Role Of Protease-activated Receptor-2 Research Articles

Coagulation Protease-Driven Cancer Immune Evasion: Potential Targets for Cancer Immunotherapy.

Blood coagulation and cancer are intrinsically connected, hypercoagulation-associated thrombotic complications are commonly observed in certain types of cancer, often leading to decreased survival in cancer patients. Apart from the common role in coagulation, coagulation proteases often trigger intracellular signaling in various cancers via the activation of a G protein-coupled receptor superfamily protease: protease-activated receptors (PARs). Although the role of PARs is well-established in the development and progression of certain types of cancer, their impact on cancer immune response is only just emerging. The present review highlights how coagulation protease-driven PAR signaling plays a key role in modulating innate and adaptive immune responses. This is followed by a detailed discussion on the contribution of coagulation protease-induced signaling in cancer immune evasion, thereby supporting the growth and development of certain tumors. A special section of the review demonstrates the role of coagulation proteases, thrombin, factor VIIa, and factor Xa in cancer immune evasion. Targeting coagulation protease-induced signaling might be a potential therapeutic strategy to boost the immune surveillance mechanism of a host fighting against cancer, thereby augmenting the clinical consequences of targeted immunotherapeutic regimens.

Role of PAR1 -506 deletion/insertion polymorphism in primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a rare cholestatic liver disease characterized by inflammation of the intra- and extrahepatic bile ducts. Pathogenesis of PSC is still enigmatic but is likely to be multifactorial. Recently, we identified an interleukin-6 (IL-6)-dependent signal transducer and activator of transcription 3 (STAT3) activation in CD4+ TH1 and TH17 cells in PSC. The IL-6/STAT3 pathway was shown to be regulated by protease-activated receptor 1 (PAR1) contributing to inflammation. The role of the PAR1 -506 deletion/insertion (Del/Ins) polymorphism in PSC has not yet been investigated. Two hundred eighty four PSC patients (200 patients with inflammatory bowel diseases [IBD] and 84 without IBD) and 309 healthy controls were genotyped for PAR1 rs11267092 (-506 Del/Ins -13bp). Results were correlated with clinical characteristics and transplant-free survival. The frequency of PAR1 -506 Ins allele carriers (Del/Ins and Ins/Ins) was significantly higher in PSC patients (57.0%) compared to healthy controls (39.8%). Furthermore, carriers of PAR1 -506 Ins allele were more likely to have PSC than noncarriers (odds ratio 2.01; 95% confidence interval, 1.45-2.79). Patients with PSC carrying the PAR1 -506 Ins allele showed significantly higher alanine aminotransferase serum levels (p=0.0357) and a trend toward shorter transplant-free survival time compared to noncarriers (8.9±6.6years vs. 10.5±7.1years; p=0.076). Our study shows that PAR1 -506 Ins is significantly more frequent in people with PSC. As PAR1 -506 Ins allele carriers tended to have a shorter transplant-free survival, PAR1 might play a role in the development and course of PSC.

Role of protease-activated receptor 4 in mouse models of acute and chronic kidney injury.

