Publications by authors named "Stefan Offermanns"

242 Publications

Adhesion Receptor Adgrg2/Gpr64 Is In The Gi-Tract Selectively Expressed In Mature Intestinal Tuft Cells.

Mol Metab 2021 Apr 5:101231. Epub 2021 Apr 5.

NNF Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, 2200, Copenhagen, Denmark. Electronic address:

Objective: GPR64/ADGRG2 is an orphan Adhesion G protein-coupled receptor (ADGR) known mainly to be expressed in parathyroid gland and epididymis. The aim of this investigation was to delineate the cellular expression of GPR64 throughout the body with focus on the GI tract.

Methods: Transgenic Gpr64 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 Gpr64-positive tuft cells was analyzed by quantitative RT-PCR analysis and in situ hybridization. The Gpr64 was crossed into the general tuft cell reporter Trpm5 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 Gpr64 reporter was identified in multiple organs and specifically in olfactory microvillous cells, enteric nerves, and importantly in respiratory and gastrointestinal tuft cells. In the small intestine, cell ablation targeting Gpr64-expressing epithelial cells eliminated tuft cells. Transcriptional analysis of small intestinal Gpr64 -positive tuft cells confirmed expression of Gpr64 and the chemo-sensors Sucnr1, Gprc5c, Drd3, and Gpr41/Ffar3. Time-lapse studies of organoids from Trpm5:Gpr64 mice revealed sequential expression of initially Trpm5 and subsequently also Gpr64 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 but in the GI tract remarkedly selectively in mature versus young immunoregulatory tuft cells.
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http://dx.doi.org/10.1016/j.molmet.2021.101231DOI Listing
April 2021

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

Nat Commun 2021 02 26;12(1):1308. Epub 2021 Feb 26.

Institute of Pharmacology, University of Marburg, Marburg, Germany.

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.
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http://dx.doi.org/10.1038/s41467-021-21513-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7910479PMC
February 2021

Do dimethyl fumarate and nicotinic acid elicit common, potentially HCA -mediated adverse reactions? A combined epidemiological-experimental approach.

Br J Clin Pharmacol 2021 Feb 19. Epub 2021 Feb 19.

Research Department, Federal Institute for Drugs and Medical Devices (BfArM), Bonn, Germany.

Aim: Dimethyl fumarate and nicotinic acid activate the hydroxy-carboxylic acid receptor 2 (HCA ) and induce flushing. It is not known whether HCA mediates other adverse drug reactions (ADRs) to these two substances. This study aims to compare ADRs associated with dimethyl fumarate and nicotinic acid, and to discuss whether they are HCA -mediated.

Methods: We identified spontaneous reports of suspected ADRs to dimethyl fumarate and nicotinic acid in the European Adverse Drug Reaction Database (EudraVigilance). These reports were analysed at different hierarchical levels of the Medical Dictionary for Regulatory Activities (MedDRA). In addition, we screened murine organs for HCA expression.

Results: Similarities in the ADR profile of dimethyl fumarate and nicotinic acid included "gastrointestinal signs and symptoms" (odds ratio [OR] 0.8 [0.6-1.1]), "hepatobiliary investigations" (OR 1.3 [0.7-2.5]) and "anxiety disorders and symptoms" (OR 0.9 [0.3-2.2]) in High Level Group Terms; "diarrhoea (excluding infective)" (OR 1.2 [0.7-1.8]) and "liver function analyses" (OR 1.3 [0.7-2.6]) in High Level Terms; and "diarrhoea" (OR 1.2 [0.7-2.0]) and "vomiting" (OR 0.9 [0.4-1.7]) in Preferred Terms. In analogy, HCA was expressed in the gastrointestinal tract, liver and central nervous system (CNS) of murine organs. A discrepant ADR profile was seen for "lymphopenia" (n = 777) at the preferred term level (only reported for dimethyl fumarate) and "blood glucose increased" (more often reported for nicotinic acid; OR 0.1 [0.0-0.5]).

Conclusion: The gastrointestinal ADRs common to both substances may be mediated by HCA . Other ADRs not common to both substances are compound or indication-specific reactions and likely do not involve HCA .
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http://dx.doi.org/10.1111/bcp.14787DOI Listing
February 2021

Isoprostanes evoke contraction of the murine and human detrusor muscle via activation of the thromboxane prostanoid TP receptor and Rho kinase.

Am J Physiol Renal Physiol 2021 04 25;320(4):F537-F547. Epub 2021 Jan 25.

Institute of Translational Medicine, Semmelweis University, Budapest, Hungary.

Local or systemic inflammation can severely impair urinary bladder functions and contribute to the development of voiding disorders in millions of people worldwide. Isoprostanes are inflammatory lipid mediators that are upregulated in the blood and urine by oxidative stress and may potentially induce detrusor overactivity. The aim of the present study was to investigate the effects and signal transduction of isoprostanes in human and murine urinary bladders in order to provide potential pharmacological targets in detrusor overactivity. Contraction force was measured with a myograph in murine and human urinary bladder smooth muscle (UBSM) ex vivo. Isoprostane 8-iso-PGE and 8-iso-PGF evoked dose-dependent contraction in the murine UBSM, which was abolished in mice deficient in the thromboxane prostanoid (TP) receptor. The responses remained unaltered after removal of the mucosa or incubation with tetrodotoxin. Smooth muscle-specific deletion of Gα protein or inhibition of Rho kinase by Y-27632 decreased the contractions. In Gα-knockout mice, responses were reduced and in the presence of Y-27632 abolished completely. In human UBSM, the TP agonist U-46619 evoked dose-dependent contractions. Neither atropine nor the purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid decreased the effect, indicating that TP receptors directly mediate detrusor muscle contraction. 8-iso-PGE and 8-iso-PGF evoked dose-dependent contraction in the human UBSM, and these responses were abolished by the TP antagonist SQ-29548 and were decreased by Y-27632. Our results indicate that isoprostanes evoke contraction in murine and human urinary bladders, an effect mediated by the TP receptor. The G-Rho-Rho kinase pathway plays a significant role in mediating the contraction and therefore may be a potential therapeutic target in detrusor overactivity. Voiding disorders affect millions of people worldwide. Inflammation can impair urinary bladder functions and contribute to the development of detrusor overactivity. The effects and signal transduction of inflammatory lipid mediator isoprostanes were studied in human and murine urinary bladders ex vivo. We found that isoprostanes evoke contraction, an effect mediated by thromboxane prostanoid receptors. The G-Rho-Rho kinase signaling pathway plays a significant role in mediating the contraction and therefore may be a potential therapeutic target.
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http://dx.doi.org/10.1152/ajprenal.00400.2020DOI Listing
April 2021

