Par-4 Expression Research Articles

Protease‐activated receptor‐1 drives pancreatic cancer progression and chemoresistance

Protease activated receptor (PAR)-1 expression in tumor cells is associated with disease progression and overall survival in a variety of cancers of epithelial origin; however, the importance of PAR-1 in the tumor microenvironment remains unexplored. Utilizing an orthotopic pancreatic cancer model in which tumor cells are PAR-1 positive whereas stromal cells are PAR-1 negative, we show that PAR-1 expression in the microenvironment drives progression and induces chemoresistance of pancreatic cancer. PAR-1 enhances monocyte recruitment into the tumor microenvironment by regulating monocyte migration and fibroblast dependent chemokine production thereby inducing chemoresistance. Overall, our data identify a novel role of PAR-1 in the pancreatic tumor microenvironment and suggest that PAR-1 may be an attractive target to reduce drug resistance in pancreatic cancer.

204 Modulation of Protease Activated Receptor Gene Expression in Endothelial Cells

IntroductionProtease activated receptors (PARs) form a distinct group of G-protein coupled receptors with key roles in several pathophysiological processes including inflammation and atherothrombotic disease. This study aimed to investigate the...

Protease-activated receptor 2 (PAR2) is upregulated by Acanthamoeba plasminogen activator (aPA) and induces proinflammatory cytokine in human corneal epithelial cells.

Acanthamoeba plasminogen activator (aPA) is a serine protease elaborated by Acanthamoeba trophozoites that facilitates the invasion of trophozoites to the host and contributes to the pathogenesis of Acanthamoeba keratitis (AK). The aim of this study was to explore if aPA stimulates proinflammatory cytokine in human corneal epithelial (HCE) cells via the protease-activated receptors (PARs) pathway. Acanthamoeba castellanii trophozoites were grown in peptone-yeast extract glucose for 7 days, and the supernatants were collected and centrifuged. The aPA was purified using the fast protein liquid chromatography system, and aPA activity was determined by zymography assays. Human corneal epithelial cells were incubated with or without aPA (100 μg/mL), PAR1 agonists (thrombin, 10 μM; TRAP-6, 10 μM), and PAR2 agonists (SLIGRL-NH2, 100 μM; AC 55541, 10 μM) for 24 and 48 hours. Inhibition of PAR1 and PAR2 involved preincubating the HCE cells for 1 hour with the antagonist of PAR1 (SCH 79797, 60 μM) and PAR2 (FSLLRY-NH2, 100 μM) with or without aPA. Human corneal epithelial cells also were preincubated with PAR1 and PAR2 antagonists and then incubated with or without PAR1 agonists (thrombin and TRAP-6) and PAR2 agonists (SLIGRL-NH2 and AC 55541). Expression of PAR1 and PAR2 was examined by quantitative RT-PCR (qRT-PCR), flow cytometry, and immunocytochemistry. Interleukin-8 expression was quantified by qRT-PCR and ELISA. Human corneal epithelial cells constitutively expressed PAR1 and PAR2 mRNA. Acanthamoeba plasminogen activator and PAR2 agonists significantly upregulated PAR2 mRNA expression (1- and 2-fold, respectively) (P < 0.05). Protease-activated receptor 2 antagonist significantly inhibited aPA, and PAR2 agonists induced PAR2 mRNA expression in HCE cells (P < 0.05). Protease-activated receptor 1 agonists, but not aPA, significantly upregulated PAR1 mRNA expression, which was significantly inhibited by PAR1 antagonist in HCE cells. Acanthamoeba plasminogen activator and PAR2 agonists stimulated IL-8 mRNA expression and protein production, which is significantly diminished by PAR2 antagonist (P < 0.05). Protease-activated receptor 1 antagonist did not alter aPA-stimulated IL-8 mRNA expression and protein production in HCE cells. Flow cytometry and immunocytochemistry showed that aPA and SLIGRL-NH2 (PAR2 agonist) upregulated PAR2 surface protein as compared to that in unstimulated HCE cells. Thrombin, but not aPA, stimulated PAR1 surface protein in HCE cells. Acanthamoeba plasminogen activator specifically induces expression and production of IL-8 in HCE cells via PAR2 pathway, and PAR2 antagonists may be used as a therapeutic target in AK.

