PR3 and Elastase Alter PAR1 Signaling and Trigger vWF Release via a Calcium-Independent Mechanism from Glomerular Endothelial Cells

Neutrophil proteases, proteinase-3 (PR3) and elastase play key roles in glomerular endothelial cell (GEC) injury during glomerulonephritis. Endothelial protease-activated receptors (PARs) are potential serine protease targets in glomerulonephritis. We investigated whether PAR1/2 are required for alterations in GEC phenotype that are mediated by PR3 or elastase during active glomerulonephritis. Endothelial PARs were assessed by flow cytometry. Thrombin, trypsin and agonist peptides for PAR1 and PAR2, TFLLR-NH(2) and SLIGKV-NH(2,) respectively, were used to assess alterations in PAR activation induced by PR3 or elastase. Endothelial von Willebrand Factor (vWF)release and calcium signaling were used as PAR activation markers. Both PR3 and elastase induced endothelial vWF release, with elastase inducing the highest response. PAR1 peptide induced GEC vWF release to the same extent as PR3. However, knockdown of PARs by small interfering RNA showed that neither PAR1 nor PAR2 activation caused PR3 or elastase-mediated vWF release. Both proteases interacted with and disarmed surface GEC PAR1, but there was no detectable interaction with cellular PAR2. Neither protease induced a calcium response in GEC. Therefore, PAR signaling and serine protease-induced alterations in endothelial function modulate glomerular inflammation via parallel but independent pathways.     

Porphyromonas gingivalis selectively up-regulates the HIV-1 coreceptor CCR5 in oral keratinocytes

Primary infection of oral epithelial cells by HIV-1, if it occurs, could promote systemic infection. Most primary systemic infections are associated with R5-type HIV-1 targeting the R5-specific coreceptor CCR5, which is not usually expressed on oral keratinocytes. Because coinfection with other microbes has been suggested to modulate cellular infection by HIV-1, we hypothesized that oral keratinocytes may up-regulate CCR5 in response to the oral endogenous pathogen Porphyromonas gingivalis by cysteine-protease (gingipains) activation of the protease-activated receptors (PARs) or LPS signaling through the TLRs. The OKF6/TERT-2-immortalized normal human oral keratinocyte line expressed CXCR4, whereas CCR5 was not detectable. When exposed to P. gingivalis ATCC 33277, TERT-2 cells induced greater time-dependent expression of CCR5-specific mRNA and surface coreceptors than CXCR4. By comparing arg- (Rgp) and lys-gingipain (Kgp) mutants, a mutant deficient in both proteases, and the action of trypsin, P. gingivalis Rgp was strongly suggested to cleave PAR-1 and PAR-2 to up-regulate CCR5. CCR5 was also slightly up-regulated by an isogenic gingipain-deficient mutant, suggesting the presence of a nongingipain-mediated mechanism. Purified P. gingivalis LPS also up-regulated CCR5. Blocking TLR2 and TLR4 receptors with Abs attenuated induction of CCR5, suggesting LPS signaling through TLRs. P. gingivalis, therefore, selectively up-regulated CCR5 by two independent signaling pathways, Rgp acting on PAR-1 and PAR-2, and LPS on TLR2 and TLR4. By inducing CCR5 expression, P. gingivalis coinfection could promote selective R5-type HIV-1 infection of oral keratinocytes.     

Induction of IL-4 release and upregulated expression of protease activated receptors by GM-CSF in P815 cells

