meals and empties into the duodenum upon demand and is thereby exposed to the intestinal microbiome. This exposure raises the need for antimicrobial factors, among them, mucins produced by cholangiocytes, the dominant epithelial cell type in the gallbladder. The role of the much less frequent biliary tuft cells is still unknown. We here show that propionate, a major metabolite of intestinal bacteria, activates tuft cells via the short-chain free fatty acid receptor 2 and downstream signaling involving the cation channel transient receptor potential cation channel subfamily M member 5. This results in corelease of acetylcholine and cysteinyl leukotrienes from tuft cells and evokes synergistic paracrine effects upon the epithelium and the gallbladder smooth muscle, respectively. Acetylcholine triggers mucin release from cholangiocytes, an epithelial defense mechanism, through the muscarinic acetylcholine receptor M3. Cysteinyl leukotrienes cause gallbladder contraction through their cognate receptor CysLTR1, prompting emptying and closing. Our results establish gallbladder tuft cells as sensors of the microbial metabolite propionate, initiating dichotomous innate defense mechanisms through simultaneous release of acetylcholine and cysteinyl leukotrienes.

Activation of lactate receptor HCAR1 down-modulates neuronal activity in rodent and human brain tissue.

Lactate can be used by neurons as an energy substrate to support their activity. Evidence suggests that lactate also acts on a metabotropic receptor called HCAR1, first described in the adipose tissue. Whether HCAR1 also modulates neuronal circuits remains unclear. In this study, using qRT-PCR, we show that HCAR1 is present in the human brain of epileptic patients who underwent resective surgery. In brain slices from these patients, pharmacological HCAR1 activation using a non-metabolized agonist decreased the frequency of both spontaneous neuronal Ca2+ spiking and excitatory post-synaptic currents (sEPSCs). In mouse brains, we found HCAR1 expression in different regions using a fluorescent reporter mouse line and in situ hybridization. In the dentate gyrus, HCAR1 is mainly present in mossy cells, key players in the hippocampal excitatory circuitry and known to be involved in temporal lobe epilepsy. By using whole-cell patch clamp recordings in mouse and rat slices, we found that HCAR1 activation causes a decrease in excitability, sEPSCs, and miniature EPSCs frequency of granule cells, the main output of mossy cells. Overall, we propose that lactate can be considered a neuromodulator decreasing synaptic activity in human and rodent brains, which makes HCAR1 an attractive target for the treatment of epilepsy.

Deletion of Gαq/11 or Gαs Proteins in Gonadotropes Differentially Affects Gonadotropin Production and Secretion in Mice.

Gonadotropin-releasing hormone (GnRH) regulates gonadal function via its stimulatory effects on gonadotropin production by pituitary gonadotrope cells. GnRH is released from the hypothalamus in pulses and GnRH pulse frequency differentially regulates follicle-stimulating hormone (FSH) and luteinizing hormone (LH) synthesis and secretion. The GnRH receptor (GnRHR) is a G protein-coupled receptor that canonically activates Gα q/11-dependent signaling on ligand binding. However, the receptor can also couple to Gα s and in vitro data suggest that toggling between different G proteins may contribute to GnRH pulse frequency decoding. For example, as we show here, knockdown of Gα s impairs GnRH-stimulated FSH synthesis at low- but not high-pulse frequency in a model gonadotrope-derived cell line. We next used a Cre-lox conditional knockout approach to interrogate the relative roles of Gα q/11 and Gα s proteins in gonadotrope function in mice. Gonadotrope-specific Gα q/11 knockouts exhibit hypogonadotropic hypogonadism and infertility, akin to the phenotypes seen in GnRH- or GnRHR-deficient mice. In contrast, under standard conditions, gonadotrope-specific Gα s knockouts produce gonadotropins at normal levels and are fertile. However, the LH surge amplitude is blunted in Gα s knockout females and postgonadectomy increases in FSH and LH are reduced both in males and females. These data suggest that GnRH may signal principally via Gα q/11 to stimulate gonadotropin production, but that Gα s plays important roles in gonadotrope function in vivo when GnRH secretion is enhanced.

LPL/AQP7/GPD2 promotes glycerol metabolism under hypoxia and prevents cardiac dysfunction during ischemia.

