Par-4 Expression Research Articles

DOP51 Investigation on the role of Par4-associated cell polarity and associated barrier defects in inflammatory bowel diseases

Abstract Background In inflammatory bowel diseases (IBD), epithelial barrier defects occur as a consequence of chronic inflammation. Recent research suggested that cell polarity alterations may be upstream of barrier defects and additionally play a role in IBD-associated carcinogenesis (colitis-associated and small intestinal carcinoma). Par4 is a gene encoding a protein crucial in the development of cell polarity. LKB1, its human homologue, is mutated in Peutz–Jeghers syndrome (PJS), a genetic condition characterised by a higher risk of epithelial cancers. While the pivotal role of Par4/LKB1 in the development of epithelial cell polarity is established, its involvement in IBD-associated barrier defects and carcinogenesis is yet to be defined. Methods Endoscopic bowel mucosa samples from patients with Crohn’s disease, ulcerative colitis, PJS and controls were analysed to assess expression and localisation of Par4/LKB1. Cryosections were immunostained for Par4/LKB-1 along with a diverse set of markers of cell polarity and intercellular adhesion. The analysis was performed by confocal laser scanning microscopy in order to compare the expression of mucosal as well as the subcellular localisation of Par4/LKB1 in the gut mucosa. A quantitative analysis of protein expression with western blotting was performed as well. The function of Par4/LKB1 in intestinal epithelial cells (IEC) was evaluated by means of a Par4-deficient IEC model as well as in an IEC Par4-overexpression model. Moreover, a preliminary assessment of the possible role of Par4/LKB1-related polarity processes in IBD-associated carcinomas was performed through scanning genetic data from colitis-associated carcinomas for potential Par4-related mutations and altered Par4/LKB1 expression. Results The immunofluorescent staining allowed visualisation of intracellular expression of Par4 in epithelia from PJS, IBD patients and controls. In PJS-polyps, despite the alteration of regular tissue architecture typical of these lesions, the polarity of epithelial cells was maintained - contrary to control tissue, a punctate pattern of the Par4 staining was shown. In IBD tissue, no relevant differences in Par4 expression at confocal microscopy, as well as a quantitative assessment, were observed as compared with controls. No differences were observed in PJS or IBD samples as regards the expression of cell polarity markers such as Par3, CD71, crb3 or adhesion molecules such as ZO-1, e-cadherin, occludin, JAM-A. As regards the IEC model, we observed that Caco-2/BBe cells overexpressing Par4/LKB1 showed enhanced Par4/LKB1 cytosolic staining at immunofluorescence. In this model, a defective epithelial polarity and organisation on permeable supports could be observed in Caco2/BBe cysts in parallel with reduced expression of native LKB1 as seen in western blots. Preliminary analysis of data from our colitis-associated carcinomas database suggested various pathways potentially involving Par4/LKB1, yet no direct analysis of tissue samples for Par4-related mutations was performed yet. Conclusions Altered expression of Par4 in epithelial cells may influence the development of a functional epithelial barrier as suggested by IEC models. Par4/LKB1 as a known oncosuppressor gene is involved in a number of pathways potentially relevant for carcinogenesis in IBD; however, a direct connection between Par4-related alteration of the epithelial barrier and carcinogenesis is yet to be established.

Thrombin receptor PAR4 drives canonical NLRP3 inflammasome signaling in the heart.

The deleterious effects of diabetes in the heart are increasingly attributed to inflammatory signaling through the NLRP3 (NOD, LRR and PYD domains-containing protein 3) inflammasome. Thrombin antagonists reduce cardiac remodeling and dysfunction in diabetic mice, in part by suppressing fibrin-driven inflammation. The role of cellular thrombin receptor subtypes in this context is not known. We sought to determine the causal involvement of protease-activated receptors (PAR) in inflammatory signaling of the diabetic heart. Mice with diet-induced diabetes showed increased abundance of pro-caspase-1 and pro-interleukin (IL)-1β in the left ventricle (LV), indicating transcriptional NLRP3 inflammasome priming, and augmented cleavage of active caspase-1 and IL-1β, pointing to canonical NLRP3 inflammasome activation. Caspase-11 activation, which mediates non-canonical NLRP3 inflammasome signaling, was not augmented. Formation of the plasma membrane pore-forming protein N-terminal gasdermin D (GDSMD), a prerequisite for IL-1β secretion, was also higher in diabetic vs. control mouse LV. NLRP3, ASC and IL-18 expression did not differ between the groups, nor did expression of PAR1 or PAR2. PAR3 was nearly undetectable. LV abundance of PAR4 by contrast increased with diabetes and correlated positively with active caspase-1. Genetic deletion of PAR4 in mice prevented the diet-induced cleavage of caspase-1, IL-1β and GDSMD. Right atrial appendages from patients with type 2 diabetes also showed higher levels of PAR4, but not of PAR1 or PAR2, than non-diabetic atrial tissue, along with increased abundance of cleaved caspase-1, IL-1β and GSDMD. Human cardiac fibroblasts maintained in high glucose conditions to mimic diabetes also upregulated PAR4 mRNA and protein, and increased PAR4-dependent IL-1β transcription and secretion in response to thrombin, while PAR1 and PAR2 expressions were unaltered. In conclusion, PAR4 drives caspase-1-dependent IL-1β production through the canonical NLRP3 inflammasome pathway in the diabetic heart, providing mechanistic insights into diabetes-associated cardiac thromboinflammation. The emerging PAR4-selective antagonists may provide a feasible approach to prevent cardiac inflammation in patients with diabetes.

