Par-4 Expression Research Articles

Platelet tissue factor pathway inhibitor-α dampens cardiac thrombosis and associated fibrosis in mice

BackgroundTissue factor pathway inhibitor (TFPI) is the primary inhibitor of events initiating the blood coagulation pathway. Tfpi−/− mice die during embryonic development. The absence of protease–activated receptor (PAR) 4, the major thrombin receptor on mouse platelets, rescues Tfpi−/−mice to adulthood. Among the 3 TFPI isoforms in mice, TFPIα is the only isoform within platelets (pltTFPIα) and the only isoform that inhibits prothrombinase, the enzymatic complex that converts prothrombin to thrombin. ObjectivesTo determine biological functions of pltTFPIα. MethodsTfpi−/−/Par4−/− mice were irradiated and transplanted with bone marrow from mice lacking or containing pltTFPIα. Thus, PAR4 expression was restored in the recipient mice, which differed selectively by the presence or absence of pltTFPIα and lacked other forms of TFPI. ResultsRecipient mice lacking pltTFPIα had reduced survival over the 200-day posttransplant period. Necropsy revealed radiation injury associated with large intraventricular platelet-rich thrombi, whereas other organs were not affected. Thrombi were associated with fibrotic presentations, including increased collagen deposition, periostin–positive activated fibroblasts, myofibroblasts, and macrophage infiltrates. Recipient mice containing pltTFPIα showed evidence of radiation injury but lacked heart pathology. ConclusionsTfpi−/−/Par4−/− mice develop severe cardiac fibrosis following irradiation and transplantation with bone marrow lacking pltTFPIα. This pathology is markedly reduced when the mice are transplanted with bone marrow containing pltTFPIα. Thus, in this model system pltTFPIα has an important physiological role in dampening pathological responses mediated by activated platelets within the heart tissue.

Blood-nerve barrier disruption and coagulation system activation induced by mechanical compression injury participate in the peripheral sensitization of trigeminal neuralgia.

The aim of this study was to investigate the effect and possible mechanisms of the blood-nerve barrier (BNB) and the coagulation-anticoagulation system in modulating the mechanical allodynia in a trigeminal neuralgia (TN) rat model induced by chronic compression of the trigeminal root entry zone (TREZ). Von Frey filaments were applied to determine the orofacial mechanical allodynia threshold. The BNB permeability was evaluated by Evans blue extravasation test. Immunohistochemical staining and laser confocal microscopy were used to measure the length of the depletion zones of the nodes of Ranvier in the TREZ, the diameter of nerve fibers and the length of the nodal gap. The transcriptional levels of prothrombin and endogenous thrombin inhibitor protease nexin-1 (PN-1) in the TREZ of TN rats were assessed by RT-qPCR. A Western blotting assay was performed to detect the expression of paranodal proteins neurofascin-155 (NF155) and neurofascin-125 (NF125) in the TREZ. The spatiotemporal expression pattern of thrombin activated receptor (i.e. protease activated receptor 1, PAR1) in TREZ were defined by immunostaining and immunoblotting assays. PAR1 receptor inhibitors SCH79797 were administrated to TN rats to analyze the effect of thrombin-PAR1 on orofacial hyperalgesia. A compression injury of a rat's TREZ successfully induced TN-like behavior and was accompanied by the destruction of the permeability of the BNB and the promotion of prothrombin and thrombin inhibitor protease nexin-1 (PN-1) expression. The expression of the paranodal proteins neurofascin-155 (NF155) and neurofascin-125 (NF125) was increased, while the nodal gap length of the nodes of Ranvier was widened and the length of node-depleted zones was shortened. Moreover, the expression of PAR1 within the TREZ was upregulated at an early stage of TN, and administration of the PAR1 antagonist SCH79797 effectively ameliorated orofacial mechanical allodynia. A compression injury of the TREZ increased the permeability of the BNB and induced disturbances in the local coagulation-anticoagulation system, concomitant with the structural changes in the nodes of Ranvier, thrombin-PAR1 may play a critical role in modulating orofacial mechanical hyperalgesia in a TN rat model.

Aspirin Inhibits the Inflammatory Response of Protease-Activated Receptor 1 in Pregnancy Neutrophils: Implications for Treating Women with Preeclampsia.

