Protease-activated Receptor 4 Expression Research Articles

MicroRNA-26a alleviates tubulointerstitial fibrosis in diabetic kidney disease by targeting PAR4.

Our previous study found that miR-26a alleviates aldosterone-induced tubulointerstitial fibrosis (TIF). However, the effect of miR-26a on TIF in diabetic kidney disease (DKD) remains unclear. This study clarifies the role and possible mechanism of exogenous miR-26a in controlling the progression of TIF in DKD models. Firstly, we showed that miR-26a was markedly decreased in type 2 diabetic db/db mice and mouse tubular epithelial cells (mTECs) treated with high glucose (HG, 30 mM) using RT-qPCR. We then used adeno-associated virus carrying miR-26a and adenovirus miR-26a to enhance the expression of miR-26a invivo and invitro. Overexpressing miR-26a alleviated the TIF in db/db mice and the extracellular matrix (ECM) deposition in HG-stimulated mTECs. These protective effects were caused by reducing expression of protease-activated receptor 4 (PAR4), which involved in multiple pro-fibrotic pathways. The rescue of PAR4 expression reversed the anti-fibrosis activity of miR-26a. We conclude that miR-26a alleviates TIF in DKD models by directly targeting PAR4, which may provide a novel molecular strategy for DKD therapy.

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

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

Epigenetic Regulation of F2RL3 Associates With Myocardial Infarction and Platelet Function.

DNA hypomethylation at the F2RL3 (F2R like thrombin or trypsin receptor 3) locus has been associated with both smoking and atherosclerotic cardiovascular disease; whether these smoking-related associations form a pathway to disease is unknown. F2RL3 encodes protease-activated receptor 4, a potent thrombin receptor expressed on platelets. Given the role of thrombin in platelet activation and the role of thrombus formation in myocardial infarction, alterations to this biological pathway could be important for ischemic cardiovascular disease. We conducted multiple independent experiments to assess whether DNA hypomethylation at F2RL3 in response to smoking is associated with risk of myocardial infarction via changes to platelet reactivity. Using cohort data (N=3205), we explored the relationship between smoking, DNA hypomethylation at F2RL3, and myocardial infarction. We compared platelet reactivity in individuals with low versus high DNA methylation at F2RL3 (N=41). We used an in vitro model to explore the biological response of F2RL3 to cigarette smoke extract. Finally, a series of reporter constructs were used to investigate how differential methylation could impact F2RL3 gene expression. Observationally, DNA methylation at F2RL3 mediated an estimated 34% of the smoking effect on increased risk of myocardial infarction. An association between methylation group (low/high) and platelet reactivity was observed in response to PAR4 (protease-activated receptor 4) stimulation. In cells, cigarette smoke extract exposure was associated with a 4.9% to 9.3% reduction in DNA methylation at F2RL3 and a corresponding 1.7-(95% CI, 1.2-2.4, P=0.04) fold increase in F2RL3 mRNA. Results from reporter assays suggest the exon 2 region of F2RL3 may help control gene expression. Smoking-induced epigenetic DNA hypomethylation at F2RL3 appears to increase PAR4 expression with potential downstream consequences for platelet reactivity. Combined evidence here not only identifies F2RL3 DNA methylation as a possible contributory pathway from smoking to cardiovascular disease risk but from any feature potentially influencing F2RL3 regulation in a similar manner.

Targeting Proteinase Activated Receptor-4 Reduces Mechanonociception During the Acute Inflammatory Phase but not the Chronic Neuropathic Phase of Osteoarthritis in Rats

Serine proteases are elevated in arthritic joints where they can cleave protease activated receptors (PARs) to modulate pain and inflammation. Activation of protease-activated receptor 4 (PAR4) has been implicated in inflammatory joint pain. Whether PAR4 is involved in osteoarthritis (OA) pain has not yet been explored. The aim of this study was to compare the role of PAR4 in modulating early versus late stage OA pain using two models of OA viz. monoiodoacetate (MIA) and medial meniscal transection (MMT). G-ratio calculation and electron microscopy analysis revealed saphenous nerve demyelination and structural damage during late stage but not early OA in both models. Using immunohistochemistry, neuronal expression of PAR4 was higher in early versus late OA. Systemic administration of the PAR4 antagonist pepducin P4pal10 reduced both secondary allodynia (von Frey hair algesiometry) and joint nociceptor firing (single unit recordings) in MMT and MIA animals compared to vehicle-treated animals in early OA. The PAR4 antagonist was ineffective at altering pain or joint afferent firing in post-inflammatory OA. During the acute phase of the models, joint inflammation as determined by laser speckle contrast analysis and intravital microscopy could be partially blocked by pepducin P4pal10. Compared to late-stage disease, inflammatory cytokines were elevated in early MIA and MMT rats. These findings suggest that PAR4 may be a viable target to treat the pain of early onset OA or during episodic inflammatory flares.

