Pieter B. van Loenen

S1P receptor signalling and RGS proteins; expression and function in vascular smooth muscle cells and transfected CHO cells

Sphingosine-1-phosphate (S1P) signalling via G protein-coupled receptors is important for the regulation of cell function and differentiation. Specific Regulators of G protein Signalling (RGS) proteins modulate the function of these receptors in many cell types including vascular smooth muscle cells (VSMCs). Therefore, we investigated the role of altered expression levels of RGS proteins in S1P receptor function in VSMCs and transfected CHO cells. The mRNA expression of the S1P(1) receptor, RGS4 and RGS16 were down-regulated in VSMCs during phenotypic modulation induced by culturing, whereas mRNA levels of RGS2, RGS3, S1P(2) and S1P(3) receptors were unchanged. Interestingly, the expression level of RGS5 was transiently up-regulated. Despite major alterations in RGS levels, S1P-induced calcium elevation in VSMCs was not altered. Co-transfection of RGS2, RGS3, RGS4, RGS5 and RGS16 into CHO-Flp-In cells stably expressing the S1P(1) or S1P(3) receptor did not modify S1P-induced inhibition of cAMP accumulation to a major extent. Similar results were obtained with SEW2871, a selective S1P(1) receptor agonist. However, the inhibition of cAMP accumulation by the agonist FTY720-P via the S1P(1) receptor was significantly decreased by co-transfection with RGS5. These results indicate that mRNA of the S1P(1) receptor, RGS4, RGS5 and RGS16 is differentially regulated during phenotypic modulation. However, major alterations in RGS protein expression have only limited effect on S1P receptor function.

Lack of evidence that nebivolol is a β3-adrenoceptor agonist

Nebivolol is a selective β₁-adrenoceptor antagonist which, in addition, displays endothelium-dependent vasodilating properties in humans and other species. β₃-adrenoceptors have been proposed to be a molecular target of nebivolol-induced vasodilatation. Therefore, we have investigated possible β₃-adrenoceptor agonism by nebivolol for relaxation of the human and rat urinary bladder (prototypical β₃-adrenoceptor-mediated responses) as well as for cAMP accumulation in Chinese hamster ovary cells stably transfected with the human β-adrenoceptor subtypes. Nebivolol concentration-dependently relaxed both human and rat isolated urinary bladder strips but with low potency, similar to that reported for vasodilatation. However, nebivolol-induced bladder relaxation in either species was not inhibited by the β₃-adrenoceptor antagonist SR 59,230A (10μM), although this compound inhibited the isoprenaline-induced relaxation with the expected potency. In radioligand binding studies nebivolol had lower affinity for human β₃-adrenoceptors than the other two β-adrenoceptor subtypes, but this low affinity was in line with its potency to relax the bladder or isolated blood vessels. In functional studies nebivolol even in high concentrations did not stimulate cAMP formation via any of the three cloned human β-adrenoceptors or in rat bladder smooth muscle cells. Taken together these data demonstrate that nebivolol can relax not only vascular but also urinary bladder smooth muscle. However, they do not support the hypothesis that nebivolol is an agonist at cloned human β₃-adrenoceptors or in rat or human urinary bladder.

Sphingosine-1-Phosphate Receptor-1 Controls Venous Endothelial Barrier Integrity in Zebrafish

Objective—Endothelial sphingosine-1-phosphate (S1P) receptor-1 (S1P1) affects different vascular functions, including blood vessel maturation and permeability. Here, we characterized the role of the zS1P1 ortholog in vascular development in zebrafish. Methods and Results—zS1P1 is expressed in dorsal aorta and posterior cardinal vein of zebrafish embryos at 24 to 30 hours postfertilization. zS1P1 downregulation by antisense morpholino oligonucleotide injection causes early pericardial edema, lack of blood circulation, alterations of posterior cardinal vein structure, and late generalized edema. Also, zS1P1 morphants are characterized by downregulation of vascular endothelial cadherin (VE-cadherin) and Eph receptor EphB4a expression and by disorganization of zonula occludens 1 junctions in posterior cardinal vein endothelium, with no alterations of dorsal aorta endothelium. VE-cadherin knockdown results in similar vascular alterations, whereas VE-cadherin overexpression is sufficient to rescue venous vascular integrity defects and EphB4a downregulation in zS1P1 morphants. Finally, S1P1 small interfering RNA transfection and the S1P1 antagonist (R)-3-amino-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W146) cause EPHB4 receptor down-modulation in human umbilical vein endothelial cells and the assembly of zonula occludens 1 intercellular contacts is prevented by the EPHB4 antagonist TNYL-RAW peptide in these cells. Conclusion—The data demonstrate a nonredundant role of zS1P1 in the regulation of venous endothelial barrier in zebrafish and identify a S1P1/VE-cadherin/EphB4a genetic pathway that controls venous vascular integrity.