Neutrophils expressing cyclooxygenase-2 (COX-2) extensively infiltrate maternal blood vessels in preeclampsia, associated with vascular inflammation. Because pregnancy neutrophils also express protease-activated receptor 1 (PAR-1, F2R thrombin receptor), which they do not in non-pregnant subjects, they can be activated by proteases. We tested the hypothesis that aspirin at a dose sufficient to inhibit COX-2 would reduce inflammatory responses in preeclampsia neutrophils. Neutrophils were isolated from normal pregnant and preeclamptic women at approximately 30 weeks' gestation. Normal pregnancy neutrophils were treated with elastase, a protease elevated in preeclampsia, or elastase plus aspirin to inhibit COX-2, or elastase plus pinane thromboxane, a biologically active structural analog of thromboxane and a thromboxane synthase inhibitor. Preeclamptic pregnancy neutrophils were treated with the same doses of aspirin or pinane thromboxane. Confocal microscopy with immunofluorescence staining was used to determine the cellular localization of the p65 subunit of nuclear factor-kappa B (NF-κB) and media concentrations of thromboxane were measured to evaluate the inflammatory response. In untreated neutrophils of normal pregnant women, p65 was localized to the cytosol. Upon stimulation with elastase, p65 translocated from the cytosol to the nucleus coincident with increased thromboxane production. When neutrophils were co-treated with aspirin or pinane thromboxane, elastase was not able to cause nuclear translocation of p65 or increase thromboxane. In untreated neutrophils of preeclamptic women, the p65 subunit was present in the nucleus and thromboxane production was elevated, but when preeclamptic neutrophils were treated with aspirin or pinane thromboxane, p65 was cleared from the nucleus and returned to the cytosol along with decreased thromboxane production. These findings suggest that COX-2 is a downstream mediator of PAR-1 and demonstrate that PAR-1- mediated inflammation can be inhibited by aspirin. Given the extensive and ubiquitous expression of PAR-1 and COX-2 in preeclamptic women, consideration should be given to treating women with preeclampsia using a dose of aspirin sufficient to inhibit COX-2.

Trypsin may be associated with duodenal eosinophils through the expression of PAR2 in early chronic pancreatitis and functional dyspepsia with pancreatic enzyme abnormalities.

Early chronic pancreatitis (ECP) has been reported to advance into chronic pancreatitis, it may be critical to differentiate the pathophysiology of ECP and functional dyspepsia (FD) in patients with pancreatic enzyme abnormalities (FD-P). This study aimed to clarify differences in the pathophysiology of ECP and FD-P and to determine whether duodenal inflammatory responses in the two diseases were associated with protease-activated receptor (PAR) 2, as the trypsin receptor. Eighty patients who presented with FD-P and ECP were enrolled. In duodenal specimens, PAR2 mRNA levels were determined using real-time PCR. Using immunostaining, CD68-, GLP-1-, PRG2-, and CCR2-positive cells, tight junction proteins, and PAR 2 were evaluated. There were no significant differences in clinical symptoms and gastric motility between ECP and FD-P patients. The CD68-positive cells infiltrations and occludin expression levels in the duodenal mucosa of patients with FD-P were significantly (p<0.001 and p = 0.048, respectively) lower than those in patients with ECP. Although serum trypsin levels in ECP and FD-P patents were significantly (p<0.05 and p<0.001, respectively) associated with duodenal eosinophils counts, elevated trypsin levels were not significantly associated with degranulated eosinophils, occludin, claudin-1 and ZO-1 expression levels in the duodenum of either group. PAR2 mRNA levels were increased in the duodenum of patients with ECP and FD-P. PAR2 was localized in the epithelial cells of the duodenal mucosa and the surface of degranulated eosinophils in ECP and FD-P patients. Elevated trypsin levels might be partly associated with duodenal inflammatory responses through PAR2-related degranulated eosinophils and the reduction of occludin in patients with ECP and FD-P.

IGF2BP3 promotes progression of gallbladder carcinoma by stabilizing KLK5 mRNA in N6-methyladenosine-dependent binding.