Cytoplasmic PAR-3 protein expression is associated with adverse prognostic factors in clear cell renal cell carcinoma and independently impacts survival.

Clear cell renal cell carcinomas (ccRCCs) represent 70% of renal cancers, and several clinical and histolopathological factors are implicated in their prognosis. We recently demonstrated that the overexpression of PAR-3 protein encoded by the PARD3 gene could be implicated in renal oncogenesis. The object of this work was to study the association of intratumoral PAR-3 expression with known prognostic parameters and clinical outcome. In this aim, PAR-3 expression was assessed by immunohistochemistry in ccRCC tumors of 101 patients from 2003 to 2005. The immunostaining of PAR-3 was scored either as membranous (mPAR-3) or as both membranous and cytoplasmic (cPAR-3). Cytoplasmic PAR-3 was significantly associated with worse histopathological and clinical prognostic factors: Fuhrman grades 3 and 4, tumor necrosis, sarcomatoid component, adrenal invasion, renal and hilar fat invasion, eosinophilic component, a noninactivated VHL gene, higher tumor grade, lymph node involvement, metastasis, and worse clinical Eastern Cooperative Oncology Group and S classification scores. After multivariate analysis, 2 parameters were independently associated with cPAR-3: necrosis and eosinophilic components. In addition, cPAR-3 patients had shorter overall and progression-free survivals independently from strong prognostic validated factors like metastases. A cytoplasmic expression of PAR-3 is therefore implicated in worse clinical and pathological cancer features in ccRCC and could be useful to identify patients with high-risk tumors.

Tryptase inhibitor APC 366 prevents hepatic fibrosis by inhibiting collagen synthesis induced by tryptase/protease-activated receptor 2 interactions in hepatic stellate cells.

Protease-activated receptor (PAR) 2 is a G-protein-coupled receptor that is activated by mast cell tryptase. PAR-2 activation augments profibrotic pathways through the induction of extracellular matrix proteins. PAR-2 is widely expressed in hepatic stellate cells (HSCs), but the role of tryptase/PAR-2 interaction in liver fibrosis is unclear. We studied the development of bile duct ligation (BDL)-induced hepatic fibrosis in rats treated with mast cell tryptase inhibitor APC 366, and showed that APC 366 reduced hepatic fibrosis scores, collagen content and serum biochemical parameters. Reduced fibrosis was associated with decreased expression of PAR-2 and α-smooth muscle actin (α-SMA). Our findings demonstrate that mast cell tryptase induces PAR-2 activation to augment HSC proliferation and promote hepatic fibrosis in rats. Treatment with tryptase antagonists may be a novel therapeutic approach to prevent fibrosis in patients with chronic liver disease.

PAR4 Mediates an Elevated Risk for Thrombosis in Blacks Relative to Whites (LB602)

Coronary heart disease (CHD) is the leading cause of mortality in the US amongst all races. CHD disproportionality burdens blacks relative to whites and even after adjusting for other demographic parameters, blacks have a two‐fold increase in incidence of CHD and a decrease in long‐term survival. Occlusive thrombus formation is the underlying cause of mortality associated with CHD. Due to the importance of platelets in CHD and formation of occlusive thrombi, the response of platelets from blacks and whites to thrombin and its protease‐activated receptors (PAR1 and PAR4) was characterized. Platelets isolated from blacks and whites differentially responded to PAR4 but not PAR1. Specifically, platelets from blacks were hyperaggregable to low and intermediate doses of PAR4‐activating peptide (PAR4‐AP) compared to platelets from whites. The mechanism responsible for the racial difference in PAR4 mediated platelet reactivity however is still unknown. We hypothesize that changes in expression or activity of proteins known to participate in the PAR4 pathway are responsible for the racial difference in platelet PAR4 reactivity. To test this hypothesis, the PAR4 pathway in platelets from blacks and whites was characterized. No measurable difference was observed in the surface expression of PAR4 on platelets from blacks or whites however following stimulation with low dose PAR4‐AP (50uM), platelets from blacks had an increase in PAR4 signaling components compared to whites including intracellular Ca2+ release, RAP1 activation, and integrin αIIbβ3 activation. This data suggest that the kinetics of platelet activation through PAR4‐AP is hyper‐responsive in blacks compared to whites due to a shift in threshold downstream of the receptor. As PAR1 antagonists are currently in clinical trials and PAR4 antagonists are in pre‐clinical development, this work has important clinical implication as to which patients may benefit the most from selective PAR inhibition and is the first to suggest a racial component in the prevention of occlusive thrombus formation and stroke.Grant Funding Source: RO1 MD007880

