Protease-Activated Receptor 4 Induces Bladder Pain through High Mobility Group Box-1

Pain is the significant presenting symptom in Interstitial Cystitis/Painful Bladder Syndrome (IC/PBS). Activation of urothelial protease activated receptor 4 (PAR4) causes pain through release of urothelial macrophage migration inhibitory factor (MIF). High Mobility Group Box-1 (HMGB1), a chromatin-binding protein, mediates bladder pain (but not inflammation) in an experimental model (cyclophosphamide) of cystitis. To determine if PAR4-induced bladder hypersensitivity depends on HMGB1 downstream, we tested whether: 1) bladder PAR4 stimulation affected urothelial HMGB1 release; 2) blocking MIF inhibited urothelial HMGB1 release; and 3) blocking HMGB1 prevented PAR4-induced bladder hypersensitivity. HMGB1 release was examined in immortalized human urothelial cultures (UROtsa) exposed to PAR4-activating peptide (PAR4-AP; 100 μM; 2 hours) or scrambled control peptide. Female C57BL/6 mice, pretreated with a HMGB1 inhibitor (glycyrrhizin: 50 mg/kg; ip) or vehicle, received intravesical PAR4-AP or a control peptide (100 μM; 1 hour) to determine 1) HMGB1 levels at 1 hour in the intravesical fluid (released HMGB1) and urothelium, and 2) abdominal hypersensitivity to von Frey filament stimulation 24 hours later. We also tested mice pretreated with a MIF blocker (ISO-1: 20 mg/kg; ip) to determine whether MIF mediated PAR4-induced urothelial HMGB1 release. PAR4-AP triggered HMGB1 release from human (in vitro) and mice (in vivo) urothelial cells. Intravesical PAR4 activation elicited abdominal hypersensitivity in mice that was prevented by blocking HMGB1. MIF inhibition prevented PAR4-mediated HMGB1 release from mouse urothelium. Urothelial MIF and HGMB1 represent novel targets for therapeutic intervention in bladder pain conditions.     

Expression of Protease-Activated Receptor 1 and 2 and Anti-Tubulogenic Activity of Protease-Activated Receptor 1 in Human Endothelial Colony-Forming Cells

Endothelial colony-forming cells (ECFCs) are obtained from the culture of human peripheral blood mononuclear cell (hPBMNC) fractions and are characterised by high proliferative and pro-vasculogenic potential, which makes them of great interest for cell therapy. Here, we describe the detection of protease-activated receptor (PAR) 1 and 2 amongst the surface proteins expressed in ECFCs. Both receptors are functionally coupled to extracellular signal-regulated kinase (ERK) 1 and 2, which become activated and phosphorylated in response to selective PAR1- or PAR2-activating peptides. Specific stimulation of PAR1, but not PAR2, significantly inhibits capillary-like tube formation by ECFCs in vitro, suggesting that tubulogenesis is negatively regulated by proteases able to stimulate PAR1 (e.g. thrombin). The activation of ERKs is not involved in the regulation of tubulogenesis in vitro, as suggested by use of the MEK inhibitor PD98059 and by the fact that PAR2 stimulation activates ERKs without affecting capillary tube formation. Both qPCR and immunoblotting showed a significant downregulation of vascular endothelial growth factor 2 (VEGFR2) in response to PAR1 stimulation. Moreover, the addition of VEGF (50–100 ng/ml) but not basic Fibroblast Growth Factor (FGF) (25–100 ng/ml) rescued tube formation by ECFCs treated with PAR1-activating peptide. Therefore, we propose that reduction of VEGF responsiveness resulting from down-regulation of VEGFR2 is underlying the anti-tubulogenic effect of PAR1 activation. Although the role of PAR2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade.     