BackgroundRecent studies have reported that IGF2BP3 is linked to the pathogenesis of various malignancies. Since IGF2BP3 is associated with poor outcomes of gallbladder carcinoma (GBC), we aimed to explore the association between its N6-methyladenosine (m6A) RNA methylation and GBC progression.MethodsBioinformatic analysis of GSE136982, GSE104165, and RNA-seq was performed. In vitro and in vivo gain- and loss-of-function assays were done. qPCR, Western blotting, and IHC were conducted in cells or in collected clinical tissue samples. RNA immunoprecipitation, RNA stability measurement, methylated RNA immunoprecipitation, and dual-luciferase reporter assays were performed in this study.ResultsThe expression of IGF2BP3 was higher in GBC tissues than in peritumoral tissues. Functions such as cell proliferation and migration, both in vitro and in vivo, were inhibited by downregulation of IGF2BP3. The analysis of RNA-seq indicated that KLK5 was a downstream target of IGF2BP3. The expression of KLK5 was measured in GBC cells and tumor samples. It was found to be positively correlated with IGF2BP3 level. Upon IGF2BP3 depletion, ectopic expression of KLK5 could rescue cell function in part. Mechanistically, we found that IGF2BP3 directly binds to KLK5 mRNA and regulates its stability in an m6A-dependent manner. As a result, inhibition of KLK5 decreased the expression of PAR2, and deregulated phospho-Akt. Using bioinformatic prediction combined with miRNA microarray analysis, we identified that let-7g-5p is an inhibitor of IGF2BP3, and let-7g-5p expression was negatively correlated with IGF2BP3. Overexpression of let-7g-5p affected the aggressive phenotype of GBC cells by deregulating IGF2BP3, and inhibiting the KLK5/PAR2/AKT axis.ConclusionsOur data showed that IGF2BP3 is associated with the aggressive phenotype of GBC. Mechanistically, IGF2BP3 activated the PAR2/AKT axis by stabilizing KLK5 mRNA in an m6A-dependent manner. The loss of let-7g-5p enhanced the expression of IGF2BP3 and improved GBC progression. Thus, IGF2BP3 plays a crucial role in GBC, and the let-7g-5p/IGF2BP3/KLK5/PAR2 axis may be a therapeutic target for GBC.

Proteinase-activated receptors regulate intestinal functions in a segment-dependent manner in rats

Proteinases released e.g. during inflammatory or allergic responses affect gastrointestinal functions via proteinase-activated receptors such as PAR1 and PAR2. As the gastrointestinal tract exerts pronounced gradients along its longitudinal axis, the present study focuses on the effect of PAR1 and PAR2 agonists on electrogenic ion transport (measured as short-circuit current; Isc), tissue conductance (Gt) and contractility of the longitudinal muscle layer of rats. In Ussing chamber experiments, the PAR1 agonist TFLLR-NH2, which mimics the tethered ligand liberated after cleavage of the receptor, evoked only a modest increase in Isc (<0.5 μEq·h−1·cm−2) in small intestine, but a strong increase (3–4 μEq·h−1·cm−2) in colon. Pretreatment with tetrodotoxin reduced the response of the colonic segments to the level of the small intestine. Thrombin, the natural activator of PAR1, was much less effective suggesting biased activation by this peptidase. A similar gradient along the longitudinal axis of the intestine was observed with trypsin, the endogenous activator of PAR2. Divergent actions of PAR1 activation by enzymatic cleavage or a mimetic peptide were also observed when recording isometric contractions of longitudinal muscle. For example, in the jejunum TFLLR-NH2 concentration-dependently induced a contractile response, whereas thrombin showed only inconsistent effects. The PAR2 activator AC264613 induced a concentration-dependent decrease in muscle tone combined with an inhibition of phasic spontaneous contractions. PCR experiments and immunohistochemical stainings confirmed the expression of PAR1 and PAR2. The data implies that PAR1 and PAR2 functions vary depending on the intestinal segment.

Anti-histamine effects of dipotassium glycyrrhizinate on lung fibroblasts, implicating its therapeutic mechanism for pulmonary fibrosis.

To examine the possible anti-histamine effects of dipotassium glycyrrhizinate (DG), a dipotassium salt of glycyrrhizic acid, on histamine-mediated lung fibroblast activation, differentiation and proliferation; to investigate the potential and underlying mechanisms for pulmonary fibrosis (PF) treatment. Rat primary lung fibroblasts were extracted to establish cell models; histamine, DG and loratadine (LTD, a histamine receptor antagonist) were applied. Cell proliferation, migration and cell cycle were explored; intracellular signal proteins were detected; mitochondrial membrane potential was examined. The anti-histamine effects of DG were found in a similar pattern of LTD on lung fibroblasts. DG inhibited histamine-induced cell activation, proliferation and migration; DG altered histamine-mediated mitochondrial membrane potentials. DG reduced the histamine-induced PAR-2 (a tryptase receptor) expression to impair mast cell tryptase co-working. Histamine-induced expressions of MMP-2, FAK, TNF-α, P38, iNOS were decreased by DG, while Bax and caspase-3, P53 were increased by DG against histamine effects. Histamine drove cells from G0/G1 to S phases, whereas DG rested cells by inhibiting G0/G1 and G2/M phases. This study provided the evidences that DG can inhibit histamine-induced effects on lung fibroblasts and promote apoptosis of abnormally activated lung fibroblasts, implicating its potential therapeutic mechanisms against PF development, also for those histamine-related diseases.