RAS-mediated suppression of PAR3 and its effects on SCC initiation and tissue architecture occur independently of hyperplasia.

Proper epithelial development and homeostasis depends on strict control of oriented cell division. Current evidence shows that this process is regulated by intrinsic polarity factors and external spatial cues. Owing to the lack of an appropriate model system that can recapitulate the architecture of the skin, deregulation of spindle orientation in human epithelial carcinoma has never been investigated. Here, using an inducible model of human squamous cell carcinoma (SCC), we demonstrate that RAS-dependent suppression of PAR3 (encoded by PARD3) accelerates epithelial disorganization during early tumorigenesis. Diminished PAR3 led to loss of E-cadherin-mediated cell adhesion, which in turn contributed to misoriented cell division. Pharmacological inhibition of the MAPK pathway downstream of RAS activation reversed the defects in PAR3 expression, E-cadherin-mediated cell adhesion and mitotic spindle orientation. Thus, temporal analysis of human neoplasia provides a powerful approach to study cellular and molecular transformations during early oncogenesis, which allowed identification of PAR3 as a critical regulator of tissue architecture during initial human SCC development.

Increased protease activated receptors in the colon of patients with Hirschsprung's disease

PurposeThe pathophysiology of Hirschsprung's associated enterocolitis (HAEC) is not understood. Abnormal intestinal motility and altered intestinal epithelial barrier function have been suggested to play a key role in the causation of HAEC. Protease-activated receptors (PARs) 1 and 2, have been implicated in inflammatory reactions, intestinal permeability and modulation of motility in the gut. MethodsWe investigated PAR-1 and PAR-2 protein expression in aganglionic and ganglionic regions of patients with Hirschsprung's Disease (HSCR) (n = 10) versus normal control colon (n = 10). Protein distribution was assessed by using immunofluorescence and confocal microscopy. Gene and protein expression were quantified using quantitative real-time polymerase chain reaction (qPCR), western blot analysis, and densitometry. ResultsqPCR and Western blot analysis revealed that PAR-1 and PAR-2 expression was significantly increased in ganglionic and aganglionic bowel in HSCR compared to controls (p < 0.003). Confocal microscopy revealed strong PAR-1 and PAR-2 expression in smooth muscles, interstitial cells of Cajal (ICCs), platelet-derived growth factor-alpha receptor-positive (PDGFRα+) cells, enteric neurons and epithelium in the ganglionic and aganglionic bowel compared to controls. ConclusionIncreased PAR-1 and PAR-2 expression in the colon of patients with HSCR suggests that excessive local release of PAR activating proteases may trigger inflammatory responses leading to HAEC.

Thrombin inhibition by dabigatran attenuates endothelial dysfunction in diabetic mice

Diabetic patients have coagulation abnormalities, in which thrombin plays a key role. Whereas accumulating evidence suggests that it also contributes to the development of vascular dysfunction through the activation of protease-activated receptors (PARs). Here we investigated whether the blockade of thrombin attenuates endothelial dysfunction in diabetic mice. Induction of diabetes by streptozotocin (STZ) increased the expression of PAR1, PAR3, and PAR4 in the aorta. STZ-induced diabetic mice showed impairment of endothelial function, while the administration of dabigatran etexilate, a direct thrombin inhibitor, significantly attenuated endothelial dysfunction in diabetic mice with no alteration of metabolic parameters including blood glucose level. Dabigatran did not affect endothelium-independent vasodilation. Dabigatran decreased the expression of inflammatory molecules (e.g., MCP-1 and ICAM-1) in the aorta of diabetic mice. Thrombin increased the expression of these inflammatory molecules and the phosphorylation of IκBα, and decreased the phosphorylation of eNOSSer1177 in human umbilical endothelial cells (HUVEC). Thrombin significantly impaired the endothelium-dependent vascular response of aortic rings obtained from wild-type mice. Inhibition of NF-κB attenuated thrombin-induced inflammatory molecule expression in HUVEC and ameliorated thrombin-induced endothelial dysfunction in aortic rings. Dabigatran attenuated the development of diabetes-induced endothelial dysfunction. Thrombin signaling may serve as a potential therapeutic target in diabetic condition.

Thrombin Induces Secretion of Multiple Cytokines and Expression of Protease-Activated Receptors in Mouse Mast Cell Line.