Vascular biotransformation of organic nitrates is independent of cytochrome P450 monooxygenases.

Br J Pharmacol 2021 Apr 18;178(7):1495-1506. Epub 2021 Feb 18.

Institute for Cardiovascular Physiology, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany.

Background And Purpose: Organic nitrates such as nitroglycerin (NTG) or pentaerythritol tetranitrate (PETN) have been used for over a century in the treatment of angina or ischaemic heart disease. These compounds are prodrugs which release their nitrovasodilators upon enzymic bioactivation by aldehyde dehydrogenase (ALDH2) or cytochromes P450 (CYP). Whereas ALDH2 is known to directly activate organic nitrates in vessels, the contribution of vascular CYPs is unknown and was studied here.

Experimental Approach: As all CYPs depend on cytochrome P450 reductase (POR) as electron donor, we generated a smooth muscle cell-specific, inducible knockout mouse of POR (smcPOR ) to investigate the contribution of POR/CYP to vascular biotransformation of organic nitrates.

Key Results: Microsomes containing recombinant CYPs expressed in human vascular tissues released nitrite from NTG and PETN with CYP2C9 and CYP2C8 being most efficient. SFK525, a CYP suicide inhibitor, blocked this effect. smcPOR mice exhibited no obvious cardiovascular phenotype (normal cardiac weight and endothelium-dependent relaxation) and plasma and vascular nitrite production was similar to control (CTL) animals. NTG- and PETN-induced relaxation of isolated endothelium-intact or endothelium-denuded vessels were identical between CTL and smcPOR . Likewise, nitrite release from organic nitrates in aortic rings was not affected by deletion of POR in smooth muscle cells (SMCs). In contrast, inhibition of ALDH2 by benomyl (10 μM) inhibited NTG-induced nitrite production and relaxation. Deletion of POR did not modulate this response.

Conclusions And Implications: Our data suggest that metabolism by vascular CYPs does not contribute to the pharmacological function of organic nitrates.
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http://dx.doi.org/10.1111/bph.15362DOI Listing
April 2021

YAP/TAZ Are Required to Suppress Osteogenic Differentiation of Vascular Smooth Muscle Cells.

iScience 2020 Dec 26;23(12):101860. Epub 2020 Nov 26.

Max Planck Institute for Heart and Lung Research, Department of Pharmacology, Bad Nauheim 61231, Germany.

Vascular smooth muscle cells (VSMCs) represent the prevailing cell type of arterial vessels and are essential for blood vessel structure and homeostasis. They have substantial potential for phenotypic plasticity when exposed to various stimuli in their local microenvironment. How VSMCs maintain their differentiated contractile phenotype is still poorly understood. Here we demonstrate that the Hippo pathway effectors YAP and TAZ play a critical role in maintaining the differentiated contractile phenotype of VSMCs. In the absence of YAP/TAZ, VSMCs lose their differentiated phenotype and undergo osteogenic differentiation, which results in vascular calcification. Osteogenic transdifferentiation was accompanied by the upregulation of Wnt target genes. The absence of YAP/TAZ in VSMCs led to Disheveled 3 (DVL3) nuclear translocation and upregulation of osteogenesis-associated genes independent of canonical Wnt/β-catenin signaling activation. Our data indicate that cytoplasmic YAP/TAZ interact with DVL3 to avoid its nuclear translocation and osteogenic differentiation, thereby maintaining the differentiated phenotype of VSMCs.
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http://dx.doi.org/10.1016/j.isci.2020.101860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726335PMC
December 2020

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

Circulation 2021 Mar 14;143(9):935-948. Epub 2020 Dec 14.

Institute for Vascular Signalling (S-I.B., J.H., J.W., M.K.D., V.R., F.D.L., B.F., S.Z., A.K., A.F.O.J., I.F.), Goethe University, Frankfurt am Main, Germany.

Background: In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (HS), that exert their biological actions via cysteine -sulfhydration of target proteins. This study set out to map the "-sulfhydrome" (ie, the spectrum of proteins targeted by HS) in human endothelial cells.

Methods: Liquid chromatography with tandem mass spectrometry was used to identify -sulfhydrated cysteines in endothelial cell proteins and β3 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 HS donor, SG1002. The endothelial cell "-sulfhydrome" consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on β3 integrin in detail we found that -sulfhydration affected intraprotein disulfide bond formation and was required for the maintenance of an extended-open conformation of the β leg. β3 integrin -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 -sulfhydration impaired interactions between β3 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 HS generation, impaired flow-induced dilatation, and failure to detect β3 integrin -sulfhydration, all of which were rescued after the administration of an HS supplement.

Conclusions: Vascular disease is associated with marked changes in the -sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short-term HS supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.051877DOI Listing
March 2021

Disturbed flow-induced Gs-mediated signaling protects against endothelial inflammation and atherosclerosis.