Abstract A12: Inhibition of protease-activated receptor 4 (PAR4) suppresses C4a-induced invasion of lung cancer cells in vitro

Abstract It has been well established that chronic inflammation can cause neoplasm development and cancer cell dissemination. The complement system, an essential component of innate immunity, contains some of the most powerful proinflammatory molecules upon its activation. Recent studies have demonstrated that complement activation components, anaphylatoxins C3a and C5a, can regulate the fate of cancer cells through direct or regulatory effects on cancer cells or immune system. C4a (77 aa) is released from complement component C4 upon the activation of the classical and lectin pathways of the complement system. Our recent study provided the data that C4a acts as an untethered ligand for G protein-coupled receptors, protease activated receptor 1 (PAR1) and PAR4. It is still unclear whether C4a generates any effects on cancer cells through binding to its receptors. In our present study, we want to determine the effect of C4a on the invasive ability of lung cancer cells in vitro, which is supposed to be correlated with lung cancer cell metastasis in vivo. First, we checked the levels of C4a in the plasma from patients of different stages of lung cancer, and our results revealed that the plasma level of C4a from stage IV lung cancer patients is significantly higher than that from stage I lung cancer patients, suggesting C4a might be involved in the metastasis of late-stage lung cancer. In addition, our in silico analysis of data from lung adenocarcinoma and squamous cell carcinoma patients using Kalpan-Meier Plotter online database revealed a negative correlation between PAR4 expression and the overall survival and progression-free survival. Moreover, our in vitro transwell invasion experiments demonstrated that C4a could significantly enhance lung cancer cell invasion in a concentration-dependent fashion, and the blockade of PAR4 by its specific antagonist (ML354) significantly inhibited C4a-induced lung cancer cell invasion in vitro. Our data provide the direct evidence that complement activation fragment C4a induces lung cancer cell invasion through acting on its receptor PAR4, suggesting that targeting PAR4 as a therapeutic intervention might be beneficial for impeding cancer cell metastasis in late-stage lung cancer patients. Citation Format: Hongbin Wang, Lingxin Xiong, Zhibin Wang, Yuanyuan Wang, Ling Xu, Weidong Zhang. Inhibition of protease-activated receptor 4 (PAR4) suppresses C4a-induced invasion of lung cancer cells in vitro [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A12.

Expression and Purification of Protease-Activated Receptor 4 (PAR4) and Analysis with Histidine Hydrogen-Deuterium Exchange.

Protease-activated receptors (PARs) are G-protein-coupled receptors that are activated by proteolysis of the N-terminus, which exposes a tethered ligand that interacts with the receptor. Numerous studies have focused on the signaling pathways mediated by PARs. However, the structural basis for initiation of these pathways is unknown. Here, we describe a strategy for the expression and purification of PAR4. This is the first PAR family member to be isolated without stabilizing modifications for biophysical studies. We monitored PAR4 activation with histidine hydrogen-deuterium exchange. PAR4 has nine histidines that are spaced throughout the protein, allowing a global view of solvent accessible and nonaccessible regions. Peptides containing each of the nine His residues were used to determine the t1/2 for each His residue in apo or thrombin-activated PAR4. The thrombin-cleaved PAR4 exhibited a 2-fold increase (p > 0.01) in t1/2 values observed for four histidine residues (His180, His229, His240, and His380), demonstrating that these regions have decreased solvent accessibility upon thrombin treatment. In agreement, thrombin-cleaved PAR4 also was resistant to thermolysin digestion. In contrast, the rate of thermolysin proteolysis following stimulation with the PAR4 activation peptide was the same as that of unstimulated PAR4. Further analysis showed the C-terminus is protected in thrombin-activated PAR4 compared to uncleaved or agonist peptide-treated PAR4. The studies described here are the first to examine the tethered ligand activation mechanism for a PAR family member biophysically and shed light on the overall conformational changes that follow activation of PARs by a protease.