Background Thrombin could elicit degranulation of mast cells involved in numerous physiologic and pathologic processes; however, the detailed scrutiny of this procedure and further research of possible cell signaling pathways are lacking. Methods P815 mouse mast cells were exposed to various concentrations of thrombin for 16 h. Expression of protease-activated receptor (PAR)1, PAR2, PAR3, and PAR4 mRNA in P815 was analyzed by quantitative real-time PCR (qRT-PCR) and the fittest concentration of thrombin was decided. Then, secretions of mediators from P815 stimulated by thrombin 0.2 U/ml were determined using enzyme-linked immunosorbent assay (ELISA) and Luminex liquichip; the possible cell signaling pathways were measured by immunoblotting. Furthermore, inhibition of thrombin inhibitor (hirudin), PAR1 inhibitor (SCH79797), and MAPK inhibitors (SB203580, PD98059, and SP600125) on the mediator section was evaluated by ELISA and Luminex liquichip. Results Thrombin 0.2 U/ml induced the elevated expression of PAR1, PAR2, PAR3, and PAR4, as well as the increasing level of phospho-IκBα, phospho-SAPK/JNK MAPK, phospho-P38 MAPK (Thr180/Tyr182), and phospho-ERK1/2 MAPK (p44/42) in P815. Secretion of vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α), interleukin- (IL-) 2, IL-6, chemokine ligand- (CCL-) 2, chemokine (C-X-C motif) ligand- (CXCL-) 1, and CXCL-5 from P815 increased apparently; this effect could be diminished by hirudin, whereas SCH79797 and MAPK inhibitors (SB203580, PD98059, and SP600125) diminish the secretions with weaker effect. Conclusion We found the expression of PAR mRNA in P815, activation of signaling pathways of nuclear factor-kappaB (NF-κB), and mitogen-activated protein kinases (MAPKs) including C-Jun NH2-terminal kinase (JNK), P38, and extracellular signal-regulated kinase 1/2 (ERK1/2), and the release of multiple inflammatory mediators stimulated by thrombin, as well as the inhibition of the inflammatory releases by hirudin, SCH79797, and MAPK inhibitors including SB203580, PD98059, and SP600125.

Quiescence/Mobilization of Hematopoietic Immature Cells Induced By Polyphenols through Modulation of APC/EPCR/PAR-1 Axis

The mechanism underlying quiescence and/or mobilization of hematopoietic stem cells and their bone marrow progenitors (HSPC) into circulation are tightly regulated for the continuous supply of peripheral blood cells; however, non-physiological or stress conditions, such as infections, can accelerate these mechanisms. Our results have shown that polyphenols modulate quiescence/mobilization of HSPC, but do not affect mature populations. Thrombin has been reported to induce the rapid HSPC mobilization through coagulation thrombin/PAR-1 axis, and quiescence is maintained across the APC/EPCR/PAR-1 axis (Nat. Med. 2015, 21:1307-17). Our objective was to investigate the effect of polyphenols on thrombin/PAR-1 and APC/EPCR/PAR-1 axis. C57BL/6J mice (6-8 weeks old) were treated with polyphenols from green tea extract (250 mg/kg body weight) orally (gavage) once every seven days and injected (i.p.) at day 7 with lipopolysaccharide (LPS) (100μg;Sigma) (n=6). The control group received vehicle and was injected with LPS (n=6). After 24h of LPS injection, mice were anesthetized for blood collection, and then sacrificed for bone marrow collection. PAR-1 and EPCR expression, nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) phosphorylation were evaluated in HSPC by flow cytometry. The functional ability of HSC was assessed by competitive repopulation assay. Vascular permeability was studied using Evans blue. After LPS injection, mice showed reduced expression of EPCR in bone marrow LSK parallel to an increase of PAR-1 expression in circulating immature and mature cells. Treatment of these mice with polyphenols partially prevented the reduced expression of EPCR in bone marrow LSK (13±3 vs 54±12; p&lt;0.05), but did not affect the increased PAR-1 expression in circulating immature and mature cells. Evans blue assay revealed a reduction in the vascular permeability of the bone marrow of LPS-injected mice treated with polyphenols (3.9±0.5 vs 2.1±0.1; p&lt;0.05). To assess whether polyphenols altered NO production, we measured NO levels and eNOS phosphorylation in immature LSK EPCRhigh (or LT-HSC) cells. NO production is activated by eNOS phosphorylation at Ser1177 and negatively regulated by eNOS phosphorylation at Thr495. LPS injection rapidly increased NO levels and eNOS phosphorylation at Ser1177 in bone marrow LSK of mice. Treatment of these mice with polyphenols reduced the percentage of bone marrow LSK EPCRhigh cells with higher intracellular NO (52±2.8 vs 28±5.6; p&lt;0.01) and increased eNOS phosphorylation at Thr495 in immature LSK. In order to evaluate the action of polyphenols on the functional ability of HSC, a competitive bone marrow repopulation assay was performed. Donor mice (C57BL/6J) received or not polyphenols followed by LPS injection (treated group: Polyphenols+LPS; control group: LPS), and bone marrow cells were transplanted (1:1) together with bone marrow cells of competitors (B6.SJL-PtprcaPepcb/BoyJ) in lethally irradiated recipients (B6.SJL-PtprcaPepcb/BoyJ). Mice were followed for 16 weeks and hematological analysis revealed no difference in circulating leukocytes, platelets or hemoglobin levels. Transplanted mice (recipients) presented a higher percentage of CD45+ cells from Polyphenols+LPS donors (33.7±13 vs 78.6±0.9; p&lt;0.05) in the peripheral blood, as well as increased number of T lymphocytes (6.7±4.5 vs 37.2±2.9; p&lt;0.05) and myeloid cells (68.5±1.7 vs 82.5±3.5; p&lt;0.05) from Polyphenols+LPS group. After 16 weeks, mice were euthanized and a higher percentage of LSK (or HSC) and LSK EPCRhigh (or LT-HSC) cells from Polyphenols+LPS donors were detected in the bone marrow, although only the percentage of LSK EPCRhigh was statistically different (0.0014±0.0001 vs 0.0032±0.001; p&lt;0.05). Taken together, our results indicate that polyphenols increased the functional ability of HSC in LPS-injected mice showing increased percentage of bone marrow LSK EPCRhigh cells, which are the most quiescent stem cells with strong self-renewal ability. Polyphenols reduced EPCR expression and NO production in immature cells of LPS-injected mice, exhibiting an anti-inflammatory effect that leads to the maintenance of barrier integrity and quiescence of cells, which was corroborated by reducing vascular permeability in the bone marrow. Thus, polyphenols appear to modulate quiescence/mobilization of HSPC through APC/EPCR/PAR-1 axis. Disclosures No relevant conflicts of interest to declare.