Protease-activated receptor 4 (PAR4) is a G protein-coupled receptor activated by thrombin. In the platelet, response to thrombin PAR4 contributes to the predominant procoagulant microparticle formation, increased fibrin deposition, and initiation of platelet-stimulated inflammation. In addition, PAR4 is expressed in other cell types, including endothelial cells. Under inflammatory conditions, PAR4 is overexpressed via epigenetic demethylation of the PAR4 gene, F2RL3. PAR4 knockout (KO) studies have determined a role for PAR4 in ischemia-reperfusion injury in the brain, and PAR4 KO mice display normal cardiac function but present less myocyte death and cardiac dysfunction in response to acute myocardial infarction. Although PAR4 has been reported to be expressed within the kidney, the contribution of PAR4 to acute kidney injury (AKI) and chronic kidney disease (CKD) is not well understood. Here we report that PAR4 KO mice are protected against kidney injury in two mouse models. First, PAR4 KO mice are protected against induction of markers of both fibrosis and inflammation in two different models of kidney injury: 1) 7 days following unilateral ureter obstruction (UUO) and 2) an AKI-CKD model of ischemia-reperfusion followed by 8 days of contralateral nephrectomy. We further show that PAR4 expression in the kidney is low in the control mouse kidney but induced over time following UUO. PAR4 KO mice are protected against blood urea nitrogen (BUN) and glomerular filtration rate (GFR) kidney function pathologies in the AKI-CKD model. Following the AKI-CKD model, PAR4 is expressed in the collecting duct colocalizing with Dolichos biflorus agglutinin (DBA), but not in the proximal tubule with Lotus tetragonolobus lectin (LTL). Collectively, the results reported in this study implicate PAR4 as contributing to the pathology in mouse models of acute and chronic kidney injury.NEW & NOTEWORTHY The contribution of the thrombin receptor protease-activated receptor 4 (PAR4) to acute kidney injury (AKI) and chronic kidney disease (CKD) is not well understood. Here we report that PAR4 expression is upregulated after kidney injury and PAR4 knockout (KO) mice are protected against fibrosis following kidney injury in two mouse models. First, PAR4 KO mice are protected against unilateral ureter obstruction. Second, PAR4 KO mice are protected against an AKI-CKD model of ischemia-reperfusion followed by contralateral nephrectomy.

Role of protease-activated receptor-1 in the inflammatory response in a coculture model of pig endothelial cells and human monocytes

Role of protease-activated receptor-1 in the inflammatory response in a coculture model of pig endothelial cells and human monocytes

Bcl-xL mediates interferon-beta secretion by protease-activated receptor 2 deficiency through the mitochondrial permeability transition pore in colorectal cancer metastasis

Cellular plasticity and immune escape are synergistic drivers of tumor colonization in metastatic organs. Activation of protease-activated receptor 2 (PAR2) signaling promotes metastasis of colorectal carcinoma (CRC). The role of PAR2 in regulating the immune microenvironment and cancer progression remains unclear. We demonstrated that the regulation of liver metastasis by PAR2 requires a competent immune system. PAR2 knockdown enhanced liver infiltration of activated CD8+ T cells prior to metastatic foci formation in an interferon receptor-dependent manner. PAR2 depletion increased interferon (IFN)-β production via the cGAS-STING and RIG-1 pathways. PAR2 inhibition increased mitochondrial permeability and cytosolic accumulation of mitochondrial DNA, which was reversed by Bcl-xL expression. Strikingly, shRNA against PAR2 with an immune checkpoint blocker (ICB) acted synergistically to suppress liver metastasis. Analysis of single-cell sequence data and 24 paired samples confirmed the regulatory effect of PAR2 on the metastatic immune environment in human CRC. Therefore, PAR2 signaling is involved in stabilizing the mitochondrial membrane and regulating the immune microenvironment through IFN-β during liver metastasis in CRC. The synergistic effect of the PAR2 inhibitor and ICB provides a potential therapeutic strategy for metastatic CRC treatment.

PAR2 deficiency tunes inflammatory microenvironment to magnify STING signalling for mitigating cancer metastasis via anionic CRISPR/Cas9 nanoparticles

Metastasis is one of the most significant causes for deterioration of breast cancer, contributing to the clinical failure of anti-tumour drugs. Excessive inflammatory responses intensively promote the occurrence and development of tumour, while protease-activated receptor 2 (PAR2) as a cell membrane receptor actively participates in both tumour cell functions and inflammatory responses. However, rare investigations linked PAR2-mediated inflammatory environment to tumour progression. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology is an emerging and powerful gene editing technique and can be applied for probing the new role of PAR2 in breast cancer metastasis, but it still needs the development of an efficient and safe delivery system. This work constructed anionic bovine serum albumin (BSA) nanoparticles to encapsulate CRISPR/Cas9 plasmid encoding PAR2 sgRNA and Cas9 (tBSA/Cas9-PAR2) for triggering PAR2 deficiency. tBSA/Cas9-PAR2 remarkably promoted CRISPR/Cas9 to enter and transfect both inflammatory and cancer cells, initiating precise PAR2 gene editing in vitro and in vivo. PAR2 deficiency by tBSA/Cas9-PAR2 effectively suppressed NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome signalling in inflammatory microenvironment to magnify stimulator of interferon genes (STING) signalling, reactive oxygen species (ROS) accumulation and epithelial-mesenchymal transition (EMT) reversal, consequently preventing breast cancer metastasis. Therefore, this study not only demonstrated the involvement and underlying mechanism of PAR2 in tumour progression via modulating inflammatory microenvironment, but also suggested PAR2 deficiency by tBSA/Cas9-PAR2 as an attractive therapeutic strategy candidate for breast cancer metastasis.