Effects of salvianolic acid A and salvianolic acid B in renal interstitial fibrosis via PDGF-C/PDGFR-α signaling pathway

BackgroundRenal interstitial fibrosis (RIF) is the main pathological feature of end-stage renal disease (ESRD) caused by various chronic kidney diseases (CKD), and is closely related to renal dysfunction and patient prognosis. Salvianolic acid A (Sal A) and salvianolic acid B (Sal B), isolated from traditional Chinese medicine Salviae miltiorrhizae, have been confirmed to have anti-fibrotic effects on liver, cardiac and kidney. However, the precise molecular mechanism underlying the nephroprotective effects of Sal A and Sal B, and whether there is a difference between the two in RIF are still unclear. PurposeThis study investigated the pharmacological effects of Sal A and Sal B in RIF and explore the underlying mechanisms by in vivo and in vitro experiments. MethodsThe nephroprotective effects of Sal A, Sal B and Sal A+B were evaluated by assessing the parameters related to kidney function such as renal histology, renal function, urinary protein NAG, urinary β2 microglobulin. In addition, RIF-related markers such as CTCF and Par3 were also detected. Thereafter, the related protein or gene levels of PDGF-C/PDGFR-α signaling pathways, apoptosis and endoplasmic reticulum stress (ERS) were determined by western blot, real-time PCR, flow cytometry or immunofluorescence staining. ResultsIn vivo, the results showed that Sal A, Sal B and Sal A+B partially improved kidney dysfunction, increased the expression of Par-3 and reduced the expression of CTGF, PDGF-C and PDGFR-α. In vitro, the results also showed that Sal A, Sal B and Sal A+B reversed apoptosis and ERS in HSA-induced HK-2 cells via regulating PDGF-C/PDGFR-α signaling pathway. ConclusionThis article revealed a novel mechanism linking PDGF-C/PDGFR-α signaling pathway to RIF and suggested that Sal A, Sal B and Sal A+B were considered as potential therapeutic agents for the amelioration of RIF.

Protease-activated receptor-2: Role in asthma pathogenesis and utility as a biomarker of disease severity.

PAR2, a receptor activated by serine proteases, has primarily pro-inflammatory roles in the airways and may play a role in asthma pathogenesis. PAR2 exerts its effects in the lungs through activation of a variety of airway cells, but also activation of circulating immune cells. There is evidence that PAR2 expression increases in asthma and other inflammatory diseases, although the regulation of PAR2 expression is not fully understood. Here we review the available literature on the potential role of PAR2 in asthma pathogenesis and propose a model of PAR2-mediated development of allergic sensitization. We also propose, based on our previous work, that PAR2 expression on peripheral blood monocyte subsets has the potential to serve as a biomarker of asthma severity and/or control.

A novel pathogenetic factor of laryngeal attack in hereditary angioedema? Involvement of protease activated receptor 1

BackgroundHereditary angioedema (HAE) is a rare, life-threatening disease. The knowledge about the molecular pathogenesis of HAE has derived mainly from investigating blood samples. However, limited data are available on the role of the molecular mechanisms in the affected tissues during HAE attack.ObjectiveThe aim of our study was to explore the histological changes occurring in HAE attacks.MethodsPost mortem macro-, microscopic and immunohistological assessment of upper airway tissues of a patient with HAE due to C1 inhibitor deficiency (C1-INH-HAE) type 2 who died from laryngeal HAE attack was compared with a non-HAE patient who died from other condition without any signs of angioedema.ResultsCompared to the control patient, we demonstrated stronger T cell/monocyte infiltration and a more intense C1-INH staining in the C1-INH-HAE patient. The expression of both bradykinin receptors (B1/B2) was observed with a slightly lower level in the C1-INH-HAE patient than in the control patient. PAR1 expression was strongly reduced in the C1-INH-HAE patient suggesting overactivation of this hyperpermeability inducing receptor.ConclusionOur unique case and novel results correspond to the knowledge about C1-INH and BDKRs observed in plasma; however, it revealed new information about the pathomechanism of HAE attack focusing on the potential involvement of PAR1 in edema formation. This observation, if it is verified by subcutaneous biopsy studies, may designate a new therapeutic target in HAE.

Study on the antipruritic mechanism of Zanthoxylum bungeanum and Zanthoxylum schinifolium volatile oil on chronic eczema based on H1R and PAR-2 mediated GRPR pathway.