3308The critical roles of coagulation factors in inducing brown adipose tissue dysfunction and systemic metabolic disorder in obesity

Abstract Obese individuals are predisposed to cardio-metabolic disorders. Brown adipose tissue (BAT) is an active metabolic organ abundant with mitochondria, and studies suggest a potential role of BAT in the maintenance of metabolic health in rodents and humans. Metabolic stress causes BAT dysfunction, but the underlying mechanisms are largely unknown. Coagulation factor Xa (FXa) is critically involved in a coagulation cascade, and it is also known to mediate biological effects by the activation of protease-activated receptor (PAR)-signaling. Accumulating evidence shows that PAR1 contributes to tissue remodeling in cardiovascular system. Here we show a previously unknown role of FXa-PAR signaling in promoting BAT dysfunction and systemic metabolic disorder in a murine dietary obese model. Imposing a high fat diet (HFD) on C57BL/6NCr mice led to a marked increase in tissue factor (TF), coagulation factor VII and FXa in BAT. TF-FVIIa (activated form of FVII)-FXa complex is known to activate PAR1, and we found a significant increase in PAR1 expression in BAT upon metabolic stress. Administration of a FXa inhibitor ameliorated BAT whitening, improved thermogenic response and systemic glucose intolerance upon dietary obesity. In contrast, administration of warfarin did not show any phenotype in BAT. BAT specific TF and PAR1 over-expression model showed significant whitening of this tissue, which was associated with systemic glucose intolerance. BAT specific PAR1 KO mice improved glucose intolerance and thermogenic response under a metabolically stressed condition. In differentiated brown adipocytes, FXa markedly increased mitochondrial reactive oxygen species (ROS) and reduced mitochondrial membrane potential. Inhibition of PAR1 ameliorated FXa-induced mitochondrial ROS production and reduction in membrane potential. We also found that plasma FXa level did not increase in obese mice as well as in obese individuals. These results suggest the previously unknown role of coagulation systems in promoting BAT dysfunction, leading to systemic metabolic disorders. Maintenance of BAT homeostasis through the suppression of FXa-PAR1 signaling would become a new therapeutic target for obesity and diabetes.

P2563Effects of rivaroxaban on cardiac remodeling after experimental myocardial infarction in mice