Genome editing of PAR2 through targeted delivery of CRISPR-Cas9 system for alleviating acute lung inflammation via ERK/NLRP3/IL-1β and NO/iNOS signalling

Excessive and uncontrollable inflammatory responses in alveoli can dramatically exacerbate pulmonary disease progressions through vigorous cytokine releases, immune cell infiltration and protease-driven tissue damages. It is an urgent need to explore potential drug strategies for mitigating lung inflammation. Protease-activated receptor 2 (PAR2) as a vital molecular target principally participates in various inflammatory diseases via intracellular signal transduction. However, it has been rarely reported about the role of PAR2 in lung inflammation. This study applied CRISPR-Cas9 system encoding Cas9 and sgRNA (pCas9-PAR2) for PAR2 knockout and fabricated an anionic human serum albumin-based nanoparticles to deliver pCas9-PAR2 with superior inflammation-targeting efficiency and stability (TAP/pCas9-PAR2). TAP/pCas9-PAR2 robustly facilitated pCas9-PAR2 to enter and transfect inflammatory cells, eliciting precise gene editing of PAR2 in vitro and in vivo. Importantly, PAR2 deficiency by TAP/pCas9-PAR2 effectively and safely promoted macrophage polarization, suppressed pro-inflammatory cytokine releases and alleviated acute lung inflammation, uncovering a novel value of PAR2. It also revealed that PAR2-mediated pulmonary inflammation prevented by TAP/pCas9-PAR2 was mainly dependent on ERK-mediated NLRP3/IL-1β and NO/iNOS signalling. Therefore, this work indicated PAR2 as a novel target for lung inflammation and provided a potential nanodrug strategy for PAR2 deficiency in treating inflammatory diseases.

Date palm seed extract and herbal mixture mitigate gentamicin-induced renal injury in mice: Role of Protease-activated receptors (PARs) and Retinoid X receptor alpha (RXR-α)

Introduction: Gentamicin (Gen) causes renal toxicity by inhibiting protein synthesis in kidney cells, causing proximal tubule cell necrosis and renal failure. Herein, we examined the nephroprotective effect of date palm seed extract (DPSE) and one herbal mixture (HM; composed of Tribulus terrestris, Aerva lanata, Andrographis paniculata, and Raphanus sativus) against Gen-induced renal toxicity in mice with special reference to the possible role of retinoid X receptor alpha (RXR-α) and protease-activated receptor 2 (PAR-2) in this effect. Methods: Thirty-two male Balb/c mice divided randomly into four groups were either treated with saline, Gen (225 mg/kg/i.p., daily from day 3 to day 10), Gen (225 mg/kg i.p.) daily from day 3 to day 10 and DPSE (100 mg/kg/p.o.) daily for 10 days, or Gen (225 mg/kg i.p.) daily from day 3 to day 10 and HM (100 mg/kg/p.o., daily for 10 days). Mice were sacrificed 24 hours after the last dose administration, and kidney tissues were dissected out, weighed, and subjected to histological, immunofluorescence, and biochemical assays. Results: The Gen-induced renal toxicity group demonstrated a significant decrease in RXR-α and a significant increase in PAR-2 protein expression. Treatment with DPSE or HM significantly improved Gen-induced effects on serum creatinine, blood urea nitrogen (BUN), white blood cells (WBCs), platelets, RXR-α extracellular matrix deposition, and PAR-2. Conclusion: The present study stated the nephroprotective effects of DPSE and HM and revealed, for the first time, the involvement of retinoid receptors and PAR-2 in Gen-induced renal toxicity as well as in the protective effects of the two tested natural products.