To observe the antipruritic effect and mechanism of the volatile oil of Zanthoxylum bungeanum and Zanthoxylum schinifolium on chronic eczema to provide data support for clinical application and new drug development of Zanthoxylum bungeanum and Zanthoxylum schinifolium. The model of chronic eczema was established by using 2-dinitrochlorobenzene (DNCB), and the composition and content of volatile oil in Zanthoxylum schinifolium and Zanthoxylum bungeanum was determined by gas chromatography-mass spectrometry (GC-MS). The antipruritic effect by (EASI) score of eczema area and severity index and scratching times was then evaluated. Then, the contents of histamine, gastrin-releasing peptide (GRP), interleukin-4 (IL-4), and immunoglobulin E (IgE) in serum of rats was determined by enzyme-linked immunosorbent assay (ELISA). The tissue morphology was observed by HE staining. The expressions of H1R, PAR-2, TRPV1, TRPA1, and GRPR was then detected by immunohistochemistry, Western blot, and QRT-PCR. The results revealed that there were differences in the composition of volatile oil between Zanthoxylum bungeanum and Zanthoxylum schinifolium. Compared to the model group, the medium-dose group of Zanthoxylum bungeanum and Zanthoxylum schinifolium group significantly increased the difference of EASI score and scratching times, significantly decreased the concentrations of IL-4, IgE, GRP, and histamine, and significantly decreased the expression levels of H1R, PAR-2, TRPV1, and GRPR. The degree of inhibition on the pathological manifestations of chronic eczema was evident. There was no significant difference in antipruritic effect between the two groups. The expression of TRPA1 was inconsistent at the protein and gene level, which needs to be further researched. The volatile oil of Zanthoxylum bungeanum and Zanthoxylum schinifolium can reduce the expression of H1R, PAR-2, TRPV1, and GRPR. The mechanism may be through the H1R and PAR-2-mediated GRPR pathway intervention to achieve the effect, both of which have the same antipruritic effect.

Candida albicans Sap6 Initiates Oral Mucosal Inflammation via the Protease Activated Receptor PAR2.

Candida albicans Sap6, a secreted aspartyl protease (Sap), contributes to fungal virulence in oral candidiasis. Beside its protease activity, Sap6 contains RGD (RGDRGD) motif required for its binding to host integrins. Sap6 activates immune cells to induce proinflammatory cytokines, although its ability to interact and activate human oral epithelial cells (OECs) remain unknown. Addition of purified recombinant Sap6 (rSap6) to OECs resulted in production of IL-1β and IL-8 cytokines similar to live hyphal C. albicans. OECs exposed to rSap6 showed phosphorylation of p38 and MKP1 and expression of c-Fos not found with C. albicans Δsap6, heat-inactivated Sap6, or rSap6ΔRGD . Heat inactivated rSap6 was able to induce IL-1β but not IL-8 in OECs, while rSap6ΔRGD induced IL-8 but not IL-1β suggesting parallel signaling pathways. C. albicans hyphae increased surface expression of Protease Activated Receptors PAR1, PAR2 and PAR3, while rSap6 increased PAR2 expression exclusively. Pretreatment of OECs with a PAR2 antagonist blocked rSap6-induced p38 MAPK signaling and IL-8 release, while rSap6ΔRGD had reduced MKP1 signaling and IL-1β release independent from PAR2. OECs exposed to rSap6 exhibited loss of barrier function as measured by TEER and reduction in levels of E-cadherin and occludin junctional proteins that was prevented by pretreating OECs with a PAR2 antagonist. OECs treated with PAR2 antagonist also showed reduced rSap6-mediated invasion by C. albicans cells. Thus, Sap6 may initiate OEC responses mediated both through protease activation of PAR2 and by its RGD domain. This novel role of PAR2 suggests new drug targets to block C. albicans oral infection.

Protease‐Activated Receptor‐2 Differentially Regulates Vascular Smooth Muscle Cell Proliferation in Cyclic AMP‐Dependent Protein Kinase/Phosphoinositide 3‐Kinase‐Dependent Manner

Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality worldwide, despite decades of extensive research. One key facet in CVD pathogenesis is the phenotypic transition of affected vascular smooth muscle (VSM) from a quiescent, contractile state to a synthetic, proliferative state. Protease‐activated receptors (PARs) are of emerging interest in CVD; however, their potential impact on VSM phenotypic switching inherent in CVD is unclear. The overall objective of the current study was to determine the role of PAR2 and PAR4 in phenotypic switching and VSM growth. We hypothesized that PAR2 and PAR4 increase following arterial injury in vivo and after growth stimulation in vitro, that they serve to protect against VSM cell growth, and that they signal primarily through specific protein kinases in phenotype‐specific fashion. In rat carotid arteries, PAR2 expression and activity increased following mechanical distension injury compared to uninjured controls. In rat primary VSM cells, PAR2 expression also increased in response to growth stimulation. Differential expression of cyclic GMP‐dependent protein kinase PKG‐1 and the PKG target VASP distinguished low passage (P3‐P6) from high P (P10‐P15) VSM cells, with low P cells showing elevated PKG‐1, phosphorylated (Ser239) VASP and total VASP compared to high P cells. Pharmacologic PAR2 agonism promoted DNA synthesis and proliferation in low P cells but decreased DNA synthesis and proliferation in high P cells. Western blotting analyses showed increased expression of MEK1/2 MAPK, PKA, and PI3K in low P cells versus high P cells. Specific protein kinase blockade revealed that PAR2 differentially controls proliferation of phenotypically distinct low versus high P VSM cells predominantly through PKA and PI3K. Our findings highlight potential (patho)physiological relevance of PAR2 and PAR2‐driven PKA/PI3K in the control of VSM cell growth and provide evidence for continued study of VSM PAR2 in CVD.

Platelet CFTR inhibition enhances arterial thrombosis via increasing intracellular Cl- concentration and activation of SGK1 signaling pathway.

Platelet hyperactivity is essential for thrombus formation in coronary artery diseases (CAD). Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) in patients with cystic fibrosis elevates intracellular Cl- levels ([Cl-]i) and enhanced platelet hyperactivity. In this study, we explored whether alteration of [Cl-]i has a pathological role in regulating platelet hyperactivity and arterial thrombosis formation. CFTR expression was significantly decreased, while [Cl-]i was increased in platelets from CAD patients. In a FeCl3-induced mouse mesenteric arteriole thrombosis model, platelet-specific Cftr-knockout and/or pre-administration of ion channel inhibitor CFTRinh-172 increased platelet [Cl-]i, which accelerated thrombus formation, enhanced platelet aggregation and ATP release, and increased P2Y12 and PAR4 expression in platelets. Conversely, Cftr-overexpressing platelets resulted in subnormal [Cl-]i, thereby decreasing thrombosis formation. Our results showed that clamping [Cl-]i at high levels or Cftr deficiency-induced [Cl-]i increasement dramatically augmented phosphorylation (Ser422) of serum and glucocorticoid-regulated kinase (SGK1), subsequently upregulated P2Y12 and PAR4 expression via NF-κB signaling. Constitutively active mutant S422D SGK1 markedly increased P2Y12 and PAR4 expression. The specific SGK1 inhibitor GSK-650394 decreased platelet aggregation in wildtype and platelet-specific Cftr knockout mice, and platelet SGK1 phosphorylation was observed in line with increased [Cl-]i and decreased CFTR expression in CAD patients. Co-transfection of S422D SGK1 and adenovirus-induced CFTR overexpression in MEG-01 cells restored platelet activation signaling cascade. Our results suggest that [Cl-]i is a novel positive regulator of platelet activation and arterial thrombus formation via the activation of a [Cl-]i-sensitive SGK1 signaling pathway. Therefore, [Cl-]i in platelets is a novel potential biomarker for platelet hyperactivity, and CFTR may be a potential therapeutic target for platelet activation in CAD.

The Outer Membrane Lipoprotein Tp0136 Stimulates Human Platelet Activation and Aggregation Through PAR1 to Enhance Gq/Gi Signaling.

BackgroundChancre self-healing, a typical clinical phenomenon of primary syphilis, is essentially wound healing. The first response to a wound is constriction of the injured blood vessels and activation of platelets to form a fibrin clot. However, the role of Treponema pallidum in platelet activation and clot formation remains unclear.ObjectivesWe aimed to elucidate the role of the outer membrane Treponema pallidum lipoprotein Tp0136 in human platelet activation and aggregation and explore the related mechanism.MethodsA series of experiments were performed to assess the effects of Tp0136 on human platelet activation and aggregation in vitro. The effect of Tp0136 on platelet receptors was studied by detecting PAR1 protein levels and studying related receptor sites. The involvement of the Gq/Gi signaling pathway downstream of PAR1 was explored.ResultsTp0136 significantly accelerated the formation of human platelet clots as well as platelet adhesion to and diffusion on fibrinogen to promote platelet aggregation. Tp0136 also potentiated P-selectin expression and PF4 release to promote platelet activation and downregulated PAR1 expression. The activation and aggregation induced by Tp0136 were reverted by the specific PAR1 antagonist RWJ56110 and the human PAR1 antibody. In addition, Tp0136 significantly enhanced Gq and Gi signaling activation, thereby triggering p38 phosphorylation and Akt-PI3K activation, increasing the release of intraplatelet Ca2+ and attenuating the release of cytosolic cAMP. Furthermore, the specific PAR1 antagonist RWJ56110 significantly suppressed Gq and Gi signaling activation.ConclusionsOur results showed that the Treponema pallidum Tp0136 protein stimulated human platelet activation and aggregation by downregulating PAR1 and triggered PAR1-dependent Gq and Gi pathway activation. These findings may contribute to our understanding of the self-healing of chancroid in early syphilis.