JCI Insight 2020 12 3;5(23). Epub 2020 Dec 3.

Max Planck Institute for Heart and Lung Research, Department of Pharmacology, Bad Nauheim, Germany.

Atherosclerosis develops preferentially in areas of the arterial system, in which blood flow is disturbed. Exposure of endothelial cells to disturbed flow has been shown to induce inflammatory signaling, including NF-κB activation, which leads to the expression of leukocyte adhesion molecules and chemokines. Here, we show that disturbed flow promotes the release of adrenomedullin from endothelial cells, which in turn activates its Gs-coupled receptor calcitonin receptor-like receptor (CALCRL). This induces antiinflammatory signaling through cAMP and PKA, and it results in reduced endothelial inflammation in vitro and in vivo. Suppression of endothelial expression of Gαs, the α subunit of the G-protein Gs; CALCRL; or adrenomedullin leads to increased disturbed flow-induced inflammatory signaling in vitro and in vivo. Furthermore, mice with induced endothelial-specific deficiency of Gαs, CALCRL, or adrenomedullin show increased atherosclerotic lesions. Our data identify an antiinflammatory signaling pathway in endothelial cells stimulated by disturbed flow and suggest activation of the endothelial adrenomedullin/CALCRL/Gs system as a promising approach to inhibit progression of atherosclerosis.
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http://dx.doi.org/10.1172/jci.insight.140485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7714404PMC
December 2020

YAP and TAZ protect against white adipocyte cell death during obesity.

Nat Commun 2020 10 28;11(1):5455. Epub 2020 Oct 28.

Department of Pharmacology, Max Planck Institute for Heart and Lung Research, Bad Nauheim, 61231, Germany.

The expansion of the white adipose tissue (WAT) in obesity goes along with increased mechanical, metabolic and inflammatory stress. How adipocytes resist this stress is still poorly understood. Both in human and mouse adipocytes, the transcriptional co-activators YAP/TAZ and YAP/TAZ target genes become activated during obesity. When fed a high-fat diet (HFD), mice lacking YAP/TAZ in white adipocytes develop severe lipodystrophy with adipocyte cell death. The pro-apoptotic factor BIM, which is downregulated in adipocytes of obese mice and humans, is strongly upregulated in YAP/TAZ-deficient adipocytes under HFD, and suppression of BIM expression reduces adipocyte apoptosis. In differentiated adipocytes, TNFα and IL-1β promote YAP/TAZ nuclear translocation via activation of RhoA-mediated actomyosin contractility and increase YAP/TAZ-mediated transcriptional regulation by activation of c-Jun N-terminal kinase (JNK) and AP-1. Our data indicate that the YAP/TAZ signaling pathway may be a target to control adipocyte cell death and compensatory adipogenesis during obesity.
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http://dx.doi.org/10.1038/s41467-020-19229-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7595161PMC
October 2020

Dissecting G-Mediated Plasma Membrane Translocation of Sphingosine Kinase-1.

Cells 2020 09 29;9(10). Epub 2020 Sep 29.

Institut für Allgemeine Pharmakologie und Toxikologie, Universitätsklinikum, Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany.

Diverse extracellular signals induce plasma membrane translocation of sphingosine kinase-1 (SphK1), thereby enabling inside-out signaling of sphingosine-1-phosphate. We have shown before that G-coupled receptors and constitutively active Gα specifically induced a rapid and long-lasting SphK1 translocation, independently of canonical G/phospholipase C (PLC) signaling. Here, we further characterized G regulation of SphK1. SphK1 translocation by the M receptor in HEK-293 cells was delayed by expression of catalytically inactive G-protein-coupled receptor kinase-2, p63Rho guanine nucleotide exchange factor (p63RhoGEF), and catalytically inactive PLCβ, but accelerated by wild-type PLCβ and the PLCδ PH domain. Both wild-type SphK1 and catalytically inactive SphK1-G82D reduced M receptor-stimulated inositol phosphate production, suggesting competition at Gα. Embryonic fibroblasts from Gα double-deficient mice were used to show that amino acids W263 and T257 of Gα, which interact directly with PLCβ and p63RhoGEF, were important for bradykinin B receptor-induced SphK1 translocation. Finally, an AIXXPL motif was identified in vertebrate SphK1 (positions 100-105 in human SphK1a), which resembles the Gα binding motif, ALXXPI, in PLCβ and p63RhoGEF. After M receptor stimulation, SphK1-A100E-I101E and SphK1-P104A-L105A translocated in only 25% and 56% of cells, respectively, and translocation efficiency was significantly reduced. The data suggest that both the AIXXPL motif and currently unknown consequences of PLCβ/PLCδ(PH) expression are important for regulation of SphK1 by G.
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http://dx.doi.org/10.3390/cells9102201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7599897PMC
September 2020

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

ACS Pharmacol Transl Sci 2020 Aug 24;3(4):676-689. Epub 2020 Jun 24.

Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, 61231, Germany.

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, expression is enriched in the hemogenic endothelium (HE), and display an increased expression of HE and hematopoietic stem cell (HSC) marker genes. Notably, we find an increased number of myeloid cells in 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 compared to wild-type. 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.
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http://dx.doi.org/10.1021/acsptsci.0c00020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432686PMC
August 2020

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

Nat Commun 2020 07 15;11(1):3547. Epub 2020 Jul 15.

Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.

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.
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http://dx.doi.org/10.1038/s41467-020-17402-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363928PMC
July 2020

Angiopoietin-2-integrin α5β1 signaling enhances vascular fatty acid transport and prevents ectopic lipid-induced insulin resistance.

Nat Commun 2020 06 12;11(1):2980. Epub 2020 Jun 12.

Center for Vascular Research, Institute for Basic Science, Daejeon, 34141, Republic of Korea.

