Par-4 Expression Research Articles

Icaritin Reverses Multidrug Resistance of Multiple Myeloma Cell Line KM3/BTZ

To investigate the reversing effect of icaritin on multidrug resistance of multiple myeloma cell lines KM3/BTZ and its underlying mechanism. KM3/BTZ cells were established by a gradually ascending gradient induction of bortezomib (BTZ). The sensitivities of KM3 and KM3/BTZ cells to 7 chemotherapeutic drugs, the inhibition and reversal effects of icaritin on proliferation and drug-resistance of KM3/BTZ cells were analyzed by MTT. The apoptosis was analyzed by flow cytometry, and the expression of Par-4, HSP27 and P-gp were detected by Western blot. KM3/BTZ cells were not only resistant to BTZ, but also to other 6 chemotherapeutic drugs. The resistance index (RI) to BTZ was 17.84, and higher than that of other chemotherapeutic drugs. Icaritin inhibited the proliferation and induced the apoptosis of KM3/BTZ cells. The IC50 value of BTZ decreased from 0.345 µg/ml to 0.149 µg/ml, and the reversal index was 2.38 (P<0.05). The expression of Par-4 protein increased in a concentration-dependent manner, while the expression of HSP27 and P-gp were down-regulated. Icaritin can inhibit cell proliferation and induce apoptosis of KM3/BTZ cells, moreover, can effectively reverse the multidrug resistance of KM3/BTZ cells. The mechanism may be related with down-regulation of HSP27 and P-gp expression, and up-regulation of Par-4 expression.

Differential roles of kallikrein-related peptidase 6 in malignant transformation and ΔNp63β-mediated epithelial-mesenchymal transition of oral squamous cell carcinoma

We previously reported that epithelial-to-mesenchymal transition (EMT) was mediated by ΔNp63β in oral squamous cell carcinoma (OSCC). In this study, DNA microarray analyses were performed using ΔNp63β-overexpressing OSCC cells to identify genes associated with ΔNp63β-mediated EMT. Thereby, we focused on kallikrein-related peptidase (KLK) 6, most up-regulated following ΔNp63β-overexpression, that activates protease-activated receptors (PARs). In RT-PCR analyses, ΔNp63 was positively associated with KLK6 and PAR2 and negatively with PAR1 in OSCC cells. By ΔNp63 knockdown, KLK6 and PAR2 expression was decreased and PAR1 was increased. Furthermore, KLK6 knockdown led to enhancing migration and invasion, and inhibiting proliferation, suggesting EMT-phenotypes. Although, in the KLK6 or PAR2 knockdown cells, phosphorylation of ERK was reduced, it was restored in the KLK6 knockdown OSCC cells treated with recombinant KLK6 proteins. Immunohistochemistry showed ΔNp63, KLK6, and PAR2 were more strongly expressed in the epithelial dysplasia and central region of OSCC than normal oral epithelium, whereas PAR1 expression was undetectable. Interestingly, at the invasive front of OSCC, ΔNp63, KLK6, and PAR2 were reduced, but PAR1 was elevated. In addition, the OSCC patients with decreasing KLK6 expression at the invasive front had more unfavourable prognosis. These results suggested differential roles of KLK6 in malignant transformation and EMT; high ΔNp63β expression up-regulates KLK6-PAR2 and down-regulates PAR1, inducing malignant transformation in oral epithelium with stimulating proliferation through ERK signal activation. Moreover, KLK6-PAR2 expression is down-regulated and PAR1 is up-regulated when ΔNp63β expression is decreased, leading to EMT with enhancing migration and invasion through ERK signal reduction at the invasive front.

Tissue Factor-Expressing Tumor-Derived Extracellular Vesicles Activate Quiescent Endothelial Cells via Protease-Activated Receptor-1.