Vascular protease‐activated receptor 4 upregulation, increased platelet aggregation, and coronary lipid deposits induced by long‐term dabigatran administration – results from a diabetes animal model

Essentials The impact of long-term thrombin inhibition outside the coagulation cascade is far from clear. We aimed to assess the impact of dabigatran etexilate (DE) in diabetic and control rats. In diabetic rats, DE increased platelet aggregation and lead to coronary lipid deposits. Long-term thrombin inhibition may increase atherosclerotic and atherothrombotic risk. SUMMARY: Background Besides its role in the coagulation cascade, thrombin contributes to platelet aggregation and to a plethora of non-hemostatic functions. Objectives To assess the impact of long-term thrombin inhibition with dabigatran etexilate (DE) on platelet aggregation and on extrahemostatic thrombin-related functions in diabetic and control rats. Methods Markers of inflammation, endothelial dysfunction, oxidative stress, angiogenesis and cell adhesion molecules were quantified in control rats (Control; n = 6), DE-treated control rats (Control-Dabi; n = 8), diabetic rats (Diabetes; n = 5), and DE-treated diabetic rats (Diabetes-Dabi; n = 8). Agonist-induced platelet aggregation, aortic and coronary lipid deposits and aortic protease-activated receptor 4 (PAR4) expression were also assessed. Results Control-Dabi rats showed significantly higher high-sensitivity C-reactive protein, von Willebrand factor (VWF), vascular endothelial growth factor (VEGF) and fibronectin levels, and significantly lower PAR4 agonist-induced aggregation, than Control rats. Control-Dabi rats also showed mild aortic lipid deposits, whereas no such changes were observed in Control rats. Diabetes-Dabi rats showed significantly higher VWF, VEGF and fibronectin levels than Diabetes rats, and similar PAR4 agonist-induced aggregation as Diabetes rats, and significantly higher ADP-induced aggregation than Diabetes rats. Coronary lipid deposits were observed in 75% of Diabetes-Dabi rats and in none of the Diabetes rats. PAR4 expression was 20.4% higher in Control-Dabi rats and 27.4% higher in Diabetes-Dabi rats than in their non-treated peers. Conclusions This study indicates that long-term thrombin inhibition increases vascular PAR4 expression, promotes atherosclerosis-related mechanisms, and, in diabetic rats, increases platelet aggregation and favors the occurrence of coronary lipid deposits. These experimental data suggest that long-term thrombin inhibition may increase atherosclerotic and atherothrombotic risk, particularly in the presence of diabetes.

PAR4 overexpression promotes colorectal cancer cell proliferation and migration.

Protease-activated receptor 4 (PAR4), a member of the G-protein-coupled receptor family, was previously identified to be involved in the progression of cancer. Previous study revealed that the expression of PAR4 was increased in colorectal cancer tissues compared with the associated normal tissues, particularly in positive lymph node and poorly differentiated types of cancer. We hypothesized that PAR4 serves a function in the progression of colorectal cancer. In the present study, overexpression of PAR4 in colorectal cancer LoVo cells promoted proliferation, anchorage-independent growth and migration. In vivo, PAR4 increased LoVo cell tumorgenicity. In contrast, knockdown of PAR4 in HT-29 cells decreased proliferation, anchorage-independent growth and migration. Mechanistic studies revealed that PAR4 increased the phosphorylation of extracellular-signal-regulated kinase 1/2 in colorectal cancer cells, which is the potential molecular mechanism that promotes cellular proliferation and migration. Taken together, the results of the present study indicated that overexpression of PAR4 promoted colorectal cancer cell proliferation, survival and metastasis, indicating that PAR4 is a promising therapeutic target for preventing colon cancer progression.

Downregulation of iNOS, IL-1β, and P2X7 Expression in Mast Cells via Activation of PAR4 Contributes to the Inhibition of Visceral Hyperalgesia in Rats.