GM-CSF has been showed to be able to induce up-regulated receptor and cytokine expression in mast cells in inflammatory conditions. However, little is known of its effects on protease activated receptor (PAR) expression and Th2 cytokine secretion from mast cells. In the present study, we examined potential influence of GM-CSF on mast cell PAR expression and IL-4 and IL-10 release by using flow cytometry analysis, quantitative real time PCR, ELISA and cellular activation of signaling ELISA (CASE) techniques. The results showed that GM-CSF induced up to 3.0-fold increase in IL-4 release from P815 cells, and FSLLRY-NH₂ and trans-cinnamoyl (tc)-YPGKF-NH₂ did not affect GM-CSF induced IL-4 release. GM-CSF reduced tryptase and trypsin induced IL-4 release by up to approximately 55.8% and 70.3%, respectively. GM-CSF elicited the upregulated expression of PAR-1, PAR-2, PAR-3 and PAR-4 mRNAs, but enhanced only PAR-4 protein expression in P815 cells. U0126, PD98059 and LY204002 almost completely abolished GM-CSF induced IL-4 release when they were preincubated with P815 cells for 30 min, indicating ERK and Akt cell signaling pathways may be involved in the event. In conclusion, GM-CSF can stimulate IL-4 release from mast cells through an ERK and Akt cell signaling pathway dependent, but PAR independent mechanism. GM-CSF may serve as a regulator for IL-4 production in mast cells and through which participates in the mast cell related inflammation.     

Regulation of Feto-Maternal Barrier by Matriptase- and PAR-2-Mediated Signaling Is Required for Placental Morphogenesis and Mouse Embryonic Survival

The development of eutherian mammalian embryos is critically dependent on the selective bi-directional transport of molecules across the placenta. Here, we uncover two independent and partially redundant protease signaling pathways that include the membrane-anchored serine proteases, matriptase and prostasin, and the G protein-coupled receptor PAR-2 that mediate the establishment of a functional feto-maternal barrier. Mice with a combined matriptase and PAR-2 deficiency do not survive to term and the survival of matriptase-deficient mice heterozygous for PAR-2 is severely diminished. Embryos with the combined loss of PAR-2 and matriptase or PAR-2 and the matriptase partner protease, prostasin, uniformly die on or before embryonic day 14.5. Despite the extensive co-localization of matriptase, prostasin, and PAR-2 in embryonic epithelia, the overall macroscopic and histological analysis of the double-deficient embryos did not reveal any obvious developmental abnormalities. In agreement with this, the conditional deletion of matriptase from the embryo proper did not affect the prenatal development or survival of PAR-2-deficient mice, indicating that the critical redundant functions of matriptase/prostasin and PAR-2 are limited to extraembryonic tissues. Indeed, placentas of the double-deficient animals showed decreased vascularization, and the ability of placental epithelium to establish a functional feto-maternal barrier was severely diminished. Interestingly, molecular analysis suggested that the barrier defect was associated with a selective deficiency in the expression of the tight junction protein, claudin-1. Our results reveal unexpected complementary roles of matriptase-prostasin- and PAR-2-dependent proteolytic signaling in the establishment of placental epithelial barrier function and overall embryonic survival.     

Protease activated receptors in cardiovascular function and disease

Recent studies have shown that a novel class of protease activated receptors (PARs), which are composed of seven transmembrane G protein-coupled domains, are activated by serine proteases such as thrombin, trypsin and tryptase. Although four types (PAR 1, PAR 2, PAR 3 and PAR 4) of this class of receptors have been identified, their discrete physiological and pathological roles are still being unraveled. Extracellular proteolytic activation of PARs results in the cleavage of specific sites in the extracellular domain and formation of a new N-terminus which functions as a tethered ligand. The newly formed tethered ligand binds intramolecularly to an exposed site in the second transmembrane loop and triggers G-protein binding and intracellular signaling. Recent studies have shown that PAR-1, PAR-2 and PAR-4 have been involved in vascular development and a variety of other biological processes including apoptosis and remodeling. The use of animal model systems, mainly transgenic mice and synthetic tethered ligand domains, have contributed enormously to our knowledge of molecular signaling and the regulatory properties of various PARs in cardiomyocytes. This review focuses on the role of PARs in cardiovascular function and disease.     