The Yin-Yang roles of protease-activated receptors in inflammatory signalling and diseases.

Inflammatory diseases have become increasingly prevalent throughout the world. Coronavirus disease 2019 (COVID-19), which has recently become pandemic, also exhibits hyperinflammation and cytokine release syndrome. To address inflammation-related diseases, numerous molecular targets have been explored in preclinical studies and clinical trials. Among them, the protease-activated receptors (PARs) that belong to G protein-coupled receptors are one of the most popular classes of drug targets, participating in inflammatory signalling and diseases. PARs activation can trigger downstream intracellular signalling to modulate a variety of inflammatory responses in multiple systems, including nervous, respiratory, digestive, circulatory, urinary and immune systems. Importantly, there are the Yin-Yang effects, comprising anti- and pro-inflammatory roles, of PARs activation in different types of inflammations, and these are context-dependent. Alternatively, it was recently revealed that PARs not only modulate inflammatory-related tumour progression, but also participate in inflammatory cytokine release related to COVID-19 via direct interaction with severe acute respiratory syndrome coronavirus 2 protein, suggesting that PARs also participate in other diseases via inflammatory responses. In this review, we renew and discuss the findings of PARs as molecular targets for treating inflammatory diseases, highlighting the novel roles of PARs and facilitating a better understanding of their designated values in the specific inflammatory environment.

Protease-activated receptor-2 dependent and independent responses of bone cells to prostate cancer cell secretory products.

Metastatic prostate cancer lesions in the skeleton are frequently characterized by excessive formation of bone. Prostate cancer cells secrete factors, including serine proteases, that are capable of influencing the behavior of surrounding cells. Some of these proteases activate protease-activated receptor-2 (PAR2 ), which is expressed by osteoblasts (bone-forming cells) and precursors of osteoclasts (bone-resorbing cells). The aim of the current study was to investigate a possible role for PAR2 in regulating the behavior of bone cells exposed to metastatic prostate cancer cells. The effect of medium conditioned by the PC3, DU145, and MDA-PCa-2b prostate cancer cell lines was investigated in assays of bone cell function using cells isolated from wildtype and PAR2 -null mice. Osteoclast differentiation was assessed by counting tartrate-resistant acid phosphatase-positive multinucleate cells in bone marrow cultured in osteoclastogenic medium. Osteoblasts were isolated from calvariae of neonatal mice, and BrdU incorporation was used to assess their proliferation. Assays of alkaline phosphatase activity and quantitative PCR analysis of osteoblastic gene expression were used to assess osteoblast differentiation. Responses of osteoblasts to medium conditioned by MDA-PCa-2b cells were analyzed by RNAseq. Conditioned medium (CM) from all three cell lines inhibited osteoclast differentiation independently of PAR2 . Media from PC3 and DU145 cells had no effect on assays of osteoblast function. Medium conditioned by MDA-PCa-2b cells stimulated BrdU incorporation in both wildtype and PAR2 -null osteoblasts but increased alkaline phosphatase activity and Runx2 and Col1a1 expression in wildtype but not PAR2 -null cells. Functional enrichment analysis of RNAseq data identified enrichment of multiple gene ontology terms associated with lysosomal function in both wildtype and PAR2 -null cells in response to MDA-PCa-2b-CM. Analysis of individual genes identified osteogenesis-associated genes that were either upregulated by MDA-PCa-2b-CM selectively in wildtype cells or downregulated selectively in PAR2 -null cells. Factors secreted by prostate cancer cells influence bone cell behavior through both PAR2 -dependent and -independent mechanisms. Both PAR2 -independent suppression of osteoclast differentiation and PAR2 -dependent stimulation of osteogenesis are likely to determine the nature of prostate cancer metastases in bone.