Protease-activated receptor 4 (PAR4) is implicated in the inhibition of visceral hyperalgesia. In the present study, the effects of PAR4 activation on visceral hypersensitivity and expression of inflammatory mediators, including interleukin-1β (IL-1β), P2RX7 purinergic receptor (P2X7), inducible nitric oxide synthase (iNOS), and tryptase, in mast cells (MCs) were investigated via in vivo and in vitro studies. The numbers of tryptase-positive MCs with extensive PAR4, P2X7, and iNOS expression were increased in the colons of visceral hyperalgesia rats compared with controls. Intracolonic administration of PAR4-activating peptide (PAR4-AP) significantly attenuated the visceral hypersensitivity to colorectal distention and reduced the iNOS, IL-1β, P2X7, and tryptase protein and mRNA levels in the colonic mucosa. Treatment of rat bone marrow MCs (BMMCs) with PAR4-AP also reduced the iNOS, IL-1β, P2X7, and tryptase protein and mRNA levels. ERK1/2 and p38 activators (t-butylhydroquinone, tBHQ, and U-46619) reversed the suppressive effect of PAR4 activation on IL-1β and iNOS expression, whereas ERK1/2 and p38 inhibitors (PD98059 and SB203580) reversed the suppressive effect of PAR4 activation on P2X7 and tryptase expression. Our results indicate that the downregulation of inflammatory mediators, including iNOS, IL-1β, P2X7, and tryptase, in MCs that are mediated by PAR4 activation could inhibit visceral hyperalgesia via the mitogen-activated protein kinase (MAPK) signal pathway.

Protease-Activated Receptor 4 Variant p.Tyr157Cys Reduces Platelet Functional Responses and Alters Receptor Trafficking.

Protease-activated receptor 4 (PAR4) is a key regulator of platelet reactivity and is encoded by F2RL3, which has abundant rare missense variants. We aimed to provide proof of principle that rare F2LR3 variants potentially affect platelet reactivity and responsiveness to PAR1 antagonist drugs and to explore underlying molecular mechanisms. We identified 6 rare F2RL3 missense variants in 236 cardiac patients, of which the variant causing a tyrosine 157 to cysteine substitution (Y157C) was predicted computationally to have the greatest effect on PAR4 structure. Y157C platelets from 3 cases showed reduced responses to PAR4-activating peptide and to α-thrombin compared with controls, but no reduction in responses to PAR1-activating peptide. Pretreatment with the PAR1 antagonist vorapaxar caused lower residual α-thrombin responses in Y157C platelets than in controls, indicating greater platelet inhibition. HEK293 cells transfected with a PAR4 Y157C expression construct had reduced PAR4 functional responses, unchanged total PAR4 expression but reduced surface expression. PAR4 Y157C was partially retained in the endoplasmic reticulum and displayed an expression pattern consistent with defective N-glycosylation. Mutagenesis of Y322, which is the putative hydrogen bond partner of Y157, also reduced PAR4 surface expression in HEK293 cells. Reduced PAR4 responses associated with Y157C result from aberrant anterograde surface receptor trafficking, in part, because of disrupted intramolecular hydrogen bonding. Characterization of PAR4 Y157C establishes that rare F2RL3 variants have the potential to markedly alter platelet PAR4 reactivity particularly after exposure to therapeutic PAR1 antagonists.

Abstract WP263: Mutagenesis Studies Revealed Minimal Impact of Human A120T Variant of Protease-activated Receptor 4 on Receptor function or Pharmacological Response to a Potent and Selective Antagonist

Introduction: Protease-activated receptor 4 (PAR4) is a platelet thrombin receptor and a novel target for ischemic stroke treatment. Recent reports suggested a subtle racial difference in platelet responses to submaximal concentrations of PAR4 agonist peptide (PAR4-AP). One of the PAR4 variants, A120T, is more common in African American than white (63% vs. 19%, Edelstein et al., Blood 2014). It was suggested that this variant contributes to the difference in response to PAR4-AP and impacts the in vitro response to YD-3, a PAR4 antagonist. In this study, site-directed mutagenesis was used to evaluate the effect of the A120T variant on PAR4 function and response to a newly discovered potent and selective PAR4 antagonist, UDM-001651. Unlike YD-3, UDM-001651 inhibited thrombin-induced platelet aggregation and prevented thrombosis in a monkey model and thus served as a relevant PAR4 pharmacology tool. Methods: Human PAR4 cDNAs expressing A120 and T120 variants were stably expressed in HEK293 cells. Cell surface expression of PAR4 was analyzed by FACS. Functional responses of cells were evaluated by monitoring calcium mobilization induced by BMS PAR4-AP, which was optimized based on AYPGKF. The potency of UDM-001651 was derived from an 11-point concentration response curve in the calcium assay using PAR4-AP at the EC80 concentration. Transient transfection studies were also performed to confirm the results. Results: Comparable levels of expression and functional responses were observed between A120 and T120 expressing cells. Results from side-by-side comparison between the two cell lines demonstrated no detectable difference in the mean EC50 values (0.42±0.054 versus 0.46±0.051 uM standard error, n=9) in calcium responses to PAR4-AP stimulation. Similarly, the potency of UDM-001651 in the same assay were similar between these two cell lines. Moreover, preliminary clinical results did not show differences in PAR4-mediated platelet response between African-American and white subjects. Conclusion: In contrast to the previous report, the results reported herein from site directed mutagenesis studies indicate that the A120T variant of PAR4 has no apparent impact on calcium signaling in response to agonist stimulation or response to a PAR4 antagonist.