Tissue factor (TF)-expressing tumor-derived extracellular vesicles (EVs) can promote metastasis and pre-metastatic niche formation, but the mechanisms by which this occurs remain largely unknown. We hypothesized that generation of activated factor X (FXa) by TF expressed on tumor-derived EV could activate protease-activated receptors (PARs) on non-activated endothelial cells to induce a pro-adhesive and pro-inflammatory phenotype. We obtained EV from TF-expressing breast (MDA-MB-231) and pancreatic (BxPC3 and Capan-1) tumor cell lines. We measured expression of E-selectin and secretion of interleukin-8 (IL-8) in human umbilical vein endothelial cells after exposure to EV and various immunologic and chemical inhibitors of TF, FXa, PAR-1, and PAR-2. After 6 h of exposure to tumor-derived EV (pretreated with factor VIIa and FX) in vitro, endothelial cells upregulated E-selectin expression and secreted IL-8. These changes were decreased with an anti-TF antibody, FXa inhibitors (FPRCK and EGRCK), and PAR-1 antagonist (E5555), demonstrating that FXa generated by TF-expressing tumor-derived EV was signaling through endothelial PAR-1. Due to weak constitutive PAR-2 expression, these endothelial responses were not induced by a PAR-2 agonist peptide (SLIGKV) and were not inhibited by a PAR-2 antagonist (FSLLRY) after exposure to tumor-derived EV. In conclusion, we found that TF-expressing cancer-derived EVs activate quiescent endothelial cells, upregulating E-selectin and inducing IL-8 secretion through generation of FXa and cleavage of PAR-1. Conversion of resting endothelial cells to an activated phenotype by TF-expressing cancer-derived EV could promote cancer metastases.

MiR-17-3P regulates the proliferation and survival of colon cancer cells by targeting Par4.

Colorectal cancer (CRC) is a common malignancy worldwide. However, the pathogenesis by which CRC progression occurs remains to be elucidated. The present study investigated the role of miRNA (miR)‑17‑3P in the regulation of CRC cell survival. Firstly, miR‑17‑3P expression was aberrantly upregulated in human CRC tumor tissues compared with controls. Further results demonstrated that the proliferation capacity of human CRC SW480 and LoVo cells was significantly increased by an miR‑17‑3P specific mimic, and was inhibited by miR‑17‑3P silencing. Conversely, the apoptosis of human CRC cells was remarkably decreased by miR‑17‑3P mimic, and enhanced by miR‑17‑3P suppression compared with control. Additionally, it was observed that there was a potential binding site of miR‑17‑3P on the 3'‑untranslated region of Prostate apoptosis responde‑4 (Par4) and miR‑17‑3P may directly target Par4 mRNA. In human CRC cells, an miR‑17‑3P inhibitor significantly upregulated Par 4 expression, however the miR‑17‑3P mimic reduced Par4expression. Furthermore, Par4 expression exhibited an inhibitory effect on the proliferation of CRC cells transfected with miR‑17‑3P mimic, and exhibited a promoting role in the repressed apoptosis by miR‑17‑3P mimic. Inconclusion, the results of the present study demonstrated that miR‑17‑3P is important in CRC cell survival by targeting Par4, indicating a novel finding regarding human CRC progression.

Elevated expression of Par3 promotes prostate cancer metastasis by forming a Par3/aPKC/KIBRA complex and inactivating the hippo pathway

BackgroundProstate cancer (PCa) is one of the most frequent tumors and leading cause of cancer deaths among males worldwide. The majority of deaths are due to recurrence and subsequent development of the metastatic cancer. Although loss or dislocalization of polarity proteins has been implicated in embryogenesis deficiency and tumorigenesis, association of polarity protein expression levels with tumor metastasis remains unclear.MethodsBioinformatics, qRT-PCR, western blot and immunohistochemical (IHC) analyses were used to examine expression of Par3, a key component of polarity-associated partitioning defective (PAR) complex, in primary and metastatic clinical PCa samples. Loss-of-function and gain-of-function studies in vitro and in vivo were performed to determine the functions of Par3 during metastasis of PCa. Co-immunoprecipitation (co-IP), western blot, immunofluorescence (IF), chromatin immunoprecipitation (ChIP) and qRT-PCR analyses were conducted to investigate the underlying mechanism for the function of Par3 on PCa metastasis.ResultsIn this study, we found that elevated expression of Par3 is positively associated with PCa metastasis. Knockdown of Par3 inhibits PCa cell migration and invasion in vitro and tumor metastasis in vivo, whereas overexpression of Par3 yields the opposite results. Mechanistically, Par3 suppresses phosphorylation of LATS to inactivate the Hippo pathway and enhances nuclear translocation of YAP by sequestrating KIBRA from the KIBRA/Merlin/FRMD6 complex and forming a Par3/aPKC/KIBRA complex. Stable knockdown of Par3 leads to restoration of the KIBRA/Merlin/FRMD6 complex and activation of the Hippo pathway, and then results in an inhibition on YAP nuclear translocation. In addition, in conjunction with the TEA domain (TEAD) transcription factor family, intranuclear YAP promotes the transcription of several pro-metastatic genes such as the matrix metalloproteinase (MMP) family, Zeb1, Snail1 and Twist1. Moreover, knockdown of Par3 downregulates expression of these pro-metastatic genes.ConclusionsOur findings indicate that elevated expression of Par3 promotes PCa metastasis via KIBRA sequestration-mediated inactivation of the Hippo pathway to upregulate expression of pro-metastatic genes. Downregulation of Par3 expression may serve as a potential treatment approach for PCa metastasis by activating the Hippo pathway.