Coagulation-independent effects of thrombin and Factor Xa: role of protease-activated receptors in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a devastating disease with limited therapeutic options. Vascular remodelling of pulmonary arteries, characterized by increased proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), is a hallmark of PAH. Here, we aimed to systematically characterize coagulation-independent effects of key coagulation proteases thrombin and Factor Xa (FXa) and their designated receptors, protease-activated receptor (PAR)-1 and -2, on PASMCs in vitro and experimental PAH in vivo. In human and murine PASMCs, both thrombin and FXa were identified as potent mitogens, and chemoattractants. FXa mediated its responses via PAR-1 and PAR-2, whereas thrombin signalled through PAR-1. Extracellular-signal regulated kinases 1/2, protein kinase B (AKT), and sphingosine kinase 1 were identified as downstream mediators of PAR-1 and PAR-2. Inhibition of FXa or thrombin blunted cellular responses in vitro, but unexpectedly failed to protect against hypoxia-induced PAH in vivo. However, pharmacological inhibition as well as genetic deficiency of both PAR-1 and PAR-2 significantly reduced vascular muscularization of small pulmonary arteries, diminished right ventricular systolic pressure, and right ventricular hypertrophy upon chronic hypoxia compared to wild-type controls. Our findings indicate a coagulation-independent pathogenic potential of thrombin and FXa for pulmonary vascular remodelling via acting through PAR-1 and PAR-2, respectively. While inhibition of single coagulation proteases was ineffective in preventing experimental PAH, our results propose a crucial role for PAR-1 and PAR-2 in its pathobiology, thus identifying PARs but not their dedicated activators FXa and thrombin as suitable targets for the treatment of PAH.

PAR1, a therapeutic target for remote lung injury associated with hind limb ischemia/reperfusion: ERK5/KLF2-dependent lung capillary barrier preservation

Protease-activated receptor 1 (PAR1) is expressed in pneumocytes and endothelial cells of the alveolar barrier. Its activation by thrombin disrupts the barrier integrity dynamics and induces lung injury in in vitro and in vivo paradigms. Nonetheless, the role of PAR1, as a therapeutic target, in hind limb ischemia/reperfusion (I/R)-mediated remote lung injury has been unclear. Therefore, this study aimed to determine the potential benefit of PAR1 blockade using the selective antagonist SCH79797 in distant lung dysfunction following hind limb I/R injury with special emphasis on the extracellular signal-regulated kinase 5 (ERK5)/Krüppel-like factor 2 (KLF2) axis. Rats were subdivided into control, bilateral hind limb I/R, SCH79797, and SCH79797+BIX02189 (ERK5 inhibitor) groups. PAR1 blockade, ERK5-dependently, alleviated alveolar barrier disruption as evidenced by reductions in both pulmonary systemic leakage of surfactant protein-D and lung fluid accumulation with increase in pulmonary claudin 5, vascular endothelial cadherin, and connexin 37 levels. Such improvements are downstream targets of the ERK5/KLF2-mediated sphingosine-1-phosphate receptor 1 (S1PR1) upregulated expression and pS536-nuclear factor-κB (NF-κB) p65 inhibition. SCH79797 effectively impedes the evoked inflammatory response and oxidative burst by suppressing vascular endothelial growth factor, tumor necrosis factor-α, lipid peroxidation, and neutrophil infiltration while boosting the glutathione antioxidant defense. Accordingly, PAR1 could be a therapeutic target, where its blockade mitigated pulmonary-endothelial barrier disruption via mutual S1PR1 enhancement and NF-κB p65 inhibition following ERK5/KLF2 activation.