Protease-Activated Receptor 2 Activation Inhibits N-Type Ca2+ Currents in Rat Peripheral Sympathetic Neurons

The protease-activated receptor (PAR)-2 is highly expressed in endothelial cells and vascular smooth muscle cells. It plays a crucial role in regulating blood pressure via the modulation of peripheral vascular tone. Although several mechanisms have been suggested to explain PAR-2-induced hypotension, the precise mechanism remains to be elucidated. To investigate this possibility, we investigated the effects of PAR-2 activation on N-type Ca²⁺ currents (I_Ca-N) in isolated neurons of the celiac ganglion (CG), which is involved in the sympathetic regulation of mesenteric artery vascular tone. PAR-2 agonists irreversibly diminished voltage-gated Ca²⁺ currents (I_Ca), measured using the patch-clamp method, in rat CG neurons, whereas thrombin had little effect on I_Ca. This PAR-2-induced inhibition was almost completely prevented by ω-CgTx, a potent N-type Ca²⁺ channel blocker, suggesting the involvement of N-type Ca²⁺ channels in PAR-2-induced inhibition. In addition, PAR-2 agonists inhibited I_Ca–N in a voltage-independent manner in rat CG neurons. Moreover, PAR-2 agonists reduced action potential (AP) firing frequency as measured using the current-clamp method in rat CG neurons. This inhibition of AP firing induced by PAR-2 agonists was almost completely prevented by ω-CgTx, indicating that PAR-2 activation may regulate the membrane excitability of peripheral sympathetic neurons through modulation of N-type Ca²⁺ channels. In conclusion, the present findings demonstrate that the activation of PAR-2 suppresses peripheral sympathetic outflow by modulating N-type Ca²⁺ channel activity, which appears to be involved in PAR-2-induced hypotension, in peripheral sympathetic nerve terminals.     

颗粒酶A诱导Caspases非依赖性细胞死亡

颗粒酶A(granzyme A,GzmA),是存在于细胞毒性T淋巴细胞(CTL)和天然杀伤细胞(NK细胞)的细胞毒颗粒中含量最多的一种丝氨酸蛋白酶,在穿孔素(perforin)协同作用下通过颗粒胞吐(granule exocytosis)释放进入在杀伤细胞和靶细胞之间形成的免疫突触(immunological synapse),然后进入靶细胞的细胞浆,并在细胞核聚集,诱导一种caspases非依赖性细胞死亡。GzmA靶向作用于一种与内质网结合的特殊的复合体——SET复合体,其包含3种GzmA底物：核小体装配蛋白SET、DNA结合蛋白HMG—2、具有碱基切除修复作用的核酸内切酶Apel。SET复合体还含有一种抑癌蛋白pp32和一种具有脱氧核糖核酸酶(DNase)活性的NM23—H1。当GzmA作用于SET复合体时释放出NM23—H1并激活其DNase活性,也阻断了Apel对DNA损伤的修复作用,在DNA上形成单链的缺刻。这是一种新发现的由GzmA诱导的细胞凋亡途径。     

Translocation of Exogenous FGF1 and FGF2 Protects the Cell against Apoptosis Independently of Receptor Activation

FGF1 and FGF2 bind to specific cell-surface tyrosine kinase receptors (FGFRs) and activate intracellular signaling that leads to proliferation, migration or differentiation of many cell types. Besides this classical mode of action, under stress conditions, FGF1 and FGF2 are translocated in a receptor-dependent manner via the endosomal membrane into the cytosol and nucleus of the cell. However, despite many years of research, the role of translocated FGF1 and FGF2 inside the cell remains unclear. Here, we reveal an anti-apoptotic activity of intracellular FGF1 and FGF2, which is independent of FGFR activation and downstream signaling. We observed an inhibition of cell apoptosis induced by serum starvation or staurosporine upon treatment with exogenous FGF1 or FGF2, despite the presence of highly potent FGFR inhibitors. Similar results were found when the tyrosine kinase of FGFR1 was completely blocked by a specific mutation. Moreover, the anti-apoptotic effect of the growth factors was abolished by known inhibitors of the translocation of FGF1 and FGF2 from the endosomes to the interior of the cell. Interestingly, FGF2 showed higher anti-apoptotic activity than FGF1. Since FGF2 is not phosphorylated by PKCδ and is present inside the nucleus longer than is FGF1, we speculated that the different activities could reflect their diverse nuclear export kinetics. Indeed, we observed that FGF1 mutations preventing binding to nucleolin and therefore phosphorylation in the nucleus affect the anti-apoptotic activity of FGF1. Taken together, our data indicate that the translocation of FGF1 and FGF2 protects cells against apoptosis and promotes cell survival.     

