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Plexins are widely expressed transmembrane proteins that mediate the cellular effects of semaphorins. The molecular mechanisms of plexin-mediated signal transduction are still poorly understood. Here we show that signalling via B-family plexins leading to the activation of the small GTPase RhoA requires activation of the IκB kinase (IKK)-complex. In contrast, plexin-B-dependent regulation of R-Ras activity is not affected by IKK activity. This regulation of plexin signalling depends on the kinase activity of the IKK-complex, but is independent of NF-κB activation. We confirm that the IKK-complex is active in tumour cells and osteoblasts, and we demonstrate that plexin-B-dependent tumour cell invasiveness and regulation of osteoblast differentiation require an active IKK-complex. This study identifies a novel, NF-κB-independent function of the IKK-complex and shows that IKK directs plexin-B signalling to the activation of RhoA.
Tubulogenesis is essential for the formation and function of internal organs. One such organ is the trachea, which allows gas exchange between the external environment and the lungs. However, the cellular and molecular mechanisms underlying tracheal tube development remain poorly understood. Here, we show that the potassium channel KCNJ13 is a critical modulator of tracheal tubulogenesis. We identify Kcnj13 in an ethylnitrosourea forward genetic screen for regulators of mouse respiratory organ development. Kcnj13 mutants exhibit a shorter trachea as well as defective smooth muscle (SM) cell alignment and polarity. KCNJ13 is essential to maintain ion homeostasis in tracheal SM cells, which is required for actin polymerization. This process appears to be mediated, at least in part, through activation of the actin regulator AKT, as pharmacological increase of AKT phosphorylation ameliorates the Kcnj13-mutant trachea phenotypes. These results provide insight into the role of ion homeostasis in cytoskeletal organization during tubulogenesis.
Activation of TRPC6 channels is essential for lung ischaemia–reperfusion induced oedema in mice
(2012)
Lung ischaemia–reperfusion-induced oedema (LIRE) is a life-threatening condition that causes pulmonary oedema induced by endothelial dysfunction. Here we show that lungs from mice lacking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2y/−) or the classical transient receptor potential channel 6 (TRPC6−/−) are protected from LIR-induced oedema (LIRE). Generation of chimeric mice by bone marrow cell transplantation and endothelial-specific Nox2 deletion showed that endothelial Nox2, but not leukocytic Nox2 or TRPC6, are responsible for LIRE. Lung endothelial cells from Nox2- or TRPC6-deficient mice showed attenuated ischaemia-induced Ca2+ influx, cellular shape changes and impaired barrier function. Production of reactive oxygen species was completely abolished in Nox2y/− cells. A novel mechanistic model comprising endothelial Nox2-derived production of superoxide, activation of phospholipase C-γ, inhibition of diacylglycerol (DAG) kinase, DAG-mediated activation of TRPC6 and ensuing LIRE is supported by pharmacological and molecular evidence. This mechanism highlights novel pharmacological targets for the treatment of LIRE.
19(S)-hydroxy-eicosatetraenoic acid (19(S)-HETE) belongs to a family of arachidonic acid metabolites produced by cytochrome P450 enzymes, which play critical roles in the regulation of cardiovascular, renal and pulmonary functions. Although it has been known for a long time that 19(S)-HETE has vascular effects, its mechanism of action has remained unclear. In this study we show that 19(S)-HETE induces cAMP accumulation in the human megakaryoblastic leukemia cell line MEG-01. This effect was concentration-dependent with an EC50 of 520 nM, insensitive to pharmacological inhibition of COX-1/2 and required the expression of the G-protein Gs. Systematic siRNA-mediated knock-down of each G-protein coupled receptor (GPCR) expressed in MEG-01 followed by functional analysis identified the prostacyclin receptor (IP) as the mediator of the effects of 19(S)-HETE, and the heterologously expressed IP receptor was also activated by 19(S)-HETE in a concentration-dependent manner with an EC50 of 567 nM. Pretreatment of isolated murine platelets with 19(S)-HETE blocked thrombin-induced platelets aggregation, an effect not seen in platelets from mice lacking the IP receptor. Furthermore, 19(S)-HETE was able to relax mouse mesenteric artery- and thoracic aorta-derived vessel segments. While pharmacological inhibition of COX-1/2 enzymes had no effect on the vasodilatory activity of 19(S)-HETE these effects were not observed in vessels from mice lacking the IP receptor. These results identify a novel mechanism of action for the CYP450-dependent arachidonic acid metabolite 19(S)-HETE and point to the existence of a broader spectrum of naturally occurring prostanoid receptor agonists.
The long-chain fatty acid receptor FFAR1 is highly expressed in pancreatic β-cells. Synthetic FFAR1 agonists can be used as antidiabetic drugs to promote glucose-stimulated insulin secretion (GSIS). However, the physiological role of FFAR1 in β-cells remains poorly understood. Here we show that 20-HETE activates FFAR1 and promotes GSIS via FFAR1 with higher potency and efficacy than dietary fatty acids such as palmitic, linoleic, and α-linolenic acid. Murine and human β-cells produce 20-HETE, and the ω-hydroxylase-mediated formation and release of 20-HETE is strongly stimulated by glucose. Pharmacological inhibition of 20-HETE formation and blockade of FFAR1 in islets inhibits GSIS. In islets from type-2 diabetic humans and mice, glucose-stimulated 20-HETE formation and 20-HETE-dependent stimulation of GSIS are strongly reduced. We show that 20-HETE is an FFAR1 agonist, which functions as an autocrine positive feed-forward regulator of GSIS, and that a reduced glucose-induced 20-HETE formation contributes to inefficient GSIS in type-2 diabetes.