A novel secreted protease from Pseudomonas aeruginosa activates NF-kappaB through protease-activated receptors

The Pseudomonas aeruginosa -derived alkaline protease (AprA), elastase A (LasA), elastase B (LasB) and protease IV are considered to play an important role in pathogenesis of this organism. Although the sequence analysis of P. aeruginosa genome predicts the presence of several genes encoding other potential proteases in the genome, little has been known about the proteases involving in pathogenesis. Recently, Porphyromonas gingivalis gingipains and Serratia marcescens serralysin have been shown to activate protease-activated receptor 2 (PAR-2), thereby modulating host inflammatory and immune responses. Accordingly, we hypothesized that unknown protease(s) from P. aeruginosa would also modulate such responses through PARs. In this study, we found that P. aeruginosa produces a novel l arge e xo p rotease (LepA) distinct from known proteases such as AprA, LasA, LasB and protease IV. Sequence analysis of LepA showed a molecular feature of the proteins transported by the two-partner secretion pathway. Our results indicated that LepA activates NF-κB-driven promoter through human PAR-1, -2 or -4 and cleaves the peptides corresponding to the tethered ligand region of human PAR-1, -2 and -4 at a specific site with exposure of their tethered ligands. Considered together, these results suggest that LepA would require PARs to modulate various host responses against bacterial infection.     

Kallikrein-related peptidase 4 (KLK4) initiates intracellular signaling via protease-activated receptors (PARs). KLK4 and PAR-2 are co-expressed during prostate cancer progression

Kallikrein-related peptidase 4 (KLK4) is one of the 15 members of the human KLK family and a trypsin-like, prostate cancer-associated serine protease. Signaling initiated by trypsin-like serine proteases are transduced across the plasma membrane primarily by members of the protease-activated receptor (PAR) family of G protein-coupled receptors. Here we show, using Ca(2+) flux assays, that KLK4 signals via both PAR-1 and PAR-2 but not via PAR-4. Dose-response analysis over the enzyme concentration range 0.1-1000 nM indicated that KLK4-induced Ca(2+) mobilization via PAR-1 is more potent than via PAR-2, whereas KLK4 displayed greater efficacy via the latter PAR. We confirmed the specificity of KLK4 signaling via PAR-2 using in vitro protease cleavage assays and anti-phospho-ERK1/2/total ERK1/2 Western blot analysis of PAR-2-overexpressing and small interfering RNA-mediated receptor knockdown cell lines. Consistently, confocal microscopy analyses indicated that KLK4 initiates loss of PAR-2 from the cell surface and receptor internalization. Immunohistochemical analysis indicated the co-expression of agonist and PAR-2 in primary prostate cancer and bone metastases, suggesting that KLK4 signaling via this receptor will have pathological relevance. These data provide insight into KLK4-mediated cell signaling and suggest that signals induced by this enzyme via PARs may be important in prostate cancer.     

Studies on the receptors mediating responses of osteoblasts to thrombin

The serine protease thrombin stimulates proliferation in osteoblasts, but decreases alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation. Three thrombin receptors have been identified, protease activated receptor (PAR)-1, PAR-3 and PAR-4; we have previously demonstrated that mouse osteoblasts express PAR-1 and PAR-4. The effect of thrombin on osteoblast proliferation and differentiation was studied to determine which of the thrombin receptors is responsible for the primary effects of thrombin. Primary mouse calvarial osteoblasts from PAR-1-null and wild-type mice, and synthetic peptides that specifically activate PAR-1 (TFFLR-NH2) and PAR-4 (AYPGKF-NH2) were used. Both the PAR-1-activating peptide and thrombin stimulated incorporation of 5-bromo-2'-deoxyuridine (two to four-fold, P < 0.001) and reduced alkaline phosphatase activity (approximately three-fold, P < 0.05) in cells from wild-type mice. The PAR-4-activating peptide, however, had no effect on either alkaline phosphatase activity or proliferation in these cells. Neither thrombin nor PAR-4-activating peptide was able to affect osteoblast proliferation or alkaline phosphatase activity in cells isolated from PAR-1-null mice. The results demonstrate that thrombin stimulates proliferation and inhibits differentiation of osteoblasts through activation of PAR-1. No other thrombin receptor appears to be involved in these effects.     