PAR4 Mediates an Elevated Risk for Thrombosis in Blacks Relative to Whites (425.1)

Coronary heart disease (CHD) is the leading cause of mortality in the US amongst all races. CHD disproportionality burdens blacks relative to whites and even after adjusting for other demographic parameters, blacks have a two‐fold increase in incidence of CHD and a decrease in long‐term survival. Occlusive thrombus formation is the underlying cause of mortality associated with CHD. Due to the importance of platelets in CHD and formation of occlusive thrombi, the response of platelets from blacks and whites to thrombin and its protease‐activated receptors (PAR1 and PAR4) was characterized. Platelets isolated from blacks and whites differentially responded to PAR4 but not PAR1. Specifically, platelets from blacks were hyperaggregable to low and intermediate doses of PAR4‐activating peptide (PAR4‐AP) compared to platelets from whites. The mechanism responsible for the racial difference in PAR4 mediated platelet reactivity however is still unknown. We hypothesize that changes in expression or activity of proteins known to participate in the PAR4 pathway are responsible for the racial difference in platelet PAR4 reactivity. To test this hypothesis, the PAR4 pathway in platelets from blacks and whites was characterized. No measurable difference was observed in the surface expression of PAR4 on platelets from blacks or whites however following stimulation with low dose PAR4‐AP (50uM), platelets from blacks had an increase in PAR4 signaling components compared to whites including intracellular Ca2+ release, RAP1 activation, and integrin αIIbβ3 activation. This data suggest that the kinetics of platelet activation through PAR4‐AP is hyper‐responsive in blacks compared to whites due to a shift in threshold downstream of the receptor. As PAR1 antagonists are currently in clinical trials and PAR4 antagonists are in pre‐clinical development, this work has important clinical implication as to which patients may benefit the most from selective PAR inhibition and is the first to suggest a racial component in the prevention of occlusive thrombus formation and stroke.Grant Funding Source: RO1 MD007880

Environmental changes could enhance the biological effect of Hop J pollens on human airway epithelial cells

Environmental changes could enhance the biological effect of Hop J pollens on human airway epithelial cells

Thrombin promotes proliferation of human lung fibroblasts via protease activated receptor‐1‐dependent and NF‐κB‐independent pathways

Acute and chronic respiratory diseases are associated with abnormal coagulation regulation and fibrolysis. However, the detailed mechanism by which coagulation regulation and fibrolysis affect the occurrence and development of lung diseases remain to be elucidated. Protease activated receptor-1 (PAR-1), a major high-affinity thrombin receptor, and nuclear factor kappa B (NF-κB), a transcription factor, are involved in cell survival, differentiation, and proliferation. We have investigated the potential mechanism of thrombin-induced fibroblast proliferation and roles of PAR-1 and NF-κB signalling in this process. The effect of thrombin on proliferation of human pulmonary fibroblasts (HPF) was assessed by 5-bromo-2-deoxyuridine (BrdU) incorporation assay. The expression of PAR1 and NF-κB subunit p65 protein was detected by Western blot. Nuclear translocation of p65 was examined by laser scanning confocal microscopy. We show that thrombin significantly increased proliferation of HPF as determined by induction of BrdU-positive incorporation ratio. Induced PAR1 protein expression was also seen in HPF cells treated with thrombin. However, thrombin had no significant effect on expression and translocation of NF-κB p65 in HPF cells. The results indicate that, by increasing protein expression and interacting with PAR1, thrombin promotes HPF proliferation. NF-κB signalling appears to play no role in this process.

Hep par-1: a novel immunohistochemical marker for differentiating hepatocellular carcinoma from metastatic carcinoma.