Dysregulation of lysophosphatidic acids in multiple sclerosis and autoimmune encephalomyelitis
(2017)
Bioactive lipids contribute to the pathophysiology of multiple sclerosis. Here, we show that lysophosphatidic acids (LPAs) are dysregulated in multiple sclerosis (MS) and are functionally relevant in this disease. LPAs and autotaxin, the major enzyme producing extracellular LPAs, were analyzed in serum and cerebrospinal fluid in a cross-sectional population of MS patients and were compared with respective data from mice in the experimental autoimmune encephalomyelitis (EAE) model, spontaneous EAE in TCR1640 mice, and EAE in Lpar2 -/- mice. Serum LPAs were reduced in MS and EAE whereas spinal cord LPAs in TCR1640 mice increased during the ‘symptom-free’ intervals, i.e. on resolution of inflammation during recovery hence possibly pointing to positive effects of brain LPAs during remyelination as suggested in previous studies. Peripheral LPAs mildly re-raised during relapses but further dropped in refractory relapses. The peripheral loss led to a redistribution of immune cells from the spleen to the spinal cord, suggesting defects of lymphocyte homing. In support, LPAR2 positive T-cells were reduced in EAE and the disease was intensified in Lpar2 deficient mice. Further, treatment with an LPAR2 agonist reduced clinical signs of relapsing-remitting EAE suggesting that the LPAR2 agonist partially compensated the endogenous loss of LPAs and implicating LPA signaling as a novel treatment approach.
Formation of specialized pro-resolving lipid mediators (SPMs) such as lipoxins or resolvins usually involves arachidonic acid 5-lipoxygenase (5-LO, ALOX5) and different types of arachidonic acid 12- and 15-lipoxygenating paralogues (15-LO1, ALOX15; 15-LO2, ALOX15B; 12-LO, ALOX12). Typically, SPMs are thought to be formed via consecutive steps of oxidation of polyenoic fatty acids such as arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid. One hallmark of SPM formation is that reported levels of these lipid mediators are much lower than typical pro-inflammatory mediators including the monohydroxylated fatty acid derivatives (e.g., 5-HETE), leukotrienes or certain cyclooxygenase-derived prostaglandins. Thus, reliable detection and quantification of these metabolites is challenging. This paper is aimed at critically evaluating i) the proposed biosynthetic pathways of SPM formation, ii) the current knowledge on SPM receptors and their signaling cascades and iii) the analytical methods used to quantify these pro-resolving mediators in the context of their instability and their low concentrations. Based on current literature it can be concluded that i) there is at most, a low biosynthetic capacity for SPMs in human leukocytes. ii) The identity and the signaling of the proposed G-protein-coupled SPM receptors have not been supported by studies in knock-out mice and remain to be validated. iii) In humans, SPM levels were neither related to dietary supplementation with their ω-3 polyunsaturated fatty acid precursors nor were they formed during the resolution phase of an evoked inflammatory response. iv) The reported low SPM levels cannot be reliably quantified by means of the most commonly reported methodology. Overall, these questions regarding formation, signaling and occurrence of SPMs challenge their role as endogenous mediators of the resolution of inflammation.
Vascular biotransformation of organic nitrates is independent of cytochrome P450 monooxygenases
(2021)
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.
GPR116 (ADGRF5) and ELTD1 (ADGRL4) belong to different subfamilies of the adhesion G-protein-coupled receptor group but are both expressed in endothelial cells. We therefore analyzed their functions in mice lacking these receptors. While loss of GPR116 or ELTD1 alone had no obvious effect on cardiovascular or kidney function, mice lacking both, GPR116 and ELTD1, showed malformations of the aortic arch arteries and the cardiac outflow tract leading to perinatal lethality in about 50% of the mutants. In addition to cardiovascular malformations, surviving mice developed renal thrombotic microangiopathy as well as hemolysis and splenomegaly, and their lifespan was significantly reduced. Loss of GPR116 and ELTD1 specifically in endothelial cells or neural crest-derived cells did not recapitulate any of the phenotypes observed in GPR116-ELTD1 double deficient mice, indicating that loss of GPR116 and ELTD1 expressed by other cells accounts for the observed cardiovascular and renal defects.
The mouse bitter taste receptors Tas2r143, Tas2r135, and Tas2r126 are encoded by genes that cluster on chromosome 6 and have been suggested to be expressed under common regulatory elements. Previous studies indicated that the Tas2r143/Tas2r135/Tas2r126 cluster is expressed in the heart, but other organs had not been systematically analyzed. In order to investigate the expression of this bitter taste receptor gene cluster in non-gustatory tissues, we generated a BAC (bacterial artificial chromosome) based transgenic mouse line, expressing CreERT2 under the control of the Tas2r143 promoter. After crossing this line with a mouse line expressing EGFP after Cre-mediated recombination, we were able to validate the Tas2r143-CreERT2 transgenic mouse line and monitor the expression of Tas2r143. EGFP-positive cells, indicating expression of members of the cluster, were found in about 47% of taste buds, and could also be found in several other organs. A population of EGFP-positive cells was identified in thymic epithelial cells, in the lamina propria of the intestine and in vascular smooth muscle cells of cardiac blood vessels. EGFP-positive cells were also identified in the epithelium of organs readily exposed to pathogens including lower airways, the gastrointestinal tract, urethra, vagina, and cervix. With respect to the function of cells expressing this bitter taste receptor cluster, RNA-seq analysis in EGFP-positive cells isolated from the epithelium of trachea and stomach showed expression of genes related to innate immunity. These data further support the concept that bitter taste receptors serve functions outside the gustatory system.