Increased expression of protease-activated receptor 4 and Trefoil factor 2 in human colorectal cancer.

Protease-activated receptor 4 (PAR4), a member of G-protein coupled receptors family, was recently reported to exhibit decreased expression in gastric cancer and esophageal squamous cancer, yet increased expression during the progression of prostate cancer. Trefoil factor 2 (TFF2), a small peptide constitutively expressed in the gastric mucosa, plays a protective role in restitution of gastric mucosa. Altered TFF2 expression was also related to the development of gastrointestinal cancer. TFF2 has been verified to promote cell migration via PAR4, but the roles of PAR4 and TFF2 in the progress of colorectal cancer are still unknown. In this study, the expression level of PAR4 and TFF2 in colorectal cancer tissues was measured using real-time PCR (n = 38), western blotting (n=38) and tissue microarrays (n = 66). The mRNA and protein expression levels of PAR4 and TFF2 were remarkably increased in colorectal cancer compared with matched noncancerous tissues, especially in positive lymph node and poorly differentiated cancers. The colorectal carcinoma cell LoVo showed an increased response to TFF2 as assessed by cell invasion upon PAR4 expression. However, after intervention of PAR4 expression, PAR4 positive colorectal carcinoma cell HT-29 was less responsive to TFF2 in cell invasion. Genomic bisulfite sequencing showed the hypomethylation of PAR4 promoter in colorectal cancer tissues and the hypermethylation in the normal mucosa that suggested the low methylation of promoter was correlated to the increased PAR4 expression. Taken together, the results demonstrated that the up-regulated expression of PAR4 and TFF2 frequently occurs in colorectal cancer tissues, and that overexpression of PAR4 may be resulted from promoter hypomethylation. While TFF2 promotes invasion activity of LoVo cells overexpressing PAR4, and this effect was significantly decreased when PAR4 was knockdowned in HT-29 cells. Our findings will be helpful in further investigations into the functions and molecular mechanisms of Proteinase-activated receptors (PARs) and Trefoil factor factors (TFFs) during the progression of colorectal cancer.

Development and characterization of monoclonal antibodies against Protease Activated Receptor 4 (PAR4)

BackgroundProtease activated receptor 4 (PAR4) is a G protein coupled receptor (GPCR) which is activated by proteolytic cleavage of its N-terminal exodomain. This generates a tethered ligand that activates the receptor and triggers downstream signaling events. With the current focus in the development of anti-platelet therapies shifted towards PARs, new reagents are needed for expanding the field’s knowledge on PAR4. Currently, there are no PAR4 reagents which are able to detect activation of the receptor. MethodsMonoclonal PAR4 antibodies were purified from hybridomas producing antibody that were generated by fusing splenocytes with NS-1 cells. Immunoblotting, immunofluorescence, and flow cytometry were utilized to detect the epitope for each antibody and to evaluate the interaction of the antibodies with cells. ResultsHere, we report the successful generation of three monoclonal antibodies to the N-terminal extracellular domain of PAR4: 14H6, 5F10, and 2D6. We mapped the epitope on PAR4 of 14H6, 5F10, and 2D6 antibodies to residues (48-53), (41-47), and (73-78), respectively. Two of the antibodies (14H6 and 5F10) interacted close to the thrombin cleavage and were sensitive to α-thrombin cleavage of PAR4. In addition, 5F10 was able to partially inhibit the cleavage of PAR4 expressed in HEK293 cells by α-thrombin. ConclusionsThese new antibodies provide a means to monitor endogenous PAR4 expression and activation by proteases on cells.