To evaluate the diagnostic utility of Hep par-1 in differentiating hepatocellular carcinoma from metastatic carcinoma taking histopathology as a gold standard. Comparative cross-sectional study. Pathology Department, Shaukat Khanum Memorial Cancer Hospital and Research Centre, Lahore, from April 2007 to February 2008. Hep par-1 immunohistochemical stain was performed on 60 cases of liver carcinoma, 30 cases each of metastatic and hepatocellular carcinoma. Information regarding patient age, gender, sign and symptoms, radiographic findings, histological grade of tumour, and expression of Hep par-1 on hepatocellular and metastatic carcinoma were recorded on proforma sheet. Sensitivity, specificity, positive and negative predictive values, and accuracy of Hep par-1 were calculated using the formulas. Hep par-1 expression was noted in 25 out of 30 cases of hepatocellular carcinoma (83%). Out of 30 cases of metastatic carcinoma, only one case expressed staining in < 5% tumour cells and remaining 29 cases showed no reactivity. The age of the patients with hepatocellular carcinoma ranged from 40 to 76 years with a median age of 60.5 years and 40 - 75 years for metastatic carcinomas with a median age of 57.5 years. Hep par-1 is a reliable immunohistochemical marker for cases of hepatocellular carcinoma (HCC). It can be used along with other markers in morphologically difficult cases when differential diagnosis lies between poorly differentiated HCC and metastatic carcinoma of liver.

Tissue Kallikrein Mediates Pro-Inflammatory Pathways and Activation of Protease-Activated Receptor-4 in Proximal Tubular Epithelial Cells

Tissue kallikrein (KLK1) expression is up-regulated in human diabetic kidney tissue and induced by high glucose (HG) in human proximal tubular epithelial cells (PTEC). Since the kallikrein-kinin system (KKS) has been linked to cellular inflammatory process in many diseases, it is likely that KLK1 expression may mediate the inflammatory process during the development of diabetic nephropathy. In this study, we explored the role of KLK1 in tubular pro-inflammatory responses under the diabetic milieu. Recombinant KLK1 stimulated the production of inflammatory cytokines in PTEC via the activation of p42/44 and p38 MAPK signaling pathways. Molecular knockdown of endogenous KLK1 expression by siRNA transfection in PTEC attenuated advanced glycation end-products (AGE)-induced IL-8 and ICAM-1 productions in vitro. Interestingly, exposure of PTEC to KLK1 induced the expression of protease-activated receptors (PARs). There was a 2.9-fold increase in PAR-4, 1.4-fold increase in PAR-1 and 1.2-fold increase in PAR-2 mRNA levels. Activation of PAR-4 by a selective agonist was found to elicit the pro-inflammatory and pro-fibrotic phenotypes in PTEC while blockade of the receptor by specific antagonist attenuated high glucose-induced IL-6, CCL-2, CTGF and collagen IV expression. Calcium mobilization by the PAR-4 agonist in PTEC was desensitized by pretreatment with KLK1. Consistent with these in vitro findings, there was a markedly up-regulation of tubular PAR-4 expression in human diabetic renal cortical tissues. Together, these results suggest that up-regulation of KLK1 in tubular epithelial cells may mediate pro-inflammatory pathway and PAR activation during diabetic nephropathy and provide a new therapeutic target for further investigation.

Expression of protease‐activated receptors in allergic fungal rhinosinusitis

The etiology of the intense inflammatory response showed by patients with allergic fungal rhinosinusitis (AFRS) remains a mystery. Potential sources of this inflammation may include fungal proteases. Protease-activated receptors (PARs) are components of the innate immune response that are modulated by proteolytic activity and are involved in potentiating T helper 2 (Th2) responses. The objective of the study was to determine whether there is differential expression of PARs in patients with AFRS compared to controls. The study was designed as a comparison of gene expression profiles in patients with AFRS vs diseased and nondiseased controls. Twenty-five patients were enrolled. Patients with AFRS (n = 15) were compared to nondiseased controls (n = 5) undergoing minimally invasive pituitary surgery (MIPS) and patients with chronic rhinosinusitis with nasal polyps (CRSwNP, n = 5) undergoing functional endoscopic sinus surgery (FESS). Ethmoid mucosa RNA was hybridized to 4 × 44 K microarray chips. Four gene probes (PAR1, PAR2, PAR3, and PAR4) were used to assess for differential expression. A linear-mixed model was used to account for some patients having multiple samples. Significance level was determined at p < 0.05. Of the 4 probes, only PAR3 showed statistically significant differential expression between AFRS and nondiseased control samples (p = 0.03) as well as a 2.21-fold change. No additional statistical difference in PAR expression among the comparison groups was noted. PARs have been shown to enhance production of inflammatory cytokines and potentiate Th2 responses. In this initial report, patients with AFRS have a significantly increased expression of PAR3 compared to nondiseased controls.