Possible role of rivaroxaban in attenuating pressure-overload-induced atrial fibrosis and fibrillation

BackgroundCoagulation factor Xa (FXa) promotes thrombus formation and exacerbates inflammation via activation of protease-activated receptor (PAR)-2. We tested the hypothesis of whether administration of direct oral anticoagulant, rivaroxaban, would attenuate transverse aortic constriction (TAC)-induced atrial inflammatory fibrosis and vulnerability to atrial fibrillation (AF) in mice. MethodsTen-week-old male CL57/B6 mice were divided into a sham-operation (CNT) group and TAC-surgery group. These two groups were then subdivided into vehicle (VEH) and rivaroxaban (RVX) treatment (30μg/g/day) groups. We assessed PAR-2 expression in response to TAC-related stimulation using rat cultured cells. ResultsTAC-induced left atrial thrombus formation was not observed in the TAC-RVX group. Cardiac PAR-2 upregulation was observed in both TAC groups. In the quantitative analysis of mRNA levels, cardiac PAR-2 upregulation was attenuated in the TAC-RVX group compared to TAC-VEH group. In histological evaluation, the TAC-VEH group showed cardiac inhomogeneous interstitial fibrosis and abundant infiltration of macrophages, which were attenuated by RVX administration. Electrophysiological examination revealed that AF duration in the TAC group was shortened by RVX administration. TAC-induced protein overexpression of monocyte chemoattractant protein-1, and mRNA overexpression of tumor necrosis factor-α, interleukin (IL)-1β and IL-6 in the left atrium was suppressed by RVX treatment. In cardiac fibroblasts, persistent intermittent stretch upregulated PAR-2, which was suppressed by RVX pre-incubation. ConclusionsThese observations demonstrate that coagulation FXa inhibitor probably has a cardioprotective effect against pressure-overload-induced atrial remodeling.

HAT1 induces lung cancer cell apoptosis via up regulating Fas.

The dysfunction of apoptosis is one of the factors contributing to lung cancer (LC) growth. Histone acetyltransferase HAT1 can up regulate cell apoptosis. This study aims to investigate the mechanism by which HAT1 induces LC cell (LCC) apoptosis via up regulating the expression of Fas. In this study, the surgically removed human LC tissues were collected. LCCs were isolated from the LC tissues and analyzed for the expression of HAT1 and Fas by RT-qPCR and Western blotting. We observed that the expression of Fas was negatively correlated with PAR2 in LCCs. Activation of PAR2 suppressed the expression of Fas in normal lung epithelial cells. The expression of HAT1 was lower and positively correlated with Fas expression and negatively correlated with PAR2 expression in LCCs. Activation of PAR2 suppressed Fas expression in lung epithelial cells via inhibiting HAT1. Restoration of HAT1 expression restored Fas expression in LCCs and induced LCC apoptosis. In conclusion, less expression of HAT1 in LCCs was associated with the pathogenesis of LC. Up regulation of HAT1 expression in LCCs can induce LCCs apoptosis, which may be a potential novel therapy for the treatment of LC.