Proper storage of excessive dietary fat into subcutaneous adipose tissue (SAT) prevents ectopic lipid deposition-induced insulin resistance, yet the underlying mechanism remains unclear. Here, we identify angiopoietin-2 (Angpt2)-integrin α5β1 signaling as an inducer of fat uptake specifically in SAT. Adipocyte-specific deletion of Angpt2 markedly reduced fatty acid uptake and storage in SAT, leading to ectopic lipid accumulation in glucose-consuming organs including skeletal muscle and liver and to systemic insulin resistance. Mechanistically, Angpt2 activated integrin α5β1 signaling in the endothelium and triggered fatty acid transport via CD36 and FATP3 into SAT. Genetic or pharmacological inhibition of the endothelial integrin α5β1 recapitulated adipocyte-specific Angpt2 knockout phenotypes. Our findings demonstrate the critical roles of Angpt2-integrin α5β1 signaling in SAT endothelium in regulating whole-body fat distribution for metabolic health and highlight adipocyte-endothelial crosstalk as a potential target for prevention of ectopic lipid deposition-induced lipotoxicity and insulin resistance.
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http://dx.doi.org/10.1038/s41467-020-16795-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293240PMC
June 2020

Long non-coding RNA LASSIE regulates shear stress sensing and endothelial barrier function.

Commun Biol 2020 May 26;3(1):265. Epub 2020 May 26.

Dept. of Physiology, Amsterdam Cardiovascular Sciences (ACS), Amsterdam UMC, VU University Medical Center, Amsterdam, The Netherlands.

Blood vessels are constantly exposed to shear stress, a biomechanical force generated by blood flow. Normal shear stress sensing and barrier function are crucial for vascular homeostasis and are controlled by adherens junctions (AJs). Here we show that AJs are stabilized by the shear stress-induced long non-coding RNA LASSIE (linc00520). Silencing of LASSIE in endothelial cells impairs cell survival, cell-cell contacts and cell alignment in the direction of flow. LASSIE associates with junction proteins (e.g. PECAM-1) and the intermediate filament protein nestin, as identified by RNA affinity purification. The AJs component VE-cadherin showed decreased stabilization, due to reduced interaction with nestin and the microtubule cytoskeleton in the absence of LASSIE. This study identifies LASSIE as link between nestin and VE-cadherin, and describes nestin as crucial component in the endothelial response to shear stress. Furthermore, this study indicates that LASSIE regulates barrier function by connecting AJs to the cytoskeleton.
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http://dx.doi.org/10.1038/s42003-020-0987-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7251106PMC
May 2020

Attenuated Epigenetic Suppression of Muscle Stem Cell Necroptosis Is Required for Efficient Regeneration of Dystrophic Muscles.

Cell Rep 2020 05;31(7):107652

Department of Cardiac Development and Remodeling, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany; German Center for Cardiovascular Research (DZHK); German Center for Lung Research (DZL). Electronic address:

Somatic stem cells expand massively during tissue regeneration, which might require control of cell fitness, allowing elimination of non-competitive, potentially harmful cells. How or if such cells are removed to restore organ function is not fully understood. Here, we show that a substantial fraction of muscle stem cells (MuSCs) undergo necroptosis because of epigenetic rewiring during chronic skeletal muscle regeneration, which is required for efficient regeneration of dystrophic muscles. Inhibition of necroptosis strongly enhances suppression of MuSC expansion in a non-cell-autonomous manner. Prevention of necroptosis in MuSCs of healthy muscles is mediated by the chromatin remodeler CHD4, which directly represses the necroptotic effector Ripk3, while CHD4-dependent Ripk3 repression is dramatically attenuated in dystrophic muscles. Loss of Ripk3 repression by inactivation of Chd4 causes massive necroptosis of MuSCs, abolishing regeneration. Our study demonstrates how programmed cell death in MuSCs is tightly controlled to achieve optimal tissue regeneration.
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http://dx.doi.org/10.1016/j.celrep.2020.107652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7242912PMC
May 2020

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

Nat Commun 2020 05 8;11(1):2303. Epub 2020 May 8.

Max Planck Institute for Heart and Lung Research, Department of Pharmacology, Ludwigstr. 43, 61231, Bad Nauheim, Germany.

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.
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http://dx.doi.org/10.1038/s41467-020-16026-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7211025PMC
May 2020

Chemosensory Cell-Derived Acetylcholine Drives Tracheal Mucociliary Clearance in Response to Virulence-Associated Formyl Peptides.

Immunity 2020 04;52(4):683-699.e11

Institute for Anatomy and Cell Biology, German Center for Lung Research, Justus Liebig University, 35385 Giessen, Germany. Electronic address:

Mucociliary clearance through coordinated ciliary beating is a major innate defense removing pathogens from the lower airways, but the pathogen sensing and downstream signaling mechanisms remain unclear. We identified virulence-associated formylated bacterial peptides that potently stimulated ciliary-driven transport in the mouse trachea. This innate response was independent of formyl peptide and taste receptors but depended on key taste transduction genes. Tracheal cholinergic chemosensory cells expressed these genes, and genetic ablation of these cells abrogated peptide-driven stimulation of mucociliary clearance. Trpm5-deficient mice were more susceptible to infection with a natural pathogen, and formylated bacterial peptides were detected in patients with chronic obstructive pulmonary disease. Optogenetics and peptide stimulation revealed that ciliary beating was driven by paracrine cholinergic signaling from chemosensory to ciliated cells operating through muscarinic M3 receptors independently of nerves. We provide a cellular and molecular framework that defines how tracheal chemosensory cells integrate chemosensation with innate defense.
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http://dx.doi.org/10.1016/j.immuni.2020.03.005DOI Listing
April 2020

Digenic inheritance of mutations in EPHA2 and SLC26A4 in Pendred syndrome.

