Protease-Activated Receptors (PARs) belong to an unusual family of G Protein Coupled Receptors (GPCRs). Each of the four known members is activated by its own N-terminus exposed by proteolytic cleavage and there is no other endogenous agonist known to date. PAR2 is the second member of the family and it has been implicated in wide range of pathophysiological conditions, particularly in various inflammatory diseases and cancers. In contrast, very little is known about the PAR2 receptor itself despite having been discovered more than 10 years ago. The purpose of this project was to improve our understanding of PAR2 regulation by discovering new agonists and antagonists and using them to probe the structural and functional properties of the receptor. Chapter 1 provides a brief literature overview of the initial discovery of PAR2, what is known about the mechanism of receptor activation, information on the structures and properties of current agonists and an antagonist for PAR2, and the putative physiological roles of human PAR2. As well, it summarizes the aims of this thesis. Chapter 2 investigates the regulation of gene expression by two different agonists of PAR2, a synthetic hexapeptide, 2f-LIGRLO-NH2, and the endogenous activator, trypsin, the idea being that genes up- or down- regulated by both agonists may more accurately profile PAR2-selective events. The effects of PAR2 activation on gene transcription in the human kidney HEK293 cell line were studied using a DNA microarray consisting of 19,000 human genes in an attempt to broadly cover the human genome and associated cell pathways with PAR2 activation. About 2,500 genes were regulated similarly by both agonists and, for genes expressed more than 5-fold, the mRNA results were further analyzed by quantitative RT-PCR techniques. PAR2 activation was shown to be associated with cellular metabolism, cell cycle, mitogen-activated protein kinase pathways, histone deacetylase and sirtuin enzymes, inflammatory cytokines and anti-complement function. Chapter 3 described a range of molecular events surrounding the activation of the receptor. PAR2 mRNA expression was quantitated by qRT-PCR and cross-checked with an intracellular Ca2+ assay. In this way whole cell PAR2 could be correlated with cell surface expression of PAR2. Three cell lines expressing high levels of PAR2 were chosen for subsequent experiments, these being colorectal carcinoma HT29, lung carcinoma A549 and human embryonic kidney HEK293 cells. Receptor activation, internalization, desensitization and resensitization assays were carried out on these cell lines to define some key functions relevant for investigating inhibitors in subsequent chapters. Chapter 4 reports a PAR2 mutagenesis study designed to identify the location of the binding site on PAR2 for a specific peptide agonist. A homology model of PAR2 based on bovine rhodopsin was used for docking of an agonist ligand, and the docking results were then investigated via two successive rounds of PAR2 mutagenesis in which the effect of each mutation (20 in all) was separately investigated by changes in agonist potency in the intracellular calcium release assay. Five PAR2 mutants showed more than a 5-fold reduction in agonist potency, while three others showed up to a 7-fold reduction. Mutations found to be important for agonist activity were mapped back to the model. Because there was extensive clustering of these key mutated amino acids, it is likely that this study has pinpointed the precise binding site of the agonist peptide in PAR2. Interestingly, this site is within the transmembrane region of the receptor. Chapter 5 reports the design, discovery and development of novel PAR2 agonists and antagonists and their regulatory effects in a diverse array of cell types. Structure-activity relationships were used to examine influences on the first, sixth and seventh positions of a PAR2 agonist peptide. At least five compounds were found herein to be equiopotent with the most potent PAR2 agonist reported. Knowledge obtained from this study was then used to create the first non-peptidic agonists for PAR2. The most potent nonpeptidic agonist (retaining one natural amino acid) was at least equipotent with the best peptide agonists. Conversion to nonpeptidic antagonists proved to be successful and this chapter reports the most potent known nonpeptide antagonist, which was selective for PAR2 and active at low micromolar concentrations. It inhibited intracellular Ca2+ release induced by different PAR2 agonists (trypsin, 2f-LIGRLO-NH2, nonpeptide agonists) in multiple cell lines (HT29, Panc-1, A549, MKN1, MKN45, MDA-MB-231, HUVEC) that have been physiologically associated with PAR2. It also inhibited release of inflammatory cytokines IL-8 and IL-6 and shows antiproliferative activity against primary human cells. The antagonist is competitive, reversible and surmountable (pA2 6.11). This thesis summarizes a large body of work that provides valuable molecular insights to PAR2 regulation, and lays the groundwork for rational design and development of novel nonpeptidic agonists and antagonists of PAR2 as potentially valuable pharmacological probes in vivo and as useful leads to development of therapeutics for inflammatory diseases and cancers.