Sphingosine‐1‐Phosphate Receptor 1 Regulates Cardiac Function by Modulating Ca2+ Sensitivity and Na+/H+ Exchange and Mediates Protection by Ischemic Preconditioning

Background Sphingosine‐1‐phosphate plays vital roles in cardiomyocyte physiology, myocardial ischemia–reperfusion injury, and ischemic preconditioning. The function of the cardiomyocyte sphingosine‐1‐phosphate receptor 1 (S1P1) in vivo is unknown. Methods and Results Cardiomyocyte‐restricted deletion of S1P1 in mice (S1P1 α MHCC re) resulted in progressive cardiomyopathy, compromised response to dobutamine, and premature death. Isolated cardiomyocytes from S1P1 α MHCC re mice revealed reduced diastolic and systolic Ca2+ concentrations that were secondary to reduced intracellular Na+ and caused by suppressed activity of the sarcolemmal Na+/H+ exchanger NHE‐1 in the absence of S1P1. This scenario was successfully reproduced in wild‐type cardiomyocytes by pharmacological inhibition of S1P1 or sphingosine kinases. Furthermore, Sarcomere shortening of S1P1 α MHCC re cardiomyocytes was intact, but sarcomere relaxation was attenuated and Ca2+ sensitivity increased, respectively. This went along with reduced phosphorylation of regulatory myofilament proteins such as myosin light chain 2, myosin‐binding protein C, and troponin I. In addition, S1P1 mediated the inhibitory effect of exogenous sphingosine‐1‐phosphate on β‐adrenergic–induced cardiomyocyte contractility by inhibiting the adenylate cyclase. Furthermore, ischemic precondtioning was abolished in S1P1 α MHCC re mice and was accompanied by defective Akt activation during preconditioning. Conclusions Tonic S1P1 signaling by endogenous sphingosine‐1‐phosphate contributes to intracellular Ca2+ homeostasis by maintaining basal NHE‐1 activity and controls simultaneously myofibril Ca2+ sensitivity through its inhibitory effect on adenylate cyclase. Cardioprotection by ischemic precondtioning depends on intact S1P1 signaling. These key findings on S1P1 functions in cardiac physiology may offer novel therapeutic approaches to cardiac diseases.

Agonist-dependent effects of mutations in the sphingosine-1-phosphate type 1 receptor.

The sphingosine-1-phosphate type 1 (S1P(1)) receptor is a new target in the treatment of auto-immune diseases as evidenced by the recent approval of FTY720 (Fingolimod). The ligand-binding pocket of the S1P(1) receptor has been generally characterised but detailed insight into ligand-specific differences is still lacking. The aim of the current study is to determine differences in ligand-induced S1P(1) receptor activation using an in silico guided site-directed mutagenesis strategy. S1P(1) mutant receptors (modifications of residues Y98(2.57), R120(3.28), F125(3.33)) were probed with a chemically diverse set of S1P(1) agonists (S1P, dihydro-S1P (dhS1P), R-, S- and racemic FTY720-P, VPC24191, SEW2871). Mutation of the R(3.28) residue generally results in a reduction of the potency of all ligands although the synthetic ligands including FTY720-P are less sensitive to these mutations. The Y(2.57)F mutation does not affect the potency of any of the ligands tested, but for all ligands except FTY720-P a significant decrease in potency is observed at the Y(2.57)A mutant. The F(3.33)A mutation significantly decreased the potency of FTY720-P and is detrimental for SEW2871 and VPC24191. The non-aromatic endogenous ligands S1P and dhS1P are less affected by this mutation. Our in silico guided mutagenesis studies identified new molecular determinants of ligand-induced S1P(1) receptor activation: 1) the flexibility of the polar head of the agonist to maintain a tight H-bond network with R(3.28) and 2) the ability of the agonist to make aromatic π-stacking interactions with F(3.33). Interestingly, FTY720-P has both chemical properties and is the only ligand that can efficiently activate the Y(2.57)A mutant.