Direct thrombin inhibition with dabigatran attenuates pressure overload-induced cardiac fibrosis and dysfunction in mice

IntroductionThe multifunctional serine protease thrombin exerts proinflammatory and profibrotic cellular effects that may contribute to cardiac remodeling. This study was designed to investigate whether direct thrombin inhibition with dabigatran attenuates myocardial injury in the setting of pressure overload-induced heart failure. Material and methodsTransverse aortic constriction (TAC) surgery was performed on C57Bl/6J male mice to elicit cardiac hypertrophy. TAC, or sham, mice were randomly assigned to receive chow supplemented with the oral anticoagulant, dabigatran etexilate, or placebo. ResultsDabigatran did not affect cardiac hypertrophy, as measured by heart weight-to-body weight or the heart weight-to-tibia length, although a non-significant reduction in myocardial hypertrophic markers (ANP, BNP and MHC) occurred. Dabigatran reduced perivascular fibrosis by 25%, interstitial fibrosis by 54%, and the expression of myocardial fibrosis markers collagen I & III, MMP9, SMA, and PAR-1. These changes were associated with significant improvement in both coronary flow reserve and global left ventricular function. In cultured cardiac fibroblasts, dabigatran decreased thrombin and PAR-1-mediated collagen deposition by 30% and 37%, respectively. ConclusionsDabigatran attenuates cardiac fibrosis in the setting of pressure overload and improves coronary flow reserve and global cardiac function possibly by inhibiting thrombin activity and down-regulating PAR-1 expression in the absence of an effect on cardiomyocyte hypertrophy.

PAR-1 and PAR-2 Expression Is Enhanced in Inflamed Odontoblast Cells

Protease-activated receptors (PARs) are G protein–coupled receptors, which are activated by proteolytical cleavage of the amino-terminus and act as sensors for extracellular proteases. We hypothesized that PAR-1 and PAR-2 can be modulated by inflammatory stimulus in human dental pulp cells. PAR-1 and PAR-2 gene expression in human pulp tissue and MDPC-23 cells were analyzed by quantitative polymerase chain reaction. Monoclonal PAR-1 and PAR-2 antibodies were used to investigate the cellular expression of these receptors using Western blot, flow cytometry, and confocal microscopy in MDPC-23 cells. Immunofluorescence assays of human intact and carious teeth were performed to assess the presence of PAR-1 and PAR-2 in the dentin-pulp complex. The results show for the first time that human odontoblasts and MDPC-23 cells constitutively express PAR-1 and PAR-2. PAR-2 activation increased significantly the messenger RNA expression of matrix metalloproteinase (MMP)–2, MMP-9, MMP-13, and MMP-14 in MDPC-23 cells (P < 0.05), while the expression of these enzymes decreased significantly in the PAR-1 agonist group (P < 0.05). The high-performance liquid chromatography and matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry analysis showed the presence of MMP-13 activity cleaving PAR-1 at specific, noncanonical site TLDPRS42↓F43LL in human dental pulp tissues. Also, we detected a presence of a trypsin-like activity cleaving PAR-2 at canonical site SKGR20↓S21LIGRL in pulp tissues. Confocal microscopy analysis of human dentin-pulp complex showed intense positive staining of PAR-1 and PAR-2 in the odontoblast processes in dentinal tubules of carious teeth compared to intact ones. The present results support the hypothesis of activation of the upregulated PAR-1 and PAR-2 by endogenous proteases abundant during the inflammatory response in dentin-pulp complex.

Platelet function and microparticle levels in atrial fibrillation: Changes during the acute episode