Effects of non-iron metalloporphyrins on growth and gene expression of Porphyromonas gingivalis

The oral anaerobic bacterium Porphyromonas gingivalis, which is implicated as an important pathogen for chronic periodontitis, requires heme for its growth. Non-iron metalloporphyrins, In-PPIX and Ga-PPIX, were examined for antibacterial effects on P. gingivalis. Both In-PPIX and Ga-PPIX caused retardation of P. gingivalis growth in a dose-dependent fashion. Microarray and qPCR analyses revealed that In-PPIX treatment upregulated the expression of several genes encoding proteins including ClpB and ClpC, which are members of the Clp (caseinolytic protease, Hsp100) family, and aRNR, aRNR-activating protein and thioredoxin reductase, whereas In-PPIX treatment had no effect on the expression of genes encoding proteins involved in heme uptake pathways, Hmu-mediated, Iht-mediated and Tlr-mediated pathways. P. gingivalis ihtA and ihtB mutants were more resistant to In-PPIX than was the wild-type parent, whereas hmuR and tlr mutants did not show such resistance to In-PPIX. The results suggest that In-PPIX is incorporated by the Iht-mediated heme uptake pathway and that it influences protein quality control and nucleotide metabolism and retards growth of P. gingivalis.     

Protease activated receptors in cardiovascular function and disease

Recent studies have shown that a novel class of protease activated receptors (PARs), which are composed of seven transmembrane G protein-coupled domains, are activated by serine proteases such as thrombin, trypsin and tryptase. Although four types (PAR 1, PAR 2, PAR 3 and PAR 4) of this class of receptors have been identified, their discrete physiological and pathological roles are still being unraveled. Extracellular proteolytic activation of PARs results in the cleavage of specific sites in the extracellular domain and formation of a new N-terminus which functions as a tethered ligand. The newly formed tethered ligand binds intramolecularly to an exposed site in the second transmembrane loop and triggers G-protein binding and intracellular signaling. Recent studies have shown that PAR-1, PAR-2 and PAR-4 have been involved in vascular development and a variety of other biological processes including apoptosis and remodeling. The use of animal model systems, mainly transgenic mice and synthetic tethered ligand domains, have contributed enormously to our knowledge of molecular signaling and the regulatory properties of various PARs in cardiomyocytes. This review focuses on the role of PARs in cardiovascular function and disease. (Mol Cell Biochem 263: 227–239, 2004)     

Determinants of the specificity of protease-activated receptors 1 and 2 signaling by factor Xa and thrombin

Factor Xa (FXa) elicits intracellular signaling responses through the activation of protease-activated receptor 2 (PAR2) and possibly also through PAR1 in endothelial cells. In this study, we investigated FXa signaling in endothelial cells when the protease was either in free form or assembled into the prothrombinase complex. Furthermore, we prepared several wild-type and mutant PAR1 and PAR2 cleavage-reporter constructs in which their exodomains were fused to cDNA encoding for a soluble alkaline phosphatase (ALP). In the mutants, P2 residues were exchanged between PAR1 and PAR2 cleavage-reporter constructs and the hirudin-like binding site (HLBS) of PAR1 was inserted into the homologous site of PAR2. In non-transfected cells, FXa elicited a protective response which could be blocked by a specific anti-PAR2 but not by an anti-PAR1 antibody. A similar protective activity was observed for FXa in the prothrombinase complex. Further studies revealed that neither the Gla- nor EGF1-domain of FXa is required for its signaling activity, however, the N-terminus Arg-86 and Lys-87 of the EGF2-domain were essential. In the cleavage-reporter transfected cells, FXa cleaved the PAR2 construct effectively, however, replacing its P2-Gly with P2-Pro of PAR1 impaired its cleavage by FXa but improved it by thrombin. A PAR2 construct containing both P2-Pro and HLBS of PAR1 was poorly cleaved by FXa, but effectively by thrombin. A PAR1 construct containing P2 and P3 residues of PAR2 was poorly cleaved by thrombin but effectively by FXa. These results provide new insight into mechanisms through which coagulation proteases specifically interact with their target PAR receptors.     