Increased expression of protease-activated receptor 2 and 4 within dorsal root ganglia in a rat model of bone cancer pain.

In an effort to understand the underlying mechanisms of cancer-induced bone pain, we investigated the presence of two protease-activated receptors, protease-activated receptor 2 (PAR2), and protease-activated receptor 4 (PAR4), in dorsal root ganglia (DRGs) neurons in an animal model of bone cancer pain. Female Wistar rats were randomized into three groups: tumor-bearing animals killed after 14days (D14) and tumor-bearing animals killed after 21days (D21) group and a sham operation group. After establishment of the Walker 256 carcinoma bone cancer pain model, behavioral tests were carried out to determine both the spontaneous nocifensive behavior and the paw withdrawal threshold (PWT) of mechanical and thermal hyperalgesia in these rats. Subsequently, real-time RT-PCR, Western bolt, and immunofluorescence were used to determine the messenger RNA (mRNA) and protein expression of PAR2 and PAR4 in the ipsilateral lumbar 4-5 DRG neurons. Rats in the D21 treatment group displayed a significant increase in spontaneous nocifensive behavior scores compared with the sham group as well as a considerably decreased withdrawal threshold in mechanical allodynia and thermal stimulation. Compared to sham group, the relative mRNA and protein expression of PAR2 and PAR4 was significantly upregulated in the D14 group and D21 groups, concurrent with tumor growth and proliferation. In addition, we identified the co-expression of PAR2 and PAR4 in the DRG neurons. The upregulation of mRNA and protein levels as well as the co-localization of PAR2 and PAR4 in DRG neurons suggests their novel involvement in the development and maintenance of bone cancer pain.

Interleukin-1β Increased the Expression of Protease-Activated Receptor 4 mRNA and Protein in Dorsal Root Ganglion Neurons

Protease-activated receptor-4 (PAR4) is localized in primary sensory neurons and is believed to implicate in the modulation of nociceptive mechanisms. The pro-inflammatory cytokine interleukin-1β (IL-1β) is involved in the generation of hyperalgesia in pathological states such as neuropathy and inflammation. Previous studies have shown that IL-1β enhances the expression of PAR4 in many cell types but the effect of this cytokine on primary sensory neuron PAR4 expression is less clear. In the present study, we evaluated in rat dorsal root ganglion (DRG) neurons the influence of IL-1β on PAR4 mRNA and protein levels after IL-1β intraplantar injection into the hind-paw or treatment of cultured DRG neurons. The expression of PAR4 in cultured DRG neurons was also assessed after treatment with IL-1β with pre-addition of phorbol-12-myristate 13-acetate (PMA, a PKC activator) or chelerythrine chloride (a PKC inhibitor). We found that IL-1β intraplantar injection into the hind-paw or long-term exposure of cultured DRG neurons to IL-1β significantly increased the proportion of DRG neurons expressing PAR4 immunoreactivity. Real-time PCR and western blotting showed that IL-1β treatment also significantly elevated PAR4 mRNA and protein levels in DRG neurons. This IL-1β effect was enhanced in DRG neurons when DRG cultures were pre-treatment with the PMA. But pre-incubation with chelerythrine chloride strongly inhibited the IL-1β-induced increase of PAR4 mRNA and protein levels. These results demonstrate that the expression of PAR4 mRNA and protein induced by IL-1β is PKC signaling pathway dependent.

Capsaicin Up-Regulates Protease-Activated Receptor-4 mRNA and Protein in Primary Cultured Dorsal Root Ganglion Neurons