Proteinase-activated receptor-2 (PAR-2) is a member of a family of G-protein-coupled, seven-transmembrane domain receptors that are activated by proteolytic cleavage. The receptor is expressed in a number of different tissues and potential physiological activators identified thus far include trypsin and mast cell tryptase. Acrosin, a trypsin-like serine proteinase found in spermatozoa of all mammals, was found to cleave a model peptide fluorescent quenched substrate representing the cleavage site of PAR-2. This substrate was cleaved with kinetics similar to those of the known PAR-2 activators, trypsin and mast cell tryptase. Acrosin was also shown to induce significant intracellular calcium responses in Chinese hamster ovary cells stably expressing intact human PAR-2, most probably due to activation of the receptor. Immunohistochemical studies using PAR-2 specific antibodies indicated that the receptor is expressed by mouse oocytes, which suggests that acrosin may play additional role(s) in the fertilization process via the activation of PAR-2 on oocytes.

No description available.

Inflammation involves a complex interplay of immunological responses in reaction to an infection from invading pathogens and to tissue injury. However, if the inflammatory stimulus is not eliminated or if there is an aberrant response, chronic inflammation and disease can result. Most inflammatory diseases are targeted by drugs that provide only symptomatic relief by reducing the associated pain and swelling, without combating the underlying cause and progression of the disease. The purpose of this thesis was to investigate potential drug targets in different sub-cellular compartments to learn more about inflammatory pathways and the consequences of inhibiting them using different classes of small-molecule agents. Chapter 1 is a brief overview of inflammation, outlining differences between acute and chronic inflammation in relation to normal and aberrant immune responses. Major drug classes that are currently in use are briefly discussed, and some potential new inflammatory drug targets in the nucleus, cytoplasm, plasma membrane and serum that are to be investigated in the thesis are described. Chapter 2 addresses histone deacetylase (HDAC) enzymes that modulate gene transcription, and investigates the regulatory potential of HDACs and their inhibitors in macrophages. Mainly murine macrophages were investigated, but preliminary data on human macrophages is also presented. Four kinds of small-molecule inhibitors of HDAC enzymes are described in relation to their capacity to exhibit pro- or anti- inflammatory gene expression. Broad-spectrum inhibitors, those that do not discriminate between different isoforms of HDACs, were found to induce expression of a number of pro-inflammatory mediators. This was also the case for compounds that selectively inhibit class-I HDACs. On the other hand, compounds that are known to selectively inhibit class-II HDACs did not regulate pro-inflammatory genes and could be classified as anti-inflammatory based on their profiles of gene expression. An inhibitor of class-III HDACs, the sirtuins, was also briefly investigated for anti-inflammatory gene expression. This separation of the pro- and anti-inflammatory responses suggested that specific inhibitors of class II HDACs might be promising for treating inflammatory conditions. Chapters 3 and 4 discuss a group of extracellular serum proteins that are produced through the complement pathway. Complement proteins circulate in plasma while some are formed on membranes through serine protease action, and effect both innate and adaptive immune responses. Chapter 3 deals with the alternative pathway (AP) of complement activation, in particular with the protease Factor B which plays an important role as part of a protein complex (C3 convertase) in the initiation and propagation of complement. Factor B is an inactive zymogen at pH 7, but is catalytically active at alkaline pH. Substrate-based inhibitors were developed for Factor B using a chromogenic assay previously optimised in our lab. Over 60 peptide aldehydes were evaluated, with two potent inhibitors of Factor B studied further, and were found to inhibit C3 convertase by blocking cleavage of the native substrate C3 as measured by SDS-PAGE. Chapter 4 examined the properties of a homologue of Factor B found in the classical pathway (CP), the protease C2. C2 was found herein to be an inactive zymogen at pH 7. Under alkaline conditions C2 cleaved both C3 and short