A genomewide screen for suppressors of par-2 uncovers potential regulators of PAR protein-dependent cell polarity in Caenorhabditis elegans

The PAR proteins play an essential role in establishing and maintaining cell polarity. While their function is conserved across species, little is known about their regulators and effectors. Here we report the identification of 13 potential components of the C. elegans PAR polarity pathway, identified in an RNAi-based, systematic screen to find suppressors of par-2(it5ts) lethality. Most of these genes are conserved in other species. Phenotypic analysis of double-mutant animals revealed that some of the suppressors can suppress lethality associated with the strong loss-of-function allele par-2(lw32), indicating that they might impinge on the PAR pathway independently of the PAR-2 protein. One of these is the gene nos-3, which encodes a homolog of Drosophila Nanos. We find that nos-3 suppresses most of the phenotypes associated with loss of par-2 function, including early cell division defects and maternal-effect sterility. Strikingly, while PAR-1 activity was essential in nos-3; par-2 double mutants, its asymmetric localization at the posterior cortex was not restored, suggesting that the function of PAR-1 is independent of its cortical localization. Taken together, our results identify conserved components that regulate PAR protein function and also suggest a role for NOS-3 in PAR protein-dependent cell polarity.     

Phosphorylation of Protease-activated Receptor-2 Differentially Regulates Desensitization and Internalization

Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor irreversibly activated by extracellular proteases. Activated PAR2 couples to multiple heterotrimeric G-protein subtypes including Gα_q, Gα_i, and Gα_12/13. Most activated G protein-coupled receptors are rapidly desensitized and internalized following phosphorylation and β-arrestin binding. However, the role of phosphorylation in regulation of PAR2 signaling and trafficking is not known. To investigate the function of phosphorylation, we generated a PAR2 mutant in which all serines and threonines in the C-tail were converted to alanines and designated it PAR2 0P. In mammalian cells, the addition of agonist induced a rapid and robust increase in phosphorylation of wild-type PAR2 but not the 0P mutant, suggesting that the major sites of phosphorylation occur within the C-tail domain. Moreover, desensitization of PAR2 0P signaling was markedly impaired compared with the wild-type receptor. Wild-type phosphorylated PAR2 internalized through a canonical dynamin, clathrin- and β-arrestin-dependent pathway. Strikingly, PAR2 0P mutant internalization proceeded through a dynamin-dependent but clathrin- and β-arrestin-independent pathway in both a constitutive and agonist-dependent manner. Collectively, our studies show that PAR2 phosphorylation is essential for β-arrestin binding and uncoupling from heterotrimeric G-protein signaling and that the presence of serine and threonine residues in the PAR2 C-tail hinder constitutive internalization through a non-canonical pathway. Thus, our studies reveal a novel function for phosphorylation that differentially regulates PAR2 desensitization and endocytic trafficking.     

Gingipains inactivate a cell surface ligand on Porphyromonas gingivalis that induces TLR2-and TLR4-independent signaling

Arginine-specific gingipain and lysine-specific gingipain are two major cysteine proteinases produced by Porphyromonas gingivalis. To clarify the role of gingipains in the interaction between P. gingivalis and the innate immune system, CHO reporter cells expressing TLR2 or TLR4 were stimulated with wildtype or gingipain-deficient P. gingivalis cells and activation of nuclear factor-kappaB in these cells was examined. While CHO/CD14 cells and 7.19 cells, an MD-2-defective mutant derived from CHO/CD14 cells, failed to respond to wild-type P. gingivalis, they responded to gingipain-deficient P. gingivalis. On the other hand, CHO/CD14/TLR2 cells responded to both wild-type and gingipain-deficient P. gingivalis. These results suggested that gingipains have no effects on TLR2-dependent signaling from P. gingivalis but have inhibitory effects on TLR2-and TLR4-independent signaling in CHO cells. Indeed, the activity of gingipain-deficient P. gingivalis to induce the activation of 7.19 cells was diminished after treatment of the bacterial cells with gingipains. We next partially purified bacterial cell components activating 7.19 cells from gingipain-deficient P. gingivalis. The activity of the partially purified components was diminished by treatment with heat or gingipains. It is also noteworthy that anti-CD14 mAb inhibited the activation of 7.19 cells induced by the partially purified components. These results indicated that the components of P. gingivalis that were able to induce TLR2-and TLR4-independent signaling were inactivated by gingipains before being recognized by CD14. The inactivation of the components would be helpful for P. gingivalis to escape from the innate immune system.     