Nat Commun 2020 03 12;11(1):1343. Epub 2020 Mar 12.

Laboratory for Cell Polarity and Organogenesis, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany.

Enlarged vestibular aqueduct (EVA) is one of the most commonly identified inner ear malformations in hearing loss patients including Pendred syndrome. While biallelic mutations of the SLC26A4 gene, encoding pendrin, causes non-syndromic hearing loss with EVA or Pendred syndrome, a considerable number of patients appear to carry mono-allelic mutation. This suggests faulty pendrin regulatory machinery results in hearing loss. Here we identify EPHA2 as another causative gene of Pendred syndrome with SLC26A4. EphA2 forms a protein complex with pendrin controlling pendrin localization, which is disrupted in some pathogenic forms of pendrin. Moreover, point mutations leading to amino acid substitution in the EPHA2 gene are identified from patients bearing mono-allelic mutation of SLC26A4. Ephrin-B2 binds to EphA2 triggering internalization with pendrin inducing EphA2 autophosphorylation weakly. The identified EphA2 mutants attenuate ephrin-B2- but not ephrin-A1-induced EphA2 internalization with pendrin. Our results uncover an unexpected role of the Eph/ephrin system in epithelial function.
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http://dx.doi.org/10.1038/s41467-020-15198-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7067772PMC
March 2020

Orphan G Protein-Coupled Receptor GPRC5B Controls Smooth Muscle Contractility and Differentiation by Inhibiting Prostacyclin Receptor Signaling.

Circulation 2020 04 16;141(14):1168-1183. Epub 2020 Jan 16.

Department of Pharmacology (J.C., R.C., R.L., H.K., W.Z., S.T., S.O., N.W.), Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany.

Background: G protein-coupled receptors are important regulators of contractility and differentiation in vascular smooth muscle cells (SMCs), but the specific function of SMC-expressed orphan G protein-coupled receptor class C group 5 member B (GPRC5B) is unclear.

Methods: We studied the role of GPRC5B in the regulation of contractility and dedifferentiation in human and murine SMCs in vitro and in iSM--KO (tamoxifen-inducible, SMC-specific knockout) mice under conditions of arterial hypertension and atherosclerosis in vivo.

Results: Mesenteric arteries from SMC-specific -KOs showed ex vivo significantly enhanced prostacyclin receptor (IP)-dependent relaxation, whereas responses to other relaxant or contractile factors were normal. In vitro, knockdown of GPRC5B in human aortic SMCs resulted in increased IP-dependent cAMP production and consecutive facilitation of SMC relaxation. In line with this facilitation of IP-mediated relaxation, iSM--KO mice were protected from arterial hypertension, and this protective effect was abrogated by IP antagonists. Mechanistically, we show that knockdown of GPRC5B increased the membrane localization of IP both in vitro and in vivo and that GPRC5B, but not other G protein-coupled receptors, physically interacts with IP. Last, we show that enhanced IP signaling in GPRC5B-deficient SMCs not only facilitates relaxation but also prevents dedifferentiation during atherosclerosis development, resulting in reduced plaque load and increased differentiation of SMCs in the fibrous cap.

Conclusions: Taken together, our data show that GPRC5B regulates vascular SMC tone and differentiation by negatively regulating IP signaling.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.043703DOI Listing
April 2020

Impaired endothelium-mediated cerebrovascular reactivity promotes anxiety and respiration disorders in mice.

Proc Natl Acad Sci U S A 2020 01 2;117(3):1753-1761. Epub 2020 Jan 2.

Institute for Experimental and Clinical Pharmacology and Toxicology, University of Lübeck, 23562 Lübeck, Germany;

Carbon dioxide (CO), the major product of metabolism, has a strong impact on cerebral blood vessels, a phenomenon known as cerebrovascular reactivity. Several vascular risk factors such as hypertension or diabetes dampen this response, making cerebrovascular reactivity a useful diagnostic marker for incipient vascular pathology, but its functional relevance, if any, is still unclear. Here, we found that GPR4, an endothelial H receptor, and endothelial Gα proteins mediate the CO/H effect on cerebrovascular reactivity in mice. CO/H leads to constriction of vessels in the brainstem area that controls respiration. The consequential washout of CO, if cerebrovascular reactivity is impaired, reduces respiration. In contrast, CO dilates vessels in other brain areas such as the amygdala. Hence, an impaired cerebrovascular reactivity amplifies the CO effect on anxiety. Even at atmospheric CO concentrations, impaired cerebrovascular reactivity caused longer apneic episodes and more anxiety, indicating that cerebrovascular reactivity is essential for normal brain function. The site-specific reactivity of vessels to CO is reflected by regional differences in their gene expression and the release of vasoactive factors from endothelial cells. Our data suggest the central nervous system (CNS) endothelium as a target to treat respiratory and affective disorders associated with vascular diseases.
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http://dx.doi.org/10.1073/pnas.1907467117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6983400PMC
January 2020

Sphingosine-1-Phosphate Enhances α-Adrenergic Vasoconstriction via S1P2-G-ROCK Mediated Signaling.

Int J Mol Sci 2019 Dec 17;20(24). Epub 2019 Dec 17.

Institute of Translational Medicine, Semmelweis University, 1094 Budapest, Hungary.