LIM-Only Protein FHL2 Is a Positive Regulator of Liver X Receptors in Smooth Muscle Cells Involved in Lipid Homeostasis

ABSTRACT The LIM-only protein FHL2 is expressed in smooth muscle cells (SMCs) and inhibits SMC-rich-lesion formation. To further elucidate the role of FHL2 in SMCs, we compared the transcriptomes of SMCs derived from wild-type (WT) and FHL2 knockout (KO) mice. This revealed that in addition to the previously recognized involvement of FHL2 in SMC proliferation, the cholesterol synthesis and liver X receptor (LXR) pathways are altered in the absence of FHL2. Using coimmunoprecipitation experiments, we found that FHL2 interacts with the two LXR isoforms, LXRα and LXRβ. Furthermore, FHL2 strongly enhances transcriptional activity of LXR element (LXRE)-containing reporter constructs. Chromatin immunoprecipitation (ChIP) experiments on the ABCG1 promoter revealed that FHL2 enhances the association of LXRβ with DNA. In line with these observations, we observed reduced basal transcriptional LXR activity in FHL2-KO SMCs compared to WT SMCs. This was also reflected in reduced expression of LXR target genes in intact aorta and aortic SMCs of FHL2-KO mice. Functionally, the absence of FHL2 resulted in attenuated cholesterol efflux to both ApoA-1 and high-density lipoprotein (HDL), in agreement with reduced LXR signaling. Collectively, our findings demonstrate that FHL2 is a transcriptional coactivator of LXRs and points toward FHL2 being an important determinant of cholesterol metabolism in SMCs.

Sphingosine-1-phosphate regulates RGS2 and RGS16 mRNA expression in vascular smooth muscle cells

Regulator of G protein signalling (RGS) protein expression is altered under growth promoting conditions in vascular smooth muscle cells (VSMCs). Since sphingosine-1-phosphate (S1P) is an important growth stimulatory factor, we investigated whether stimulation of VSMCs with S1P results in alterations in mRNA expression levels of several RGS proteins and which signalling components are involved. VSMCs were stimulated with S1P and mRNA expression levels of RGS2, RGS3, RGS4, RGS5 and RGS16 were measured by real-time polymerase chain reaction. S1P caused a time-dependent up-regulation of RGS2 and RGS16 mRNA expression. FTY720-P, a S1P(1)/S1P(3-5) agonist, did not regulate RGS2 mRNA levels although it did up-regulate RGS16 mRNA expression. Pertussis toxin treatment revealed that the S1P-induced RGS16 expression was G(i/o)-dependent whereas up-regulation of RGS2 mRNA was not. Phosphatidylinositol 3-kinase, protein kinase C and mitogen-activated protein kinase kinase apparently were not involved in the S1P-induced up-regulation of both RGS proteins. The present study demonstrates that S1P induces RGS2 and RGS16 mRNA expression but uses distinct S1P receptor subtypes and signalling pathways to regulate expression of these RGS proteins.

Structural and cellular mechanisms of peptidyl-prolyl isomerase Pin1-mediated enhancement of Tissue Factor gene expression, protein half-life, and pro-coagulant activity