Polarity protein Par3 sensitizes breast cancer to paclitaxel by promoting cell cycle arrest.

Paclitaxel, belongs to tubulin-binding agents (TBAs), shows a great efficacy against breast cancer via stabilizing microtubules. Drug resistance limits its clinical application. Here we aimed to explore a role of Polarity protein Par3 in improving paclitaxel effectiveness. Breast cancer specimens from 45 patients were collected to study the relationship between Par3 expression and paclitaxel efficacy. The Kaplan-Meier method was used for survival analysis. Cell viability was measured in breast cancer cells (SK-BR-3 and T-47D) with Par3 over-expression or knockdown. The flow cytometry assays were performed to measure cell apoptosis and cell cycle. BrdU incorporation assay and Hoechst 33,258 staining were performed to measure cell proliferation and cell apoptosis, respectively. Immunofluorescence was used to detect microtubule structures. Par3 expression was associated with good response of paclitaxel in breast cancer patients. Consistently, Par3 over-expression significantly sensitized breast cancer cells to paclitaxel by promoting cell apoptosis and reducing cell proliferation. In Par3 overexpressing cells upon paclitaxel treatment, we observed intensified cell cycle arrests at metaphase. Further exploration showed that Par3 over-expression stabilized microtubules of breast cancer cells in response to paclitaxel and resists to microtubules instability induced by nocodazole, a microtubule-depolymerizing agent. Par3 facilitates polymeric forms of tubulin and stabilizes microtubule structure, which aggravates paclitaxel-induced delay at the metaphase-anaphase transition, leading to proliferation inhibition and apoptosis of breast cancer cells. Par3 has a potential role in sensitizing breast cancer cells to paclitaxel, which may provide a more precise assessment of individual treatment and novel therapeutic targets.

Activated Protein C Protects against Murine Contact Dermatitis by Suppressing Protease-Activated Receptor 2.

Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with excessive inflammation and defective skin barrier function. Activated protein C (APC) is a natural anticoagulant with anti-inflammatory and barrier protective functions. However, the effect of APC on AD and its engagement with protease activated receptor (PAR)1 and PAR2 are unknown. Methods: Contact hypersensitivity (CHS), a model for human AD, was induced in PAR1 knockout (KO), PAR2KO and matched wild type (WT) mice using 2,4-dinitrofluorobenzene (DNFB). Recombinant human APC was administered into these mice as preventative or therapeutic treatment. The effect of APC and PAR1KO or PARKO on CHS was assessed via measurement of ear thickness, skin histologic changes, inflammatory cytokine levels, Th cell phenotypes and keratinocyte function. Results: Compared to WT, PAR2KO but not PAR1KO mice displayed less severe CHS when assessed by ear thickness; PAR1KO CHS skin had less mast cells, lower levels of IFN-γ, IL-4, IL-17 and IL-22, and higher levels of IL-1β, IL-6 and TGF-β1, whereas PAR2KO CHS skin only contained lower levels of IL-22 and IgE. Both PAR1KO and PAR2KO spleen cells had less Th1/Th17/Th22/Treg cells. In normal skin, PAR1 was present at the stratum granulosum and spinosum, whereas PAR2 at the upper layers of the epidermis. In CHS, however, the expression of PAR1 and PAR2 were increased and spread to the whole epidermis. In vitro, compared to WT cells, PAR1KO keratinocytes grew much slower, had a lower survival rate and higher para permeability, while PAR2KO cells grew faster, were resistant to apoptosis and para permeability. APC inhibited CHS as a therapeutic but not as a preventative treatment only in WT and PAR1KO mice. APC therapy reduced skin inflammation, suppressed epidermal PAR2 expression, promoted keratinocyte growth, survival, and barrier function in both WT and PAR1KO cells, but not in PAR2KO cells. Conclusions: APC therapy can mitigate CHS. Although APC acts through both PAR1 and PAR2 to regulate Th and mast cells, suppression of clinical disease in mice is achieved mainly via inhibition of PAR2 alone. Thus, APC may confer broad therapeutic benefits as a disease-modifying treatment for AD.