Sphingosine-1-phosphate (S1P) has been implicated recently in the physiology and pathology of the cardiovascular system including regulation of vascular tone. Pilot experiments showed that the vasoconstrictor effect of S1P was enhanced markedly in the presence of phenylephrine (PE). Based on this observation, we hypothesized that S1P might modulate α-adrenergic vasoactivity. In murine aortas, a 20-minute exposure to S1P but not to its vehicle increased the E and decreased the EC of PE-induced contractions indicating a hyperreactivity to α-adrenergic stimulation. The potentiating effect of S1P disappeared in S1P2 but not in S1P3 receptor-deficient vessels. In addition, smooth muscle specific conditional deletion of G proteins or pharmacological inhibition of the Rho-associated protein kinase (ROCK) by Y-27632 or fasudil abolished the effect of S1P on α-adrenergic vasoconstriction. Unexpectedly, PE-induced contractions remained enhanced markedly as late as three hours after S1P-exposure in wild-type (WT) and S1P3 KO but not in S1P2 KO vessels. In conclusion, the S1P-S1P2-G-ROCK signaling pathway appears to have a major influence on α-adrenergic vasoactivity. This cooperativity might lead to sustained vasoconstriction when increased sympathetic tone is accompanied by increased S1P production as it occurs during acute coronary syndrome and stroke.
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http://dx.doi.org/10.3390/ijms20246361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941080PMC
December 2019

Commensal-bacteria-derived butyrate promotes the T-cell-independent IgA response in the colon.

Int Immunol 2020 04;32(4):243-258

Division of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Science, Keio University, Tokyo, Japan.

Secretory immunoglobulin A (SIgA), the most abundant antibody isotype in the body, maintains a mutual relationship with commensal bacteria and acts as a primary barrier at the mucosal surface. Colonization by commensal bacteria induces an IgA response, at least partly through a T-cell-independent process. However, the mechanism underlying the commensal-bacteria-induced T-cell-independent IgA response has yet to be fully clarified. Here, we show that commensal-bacteria-derived butyrate promotes T-cell-independent IgA class switching recombination (CSR) in the mouse colon. Notably, the butyrate concentration in human stools correlated positively with the amount of IgA. Butyrate up-regulated the production of transforming growth factor β1 and all-trans retinoic acid by CD103+CD11b+ dendritic cells, both of which are critical for T-cell-independent IgA CSR. This effect was mediated by G-protein-coupled receptor 41 (GPR41/FFA3) and GPR109a/HCA2, and the inhibition of histone deacetylase. The butyrate-induced IgA response reinforced the colonic barrier function, preventing systemic bacterial dissemination under inflammatory conditions. These observations demonstrate that commensal-bacteria-derived butyrate contributes to the maintenance of the gut immune homeostasis by facilitating the T-cell-independent IgA response in the colon.
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http://dx.doi.org/10.1093/intimm/dxz078DOI Listing
April 2020

Helix 8 is the essential structural motif of mechanosensitive GPCRs.

Nat Commun 2019 12 19;10(1):5784. Epub 2019 Dec 19.

Walther Straub Institute of Pharmacology and Toxicology, Ludwig Maximilian University of Munich, Goethestr. 33, 80336, Munich, Germany.

G-protein coupled receptors (GPCRs) are versatile cellular sensors for chemical stimuli, but also serve as mechanosensors involved in various (patho)physiological settings like vascular regulation, cardiac hypertrophy and preeclampsia. However, the molecular mechanisms underlying mechanically induced GPCR activation have remained elusive. Here we show that mechanosensitive histamine H receptors (HRs) are endothelial sensors of fluid shear stress and contribute to flow-induced vasodilation. At the molecular level, we observe that HRs undergo stimulus-specific patterns of conformational changes suggesting that mechanical forces and agonists induce distinct active receptor conformations. GPCRs lacking C-terminal helix 8 (H8) are not mechanosensitive, and transfer of H8 to non-responsive GPCRs confers, while removal of H8 precludes, mechanosensitivity. Moreover, disrupting H8 structural integrity by amino acid exchanges impairs mechanosensitivity. Altogether, H8 is the essential structural motif endowing GPCRs with mechanosensitivity. These findings provide a mechanistic basis for a better understanding of the roles of mechanosensitive GPCRs in (patho)physiology.
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http://dx.doi.org/10.1038/s41467-019-13722-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6923424PMC
December 2019

Niacin protects against abdominal aortic aneurysm formation via GPR109A independent mechanisms: role of NAD+/nicotinamide.

Cardiovasc Res 2020 Dec;116(14):2226-2238

Department of Medicine, Medical College of Georgia, Augusta University, Augusta, GA, USA.

Aims: Chronic adventitial and medial infiltration of immune cells play an important role in the pathogenesis of abdominal aortic aneurysms (AAAs). Nicotinic acid (niacin) was shown to inhibit atherosclerosis by activating the anti-inflammatory G protein-coupled receptor GPR109A [also known as hydroxycarboxylic acid receptor 2 (HCA2)] expressed on immune cells, blunting immune activation and adventitial inflammatory cell infiltration. Here, we investigated the role of niacin and GPR109A in regulating AAA formation.

Methods And Results: Mice were supplemented with niacin or nicotinamide, and AAA was induced by angiotensin II (AngII) infusion or calcium chloride (CaCl2) application. Niacin markedly reduced AAA formation in both AngII and CaCl2 models, diminishing adventitial immune cell infiltration, concomitant inflammatory responses, and matrix degradation. Unexpectedly, GPR109A gene deletion did not abrogate the protective effects of niacin against AAA formation, suggesting GPR109A-independent mechanisms. Interestingly, nicotinamide, which does not activate GPR109A, also inhibited AAA formation and phenocopied the effects of niacin. Mechanistically, both niacin and nicotinamide supplementation increased nicotinamide adenine dinucleotide (NAD+) levels and NAD+-dependent Sirt1 activity, which were reduced in AAA tissues. Furthermore, pharmacological inhibition of Sirt1 abrogated the protective effect of nicotinamide against AAA formation.

Conclusion: Niacin protects against AAA formation independent of GPR109A, most likely by serving as an NAD+ precursor. Supplementation of NAD+ using nicotinamide-related biomolecules may represent an effective and well-tolerated approach to preventing or treating AAA.
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http://dx.doi.org/10.1093/cvr/cvz303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695356PMC
December 2020

Nicotinamide Limits Replication of Mycobacterium tuberculosis and Bacille Calmette-Guérin Within Macrophages.