Tissue Factor is a cell-surface glycoprotein expressed in various cells of the vasculature and is the principal regulator of the blood coagulation cascade and hemostasis. Notably, aberrant expression of Tissue Factor is associated with cardiovascular pathologies such as atherosclerosis and thrombosis. Here, we sought to identify factors that regulate Tissue Factor gene expression and activity. Tissue Factor gene expression is regulated by various transcription factors, including activating protein-1 and nuclear factor-κ B. The peptidyl-prolyl isomerase Pin1 is known to modulate the activity of these two transcription factors, and we now show that Pin1 augments Tissue Factor gene expression in both vascular smooth muscle cells and activated endothelial cells via activating protein-1 and nuclear factor-κ B signaling. Furthermore, the cytoplasmic domain of Tissue Factor contains a well-conserved phospho-Ser258-Pro259 amino-acid motif recognized by Pin1. Using co-immunoprecipitation and solution nuclear magnetic resonance spectroscopy, we show that the WW-domain of Pin1 directly binds the cytoplasmic domain of Tissue Factor. This interaction occurs via the phospho-Ser258-Pro259 sequence in the Tissue Factor cytoplasmic domain and results in increased protein half-life and pro-coagulant activity. Taken together, our results establish Pin1 as an upstream regulator of Tissue Factor-mediated coagulation, thereby opening up new avenues for research into the use of specific Pin1 inhibitors for the treatment of diseases characterized by pathological coagulation, such as thrombosis and atherosclerosis.

Nuclear Receptor Nur77 Limits the Macrophage Inflammatory Response through Transcriptional Reprogramming of Mitochondrial Metabolism

Summary Activation of macrophages by inflammatory stimuli induces reprogramming of mitochondrial metabolism to support the production of pro-inflammatory cytokines and nitric oxide. Hallmarks of this metabolic rewiring are downregulation of α-ketoglutarate formation by isocitrate dehydrogenase (IDH) and accumulation of glutamine-derived succinate, which enhances the inflammatory response via the activity of succinate dehydrogenase (SDH). Here, we identify the nuclear receptor Nur77 (Nr4a1) as a key upstream transcriptional regulator of this pro-inflammatory metabolic switch in macrophages. Nur77-deficient macrophages fail to downregulate IDH expression and accumulate higher levels of succinate and other TCA cycle-derived metabolites in response to inflammatory stimulation in a glutamine-independent manner. Consequently, these macrophages produce more nitric oxide and pro-inflammatory cytokines in an SDH-dependent manner. In vivo, bone marrow Nur77 deficiency exacerbates atherosclerosis development and leads to increased circulating succinate levels. In summary, Nur77 induces an anti-inflammatory metabolic state in macrophages that protects against chronic inflammatory diseases such as atherosclerosis.

Nur77 protects against adverse cardiac remodelling by limiting neuropeptide Y signalling in the sympathoadrenal-cardiac axis

Aims Cardiac remodelling and heart failure are promoted by persistent sympathetic activity. We recently reported that nuclear receptor Nur77 may protect against sympathetic agonist-induced cardiac remodelling in mice. The sympathetic co-transmitter neuropeptide Y (NPY) is co-released with catecholamines and is a known cardiac modulator and predictor of heart failure mortality. Recently, transcriptome analyses revealed NPY as a putative target of Nur77. In this study, we assess whether Nur77 modulates adverse cardiac remodelling via NPY signalling. Methods and results Nur77 represses NPY expression in the PC12 adrenal chromaffin cell line. Accordingly, NPY levels are higher in adrenal gland, plasma, and heart from Nur77-KO compared to wild-type mice. Conditioned medium from Nur77-silenced chromaffin cells and serum from Nur77-KO mice induce marked hypertrophy in cultured neonatal rat cardiomyocytes, which is inhibited by the NPY type 1 receptor (NPY1R) antagonist BIBO3304. In cardiomyocytes from Nur77-KO mice, intracellular Ca2+ is increased partially via the NPY1R. This is independent from elevated circulating NPY since cardiomyocyte-specific Nur77-deficient mice (CM-KO) do not have elevated circulating NPY, but do exhibit BIBO3304-sensitive, increased cardiomyocyte intracellular Ca2+. In vivo, this translates to NPY1R antagonism attenuating cardiac calcineurin activity and isoproterenol-induced cardiomyocyte hypertrophy and fibrosis in full-body Nur77-KO mice, but not in CM-KO mice. Conclusions The cardioprotective action of Nur77 can be ascribed to both inhibition of circulating NPY levels and to cardiomyocyte-specific modulation of NPY-NPY1R signalling. These results reveal the underlying mechanism of Nur77 as a promising modifier gene in heart failure.