Ouabain Releases Striatal Polyamines In Vivo Independently of N-Methyl-d-Aspartate Receptor Activation

Abstract: Intrastriatally infused ouabain (200 or 1,000 μ M ) markedly increased the extracellular levels of striatal spermidine and spermine in dialysis experiments in halothane-anesthetized rats. The effects of ouabain (1 m M ) on sper- midine release were rapid and unaffected by local infusion of the competitive N -methyl- d -aspartate (NMDA) antagonist 3-(2-carboxypiperazin-4-yl)propyl-1 -phosphonic acid (CPP; 100 μ M ) or by systemically administered MK-801 (0.3 mg/kg i.p.), both of which treatments markedly inhibit the effects of intrastriatally administered NMDA. The peak effects of ouabain (1 m M ) on spermine release were delayed with respect to those on spermidine release, or to the effects of NMDA, and were also insensitive to locally administered CPP (100 μ M ). However, systemically administered MK-801 (0.3 mg/kg i.p., 30 min before the striatal infusion of drugs), which totally inhibits the effects of NMDA, or CPP (10 mg/kg i.p.; 30 min before the striatal infusion of drugs) partially inhibited the effects of ouabain on spermine release, suggesting partial mediation of the delayed effects of ouabain on spermine release by indirect NMDA-receptor activation. Despite partial sensitivity of ouabain-induced spermine release to systemically administered NMDA antagonists, both spermidine and spermine can be released in vivo by sodium-pump inhibition, independently of NMDA-receptor activation.     

Murine Toll-Like Receptor 2 Activation Induces Type I Interferon Responses from Endolysosomal Compartments

Background

Toll-like receptors (TLRs) are among the first-line sentinels for immune detection and responsiveness to pathogens. The TLR2 subfamily of TLRs (TLR1, TLR2, TLR6) form heterodimers with each other and are thus able to recognize a broad range of components from several microbes such as yeast, Gram-positive bacteria and protozoa. Until now, TLR2 activation by bacterial ligands has long been associated with pro-inflammatory cytokines but not type I interferon responses.

Methodology/Principal Findings

Using a variety of transgenic mice, here we provide in vivo and in vitro data showing that TLR2 activation does in fact induce interferon-beta and that this occurs via MyD88-IRF1 and -IRF7 pathways. Interestingly, by microscopy we demonstrate that although a cell surface receptor, TLR2 dependent induction of type I interferons occurs in endolysosomal compartments where it is translocated to upon ligand engagement. Furthermore, we could show that blocking receptor internalization or endolysosomal acidification inhibits the ability of TLR2 to trigger the induction type I interferon but not pro-inflammatory responses.

Conclusion/Significance

The results indicate that TLR2 activation induces pro-inflammatory and type I interferon responses from distinct subcellular sites: the plasma membrane and endolysosomal compartments respectively. Apart from identifying and characterizing a novel pathway for induction of type I interferons, the present study offers new insights into how TLR signaling discriminates and regulates the nature of responses to be elicited against extracellular and endocytosed microbes. These findings may also have clinical implication. Excessive production of pro-inflammatory cytokines and type I IFNs following activation of TLRs is a central pathologic event in several hyper-inflammatory conditions. The discovery that the induction of pro-inflammatory and type I IFN responses can be uncoupled through pharmacological manipulation of endolysosomal acidification suggests new avenues for potential therapeutic intervention against inflammations and sepsis.     