J Infect Dis 2020 03;221(6):989-999

TB Research & Training Center, Department of Medicine, University of Washington, Seattle, Washington, USA.

Novel antimicrobials for treatment of Mycobacterium tuberculosis are needed. We hypothesized that nicotinamide (NAM) and nicotinic acid (NA) modulate macrophage function to restrict M. tuberculosis replication in addition to their direct antimicrobial properties. Both compounds had modest activity in 7H9 broth, but only NAM inhibited replication in macrophages. Surprisingly, in macrophages NAM and the related compound pyrazinamide restricted growth of bacille Calmette-Guérin but not wild-type Mycobacterium bovis, which both lack a functional nicotinamidase/pyrazinamidase (PncA) rendering each strain resistant to these drugs in broth culture. Interestingly, NAM was not active in macrophages infected with a virulent M. tuberculosis mutant encoding a deletion in pncA. We conclude that the differential activity of NAM and nicotinic acid on infected macrophages suggests host-specific NAM targets rather than PncA-dependent direct antimicrobial properties. These activities are sufficient to restrict attenuated BCG, but not virulent wild-type M. bovis or M. tuberculosis.
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http://dx.doi.org/10.1093/infdis/jiz541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7050990PMC
March 2020

Protein Kinase N Promotes Stress-Induced Cardiac Dysfunction Through Phosphorylation of Myocardin-Related Transcription Factor A and Disruption of Its Interaction With Actin.

Circulation 2019 11 30;140(21):1737-1752. Epub 2019 Sep 30.

Departments of Cardiology (T.S., M.T., K. Kato, T.T., S.E., S.I., Y.M., Y.Y., T.Y., K.U., T.O, H.I., Y.S., Y.K.B., T.M.), Nagoya University School of Medicine, Japan.

Background: Heart failure is a complex syndrome that results from structural or functional impairment of ventricular filling or blood ejection. Protein phosphorylation is a major and essential intracellular mechanism that mediates various cellular processes in cardiomyocytes in response to extracellular and intracellular signals. The RHOA-associated protein kinase (ROCK/Rho-kinase), an effector regulated by the small GTPase RHOA, causes pathological phosphorylation of proteins, resulting in cardiovascular diseases. RHOA also activates protein kinase N (PKN); however, the role of PKN in cardiovascular diseases remains unclear.

Methods: To explore the role of PKNs in heart failure, we generated tamoxifen-inducible, cardiomyocyte-specific PKN1- and PKN2-knockout mice by intercrossing the αMHC-CreERT2 line with and mice and applied a mouse model of transverse aortic constriction- and angiotensin II-induced heart failure. To identify a novel substrate of PKNs, we incubated GST-tagged myocardin-related transcription factor A (MRTFA) with recombinant GST-PKN-catalytic domain or GST-ROCK-catalytic domain in the presence of radiolabeled ATP and detected radioactive GST-MRTFA as phosphorylated MRTFA.

Results: We demonstrated that RHOA activates 2 members of the PKN family of proteins, PKN1 and PKN2, in cardiomyocytes of mice with cardiac dysfunction. Cardiomyocyte-specific deletion of the genes encoding and (cmc-PKN1/2 DKO) did not affect basal heart function but protected mice from pressure overload- and angiotensin II-induced cardiac dysfunction. Furthermore, we identified MRTFA as a novel substrate of PKN1 and PKN2 and found that MRTFA phosphorylation by PKN was considerably more effective than that by ROCK in vitro. We confirmed that endogenous MRTFA phosphorylation in the heart was induced by pressure overload- and angiotensin II-induced cardiac dysfunction in wild-type mice, whereas cmc-PKN1/2 DKO mice suppressed transverse aortic constriction- and angiotensin II-induced phosphorylation of MRTFA. Although RHOA-mediated actin polymerization accelerated MRTFA-induced gene transcription, PKN1 and PKN2 inhibited the interaction of MRTFA with globular actin by phosphorylating MRTFA, causing increased serum response factor-mediated expression of cardiac hypertrophy- and fibrosis-associated genes.

Conclusions: Our results indicate that PKN1 and PKN2 activation causes cardiac dysfunction and is involved in the transition to heart failure, thus providing unique targets for therapeutic intervention for heart failure.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.041019DOI Listing
November 2019

Myogenic vasoconstriction requires G/G and LARG to maintain local and systemic vascular resistance.

Elife 2019 09 24;8. Epub 2019 Sep 24.

Department of Pharmacology, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany.

Myogenic vasoconstriction is an autoregulatory function of small arteries. Recently, G-protein-coupled receptors have been involved in myogenic vasoconstriction, but the downstream signalling mechanisms and the in-vivo-function of this myogenic autoregulation are poorly understood. Here, we show that small arteries from mice with smooth muscle-specific loss of G/G or the Rho guanine nucleotide exchange factor ARHGEF12 have lost myogenic vasoconstriction. This defect was accompanied by loss of RhoA activation, while vessels showed normal increases in intracellular [Ca]. In the absence of myogenic vasoconstriction, perfusion of peripheral organs was increased, systemic vascular resistance was reduced and cardiac output and left ventricular mass were increased. In addition, animals with defective myogenic vasoconstriction showed aggravated hypotension in response to endotoxin. We conclude that G/G- and Rho-mediated signaling plays a key role in myogenic vasoconstriction and that myogenic tone is required to maintain local and systemic vascular resistance under physiological and pathological condition.
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http://dx.doi.org/10.7554/eLife.49374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6777979PMC
September 2019

NK2 receptor-mediated detrusor muscle contraction involves G-dependent activation of voltage-dependent Ca channels and the RhoA-Rho kinase pathway.