In the heart, fatty acid is a major energy substrate to fuel contraction under aerobic conditions. Ischemia downregulates fatty acid metabolism to adapt to the limited oxygen supply, making glucose the preferred substrate. However, the mechanism underlying the myocardial metabolic shift during ischemia remains unknown. Here, we show that lipoprotein lipase (LPL) expression in cardiomyocytes, a principal enzyme that converts triglycerides to free fatty acids and glycerol, increases during myocardial infarction (MI). Cardiomyocyte-specific LPL deficiency enhanced cardiac dysfunction and apoptosis following MI. Deficiency of aquaporin 7 (AQP7), a glycerol channel in cardiomyocytes, increased the myocardial infarct size and apoptosis in response to ischemia. Ischemic conditions activated glycerol-3-phosphate dehydrogenase 2 (GPD2), which converts glycerol-3-phosphate into dihydroxyacetone phosphate to facilitate adenosine triphosphate (ATP) synthesis from glycerol. Conversely, GPD2 deficiency exacerbated cardiac dysfunction after acute MI. Moreover, cardiomyocyte-specific LPL deficiency suppressed the effectiveness of peroxisome proliferator-activated receptor alpha (PPARα) agonist treatment for MI-induced cardiac dysfunction. These results suggest that LPL/AQP7/GPD2-mediated glycerol metabolism plays an important role in preventing myocardial ischemia-related damage.

G-protein-coupled receptor P2Y10 facilitates chemokine-induced CD4 T cell migration through autocrine/paracrine mediators.

G-protein-coupled receptors (GPCRs), especially chemokine receptors, play a central role in the regulation of T cell migration. Various GPCRs are upregulated in activated CD4 T cells, including P2Y10, a putative lysophospholipid receptor that is officially still considered an orphan GPCR, i.e., a receptor with unknown endogenous ligand. Here we show that in mice lacking P2Y10 in the CD4 T cell compartment, the severity of experimental autoimmune encephalomyelitis and cutaneous contact hypersensitivity is reduced. P2Y10-deficient CD4 T cells show normal activation, proliferation and differentiation, but reduced chemokine-induced migration, polarization, and RhoA activation upon in vitro stimulation. Mechanistically, CD4 T cells release the putative P2Y10 ligands lysophosphatidylserine and ATP upon chemokine exposure, and these mediators induce P2Y10-dependent RhoA activation in an autocrine/paracrine fashion. ATP degradation impairs RhoA activation and migration in control CD4 T cells, but not in P2Y10-deficient CD4 T cells. Importantly, the P2Y10 pathway appears to be conserved in human T cells. Taken together, P2Y10 mediates RhoA activation in CD4 T cells in response to auto-/paracrine-acting mediators such as LysoPS and ATP, thereby facilitating chemokine-induced migration and, consecutively, T cell-mediated diseases.

Protein kinase N2 mediates flow-induced endothelial NOS activation and vascular tone regulation.

Formation of NO by endothelial NOS (eNOS) is a central process in the homeostatic regulation of vascular functions including blood pressure regulation, and fluid shear stress exerted by the flowing blood is a main stimulus of eNOS activity. Previous work has identified several mechanosensing and -transducing processes in endothelial cells, which mediate this process and induce the stimulation of eNOS activity through phosphorylation of the enzyme via various kinases including AKT. How the initial mechanosensing and signaling processes are linked to eNOS phosphorylation is unclear. In human endothelial cells, we demonstrated that protein kinase N2 (PKN2), which is activated by flow through the mechanosensitive cation channel Piezo1 and Gq/G11-mediated signaling, as well as by Ca2+ and phosphoinositide-dependent protein kinase 1 (PDK1), plays a pivotal role in this process. Active PKN2 promoted the phosphorylation of human eNOS at serine 1177 and at a newly identified site, serine 1179. These phosphorylation events additively led to increased eNOS activity. PKN2-mediated eNOS phosphorylation at serine 1177 involved the phosphorylation of AKT synergistically with mTORC2-mediated AKT phosphorylation, whereas active PKN2 directly phosphorylated human eNOS at serine 1179. Mice with induced endothelium-specific deficiency of PKN2 showed strongly reduced flow-induced vasodilation and developed arterial hypertension accompanied by reduced eNOS activation. These results uncover a central mechanism that couples upstream mechanosignaling processes in endothelial cells to the regulation of eNOS-mediated NO formation, vascular tone, and blood pressure.

GPR182 is an endothelium-specific atypical chemokine receptor that maintains hematopoietic stem cell homeostasis.