Previous study has shown that there is a functional link between the transient receptor potential vanilloid type 1 (TRPV1) receptor and protease-activated receptor-4 (PAR4) in modulation of inflammation and pain. Capsaicin activation of TRPV1 is involved in enhancement of the expression of TRPV1 in mRNA and protein in dorsal root ganglion (DRG) in vivo. Whether capsaicin could influence expression of PAR4 in primary sensory neurons remains unknown. In the present study, expression of PAR4 in cultured rat DRG neurons was observed using immunofluorescence, real-time PCR and Western blots to examine whether increases in PAR4 mRNA and protein levels are induced by capsaicin treatment with or without pre-treatment of forskolin, a cyclic AMP/protein kinase A (cAMP/PKA) activator or PKA inhibitor fragment 14-22 (PKI14-22), a PKA inhibitor. Capsaicin treatment of cultured DRG neurons significantly increased the expression of PAR4 in mRNA and protein levels. The percentage of PAR4-, TRPV1-immunoreactive neurons and their co-localization in cultured DRG neurons increased significantly in the presence of capsaicin as compared with that in the absence of capsaicin. Compared with capsaicin-only group, pre-incubation with forskolin strongly enhanced the capsaicin-induced increase of PAR4 in mRNA and protein levels. Consistent with the involvement of PKA in the modulation of PAR4 expression, this evoked expression both at mRNA and protein levels was significantly inhibited after PKA was inhibited by pre-incubation with PKI14-22. Taken together, these results provide evidence that TRPV1 activation significantly increases the expression of PAR4 mRNA and protein levels in primary cultures of DRG neurons after capsaicin incubation. Effects of capsaicin on PAR4 expression appear to be mediated by cAMP/PKA signal pathways in DRG neurons.

The Catalytic Subunit of Protein Phosphatase 1 Gamma Regulates Thrombin-Induced Murine Platelet αIIbβ3 Function

BackgroundHemostasis and thrombosis are regulated by agonist-induced activation of platelet integrin αIIbβ3. Integrin activation, in turn is mediated by cellular signaling via protein kinases and protein phosphatases. Although the catalytic subunit of protein phosphatase 1 (PP1c) interacts with αIIbβ3, the role of PP1c in platelet reactivity is unclear.Methodology/Principal FindingsUsing γ isoform of PP1c deficient mice (PP1cγ−/−), we show that the platelets have moderately decreased soluble fibrinogen binding and aggregation to low concentrations of thrombin or protease-activated receptor 4 (PAR4)-activating peptide but not to adenosine diphosphate (ADP), collagen or collagen-related peptide (CRP). Thrombin-stimulated PP1cγ−/− platelets showed decreased αIIbβ3 activation despite comparable levels of αIIbβ3, PAR3, PAR4 expression and normal granule secretion. Functions regulated by outside-in integrin αIIbβ3 signaling like adhesion to immobilized fibrinogen and clot retraction were not altered in PP1cγ−/− platelets. Thrombus formation induced by a light/dye injury in the cremaster muscle venules was significantly delayed in PP1cγ−/− mice. Phosphorylation of glycogen synthase kinase (GSK3)β-serine 9 that promotes platelet function, was reduced in thrombin-stimulated PP1cγ−/− platelets by an AKT independent mechanism. Inhibition of GSK3β partially abolished the difference in fibrinogen binding between thrombin-stimulated wild type and PP1cγ−/− platelets.Conclusions/SignificanceThese studies illustrate a role for PP1cγ in maintaining GSK3β-serine9 phosphorylation downstream of thrombin signaling and promoting thrombus formation via fibrinogen binding and platelet aggregation.

Cellular expression pattern of the protease‐activated receptor 4 in the hippocampus in naïve rats and after global ischaemia

A pronounced hippocampal expression of the Protease-activated Receptor 4 (PAR4) has recently been shown. In the current study the authors define the PAR4-associated sub-cellular structures and the influence of global ischaemia on the expression of PAR4. For that purpose the authors performed double labelling with fluorescence immunohistochemistry on tissue from naïve and post-ischaemic rats. In naïve animals - apart from the expression in granular and pyramidal neurons - there was an intensive lamellar expression of PAR4 in the CA4 region. Further analysis revealed that PAR4 was localised exclusively on mossy fibre axons in CA4 as detected by double-labelling with calbindin D-28k, but there was no overlap with markers of the neuronal cell body, interneurons, and post-synaptic, pre-synaptic and dendritic structures. Three and 14 days post ischaemia, CA1 neurons were degenerated and, consequently, there was no PAR4 signal in the CA1 band. In most other hippocampal structures no change in the PAR4 expression was detectable, with the exception of the CA3 region. Here, the fibre-associated PAR4 signal was diminished and disintegrated post ischaemia. Additionally, a redistribution from the membrane-bound neuronal localisation of PAR4 in control animals to a diffuse localisation all over the cell soma was revealed in the CA3 area 14 days post ischaemia. In conclusion, the current study proves for the first time that PAR4 is localised in mossy fibre axons. The altered expression in CA3 neurons after ischaemia indicates that PAR4 may be involved in post-ischaemic adaptive mechanisms.