Factor X/Xa Elicits Protective Signaling Responses in Endothelial Cells Directly via PAR-2 and Indirectly via Endothelial Protein C Receptor-dependent Recruitment of PAR-1

We recently demonstrated that the Gla domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier-protective response if endothelial cells were preincubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a cross-talk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells.     

Interaction of mite allergens Der p3 and Der p9 with protease-activated receptor-2 expressed by lung epithelial cells.

The respiratory epithelium represents the first barrier encountered by airborne Ags. Two major dust mite Ags, Der p3 and Der p9, are serine proteases that may activate lung epithelial cells by interaction with the protease-activated receptor 2 (PAR-2). In this study both Der p3 and Der p9 cleaved the peptide corresponding to the N terminus of PAR-2 at the activation site. Both Ags sequentially stimulated phosphoinositide hydrolysis, transient cytosolic Ca(2+) mobilization, and release of GM-CSF and eotaxin in human pulmonary epithelial cells. These responses were similar to those observed with trypsin and a specific PAR-2 agonist and were related to the serine protease activity of Der p3 and Der p9. Cell exposure to the Ags resulted in a refractory period, indicating that a PAR had been cleaved. Partial desensitization to Der p3 and Der p9 by the PAR-2 agonist suggested that PAR-2 was one target of the Ags. However, PAR-2 was not the only target, because the PAR-2 agonist caused less desensitization to Der p3 and Der p9 than did trypsin. A phospholipase C inhibitor prevented the cytokine-releasing effect of the PAR-2 agonist and abolished or reduced (>70%) the cytokine-releasing effects of Der p3 and Der p9. Our results suggest that Der p 3 and Der p9 may induce a nonallergic inflammatory response in the airways through the release of proinflammatory cytokines from the bronchial epithelium and that this effect is at least in part mediated by PAR-2.     

Protease-activated receptor 2 activation of myeloid dendritic cells regulates allergic airway inflammation

Background

A common characteristic of allergens is that they contain proteases that can activate protease-activated receptor (PAR-2); however the mechanism by which PAR-2 regulates allergic airway inflammation is unclear.

Methods

Mice (wild type and PAR-2-deficient) were sensitized using German cockroach (GC) feces (frass), the isolated protease from GC frass, or through adoptive transfer of GC frass-treated bone marrow-derived dendritic cells (BMDC) and measurements of airway inflammation (cellular infiltration, cytokine expression, and mucin production), serum IgE levels and airway hyperresponsiveness (AHR) were assessed. BMDC were cultured, treated with GC frass and assessed for cytokine production. PAR-2 expression on pulmonary mDCs was determined by flow cytometry.

Results

Exposure to GC frass induced AHR and airway inflammation in wild type mice; however PAR-2-deficient mice had significantly attenuated responses. To directly investigate the role of the protease, we isolated the protease from GC frass and administered the endotoxin-free protease into the airways of mice in the presence of OVA. GC frass proteases were sufficient to promote the development of AHR, serum IgE, and Th2 cytokine production. PAR-2 expression on mDC was upregulated following GC frass exposure, but the presence of a functional PAR-2 did not alter antigen uptake. To determine if PAR-2 activation led to differential cytokine production, we cultured BMDC in the presence of GM-CSF and treated these cells ex vivo with GC frass. PAR-2-deficient BMDC released significantly less IL-6, IL-23 and TNFα compared to BMDC from wild type mice, suggesting PAR-2 activation was important in Th2/Th17 skewing cytokine production. To determine the role for PAR-2 on mDCs on the initiation of allergic airway inflammation, BMDCs from wild type and PAR-2-deficient mice were treated in the presence or absence of GC frass and then adoptively transferred into the airway of wild type mice. Importantly, GC frass-stimulated wild type BMDCs were sufficient to induce AHR and allergic airway inflammation, while GC frass-stimulated PAR-2-deficient BMDC had attenuated responses.