BackgroundThrombotic risk constitutes a major complication of atrial fibrillation (AF). Platelets and microparticles (MPs) are important for hemostasis and thrombosis, however their participation during AF is not well known. The aim of this study was to characterize platelet function and MPs procoagulant and fibrinolytic activity in AF patients and to determine the effects of an acute-AF episode. MethodsBlood was collected from paroxysmal (21) and persistent (16) AF patients referred for AF catheter ablation. Ten patients in sinus rhythm for 10days were induced in AF allowing comparisons of left atrium samples before and after induction. Platelet aggregation with ADP, TRAP, collagen, and ristocetin was studied. Platelet surface expression of PAR-1, αIIbβ3, GPIb and P-selectin were evaluated by flow cytometry, and MPs-associated procoagulant and fibrinolytic activity levels were determined by functional assays. ResultsA specific reduction in platelet aggregation to TRAP, activating the thrombin receptor PAR-1, was found in all AF patients. No differences in platelet receptor expression were found. Yet, after acute-induced AF, the platelet response was improved. Furthermore, a significant decrease of left atrium tissue factor-dependent procoagulant activity of MPs was observed. ConclusionAcute episodes of AF results in a decrease in MPs-associated tissue factor activity, possibly corresponding to consumption, which in turn favors coagulation and the local production of thrombin. A decreased platelet basal aggregation to TRAP may result from PAR1 desensitization, whereas the improved response after an induced episode of AF suggests activation of coagulation and PAR1 re-sensitization.

MiR-107 Promotes Proliferation and Inhibits Apoptosis of Colon Cancer Cells by Targeting Prostate Apoptosis Response-4 (Par4).

Colorectal cancer (CRC) is one of the most common malignancies in the world, with a high incidence and a high mortality. However, the pathogenesis of CRC carcinogenesis is still unexplored. In this study, we investigated the role of miR-107 in the regulation of CRC cell proliferation and apoptosis. First, the expression of miR-107 was observed to be aberrantly increased in human CRC tumor tissues and cell lines when compared to the colonic control tissues and colon epithelial cells. Further study showed that the proliferative and apoptotic capacities of human CRC SW480 and LoVo cells were aberrantly regulated by miR-107. The proliferation of SW480 and LoVo cells was remarkably enhanced by the miR-107 mimic but suppressed by the miR-107 inhibitor when compared to the negative control. On the contrary, the apoptotic rate of both SW480 and LoVo cells was significantly inhibited by miR-107 overexpression but increased by miR-107 inhibition. In addition, we identified prostate apoptosis response-4 (Par4) as a direct target of miR-107 with a potential binding site on the 3′-UTR of mRNA, as evaluated by bioinformatics prediction and luciferase reporter assay. Par4 expression levels were significantly inhibited by the miR-107 mimic but upregulated by the miR-107 inhibitor in both SW480 and LoVo cells. Compared to the control, the increase in Par4 expression significantly inhibited the induction role of miR-107 in the proliferation of SW480 and LoVo cells, and the apoptotic rate of cells repressed by the miR-107 mimic was also reversed by Par4 overexpression. In summary, our results demonstrated that miR-107 exerts a positive role in the survival of CRC cells by directly targeting Par4. This might reveal a novel understanding about human CRC pathogenesis.

Inhibitory effect of Par-4 combined with cisplatin on human Wilms’ tumor cells

Wilms' tumor is associated with a high treatment success rate, but there is still a risk of recurrence. Cisplatin, which is one of the chemotherapeutic agents used for its treatment, is associated with a very high rate of resistance. Par-4 (prostate apoptosis response 4) is a tumor suppressor, which is capable of sensitizing tumor cells to chemotherapy. Therefore, the aim of this study was to determine whether combined treatment with Par-4 and cisplatin is effective for inhibiting growth of Wilms' tumor. Wilms' tumor and control cell samples were collected and analyzed by immunofluorescence assay and immunohistochemistry. Total proteins extracted from cultured cells were analyzed using western blotting and flow cytometry. In addition, a mouse xenograft model was established. We discovered significantly low expression of Par-4 in the tumor tissue, which was positively correlated with high expression of GRP78 (glucose-regulated protein 78). In addition, we found that ectopic Par-4 co-localized with cell surface GRP78 and induced high expression of the endoplasmic reticulum proteins ATF4 and BAX, which activated the endoplasmic reticulum apoptosis pathway. Moreover, treatment with ectopic Par-4 and cisplatin suppressed xenograft growth in nude mice. In conclusion, our results showed that Par-4 overexpression and cisplatin had a synergistic effect on SK-NEP-1 cells, as a result of which cell growth was inhibited and cellular apoptosis was induced. Thus, in vitro and in vivo upregulation of Par-4 expression is indispensable for the trafficking of GRP78 to the cell membrane and subsequent apoptosis of cancer cells.