Prostate apoptosis response-4 mediates TGF-β-induced epithelial-to-mesenchymal transition

A growing body of evidence supports that the epithelial-to-mesenchymal transition (EMT), which occurs during cancer development and progression, has a crucial role in metastasis by enhancing the motility of tumor cells. Transforming growth factor-β (TGF-β) is known to induce EMT in a number of cancer cell types; however, the mechanism underlying this transition process is not fully understood. In this study we have demonstrated that TGF-β upregulates the expression of tumor suppressor protein Par-4 (prostate apoptosis response-4) concomitant with the induction of EMT. Mechanistic investigations revealed that exogenous treatment with each TGF-β isoform upregulates Par-4 mRNA and protein levels in parallel levels of phosphorylated Smad2 and IκB-α increase. Disruption of TGF-β signaling by using ALK5 inhibitor, neutralizing TGF-β antibody or phosphoinositide 3-kinase inhibitor reduces endogenous Par-4 levels, suggesting that both Smad and NF-κB pathways are involved in TGF-β-mediated Par-4 upregulation. NF-κB-binding sites in Par-4 promoter have previously been reported; however, using chromatin immunoprecipitation assay we showed that Par-4 promoter region also contains Smad4-binding site. Furthermore, TGF-β promotes nuclear localization of Par-4. Prolonged TGF-β3 treatment disrupts epithelial cell morphology, promotes cell motility and induces upregulation of Snail, vimentin, zinc-finger E-box binding homeobox 1 and N-Cadherin and downregulation of Claudin-1 and E-Cadherin. Forced expression of Par-4, results in the upregulation of vimentin and Snail expression together with increase in cell migration. In contrast, small interfering RNA-mediated silencing of Par-4 expression results in decrease of vimentin and Snail expression and prevents TGF-β-induced EMT. We have also uncovered a role of X-linked inhibitor of apoptosis protein in the regulation of endogenous Par-4 levels through inhibition of caspase-mediated cleavage. In conclusion, our findings suggest that Par-4 is a novel and essential downstream target of TGF-β signaling and acts as an important factor during TGF-β-induced EMT.

DOP001 Epithelial polarity is disrupted by interleukin-22

Background: Interleukin-22 (IL-22), a cytokine secreted by TH17 cells, acts specifically on epithelia via the heterodimeric IL22R1/IL10 receptor. Depending on the context it is examined, IL-22 was shown to have either protective (wound healing) or deleterious effects (induction of ileitis) on intestinal epithelia. We have previously shown that it strongly disturbs the epithelial barrier by attacking the protein complex at tight junctions (TJ). Herein epithelial polarity is analyzed using the 3D cyst model as well as IL-22 knock-out mice. Methods: Caco-2 cells, seeded in Matrigel, were grown to develop 3-dimensional cysts. These cysts were treated with IL-22 (10 ng/ml) and analyzed for the number of lumina, proliferation and apoptosis (active caspase-3). Moreover, fully developed cysts, as well as cysts in early phases of cyst formation were immunostained for apical junctional as well as polarity complex proteins and examined with confocal LSM. To examine TJ assembly, Caco2 cells on filter supports were mounted to Ussing chambers, to perform a calcium switch assay. Ilea of wild-type and IL-22 knock-out mice, previously been treated with T. gondii to induce ileitis, were mounted to Ussing chambers to measure the epithelial portion (Repi) of the transmural electrical resistance by impedance spectroscopy. Results: IL-22-treated cysts revealed a multiple lumen phenotype as seen in stages of dyspolarity. Furthermore complex cysts with branched lumina were found. IL-22 treated cysts revealed a basolateral expression of the apical membrane marker Ezrin. The expression of polarity proteins Par-3 and Dlg-1 normally expressed at the TJ and the basolateral membrane, respectively were reduced and shifted to an intracellular vesicle pool.