Conclusions

Together these data suggest an important role for allergen activation of PAR-2 on mDCs in mediating Th2/Th17 cytokine production and allergic airway responses.     

Neutrophil-mediated activation of epithelial protease-activated receptors-1 and -2 regulates barrier function and transepithelial migration

Neutrophil (PMN) infiltration and associated release of serine proteases contribute to epithelial injury during active phases of mucosal disorders such as inflammatory bowel disease. Previous studies have demonstrated that PMN contact with basolateral surfaces of intestinal epithelial cells in the presence of a chemoattractant results in disruption of barrier function even without transmigration. Similarly, serine protease-mediated activation of epithelial protease-activated receptors (PARs) has been shown to increase permeability. In this study, we assessed whether transmigrating PMNs can regulate barrier function through epithelial PAR activation. Transepithelial resistance (TER) decreased significantly after PMN contact with basolateral surfaces of T84 monolayers or after incubation with PMN elastase and proteinase-3, but not cathepsin G. Inhibition of PMN serine proteases, but not selective inhibition of elastase or cathepsin G, prevented the fall in TER induced by PMN contact and blocked PMN transepithelial migration. Basolateral, but not apical, PAR-1 and -2 activation with selective agonists also decreased TER. PAR-1 and -2 were localized intracellularly and in close proximity to lateral surfaces beneath tight junctions, and expression was increased in colonic mucosa from individuals with Crohn's disease. Combined, but not individual, transfection with small interfering RNAs targeted against epithelial PAR-1 and -2, prevented the fall in TER induced by PMN contact. Furthermore, basolateral PAR-1 and -2 activation induced phosphorylation of myosin L chain kinase and regulatory myosin L chain. Lastly, epithelial PAR-1 and -2 knockdown decreased the rate of PMN transepithelial migration. These results suggest that protease-mediated epithelial PAR-1 and -2 activation, by migrating PMNs, induces signaling events that increase epithelial permeability thereby facilitates PMN transepithelial migration.     

Mutations in ooc-5 and ooc-3 disrupt oocyte formation and the reestablishment of asymmetric PAR protein localization in two-cell Caenorhabditis elegans embryos.

The early development of Caenorhabditis elegans embryos is characterized by a series of asymmetric divisions in which the mitotic spindle is repeatedly oriented on the same axis due to a rotation of the nuclear-centrosome complex. To identify genes involved in the control of spindle orientation, we have screened maternal-effect lethal mutants for alterations in cleavage pattern. Here we describe mutations in ooc-5 and ooc-3, which were isolated on the basis of a nuclear rotation defect in the P(1) cell of two-cell embryos. These mutations are novel in that they affect the asymmetric localization of PAR proteins at the two-cell stage, but not at the one-cell stage. In wild-type two-cell embryos, PAR-3 protein is present around the entire periphery of the AB cell and prevents nuclear rotation in this cell. In contrast, PAR-2 functions to allow nuclear rotation in the P(1) cell by restricting PAR-3 localization to the anterior periphery of P(1). In ooc-5 and ooc-3 mutant embryos, PAR-3 was mislocalized around the periphery of P(1), while PAR-2 was reduced or absent. The germ-line-specific P granules were also mislocalized at the two-cell stage. Mutations in ooc-5 and ooc-3 also result in reduced-size oocytes and embryos. However, par-3 ooc double-mutant embryos can exhibit nuclear rotation, indicating that small size per se does not prevent rotation and that PAR-3 mislocalization contributes to the failure of rotation in ooc mutants. We therefore postulate that wild-type ooc-5 and ooc-3 function in oogenesis and in the reestablishment of asymmetric domains of PAR proteins at the two-cell stage.