Abstract Background Rivaroxaban, a direct activated factor X (FXa) inhibitor, has been established for prevention and treatment of arterial and venous thrombosis. Although FXa plays an important role in thrombosis, FXa also involves in inflammation via the protease-activated receptor (PAR)-1 and PAR-2 pathway. We assessed the hypothesis that rivaroxaban might protect cardiac remodeling after myocardial infarction (MI) in mice. Methods MI was induced in wild-type mice by permanent ligation of the left anterior descending coronary artery. At 1 day after MI, mice were randomly assigned to the rivaroxaban and vehicle groups. In the rivaroxaban group, the mice were provided with regular chow diet including rivaroxaban (2400ppm) after the randomization. We evaluated the cardiac function by echocardiography, expression of mRNA and protein in the infarcted and non-infarcted area 7 days after MI. Furthermore, we measured infarct size, infiltration of inflammatory cells by pathological analysis 7 days after MI. Results The fractional shortening (%FS) and Interventricular Septal thickness in diastole (IVSTd) was significantly improved 7 days after MI in the rivaroxaban group compared with the vehicle group (%FS, p=0.01; IVSTd, p=0.013). As for pathological analysis, rivaroxaban decreased infarct size (p=0.026) and the number of infiltrated macrophages in the non-infarcted area (p=0.011) compared with vehicle. The mRNA expression in tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β in the infarcted area and atrial natriuretic peptide (ANP) in the non-infarcted area was significantly lower in the rivaroxaban group compared with the vehicle (TNF-α, p=0.015; TGF-β, p=0.019; ANP, p=0.012). PAR-1 and PAR-2 mRNA expression in the infarcted area significantly decreased 7 days after MI in the rivaroxaban group compared with the vehicle (PAR-1, p=0.005; PAR-2, p=0.037). Furthermore, western blot analysis demonstrated that the phosphorylation of Extracellular Signal-regulated Kinase (ERK) and c-Jun N-terminal Kinase (JNK) in the non-infarcted area significantly decreased 7 days after MI in the rivaroxaban group compared with the vehicle (ERK, p=0.015; JNK, p=0.002). Conclusions The present study showed that rivaroxaban protected against cardiac dysfunction, probably due to the suppression of PAR-mediated increase of pro-inflammatory cytokines post-MI. Rivaroxaban might be potentially effective for improving the cardiac remodeling after MI. Acknowledgement/Funding This study was supported in part by trust-research grant from Bayer Yakuhin, Ltd.

Disruption of Robo2-Baiap2 integrated signaling drives cystic disease.

Hereditary renal cystic diseases are characterized by defects in primary cilia of renal tubular epithelial cells and abnormality of tubular epithelium, which ultimately result in the development of renal cysts. However, the mechanism leading from abnormality of the tubular epithelium to cystogenesis is not well understood. In this report, we demonstrate a critical role for Robo2 in regulating epithelial development, including ciliogenesis, polarization, and differentiation. We found that Robo2 deficiency results in cystic kidneys, and the cyst cells showed defective cilia and polarity defects in tubular epithelium. The cyst cells, less than terminally differentiated, continue to proliferate. We further established that Robo2 works with p53 as well as polarity and ciliary proteins (Par3, PKCς, ZO-2, and Claudin-2) to regulate these processes. Robo2 binds to Baiap2 (also known as IRSp53) through the IRSp53/MIM homology domain in renal epithelial cells. This binding allows Robo2 to phosphorylate MDM2 at Ser166 via Baiap2 and maintain p53 homeostasis. Disruption of the Robo2-Baiap2 complex causes MDM2 to be subjected to dephosphorylation, leading to a high level of active p53, and initiated p53-mediated cellular senescence via p21 and decreased the expression of ZO-1, ZO-2, PKCς, Par3, and Claudin-2 proteins, resulting in defects in epithelial development, including ciliogenesis, polarization, and differentiation. Importantly, double knockout of Robo2 and p53 rescued all the epithelial defects in kidneys compared with those in Robo2-knockout kidneys. Taken together, the present results demonstrate that Robo2 deficiency causes renal cystic disease, which is largely dependent on defective Robo2-Baiap2 integrated signaling in kidneys.

Sexual dimorphism in PAR2-dependent regulation of primitive colonic cells

BackgroundSexual dimorphism in biological responses is a critical knowledge for therapeutic proposals. However, gender differences in intestinal stem cell physiology have been poorly studied. Given the important role of the protease-activated receptor PAR2 in the control of colon epithelial primitive cells and cell cycle genes, we have performed a sex-based comparison of its expression and of the effects of PAR2 activation or knockout on cell proliferation and survival functions.MethodsEpithelial primitive cells isolated from colons from male and female mice were cultured as colonoids, and their number and size were measured. PAR2 activation was triggered by the addition of SLIGRL agonist peptide in the culture medium. PAR2-deficient mice were used to study the impact of PAR2 expression on colon epithelial cell culture and gene expression.ResultsColonoids from female mice were more abundant and larger compared to males, and these differences were further increased after PAR2 activation by specific PAR2 agonist peptide. The proliferation of male epithelial cells was lower compared to females but was specifically increased in PAR2 knockout male cells. PAR2 expression was higher in male colon cells compared to females and controlled the gene expression and activation of key negative signals of the primitive cell proliferation. This PAR2-dependent brake on the proliferation of male colon primitive cells was correlated with stress resistance.ConclusionsAltogether, these data demonstrate that there is a sexual dimorphism in the PAR2-dependent regulation of primitive cells of the colon crypt.

Baicalin Protects against Thrombin-Induced Cell Injury in Human Umbilical Vein Endothelial Cells.