Protease-activated receptor 2 activation induces behavioural changes associated with depression-like behaviour through microglial-independent modulation of inflammatory cytokines

RationaleMajor depressive disorder (MDD) is a leading cause of disability worldwide but currently prescribed treatments do not adequately ameliorate the disorder in a significant portion of patients. Hence, a better appreciation of its aetiology may lead to the development of novel therapies.ObjectivesIn the present study, we have built on our previous findings indicating a role for protease-activated receptor-2 (PAR2) in sickness behaviour to determine whether the PAR2 activator, AC264613, induces behavioural changes similar to those observed in depression-like behaviour.MethodsAC264613-induced behavioural changes were examined using the open field test (OFT), sucrose preference test (SPT), elevated plus maze (EPM), and novel object recognition test (NOR). Whole-cell patch clamping was used to investigate the effects of PAR2 activation in the lateral habenula with peripheral and central cytokine levels determined using ELISA and quantitative PCR.ResultsUsing a blood–brain barrier (BBB) permeable PAR2 activator, we reveal that AC-264613 (AC) injection leads to reduced locomotor activity and sucrose preference in mice but is without effect in anxiety and memory-related tasks. In addition, we show that AC injection leads to elevated blood sera IL-6 levels and altered cytokine mRNA expression within the brain. However, neither microglia nor peripheral lymphocytes are the source of these altered cytokine profiles.ConclusionsThese data reveal that PAR2 activation results in behavioural changes often associated with depression-like behaviour and an inflammatory profile that resembles that seen in patients with MDD and therefore PAR2 may be a target for novel antidepressant therapies.

The role of protease-activated receptor 1 signaling in CD8 T cell effector functions

SummaryCD8 T cells are essential for adaptive immunity against viral infections. Protease activated receptor 1 (PAR1) is expressed by CD8 T cells; however, its role in T cell effector function is not well defined. Here we show that in human CD8 T cells, PAR1 stimulation accelerates calcium mobilization. Furthermore, PAR1 is involved in cytotoxic T cell function by facilitating granule trafficking via actin polymerization and repositioning of the microtubule organizing center (MTOC) toward the immunological synapse. In vivo, PAR1−/− mice have reduced cytokine-producing T cells in response to a lymphocytic choriomeningitis virus (LCMV) infection and fail to efficiently control the virus. Specific deletion of PAR1 in LCMV GP33-specific CD8 T cells results in reduced expansion and diminished effector function. These data demonstrate that PAR1 plays a role in T cell activation and function, and this pathway could represent a new therapeutic strategy to modulate CD8 T cell effector function.

Cell type-specific roles of PAR1 in Coxsackievirus B3 infection

Protease-activated receptor 1 (PAR1) is widely expressed in humans and mice, and is activated by a variety of proteases, including thrombin. Recently, we showed that PAR1 contributes to the innate immune response to viral infection. Mice with a global deficiency of PAR1 expressed lower levels of CXCL10 and had increased Coxsackievirus B3 (CVB3)-induced myocarditis compared with control mice. In this study, we determined the effect of cell type-specific deletion of PAR1 in cardiac myocytes (CMs) and cardiac fibroblasts (CFs) on CVB3-induced myocarditis. Mice lacking PAR1 in either CMs or CFs exhibited increased CVB3 genomes, inflammatory infiltrates, macrophages and inflammatory mediators in the heart and increased CVB3-induced myocarditis compared with wild-type controls. Interestingly, PAR1 enhanced poly I:C induction of CXCL10 in rat CFs but not in rat neonatal CMs. Importantly, activation of PAR1 reduced CVB3 replication in murine embryonic fibroblasts and murine embryonic cardiac myocytes. In addition, we showed that PAR1 reduced autophagy in murine embryonic fibroblasts and rat H9c2 cells, which may explain how PAR1 reduces CVB3 replication. These data suggest that PAR1 on CFs protects against CVB3-induced myocarditis by enhancing the anti-viral response whereas PAR1 on both CMs and fibroblasts inhibits viral replication.

The Role of Protease-Activated Receptor 2 in Hepatocellular Carcinoma after Hepatectomy.