Roles of the PDZ-binding motif of HPV 16 E6 protein in oncogenic transformation of human cervical keratinocytes.

The high‐risk human papillomavirus E6 proteins have been shown to interact with and lead to degradation of PDZ‐domain‐containing proteins through its carboxy‐terminal motif. This PDZ‐binding motif plays important roles in transformation of cultured cells and carcinogenesis of E6‐transgenic mice. However, its biological effects on the natural host cells have not been elucidated. We have examined its roles in an in vitro carcinogenesis model for cervical cancer, in which E6 and E7 together with activated HRAS (HRASG 12V) can induce tumorigenic transformation of normal human cervical keratinocytes. In this model, E6Δ151 mutant, which is defective in binding to PDZ domains, almost lost tumorigenic ability, whereas E6SAT mutant, which is defective in p53 degradation showed activity close to wild‐type E6. Interestingly, we found decreased expression of PAR3 in E6‐expressing cells independently of E6AP, which has not been previously recognized. Therefore, we knocked down several PDZ‐domain containing proteins including PAR3 in human cervical keratinocytes expressing E7, HRASG 12V and E6Δ151 to examine whether depletion of these proteins can restore the tumorigenic ability. Single knockdown of SCRIB, MAGI1 or PAR3 significantly but partially restored the tumorigenic ability. The combinatorial knockdown of SCRIB and MAGI1 cooperatively restored the tumorigenic ability, and additional depletion of PAR3 further enhanced the tumorigenic ability surpassing that induced by wild‐type E6. These data highlight the importance of the carboxy‐terminal motif of the E6 protein and downregulation of PAR3 in tumorigenic transformation of human cervical keratinocytes.

Argatroban Attenuates Diabetic Cardiomyopathy in Rats by Reducing Fibrosis, Inflammation, Apoptosis, and Protease-Activated Receptor Expression.

Chronic diabetes is associated with cardiovascular dysfunctions. Diabetic cardiomyopathy (DCM) is one of the serious cardiovascular complications associated with diabetes. Despite significant efforts in understanding the pathophysiology of DCM, management of DCM is not adequate due to its complex pathophysiology. Recently, involvement of protease-activated receptors (PARs) has been postulated in cardiovascular diseases. These receptors are activated by thrombin, trypsin, or other serine proteases. Expression of PAR has been shown to be increased in cardiac diseases such as myocardial infarction, viral myocarditis, and pulmonary arterial hypertension. However, the role of PAR in DCM has not been elucidated yet. Therefore, in the present study, we have investigated the role of PAR in the condition of DCM using a pharmacological approach. We used argatroban, a direct thrombin inhibitor for targeting PAR. Type-2 diabetes mellitus (T2DM) was induced by high-fat feeding along with low dose streptozotocin (STZ 35mg/kg, i.p. single dose) in male Sprague-Dawley rats. After 16weeks of diabetes induction, animals were treated with argatroban at 0.3 and 1mg/kg dose daily for 4weeks. After 20weeks, ventricular functions were measured using ventricular catheterization. Cardiac histology, TUNEL staining, and immunoblotting were performed to evaluate cardiac fibrosis, DNA fragmentation, and expression level of different proteins, respectively. T2DM was associated with cardiac structural and functional disturbances as evidenced from impaired cardiac functional parameters and increased fibrosis. There was a significant increase in PAR expression after 20weeks of diabetes induction. Four weeks argatroban treatment ameliorated metabolic alterations (reduced plasma glucose and cholesterol), ventricular dysfunctions (improved systolic and diastolic functions), cardiac fibrosis (reduced percentage area of collagen in picro-sirius red staining), and apoptosis (reduced TUNEL positive nuclei). Reduced expression of PAR1 and PAR4 in the argatroban-treated group indicates a response towards inhibition of thrombin. In addition, AKT (Ser-473), GSK-3β (Ser-9), p-65 NFĸB phosphorylation, TGF-β, COX-2, and caspase-3 expression were reduced significantly along with an increase in SERCA expression in argatroban-treated diabetic rats which indicated the anti-fibrotic, anti-inflammatory, and anti-apoptotic potential of argatroban in DCM. This study suggests the ameliorative effects of argatroban in diabetic cardiomyopathy by improving ventricular functions and reducing fibrosis, inflammation, apoptosis, and PAR expression.