Thrombin plays a pivotal role in the pathogenesis of atherosclerosis. Baicalin, an active flavonoid compound, was shown to attenuate the development of atherosclerosis, but the mechanism remains elusive. In the present study, the role and mechanism of baicalin in thrombin-induced cell injury was investigated in human umbilical vein endothelial cells (HUVECs). Our results showed that baicalin significantly reduced thrombin-induced apoptosis of HUVECs. Additional experiments showed that baicalin inhibited thrombin-induced NF-κB activation and PAR-1 expression. In addition, baicalin decreased thrombin-induced PAR-1 expression by inhibiting ERK pathway. These results indicated that baicalin has protective effects on thrombin-induced cell injury in HUVECs possibly through inhibition of PAR-1 expression and its downstream NF-κB activation, which was mediated by ERK1/2 activation.

Small Intestine Protection of Mica Against Non-Steroidal Anti-Inflammatory Drugs-Injury Through ERK1/2 Signal Pathway in Rats.

Objective: The impact of non-steroidal anti-inflammatory drugs (NSAIDs) to damage the small intestine has been well known. Mica, one kind of natural clay, has been widely marketed in China for the treatment of gastric diseases. However, the role and mechanism of mica in small intestinal injure is still unknown. The study was designed to declare the effects of mica on intestinal injury induced by diclofenac in rats. Methods: Rats were randomly divided into control, model, PAR-2 agonist group (SLIGRL-NH2group), control peptide group (LRGILS-NH2 group), and ERK blocker group (eight mice per group). Morphological changes of mucous membrane of small intestine were observed, and the expression of tryptase, PAR-2, and p-ERK1/2 was measured by immunohistochemistry and western blot. PAR-2 mRNA was tested by qRT-PCR. Rats were also randomly divided into control, model, and mica group (eight mice per group). Morphological changes of mucous membrane were observed. The expression of tryptase, PAR-2, and p-ERK1/2 was measured by immunohistochemistry. Results: The expression of trypsin, PAR-2, and p-ERK1/2 was increased in model group compared with control. The expression of PAR-2 and p-ERK1/2 was increased in SLIGRL-NH2 group compared with model, but not LRGILS-NH2 group. The expression of PAR-2 was down-regulated in ERK blocker group compared with SLIGRL-NH2 group. Macroscopically visible lesions of mucous membrane were positively correlated with the expression of PAR-2 and p-ERK1/2. Furthermore, we also found that mica could inhibit small intestinal injure, as evidenced by the improvement of macroscopic damage. Tryptase, PAR-2, and p-ERK1/2 expression was down-regulated in mica group compared with model group. Conclusion: Mica inhibit small intestinal injury induced by NSAIDs via ERK signaling pathway.

Abstract 852: The Pathological Role of Coagulation Factors in Promoting Brown Adipose Tissue Dysfunction and Systemic Metabolic Disorder in Obesity

Obese individuals are predisposed to cardio-metabolic disorders. Brown adipose tissue (BAT) is an active metabolic organ abundant with mitochondria, and studies suggest a potential role of BAT in the maintenance of metabolic health in rodents and humans. Metabolic stress causes BAT dysfunction, but the underlying mechanisms are largely unknown. Coagulation factor Xa (FXa) is critically involved in a coagulation cascade, and it is also known to mediate biological effects by the activation of protease-activated receptor (PAR)-signaling. Accumulating evidence shows that PAR1 contributes to tissue remodeling in cardiovascular system. Here we show a previously unknown role of FXa-PAR signaling in promoting BAT dysfunction and systemic metabolic disorder in a murine dietary obese model. Imposing a high fat diet (HFD) on C57BL/6NCr mice led to a marked increase in tissue factor (TF), coagulation factor VII and FXa in BAT. TF-FVIIa (activated form of FVII)-FXa complex is known to activate PAR1, and we found a significant increase in PAR1 expression in BAT upon metabolic stress. Administration of a FXa inhibitor ameliorated BAT whitening, improved thermogenic response and systemic glucose intolerance upon dietary obesity. In contrast, administration of warfarin did not show any phenotype in BAT. BAT specific TF and PAR1 over-expression model showed significant whitening of this tissue, which was associated with systemic glucose intolerance. BAT specific PAR1 KO mice improved glucose intolerance and thermogenic response under a metabolically stressed condition. In differentiated brown adipocytes, FXa markedly increased mitochondrial reactive oxygen species (ROS) and reduced mitochondrial membrane potential. Inhibition of PAR1 ameliorated FXa-induced mitochondrial ROS production and reduction in membrane potential. We also found that plasma FXa level did not increase in obese mice as well as in obese individuals. These results suggest the previously unknown role of coagulation systems in promoting BAT dysfunction, leading to systemic metabolic disorders. Maintenance of BAT homeostasis through the suppression of FXa-PAR1 signaling would become a new therapeutic target for obesity and diabetes