Background and Objectives: Protease activated receptor-2 (PAR2) is elevated in a variety of cancers and has been promoted as a potential therapeutic target. However, the clinical and prognostic values of PAR2 in hepatocellular carcinoma (HCC) are poorly characterized. This study aimed to evaluate the expression of PAR2 in HCC tissues and examine the prognostic value of PAR2 after resection in HCC. Materials and Methods: Two hundred and eight resected specimens were collected from HCC patients at Kaohsiung Chang Gung Memorial Hospital. PAR2 protein expression was assessed by western blotting in HCC tissues and matched normal tissues. The correlation between PAR2 expression and clinicopathological parameters was analyzed. Disease-free survival (DFS) and overall survival (OS) were compared using the log-rank test. A Cox regression model was used to identify independent prognostic factors. Results: PAR2 was expressed at higher levels in HCC tissues than the paired adjacent nontumor tissues. High expression of PAR2 was associated with advanced tumor, node, metastasis (TNM )stage and histological grade. Kaplan-Meier analysis indicated high PAR2 expression was associated with poorer DFS and OS compared to low PAR2 expression. Multivariate analyses indicated high PAR2 expression [hazard ratio (HR), 1.779, p = 0.006), α-fetoprotein (AFP) (HR, 1.696, p = 0.003), liver cirrhosis (HR, 1.735, p = 0.002), and advanced TNM stage (HR, 2.061, p < 0.001) were prognostic factors for DFS, and advanced TNM stage (HR, 2.741, p < 0.001) and histological grade (HR, 2.675, p = 0.002) and high PAR2 expression (HR, 1.832, p = 0.012) were significant risk factors for OS. In subgroup analyses, the combination of PAR2 expression and serum AFP provided improved prognostic ability for OS and DFS. Conclusion: Combination PAR2 and AFP predict HCC outcomes after resection. PAR2 represents a potentially clinically relevant biomarker for HCC.

Proteinase-activated receptor-2 (PAR2) on blood pressure and electrolyte handling in the late pregnant rat.

What is the central question of this study? Pregnancy requires marked renal sodium and potassium retention and cumulative plasma volume expansion, in the setting of reduced blood pressure. Research in male rodents has shown that activation of PAR2 can produce peripheral vasodilatation, stimulate renal sodium chloride reabsorption and inhibit renal potassium secretion. Here, we investigate PAR2 activation in virgin and normal pregnant rats. What is the main finding and its importance? PAR2 expression and sensitivity to activation are increased in pregnancy. This implicates a possible role for PAR2 in supporting the renal/vascular adaptations of pregnancy required for normal maternal plasma volume expansion. A healthy pregnancy involves renal and systemic haemodynamic adaptations, which allow renal sodium and potassium retention and cumulative plasma volume expansion, accompanied by a decline in blood pressure attributable to a reduction in the total peripheral vascular resistance. When these adaptations do not occur, pregnancy is compromised. The mechanisms permitting these opposing adaptations are largely unknown. Research in male rodents has shown that activation of PAR2 can produce peripheral vasodilatation, stimulate renal sodium chloride reabsorption and inhibit renal potassium secretion. Here, we investigate PAR2 activation in female virgin and normal late pregnant (LP) rats. We measured the mRNA expression of PAR2 in the renal cortex, outer medulla and inner medulla of virgin and LP rats using quantitative real-time PCR. We also measured in vivo blood pressure, natriuretic and kaliuretic responses to PAR2-activating peptide (SLIGRL-NH2 ) in anaesthetized virgin and LP rats. We found that PAR2 mRNA was increased in the inner medulla of LP rats. We also found that LP rats had larger decreases in blood pressure and increases in net sodium retention compared with virgin rats. These findings suggest that pregnancy enhances sensitivity to the blood pressure-lowering and sodium-retaining effects of PAR2.

Targeting PAR2 Overcomes Gefitinib Resistance in Non-Small-Cell Lung Cancer Cells Through Inhibition of EGFR Transactivation.