G protein-coupled receptor 182 (GPR182) has been shown to be expressed in endothelial cells; however, its ligand and physiological role has remained elusive. We found GPR182 to be expressed in microvascular and lymphatic endothelial cells of most organs and to bind with nanomolar affinity the chemokines CXCL10, CXCL12, and CXCL13. In contrast to conventional chemokine receptors, binding of chemokines to GPR182 did not induce typical downstream signaling processes, including Gq- and Gi-mediated signaling or ß-arrestin recruitment. GPR182 showed relatively high constitutive activity in regard to ß-arrestin recruitment and rapidly internalized in a ligand-independent manner. In constitutive GPR182-deficient mice, as well as after induced endothelium-specific loss of GPR182, we found significant increases in the plasma levels of CXCL10, CXCL12, and CXCL13. Global and induced endothelium-specific GPR182-deficient mice showed a significant decrease in hematopoietic stem cells in the bone marrow as well as increased colony-forming units of hematopoietic progenitors in the blood and the spleen. Our data show that GPR182 is a new atypical chemokine receptor for CXCL10, CXCL12, and CXCL13, which is involved in the regulation of hematopoietic stem cell homeostasis.

Adhesion receptor ADGRG2/GPR64 is in the GI-tract selectively expressed in mature intestinal tuft cells.

OBJECTIVE: GPR64/ADGRG2 is an orphan Adhesion G protein-coupled receptor (ADGR) known to be mainly expressed in the parathyroid gland and epididymis. This investigation aimed to delineate the cellular expression of GPR64 throughout the body with focus on the gastrointestinal (GI) tract. METHODS: Transgenic Gpr64mCherry reporter mice were histologically examined throughout the body and reporter protein expression in intestinal tuft cells was confirmed by specific cell ablation. The GPCR repertoire of intestinal Gpr64mCherry-positive tuft cells was analyzed by quantitative RT-PCR analysis and in situ hybridization. The Gpr64mCherry was crossed into the general tuft cell reporter Trpm5GFP to generate small intestinal organoids for time-lapse imaging. Intestinal tuft cells were isolated from small intestine, FACS-purified and transcriptionally compared using RNA-seq analysis. RESULTS: Expression of the Gpr64mCherry reporter was identified in multiple organs and specifically in olfactory microvillous cells, enteric nerves, and importantly in respiratory and GI tuft cells. In the small intestine, cell ablation targeting Gpr64-expressing epithelial cells eliminated tuft cells. Transcriptional analysis of small intestinal Gpr64mCherry -positive tuft cells confirmed expression of Gpr64 and the chemo-sensors Sucnr1, Gprc5c, Drd3, and Gpr41/Ffar3. Time-lapse studies of organoids from Trpm5GFP:Gpr64mCherry mice revealed sequential expression of initially Trpm5GFP and subsequently also Gpr64mCherry in maturing intestinal tuft cells. RNA-seq analysis of small intestinal tuft cells based on these two markers demonstrated a dynamic change in expression of transcription factors and GPCRs from young to mature tuft cells. CONCLUSIONS: GPR64 is expressed in chemosensory epithelial cells across a broad range of tissues; however, in the GI tract, GPR64 is remarkably selectively expressed in mature versus young immunoregulatory tuft cells.

Mechanochemical control of epidermal stem cell divisions by B-plexins.

The precise spatiotemporal control of cell proliferation is key to the morphogenesis of epithelial tissues. Epithelial cell divisions lead to tissue crowding and local changes in force distribution, which in turn suppress the rate of cell divisions. However, the molecular mechanisms underlying this mechanical feedback are largely unclear. Here, we identify a critical requirement of B-plexin transmembrane receptors in the response to crowding-induced mechanical forces during embryonic skin development. Epidermal stem cells lacking B-plexins fail to sense mechanical compression, resulting in disinhibition of the transcriptional coactivator YAP, hyperproliferation, and tissue overgrowth. Mechanistically, we show that B-plexins mediate mechanoresponses to crowding through stabilization of adhesive cell junctions and lowering of cortical stiffness. Finally, we provide evidence that the B-plexin-dependent mechanochemical feedback is also pathophysiologically relevant to limit tumor growth in basal cell carcinoma, the most common type of skin cancer. Our data define a central role of B-plexins in mechanosensation to couple cell density and cell division in development and disease.

Mapping the Endothelial Cell S-Sulfhydrome Highlights the Crucial Role of Integrin Sulfhydration in Vascular Function.