Involvement of TLR4/ CXCL9/ PREX-2 pathway in the development of hepatocellular carcinoma (HCC) and the promising role of early administration of lactobacillus plantarum in Wistar rats

Improvement of gut microbiota may help in preventing the progression of cirrhosis. We supposed that Lactobacillus Plantarum (L. Plantarum) protects the cirrhotic liver through suppression of TLR4/ CXCL9/ PREX-2. Rats were divided into two groups. Group I, lasts for six weeks and Group II lasts for 12 weeks. Each group was subdivided into: naïve, Lactobacillus Plantarum (L. Plantarum), thioacetamide (TAA) and TAA + L. Plantarum. Liver function tests, α fetoprotein (AFP) levels, CXCL9, PREX-2 and TLR4 expression were assessed. Histological studies were performed. TAA induced significant deterioration in liver functions and increased AFP. There was periportal cirrhosis, vacuolated hepatocytes, decrease hepatocyte parrafin-1 (hep par-1) expression, increase proliferating cell nuclear antigen (PCNA) positive nuclei and cytokeratin AE1/AE3. The PCR results showed significant increase in TLR4, CXCL9 and PREX-2 expression. Early administration of L. Plantarum significantly decreased the expression of TLR4, CXCL9 and PREX-2 together with improvement in liver function and prevented the pathological changes. The cirrhotic complications induced by TAA are through activation of TLR4/ CXCL9/ PREX-2 pathway and could be prevented by the early administration of L. Plantarum.

Protease-Activated Receptors 1 and 3 are Differentially Expressed on Human Monocyte Subsets and are Upregulated by Lipopolysaccharide Ex Vivo and In Vivo

Monocytes are activated in inflammatory conditions via a variety of cytokine receptors as well as in a procoagulatory setting through thrombin, acting upon protease-activated receptors (PARs). This study investigated the expression pattern of PAR1 and PAR3 on human monocyte subsets. Furthermore, a possible regulation of the expression of PAR1 and PAR3 in these cells by inflammatory activation were studied. CD16+ monocytes showed significantly higher levels of PAR1 and PAR3 as compared with CD16- monocytes. Ex vivo treatment of whole blood with lipopolysaccharide (LPS) increased PAR1 and PAR3 messenger ribonucleic acid (mRNA) in human monocytes. In addition, increase of PAR1 was seen in all three subsets upon LPS treatment, whereas PAR3 increased significantly only in CD16- monocytes and nonclassical CD16+ monocytes. Protein levels of PAR1 and PAR3 significantly increased on monocytes in vivo in human endotoxemia 1 hour after LPS infusion. PAR1 increased significantly in CD16- monocytes and nonclassical CD16+ monocytes. In this in vivo model, PAR3 was also significantly elevated in CD16- monocytes and increased slightly albeit not significantly in CD16+ monocytes. Endotoxemia increased plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) expression in monocytes in humans. Pretreatment of healthy volunteers with the PAR1 antagonist vorapaxar blocked the increase in PAI-1 but not the increase in TF. We here provide new evidence for a critical role for monocytes as cellular mediators that contribute to the activation of coagulation in diseases characterized by an inflammatory state.

Thrombin Signaling Promotes Pancreatic Adenocarcinoma through PAR-1-Dependent Immune Evasion.

Pancreatic ductal adenocarcinoma (PDAC) is associated with robust activity of the coagulation system. To determine mechanisms by which clotting factors influence PDAC tumor progression, we generated and characterized C57Bl/6-derived KPC (KRasG12D, TRP53R172H ) cell lines. Tissue factor (TF) and protease-activated receptor-1 (PAR-1) were highly expressed in primary KPC pancreatic lesions and KPC cell lines similar to expression profiles observed in biopsies of patients with PDAC. In allograft studies, tumor growth and metastatic potential were significantly diminished by depletion of TF or Par-1 in cancer cells or by genetic or pharmacologic reduction of the coagulation zymogen prothrombin in mice. Notably, PAR-1-deleted KPC cells (KPC-Par-1KO) failed to generate sizable tumors, a phenotype completely rescued by restoration of Par-1 expression. Expression profiling of KPC and KPC-Par-1KO cells indicated that thrombin-PAR-1 signaling significantly altered immune regulation pathways. Accordingly, KPC-Par-1KO cells failed to form tumors in immune-competent mice but displayed robust tumor growth comparable to that observed with control KPC cells in immune-compromised NSG mice. Immune cell depletion studies indicated that CD8 T cells, but not CD4 cells or natural killer cells, mediated elimination of KPC-Par-1KO tumor cells in C57Bl/6 mice. These results demonstrate that PDAC is driven by activation of the coagulation system through tumor cell-derived TF, circulating prothrombin, and tumor cell-derived PAR-1 and further indicate that one key mechanism of thrombin/PAR-1-mediated tumor growth is suppression of antitumor immunity in the tumor microenvironment. SIGNIFICANCE: The tissue factor-thrombin-PAR-1 signaling axis in tumor cells promotes PDAC growth and disease progression with one key mechanism being suppression of antitumor immunity in the microenvironment.