Conformational Plasticity of Human Protease-Activated Receptor 1 upon Antagonist- and Agonist-Binding

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Okadaic Acid Induces Hyperphosphorylation of τ Independently of Mitogen-Activated Protein Kinase Activation

Abstract: Hyperphosphorylation of the microtubule-associated protein τ is a characteristic of Alzheimer brain tissue. Recent in vitro data suggest that mitogen-activated protein kinase (MAPK), a proline-directed protein kinase, phosphorylates the sites on τ common to Alzheimer's disease. Using an okadaic acid-induced τ hyperphosphorylation model, we have tested the requirement for MAPK activity, using a specific inhibitor {PD098059 [2-(2'-amino-3'-methoxyphenyl)oxanaphthalen-4-one]} of the MAPK activator Mek1. Mobility shift, phosphoepitope analysis, and direct measurement of kinase activity indicated that the Mek1 inhibitor dose-dependently blocked basal and okadaic acid-induced MAPK activation. Despite a block of MAPK activation by this inhibitor, robust τ hyperphosphorylation was observed in response to okadaic acid. In addition, activation of MAPK by phorbol 12-myristate 13-acetate did not result in τ phosphorylation, indicating that in primary cultures of cortical neurons elevated MAPK activity is not sufficient to induce τ hyperphosphorylation.     

Pharmacological Targeting of Protease-Activated Receptor 2 Affords Protection from Bleomycin-Induced Pulmonary Fibrosis

Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease that remains refractory to therapy. Despite increasing evidence that protease-activated receptor 2 (PAR-2) contributes to fibrosis, its importance in pulmonary fibrosis is under debate. We addressed whether PAR-2 deficiency persistently reduces bleomycin-induced pulmonary fibrosis or merely delays disease progression and whether pharmacological PAR-2 inhibition limits experimental pulmonary fibrosis. Bleomycin was instilled intranasally into wild-type or PAR-2–deficient mice in the presence/absence of a specific PAR-2 antagonist (P2pal-18S). Pulmonary fibrosis was consistently reduced in PAR-2–deficient mice throughout the fibrotic phase, as evident from reduced Ashcroft scores (29%) and hydroxyproline levels (26%) at d 28. Moreover, P2pal-18S inhibited PAR-2–induced profibrotic responses in both murine and primary human pulmonary fibroblasts (p < 0.05). Once daily treatment with P2pal-18S reduced the severity and extent of fibrotic lesions in lungs of bleomycin-treated wild-type mice but did not further reduce fibrosis in PAR-2–deficient mice. Importantly, P2pal-18S treatment starting even 7 d after the onset of fibrosis limits pulmonary fibrosis as effectively as when treatment was started together with bleomycin instillation. Overall, PAR-2 contributes to the progression of pulmonary fibrosis, and targeting PAR-2 may be a promising therapeutic strategy for treating pulmonary fibrosis.     

Low Energy Shock Wave Therapy Induces Angiogenesis in Acute Hind-Limb Ischemia via VEGF Receptor 2 Phosphorylation

Objectives

Low energy shock waves have been shown to induce angiogenesis, improve left ventricular ejection fraction and decrease angina symptoms in patients suffering from chronic ischemic heart disease. Whether there is as well an effect in acute ischemia was not yet investigated.

Methods

Hind-limb ischemia was induced in 10–12 weeks old male C57/Bl6 wild-type mice by excision of the left femoral artery. Animals were randomly divided in a treatment group (SWT, 300 shock waves at 0.1 mJ/mm², 5 Hz) and untreated controls (CTR), n = 10 per group. The treatment group received shock wave therapy immediately after surgery.

Results

Higher gene expression and protein levels of angiogenic factors VEGF-A and PlGF, as well as their receptors Flt-1 and KDR have been found. This resulted in significantly more vessels per high-power field in SWT compared to controls. Improvement of blood perfusion in treatment animals was confirmed by laser Doppler perfusion imaging. Receptor tyrosine kinase profiler revealed significant phosphorylation of VEGF receptor 2 as an underlying mechanism of action. The effect of VEGF signaling was abolished upon incubation with a VEGFR2 inhibitor indicating that the effect is indeed VEGFR 2 dependent.

Conclusions

Low energy shock wave treatment induces angiogenesis in acute ischemia via VEGF receptor 2 stimulation and shows the same promising effects as known from chronic myocardial ischemia. It may therefore develop as an adjunct to the treatment armentarium of acute muscle ischemia in limbs and myocardium.     