Protease-activated receptor-2 suppresses interleukin (IL)-10 expression in B cells via upregulating Bcl2L12 in patients with allergic rhinitis.

The function of interleukin (IL)-10-producing B cells (B10 cell) is compromised in patients with allergic diseases. Protease-activated receptor (PAR)-2 has immunoregulatory functions. This study aimed to elucidate the role of PAR2 in the suppression of IL-10 expression in peripheral B cells. Peripheral blood B cells were collected from patients with allergic rhinitis (AR). A correlation between the expression of Bcl2-like protein 12 (Bcl2L12) and IL-10 in the B cells was analyzed. An AR mouse model was developed. We observed that the expression of IL-10 was lower in the peripheral B cells from patients with airway allergy. A negative correlation was identified between the expression of IL-10 and PAR2 in B cells. Activation of PAR2 of B cells increased the expression of Bcl2L12 and suppression of LPS-induced IL-10 expression, which were inhibited by knocking down the Bcl2L12 gene. Treating B cells from AR patients with Bcl2L12-shRNA-carrying liposomes reversed the capability of IL-10 expression and the immunosuppressive function. Administration of Bcl2L12 shRNA-carrying liposomes attenuated experimental AR in mice. Activation of PAR2 inhibits the expression of IL-10 in B cells, which can be reversed by treating B cells with Bcl2L12 shRNA-carrying liposomes. The data suggest that regulation of Bcl2L12 may be a novel approach in the treatment for AR.

Endothelial microparticles released by activated protein C protect beta cells through EPCR/PAR1 and annexin A1/FPR2 pathways in islets.

Islet transplantation is associated with early ischaemia/reperfusion, localized coagulation and redox‐sensitive endothelial dysfunction. In animal models, islet cytoprotection by activated protein C (aPC) restores islet vascularization and protects graft function, suggesting that aPC triggers various lineages. aPC also prompts the release of endothelial MP that bear EPCR, its specific receptor. Microparticles (MP) are plasma membrane procoagulant vesicles, surrogate markers of stress and cellular effectors. We measured the cytoprotective effects of aPC on endothelial and insulin‐secreting Rin‐m5f β‐cells and its role in autocrine and paracrine MP‐mediated cell crosstalk under conditions of oxidative stress. MP from aPC‐treated primary endothelial (EC) or β‐cells were applied to H2O2‐treated Rin‐m5f. aPC activity was measured by enzymatic assay and ROS species by dihydroethidium. The capture of PKH26‐stained MP and the expression of EPCR were probed by fluorescence microscopy and apoptosis by flow cytometry. aPC treatment enhanced both annexin A1 (ANXA1) and PAR‐1 expression in EC and to a lesser extent in β‐cells. MP from aPC‐treated EC (eMaPC) exhibited high EPCR and annexin A1 content, protected β‐cells, restored insulin secretion and were captured by 80% of β cells in a phosphatidylserine and ANXA1‐dependent mechanism. eMP activated EPCR/PAR‐1 and ANXA1/FPR2‐dependent pathways and up‐regulated the expression of EPCR, and of FPR2/ALX, the ANXA1 receptor. Cytoprotection was confirmed in H2O2‐treated rat islets with increased viability (62% versus 48% H2O2), reduced apoptosis and preserved insulin secretion in response to glucose elevation (16 versus 5 ng/ml insulin per 10 islets). MP may prove a promising therapeutic tool in the protection of transplanted islets.

Mechanism of the protective effects of the combined treatment with rhynchophylla total alkaloids and sinapine thiocyanate against a prothrombotic state caused by vascular endothelial cell inflammatory damage.