DOP002 Conditional deletion of Prep1 in the intestinal epithelium alters epithelial homeostasis, intestinal development, and controls colitis susceptibility

Background: Interleukin-22 (IL-22), a cytokine secreted by TH17 cells, acts specifically on epithelia via the heterodimeric IL22R1/IL10 receptor. Depending on the context it is examined, IL-22 was shown to have either protective (wound healing) or deleterious effects (induction of ileitis) on intestinal epithelia. We have previously shown that it strongly disturbs the epithelial barrier by attacking the protein complex at tight junctions (TJ). Herein epithelial polarity is analyzed using the 3D cyst model as well as IL-22 knock-out mice. Methods: Caco-2 cells, seeded in Matrigel, were grown to develop 3-dimensional cysts. These cysts were treated with IL-22 (10 ng/ml) and analyzed for the number of lumina, proliferation and apoptosis (active caspase-3). Moreover, fully developed cysts, as well as cysts in early phases of cyst formation were immunostained for apical junctional as well as polarity complex proteins and examined with confocal LSM. To examine TJ assembly, Caco2 cells on filter supports were mounted to Ussing chambers, to perform a calcium switch assay. Ilea of wild-type and IL-22 knock-out mice, previously been treated with T. gondii to induce ileitis, were mounted to Ussing chambers to measure the epithelial portion (Repi) of the transmural electrical resistance by impedance spectroscopy. Results: IL-22-treated cysts revealed a multiple lumen phenotype as seen in stages of dyspolarity. Furthermore complex cysts with branched lumina were found. IL-22 treated cysts revealed a basolateral expression of the apical membrane marker Ezrin. The expression of polarity proteins Par-3 and Dlg-1 normally expressed at the TJ and the basolateral membrane, respectively were reduced and shifted to an intracellular vesicle pool.

Protease-activated receptors expression in gingiva in periodontal health and disease

ObjectiveProtease-activated receptors (PARs) are a unique class of receptors which are implicated in mediating inflammation, pain and other functions. The aim of this study was to elucidate the role of PARs in the pathogenesis of chronic periodontitis by differential expression analysis of PARs in the gingival tissues of chronic periodontitis patients compared with those of healthy control individuals. DesignGingival tissue specimens were collected from chronic periodontitis patients (n=20) and control individuals (n=20). The expression of PAR-1, -2, -3 and -4 was determined in these tissues by immunohistochemistry and differential expression between the two groups was investigated by quantitative real-time reverse transcription-polymerase chain reaction analysis. ResultsPAR-1, -2, -3 and -4 were expressed in all gingival tissues. A significant overexpression of PAR-3 was detected in chronic periodontitis-affected tissues compared to healthy gingival tissues. However, expression of PAR-2 was decreased in periodontal lesions. ConclusionsOur study shows that PAR-1, -2, -3 and -4 are expressed in both healthy and inflamed gingival tissues. Furthermore, PAR-2 and PAR-3 may contribute to the inflammatory responses associated with chronic periodontitis.

Association of Thrombin Generation Potential with Platelet PAR-1 Regulation and P-Selectin Expression in Patients on Dual Antiplatelet Therapy

We studied the association of thrombin generation potential with platelet protease activated receptor (PAR)-1 regulation and platelet activation in 52 stable coronary artery disease patients on continuous therapy with aspirin and clopidogrel (n = 42) or prasugrel (n = 10). Compared to controls, peak thrombin generation potential was elevated in only 11 patients (p > 0.05), while F1.2 was elevated in 26 patients (p < 0.0001). PAR-1 and thrombin inducible P-selectin expression were significantly elevated in patients compared to controls (p < 0.001). There were no significant correlations between levels of thrombin generation potential or F1.2 and PAR-1 regulation. However, there was a significant inverse correlation between levels of peak thrombin generation potential and in vitro thrombin-inducible expression of P-selectin (p = 0.002), suggesting in vivo depletion of platelet alpha granules due to ongoing platelet activation.