Drug resistance can notably restrict clinical applications of gefitinib that is a commonly used EGFR-tyrosine kinase inhibitors (EGFR-TKIs) for non-small cell lung cancer (NSCLC). The attempts in exploring novel drug targets and reversal strategies are still needed, since gefitinib resistance has not been fully addressed. Protease-activated receptor 2 (PAR2), a G protein-coupled receptor, possesses a transactivation with EGFR to initiate a variety of intracellular signal transductions, but there is a lack of investigations on the role of PAR2 in gefitinib resistance. This study established that protease-activated receptor 2 (PAR2), actively participated in NSCLC resistant to gefitinib. PAR2 expression was significantly up-regulated when NSCLC cells or tumor tissues became gefitinib resistance. PAR2 inhibition notably enhanced gefitinib to modulate EGFR transactivation, cell viability, migration and apoptosis in gefitinib-sensitive and-resistant NSCLC cells, suggesting its reversal effects in gefitinib resistance. Meanwhile, the combination of a PAR2 inhibitor (P2pal-18S) and gefitinib largely blocked ERK phosphorylation and epithelial-mesenchymal transition (EMT) compared to gefitinib alone. Importantly, we probed its underlying mechanism and uncovered that PAR2 blockade sensitized gefitinib and reversed its resistance mainly via β-arrestin-EGFR-ERK signaling axis. These effects of PAR2 inhibition were further confirmed by the in vivo study which showed that P2pal-18S reactivated gefitinib to inhibit tumor growth via restricting ERK activation. Taken together, this study could not only reveal a new mechanism of receptor-mediated transactivation to modulate drug resistance, but also provide a novel drug target and direction for overcoming gefitinib resistance in NSCLC.

Protease-activated receptor 4 plays a role in lipopolysaccharide-induced inflammatory mechanisms in murine macrophages.

The role of protease-activated receptor (PAR)4 in thrombin-induced platelet aggregation has been studied, and PAR4 blockade is thought to be useful as a new and promising approach in antiplatelet therapy in humans. In recent years, studies have been conducted to clarify the role of PAR4 in the host defense against invading microorganisms and pathogen-induced inflammation; however, to date, the role of PAR4 in mediating the LPS-induced inflammatory repertoire in macrophages remains to be elucidated. Here, we investigated the effects of the synthetic PAR4 agonist peptide (PAR4-AP) AYPGKF-NH2 on the phagocytosis of zymosan-FITC particles; NO, ROS, and iNOS expression; and cytokine production in C57/BL6 macrophages cocultured with PAR4-AP/LPS. The PAR4-AP impaired LPS-induced and basal phagocytosis, which was restored by pharmacological PAR4 blockade. Coincubation with the PAR4-AP/LPS enhanced NO and ROS production and iNOS expression; decreased IL-10, but not TNF-α, in the culture supernatant; and increased translocation of the p65 subunit of the proinflammatory gene transcription factor NF-κ-B. Our results provide evidence for a complex mechanism and new approach by which PAR4 mediates the macrophage response triggered by LPS through counter-regulating the phagocytic activity of macrophages and innate response mechanisms implicated in the killing of invading pathogens.

Protease-Activated Receptor-2 Is Associated With Adverse Outcomes in Canine Mammary Carcinoma

Protease-activated receptor-2 (PAR2) is a G protein-coupled receptor that is activated by serine proteases. In humans, PAR2 is highly expressed in various cancers, including breast cancer, and is associated with cancer progression and metastasis. However, the expression and roles of PAR2 in canine mammary carcinoma remain unclear. The purpose of this study was to examine the expression of PAR2 in canine mammary carcinoma, the association between PAR2 expression and clinical characteristics, and the role of PAR2 in the metastatic phenotypes of tumor cells. Mammary carcinoma from 31 dogs and 10 normal mammary glands were included in this study, and used for immunohistochemical analysis of PAR2 expression. Normal mammary glands did not express PAR2. In contrast, mammary carcinomas showed PAR2 immunoreactivity in the cytoplasm, and its expression level varied between specimens from negative to strongly positive. The overall survival of dogs with high PAR2 expression was shorter than that of dogs with low PAR2 expression. Moreover, PAR2 expression level was associated with the presence of lymph node involvement, advanced clinical stage, and high histopathological grade. In vitro analyses revealed that a PAR2 agonist accelerated cell migration and invasion in a canine mammary carcinoma cell line. In addition, the PAR2 agonist induced epithelial-mesenchymal transition and actin polymerization. These results suggest that PAR2 expression plays a role in tumor progression and clinical outcomes in canine mammary carcinoma.