BACKGROUND: In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (H2Sn), that exert their biological actions via cysteine S-sulfhydration of target proteins. This study set out to map the "S-sulfhydrome" (ie, the spectrum of proteins targeted by H2Sn) in human endothelial cells. METHODS: Liquid chromatography with tandem mass spectrometry was used to identify S-sulfhydrated cysteines in endothelial cell proteins and ß3 integrin intraprotein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements, and flow-induced vasodilatation in endothelial cell-specific CSE knockout mice and in a small collective of patients with endothelial dysfunction. RESULTS: Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low); (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression; and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing H2Sn donor, SG1002. The endothelial cell "S-sulfhydrome" consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on ß3 integrin in detail we found that S-sulfhydration affected intraprotein disulfide bond formation and was required for the maintenance of an extended-open conformation of the ß leg. ß3 integrin S-sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of S-sulfhydration impaired interactions between ß3 integrin and Gα13 (guanine nucleotide-binding protein subunit α 13), resulting in the constitutive activation of RhoA (ras homolog family member A) and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H2Sn generation, impaired flow-induced dilatation, and failure to detect ß3 integrin S-sulfhydration, all of which were rescued after the administration of an H2Sn supplement. CONCLUSIONS: Vascular disease is associated with marked changes in the S-sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short-term H2Sn supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.

The Orphan G-Protein Coupled Receptor 182 Is a Negative Regulator of Definitive Hematopoiesis through Leukotriene B4 Signaling.

The G protein-coupled receptor 182 (GPR182) is an orphan GPCR, the expression of which is enriched in embryonic endothelial cells (ECs). However, the physiological role and molecular mechanism of action of GPR182 are unknown. Here, we show that GPR182 negatively regulates definitive hematopoiesis in zebrafish and mice. In zebrafish, gpr182 expression is enriched in the hemogenic endothelium (HE), and gpr182 -/- display an increased expression of HE and hematopoietic stem cell (HSC) marker genes. Notably, we find an increased number of myeloid cells in gpr182 -/- compared to wild-type. Further, by time-lapse imaging of zebrafish embryos during the endothelial-to-hematopoietic transition, we find that HE/HSC cell numbers are increased in gpr182 -/- compared to wild-type. GPR182 -/- mice also exhibit an increased number of myeloid cells compared to wild-type, indicating a conserved role for GPR182 in myelopoiesis. Using cell-based small molecule screening and transcriptomic analyses, we further find that GPR182 regulates the leukotriene B4 (LTB4) biosynthesis pathway. Taken together, these data indicate that GPR182 is a negative regulator of definitive hematopoiesis in zebrafish and mice, and provide further evidence for LTB4 signaling in HSC biology.

Lactate released by inflammatory bone marrow neutrophils induces their mobilization via endothelial GPR81 signaling.

Neutrophils provide first line of host defense against bacterial infections utilizing glycolysis for their effector functions. How glycolysis and its major byproduct lactate are triggered in bone marrow (BM) neutrophils and their contribution to neutrophil mobilization in acute inflammation is not clear. Here we report that bacterial lipopolysaccharides (LPS) or Salmonella Typhimurium triggers lactate release by increasing glycolysis, NADPH-oxidase-mediated reactive oxygen species and HIF-1α levels in BM neutrophils. Increased release of BM lactate preferentially promotes neutrophil mobilization by reducing endothelial VE-Cadherin expression, increasing BM vascular permeability via endothelial lactate-receptor GPR81 signaling. GPR81-/- mice mobilize reduced levels of neutrophils in response to LPS, unless rescued by VE-Cadherin disrupting antibodies. Lactate administration also induces release of the BM neutrophil mobilizers G-CSF, CXCL1 and CXCL2, indicating that this metabolite drives neutrophil mobilization via multiple pathways. Our study reveals a metabolic crosstalk between lactate-producing neutrophils and BM endothelium, which controls neutrophil mobilization under bacterial infection.

Adipocyte Piezo1 mediates obesogenic adipogenesis through the FGF1/FGFR1 signaling pathway in mice.

White adipose tissue (WAT) expansion in obesity occurs through enlargement of preexisting adipocytes (hypertrophy) and through formation of new adipocytes (adipogenesis). Adipogenesis results in WAT hyperplasia, smaller adipocytes and a metabolically more favourable form of obesity. How obesogenic WAT hyperplasia is induced remains, however, poorly understood. Here, we show that the mechanosensitive cationic channel Piezo1 mediates diet-induced adipogenesis. Mice lacking Piezo1 in mature adipocytes demonstrated defective differentiation of preadipocyte into mature adipocytes when fed a high fat diet (HFD) resulting in larger adipocytes, increased WAT inflammation and reduced insulin sensitivity. Opening of Piezo1 in mature adipocytes causes the release of the adipogenic fibroblast growth factor 1 (FGF1), which induces adipocyte precursor differentiation through activation of the FGF-receptor-1. These data identify a central feed-back mechanism by which mature adipocytes control adipogenesis during the development of obesity and suggest Piezo1-mediated adipocyte mechano-signalling as a mechanism to modulate obesity and its metabolic consequences.