Concomitant Induction of Apoptosis and Autophagy by Prostate Apoptosis Response-4 in Hypopharyngeal Carcinoma Cells

The tumor-suppressive activity of prostate apoptosis response-4 (Par-4) has been demonstrated in a variety of human cancers. In this study, for the first time to our knowledge, we demonstrated that a higher intensity of Par-4 was significantly correlated with a better response in patients with hypopharyngeal carcinoma undergoing radiotherapy alone or concurrent chemoradiotherapy. Mechanistically, an elevated expression of Par-4 induced apoptosis of hypopharyngeal carcinoma cells and sensitized cells toward chemotherapeutic agents or X-ray irradiation. Along with apoptotic incitation, intriguingly, autophagic flux also increased on Par-4 stimulation and contributed to cell death. Moreover, the expressions of multiple common regulators involved in apoptosis and autophagy were regulated by Par-4. Taken together, our results suggested a prognostic role of Par-4 in hypopharyngeal carcinoma and showed novel activity of Par-4 in apoptosis and autophagy induction.

Expression of the PAR-1 protein on the surface of platelets in patients with chronic peripheral arterial insufficiency - preliminary report.

The activation of pro-coagulation mechanisms associated with the vascular wall's immune and inflammatory responses wall to injury plays a crucial role in the mechanisms of the induction and progression of atherosclerosis. The aim of this study was to determine the role of protease activated receptors (PAR-1) expressed on the surface of blood platelets in the pathogenesis of chronic peripheral arterial obliterative disease (PAOD) in patients with obliterative atherosclerosis (n = 24) and diabetic macroangiopathy (n = 16), as well as in the controls (n = 12). In addition to the expression of PAR-1, serum/plasma concentrations of thrombin-antithrombin complex (TAT), the von Willebrand factor (vWF), the platelet-derived growth factor, monocyte chemotactic protein, the soluble form of the platelet endothelial cell adhesion molecule, thrombin activatable fibrinolysis inhibitor and interleukin 6 (IL-6) were determined. Compared to the controls, PAOD patients were characterized by significantly higher levels of PAR-1 expression, vWF, TAT and IL-6. Individuals with diabetic macroangiopathy did not differ significantly from individuals with obliterative atherosclerosis in terms of PAR-1 expression. Upon activation with thrombin receptor antagonist peptide (TRAP), the levels of PAR-1 were comparable in all analyzed groups. In patients with diabetic macroangiopathy, a significant association was observed between the expression of PAR-1 on the surface of the platelet and the serum TAT concentration, as well as between TAT and serum IL-6 concentration. Enhanced expression of PAR-1 on the thrombocyte surface in chronic PAOD patients occurs equally in cases of diabetic macroangiopathy and in individuals free from this endocrine pathology.

The decreased expression of protease-activated receptor 4 in esophageal squamous carcinoma.

Protease-activated receptors (PARs) are a unique family of G-protein coupled receptors. PAR4, a member of PARs family, was reported to be related to the development of cancers. Whether PAR4 plays a role in the progress of esophageal squamous cancer is unknown. In this study, differential expression of PAR4 in esophageal squamous cancer was measured by real-time PCR (n = 28), western blot and tissue microarrays (n = 78). The results showed that PAR4 expression was remarkably decreased in esophageal squamous cancer tissues compared with the matched noncancerous tissues, especially in low differentiation and positive distant metastasis carcinoma tissues. Furthermore, the methylation level of PAR4 promoter in esophageal cancer cells and normal epithelial cells was determined. Human esophageal cancer cells TE-1 displayed significant hypermethylation of 19 CpG sites, but pronounced hypomethylation of the sites in esophageal epithelial cells HEEpiC. The results suggested that down-regulation expression of PAR4 occurs frequently in esophageal squamous cancers, and the loss of PAR4 expression may partly result from hypermethylation of the PAR4 promoter. That PAR4 expression difference in tumor progression possibly makes PAR4 become a molecular mark of tumor diagnosis.