Altered Expression of Par3, aPKC-λ, and Lgl1 in Hippocampus in Kainic Acid-Induced Status Epilepticus Rat Model

Introduction: Mossy fiber sprouting (MFS) is a frequent histopathological finding in temporal lobe epilepsy (TLE) and is involved in the pathology of TLE. However, molecular signals underlying MFS remain unclear. Partitioning defective 3(Par3), atypical protein kinase C-λ(aPKC-λ), and lethal giant larvae 1(Lgl1) were involved in the neuronal polarity and axon growth. The potential roles of those proteins in MFS and epileptogenesis of TLE were investigated.Material and Methods: The epileptic rat models were established by intracerebroventricular injection of kainic acid (KA). The degree of MFS was measured by using Timm staining, Neuronal loss and the expression aPKC-λ, Par3, and Lgl1 in hippocampus were measured by using immunohistochemistry and western blot analysis.Results: The neuronal loss in CA3 region was observed from 3 days to 8 weeks, while the neuronal loss in the hilar region was observed from 1 to 8 weeks in experimental group. The Timm score in the CA3 region in experimental group was significantly higher than that in the control group from 2 to 8 weeks. Compared with control group, the expressions of Par3 and Lgl1 were upregulated and the expression of aPKC-λ was downregulated in the experimental groups. Positive correlation between the Par3 expression and Timm scores, and the negative correlation between the aPKC-λ expression and Timm scores in CA3 region were discovered in experimental group.Conclusion: The findings of the present study indicated that aPKC-λ, Par3, and Lgl1 may be involved in MFS and in the epileptogenesis of temporal lobe epilepsy.

Electroacupuncture Regulates TRPV1 through PAR2/PKC Pathway to Alleviate Visceral Hypersensitivity in FD Rats.

Visceral hypersensitivity (VH) is the predominant pathogenesis of functional dyspepsia (FD). Duodenal hypersensitivity along with nausea further reduces the comfort level in gastric balloon dilatation and inhibits gastric receptive relaxation. The potential mechanism behind electroacupuncture- (EA-) mediated alleviation of VH has not been elucidated. In an FD rat model with tail clamping stress, iodine acetamide (IA) induced VH. The rats were treated with EA with or without PAR2 antagonist FSLLRY-NH2, and the body weight, gastric sensitivity, compliance, and gastrointestinal motility were determined. Mast cells and activated degranulation were stained with toluidine blue (TB) staining and visualized under a transmission electron microscope (TEM). Immunofluorescence was used to detect the expression of PAR2, PKC, and TRPV1 in the duodenum and dorsal root ganglion (DRG) and that of CGRP, SP in DRG, and c-fos in the spinal cord. EA alone and EA + antagonist enhanced the gastrointestinal motility but diminished the expression of TRPV1, CGRP, SP, and c-fos-downstream of PAR2/PKC pathway and alleviated VH in FD rats. However, there was no obvious superposition effect between the antagonists and EA + antagonists. The effect of EA alone was better than that of antagonists and EA + antagonists 2 alone. EA-induced amelioration of VH in FD rats was mediated by TRPV1 regulation through PAR2/PKC pathway. This protective mechanism involved several pathways and included several targets.

Ameloblastin promotes polarization of ameloblast cell lines in a 3-D cell culture system

Studies on animal models with mutations in ameloblastin gene have suggested that the extracellular matrix protein ameloblastin (AMBN) plays important roles in controlling cell-matrix adhesion and ameloblast polarization during amelogenesis. In order to examine the function of AMBN in cell polarization and morphology, we developed an in vitro 3D cell culture model to examine the effect of AMBN and amelogenin (AMEL) addition on ameloblast cell lines. We further used high resolution confocal microscopy to detect expression of polarization markers in response to AMBN addition. Addition of AMBN to the 3D culture matrix resulted in the clustering and elongation (higher aspect ratio) of ALC in a dose dependent manner. The molar concentration of AMEL required to exact this response from ALC was 2.75- times greater than that of AMBN. This polarization effect of ameloblastin was attributable directly to an evolutionary conserved domain within its exon 5-encoded region. The lack of exon 6-encoded region also influenced AMBN-cell interactions but to a lesser extent. The clusters formed with AMBN were polarized with expression of E-cadherin, Par3 and Cldn1 assembly at the nascent cell-cell junctions. The elongation effect was specific to epithelial cells of ameloblastic lineage ALC and LS8 cells. Our data suggest that AMBN may play critical signaling roles in the initiation of cell polarity by acting as a communicator between cell-cell and cell-matrix interactions. Our investigation has important implications for understanding the function of ameloblastin in enamel-cell matrix adhesion and the outcomes may contribute to efforts to develop strategies for enamel tissue regeneration.