The aim of the present study was to investigate the effect and the underlying mechanism of the combined treatment of rhynchophylla total alkaloids (RTA) and sinapine thiocyanate for protection against a prothrombotic state (PTS) associated with the tumor necrosis factor-alpha (TNF-α)-induced inflammatory injury of vascular endothelial cells (VECs). A TNF-α-induced VEC inflammatory injury model was established, and cell morphology of VECs was evaluated using scanning electron microscopy. In addition, reverse transcription-quantitative polymerase chain reaction and western blot analysis were performed to examine the mRNA and protein expression of coagulation-related factors, including nuclear factor-κB (NF-κB), transforming growth factor-β1 (TGF-β1), tissue factor (TF), plasminogen activator inhibitor (PAI-1), protease-activation receptors (PAR-1) and protein kinase C (PKC-α) in VECs. Combined treatment with RTA and sinapine thiocyanate was demonstrated to reduce, to a varying extent, the mRNA and protein expression of NF-κB, TGF-β1, TF, PAR-1, PKC-α and PAI-1. Furthermore, combined treatment with RTA and sinapine thiocyanate was able to downregulate the expression of coagulation-related factors in injured VECs, thereby inhibiting the PTS induced by vascular endothelial injury. The underlying mechanism is partially associated with the TF-mediated activation of the thrombin-receptor signaling pathway that suppresses coagulation during inflammation and balances fibrinolysis in order to inhibit fibrin generation and deposition.

Apigenin inhibit UV-induced melanogenesis through activation of MIF and PAR-2 expression in keratinocytes

Apigenin inhibit UV-induced melanogenesis through activation of MIF and PAR-2 expression in keratinocytes

Flow cytometry analysis reveals different activation profiles of thrombin- or TRAP-stimulated platelets in db/db mice. The regulatory role of PAR-3

IntroductionRecent studies have shown that it may be the concentration of thrombin, which is discriminative in determining of the mechanism of platelet activation via protease activated receptors (PARs). Whether the observed phenomenon of differentiated responses of mouse platelets to various thrombin concentrations in non-diabetic db/+ and diabetic db/db mice depends upon the concerted action of various PARs, remains to be established. ResultsWe found elevated reactivity of platelets, as well as the enhanced PAR-3 expression in response to both the used concentrations of AYPGKF in db/db mice, as compared to db/+ heterozygotes. At low concentration of thrombin platelets from diabetic mice demonstrated hyperreactivity, reflected by higher expression of PAR-3. For higher thrombin concentration, blood platelets from db/db mice appeared hyporeactive, compared to db/+ animals, while no significant differences in PAR-3 expression were observed between diabetic and non-diabetic mice. ConclusionsThe novel and previously unreported finding resulting from our study is that the increased expression of PAR-3 in response to either TRAP for PAR-4 or low thrombin (when PAR-4 is not the efficient thrombin receptor) may be one of the key events contributing to higher reactivity of platelets in db/db mice.

Impact of maternal and postnatal zinc dietary status on the prostate of pubescent and adult rats.

Zinc is important for cell physiology and alteration of its levels during development can modulate a series of biological events. The aim of this study was to investigate whether dietary zinc deficiency or supplementation during morphogenesis and early postnatal development could interfere in prostate maturation. Pregnant rats were exposed to a standard diet (NZ:35 mg Zn/kg chow), low-zinc diet (LZ:3 mg of Zn/kg chow) and zinc-supplemented diet (HZ:180 mg/Kg chow) from gestational day 10 (GD10) through postnatal day 21 (PND21). After weaning, male offspring were divided into three groups that were submitted to the same food conditions as their mothers until PND53. The animals were euthanized at PND53 and PND115. The ventral prostate was removed, weighed and its fragments were subjected to histological, western blot and zymography analysis. PND53: body and prostate weight were lower in LZ compared to NZ; the epithelial compartment was reduced while the stromal compartment was increased in LZ compared to NZ; there was an increase in the amount of collagen and reduction in AR and SIRT1 expression in LZ compared to NZ. PND115: body weight was lower in LZ compared to NZ and prostate weight was similar among the groups; peripheral physiological hyperplasia was observed, as well as an increased epithelial proliferation index and reduced PAR4 expression in LZ and HZ compared to NZ. Zinc deficiency during prostate morphogenesis and differentiation is potentially harmful to its morphology, however, by restoring the standard dietary environment, the gland responds to the new microenvironment independent of the previous dietary condition.