Attenuated Vasodilator Effectiveness of Protease-Activated Receptor 2 Agonist in Heterozygous par2 Knockout Mice

Studies of homozygous PAR2 gene knockout mice have described a mix of phenotypic effects in vitro and in vivo. However, there have been few studies of PAR2 heterozygous (wild-type/knockout; PAR2-HET) mice. The phenotypes of many hemi and heterozygous transgenic mice have been described as intermediates between those of wild-type and knockout animals. In our study we aimed to determine the effects of intermediary par2 gene zygosity on vascular tissue responses to PAR2 activation. Specifically, we compared the vasodilator effectiveness of the PAR2 activating peptide 2-furoyl-LIGRLO-amide in aortas of wild-type PAR2 homozygous (PAR2-WT) and PAR2-HET mice. In myographs under isometric tension conditions, isolated aortic rings were contracted by alpha 1-adrenoeceptor agonist (phenylephrine), and thromboxane receptor agonist (U46619) and then relaxation responses by the additions of 2-furoyl-LIGRLO-amide, acetylcholine, and nitroprusside were recorded. A Schild regression analysis of the inhibition by a PAR2 antagonist (GB-83) of PAR2 agonist-induced aortic ring relaxations was used to compare receptor expression in PAR2-WT to PAR2-HET. PAR2 mRNA in aortas was measured by quantitative real-time PCR. In aortas contracted by either phenylephrine or U46619, the maximum relaxations induced by 2-furoyl-LIGRLO-amide were less in PAR2-HET than in the gender-matched PAR2-WT. GB-83 was 3- to 4-fold more potent for inhibition of 2fly in PAR2-HET than in PAR2-WT. PAR2 mRNA content of aortas from PAR2-HET was not significantly different than in PAR2-WT. Acetylcholine- and nitroprusside-induced relaxations of aortas from PAR2-HET were not significantly different than in PAR2-WT and PAR2 knockout. An interesting secondary finding was that relaxations induced by agonists of PAR2 and muscarinic receptors were larger in females than in males. We conclude that the lower PAR2-mediated responses in PAR2-HET aortas are consistent with evidence of a lower quantity of functional receptor expression, despite the apparently normal PAR2 mRNA content in PAR2-HET aortas.     

Thyrsiferol Inhibits Mitochondrial Respiration and HIF-1 Activation

The cytotoxic marine red algal metabolite thyrsiferol (1) was found to inhibit hypoxia-induced hypoxia-inducible factor-1 (HIF-1) activation in T47D human breast tumor cells (66% inhibition at 3 μM). Compound 1 also suppressed hypoxic induction of HIF-1 target genes (VEGF, GLUT-1) at the mRNA level, and displayed tumor cell line-selective time-dependent inhibition of cell viability/proliferation. Mechanistic studies revealed that 1 selectively suppressed mitochondrial respiration at Complex I (IC(50) 3 μM). Thyrsiferol represents a prototypical, structurally unique electron transport chain inhibitor. The apparent rotenone-like activity may contribute to the observed cytotoxicity of 1 and play an important role in Laurencia chemical defense.     

Developmental Role of Zebrafish Protease-Activated Receptor 1 (PAR1) in the Cardio-Vascular System

Thrombin receptor, F2R or PAR1 is a G-protein coupled receptor, located in the membrane of endothelial cells. It has been initially found to transduce signals in hemostasis, but recently also known to act in cancer and in vascular development. Mouse embryos lacking PAR1 function die from hemorrhages with varying frequency at midgestation. We have performed a survey of potential PAR1 homologs in the zebrafish genome and identified a teleost ortholog of mammalian PAR1. Knockdown of par1 function in zebrafish embryos demonstrates a requirement for Par1 in cardio-vascular development. Furthermore, we show that function of Par1 requires the presence of a phylogenetically conserved proteolytic cleavage site and a second intracellular domain. Altogether our results demonstrate a high degree of conservation of PAR1 proteins in the vertebrate lineage in respect to amino acid sequence as well as protein function.