Indolylkojyl methane analogue IKM5 potentially inhibits invasion of breast cancer cells via attenuation of GRP78

More than 90% of the breast cancer deaths occur due to the metastasis of the cancer cells to secondary organ sites. Increased Glucose-regulated protein 78 (GRP78) expression is critical for epithelial-mesenchymal transition (EMT) and invasion in breast cancer resulting in poor patient survival outcomes. Therefore, there is an urgent need of potential inhibitors of GRP78 for the abrogation of invasion and metastasis in breast cancer. We investigated the effect of IKM5 (2-(1-(1H-indol-3-yl)octyl)-3-hydroxy-6-(hydroxymethyl)-4H-pyran-4-one) (a novel Indolylkojyl methane analogue) on invasion abilities of human breast cancer cells employing invadopodia formation, Matrigel invasion assays, and mouse models for metastasis. The mechanism underlying the anti-invasive effect of IKM5 was examined through molecular docking, immunoblotting, immunocytochemistry, co-immunoprecipitation analysis, siRNA silencing, and sub-cellular fractionation studies. Treatment with IKM5 at its sub-toxic concentration (200nM) suppressed invasion and invadopodia formation, and growth factor-induced cell scattering of aggressive human breast cancer MDA-MB-231, MDA-MB-468, and MCF7 cells. IKM5 spontaneously binds to GRP78 (Ki = 1.35µM) and downregulates its expression along with the EMT markers MMP-2, Twist1, and Vimentin. Furthermore, IKM5 amplified the expression and nuclear translocation of tumor suppressor Par-4 to control NF-kB-mediated pro-EMT activities. Interestingly, IKM5 disrupts the interaction between GRP78 and TIMP-1 by inhibiting GRP78 in a Par-4-dependent manner. Moreover, IKM5 inhibited tumor growth and lung metastasis at a safe dose of 30mg/kg/body weight. Our study warrants IKM5, a potential anticancer agent that can abrogate invasion and metastasis, suggesting its clinical development for the treatment of patients with advanced breast cancer.

Reconstruction of Par-dependent polarity in apolar cells reveals a dynamic process of cortical polarization.

Cellular polarization is fundamental for various biological processes. The Par network system is conserved for cellular polarization. Its core complex consists of Par3, Par6, and aPKC. However, the general dynamic processes that occur during polarization are not well understood. Here, we reconstructed Par-dependent polarity using non-polarized Drosophila S2 cells expressing all three components endogenously in the cytoplasm. The results indicated that elevated Par3 expression induces cortical localization of the Par-complex at the interphase. Its asymmetric distribution goes through three steps: emergence of cortical dots, development of island-like structures with dynamic amorphous shapes, repeating fusion and fission, and polarized clustering of the islands. Our findings also showed that these islands contain a meshwork of unit-like segments. Furthermore, Par-complex patches resembling Par-islands exist in Drosophila mitotic neuroblasts. Thus, this reconstruction system provides an experimental paradigm to study features of the assembly process and structure of Par-dependent cell-autonomous polarity.

Alteration in Uterine Protease-Activated Receptor 2 Expression in Preterm Birth Induced Experimentally in Brp-39 Null Mutant Mice.

Breast regression protein 39 (Brp-39) is a mouse homolog of human Chitinase 3-like 1, which belongs to the 18-glycosyl-hydrolase family and plays a role in inflammatory reaction and tissue remodeling. The aim of this study is to investigate the role of Brp-39 in a mouse model of preterm birth. Pregnant wild-type (WT) or Brp-39(-/-) mice were injected intraperitoneally with lipopolysaccharide (LPS) at embryonic day 15. Pregnancy outcomes were evaluated for 24 hours after LPS injection. Quantitative real-time polymerase chain reaction and immunoblotting were performed to analyze messenger RNA (mRNA) and protein expressions of cytokines and contraction-associated proteins in uterine and/or placental tissue after LPS injection. LPS injection led to preterm birth in both WT and Brp-39(-/-) mice, but the proportion of pubs delivered was reduced in Brp-39(-/-) mice, along with a longer interval from the LPS injection to delivery, compared to WT mice. Inflammatory cell infiltration and mRNA expression of cytokines and Ptgs2 in the uteri and the placentas were not significantly different between WT and Brp-39(-/-) mice. Par-2 mRNA expression in the WT uteri was increased before delivery after LPS injection and decreased after delivery, while there was no significant change in Par-2 expression in the Brp-39(-/-) uteri. Protein expressions of Par-2 and Ptgs2 were lower in the Brp-39(-/-) uteri than in the WT uteri before and after delivery. Attenuated preterm birth in Brp-39(-/-) mice indicates the significance of Brp-39 during murine preterm birth. Altered expression of Par-2 in Brp-39(-/-) uteri suggests its potential role in attenuated preterm birth of Brp-39(-/-) mice.