Am J Physiol Renal Physiol 2019 11 28;317(5):F1154-F1163. Epub 2019 Aug 28.

Institute of Translational Medicine, Semmelweis University, Budapest, Hungary.

Tachykinins (TKs) are involved in both the physiological regulation of urinary bladder functions and development of overactive bladder syndrome. The aim of the present study was to investigate the signal transduction pathways of TKs in the detrusor muscle to provide potential pharmacological targets for the treatment of bladder dysfunctions related to enhanced TK production. Contraction force, intracellular Ca concentration, and RhoA activity were measured in the mouse urinary bladder smooth muscle (UBSM). TKs and the NK2 receptor (NK2R)-specific agonist [β-Ala]-NKA(4-10) evoked contraction, which was inhibited by the NKR2 antagonist MEN10376. In Gα-deficient mice, [β-Ala]-NKA(4-10)-induced contraction and the intracellular Ca concentration increase were abolished. Although G proteins are linked principally to phospholipase Cβ and inositol trisphosphate-mediated Ca release from intracellular stores, we found that phospholipase Cβ inhibition and sarcoplasmic reticulum Ca depletion failed to have any effect on contraction induced by [β-Ala]-NKA(4-10). In contrast, lack of extracellular Ca or blockade of voltage-dependent Ca channels (VDCCs) suppressed contraction. Furthermore, [β-Ala]-NKA(4-10) increased RhoA activity in the UBSM in a G-dependent manner and inhibition of Rho kinase with Y-27632 decreased contraction force, whereas the combination of Y-27632 with either VDCC blockade or depletion of extracellular Ca resulted in complete inhibition of [β-Ala]-NKA(4-10)-induced contractions. In summary, our results indicate that NK2Rs are linked exclusively to G proteins in the UBSM and that the intracellular signaling involves the simultaneous activation of VDCC and the RhoA-Rho kinase pathway. These findings may help to identify potential therapeutic targets of bladder dysfunctions related to upregulation of TKs.
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http://dx.doi.org/10.1152/ajprenal.00106.2019DOI Listing
November 2019

The impact of Semaphorin 4C/Plexin-B2 signaling on fear memory via remodeling of neuronal and synaptic morphology.

Mol Psychiatry 2021 04 23;26(4):1376-1398. Epub 2019 Aug 23.

Institute of Pharmacology, Heidelberg University, Im Neuenheimer Feld 366, 69120, Heidelberg, Germany.

Aberrant fear is a cornerstone of several psychiatric disorders. Consequently, there is large interest in elucidation of signaling mechanisms that link extracellular cues to changes in neuronal function and structure in brain pathways that are important in the generation and maintenance of fear memory and its behavioral expression. Members of the Plexin-B family of receptors for class 4 semaphorins play important roles in developmental plasticity of neurons, and their expression persists in some areas of the adult nervous system. Here, we aimed to elucidate the role of Semaphorin 4C (Sema4C) and its cognate receptor, Plexin-B2, in the expression of contextual and cued fear memory, setting a mechanistic focus on structural plasticity and exploration of contributing signaling pathways. We observed that Plexin-B2 and Sema4C are expressed in forebrain areas related to fear memory, such as the anterior cingulate cortex, amygdala and the hippocampus, and their expression is regulated by aversive stimuli that induce fear memory. By generating forebrain-specific Plexin-B2 knockout mice and analyzing fear-related behaviors, we demonstrate that Sema4C-PlexinB2 signaling plays a crucial functional role in the recent and remote recall of fear memory. Detailed neuronal morphological analyses revealed that Sema4C-PlexinB2 signaling largely mediates fear-induced structural plasticity by enhancing dendritic ramifications and modulating synaptic density in the adult hippocampus. Analyses on signaling-related mutant mice showed that these functions are mediated by PlexinB2-dependent RhoA activation. These results deliver important insights into the mechanistic understanding of maladaptive plasticity in fear circuits and have implications for novel therapeutic strategies against fear-related disorders.
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http://dx.doi.org/10.1038/s41380-019-0491-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7985029PMC
April 2021

Shear stress-induced endothelial adrenomedullin signaling regulates vascular tone and blood pressure.

J Clin Invest 2019 06 17;129(7):2775-2791. Epub 2019 Jun 17.

Department of Pharmacology, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany.

Hypertension is a primary risk factor for cardiovascular diseases including myocardial infarction and stroke. Major determinants of blood pressure are vasodilatory factors such as nitric oxide (NO) released from the endothelium under the influence of fluid shear stress exerted by the flowing blood. Several endothelial signaling processes mediating fluid shear stress-induced formation and release of vasodilatory factors have been described. It is, however, still poorly understood how fluid shear stress induces these endothelial responses. Here we show that the endothelial mechanosensitive cation channel PIEZO1 mediated fluid shear stress-induced release of adrenomedullin, which in turn activated its Gs-coupled receptor. The subsequent increase in cAMP levels promoted the phosphorylation of endothelial NO synthase (eNOS) at serine 633 through protein kinase A (PKA), leading to the activation of the enzyme. This Gs/PKA-mediated pathway synergized with the AKT-mediated pathways leading to eNOS phosphorylation at serine 1177. Mice with endothelium-specific deficiency of adrenomedullin, the adrenomedullin receptor, or Gαs showed reduced flow-induced eNOS activation and vasodilation and developed hypertension. Our data identify fluid shear stress-induced PIEZO1 activation as a central regulator of endothelial adrenomedullin release and establish the adrenomedullin receptor and subsequent Gs-mediated formation of cAMP as a critical endothelial mechanosignaling pathway regulating basal endothelial NO formation, vascular tone, and blood pressure.
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http://dx.doi.org/10.1172/JCI123825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597232PMC
June 2019