chromogenic peptide substrates. Substrates of 7- or 8-residues, corresponding to those in the native substrate C3, were optimally processed. C2 was inhibited by inhibitors identified in Chapter 3, which also inhibited formation of the different C3 convertase formed through the CP, as well as preventing formation of the terminal membrane attack complex (MAC) as measured by an immobilized ELISA and haemolysis assay. The selectivity of small-molecule peptidic inhibitors for different serine proteases was investigated. Chapter 5 introduces two other unrelated inflammatory proteins which have been incompletely studied herein. Protease-activated receptor 2 (PAR-2) is a cell surface protein that spans the membrane like other G-protein coupled receptors. PAR-2 has been implicated in inflammatory and proliferative diseased states, though its precise roles are still unclear. Here, PAR-2 mRNA expression was measured for different human cancer and immune cells. In macrophages, PAR-2 mRNA expression was inducible by HDACi, indicating that PAR-2 is negatively regulated by HDAC enzymes. The receptor was present and functional on the surface of HDACi-treated macrophages, permitting evaluation of PAR-2 agonists and antagonists. Caspase-1 is a cytosolic cysteine protease responsible for cleaving proIL-1β and proIL-18 into their active forms that are then secreted from cells as pro-inflammatory cytokines. Using monocytic cells, novel inhibitors of caspase-1 were developed and found to dose-dependently decrease levels of IL-1β released by the cell. Inhibition of caspase-1 may be useful for modulating circulating levels of the inflammatory cytokine IL-1β. The investigation of different prospective inflammatory targets in different locations of the cell has provided a lateral overview of inflammatory mechanisms and drug intervention strategies. Specific inhibitors have also been identified for these targets; HDACs in the nucleus, caspase-1 in the cytosol, PAR-2 and MAC on the cell surface, C2 and Factor B and C3 convertase in plasma. This unconventional study of the inflammatory network provides a different viewpoint of inflammatory proteins as drug targets.

O receptor tipo 2 ativado por protease (PAR2) é um receptor pro-inflamatório que pode ser ativado por tripsina, triptase, protease 3 produzida pelo neutrófilo e pela gingipaína (produzida por Porphyromonas gingivalis). Objetivo: o objetivo do presente estudo foi investigar a expressão do PAR2 na periodontite crônica em humanos, e avaliar se esta expressão está relacionada com a presença de atividade proteolítica no fluido crevicular. Metodologia: foram coletadas amostras de fluido crevicular gengival de indivíduos do grupo controle (sítios saudáveis com profundidade de sondagem &#8804; 3mm, ausência de sangramento à sondagem; n=40), grupo periodontite crônica com destruição de leve a moderada (n=40) e grupo periodontite crônica com destruição avançada (n=40). A expressão do PAR2 foi determinada por RT-PCR e a atividade proteolítica no fluido crevicular foi analisada utilizando-se o substrato BApNA. Resultados: observou-se um aumento significativo (p<0.001) da expressão do PAR2 no grupo periodontite em relação ao grupo controle, independentemente da severidade da doença. Além disso, no grupo periodontite houve um aumento significativo na atividade proteolítica (p<0.001) presente no fluido crevicular comparado com o grupo controle. Conclusões: concluiu-se que na periodontite crônica, há um aumento da expressão do PAR2 e um aumento da atividade proteolítica do fluido crevicular. Desta forma, sugere-se que o PAR2 pode estar envolvido com a inflamação periodontal em humanos. / Proteinase-activated receptor-2 (PAR2) is a pro-inflammatory receptor that can be activated by trypsin, tryptase, neutrophil-serine protease-3 (P3), and gingipain (produced by Porphyromonas gingivalis). Objectives: the objective of the present study was to investigate the expression of PAR2 in chronic periodontitis, and to evaluate whether its expression is related to the presence of proteolytic activity at the crevicular fluid. Methodology: gingival crevicular fluid (CF) samples were collected from subjects from control group (health sites with probing depth &#8804; 3mm, absence of bleeding on probing; n=40), chronic periodontitis group with slight to moderate destruction (n=40), and chronic periodontitis group with advanced destruction (n=40). PAR2 expression was determined by reverse transcriptase-PCR, and the proteolytic activity (PA) at the CF was quantified using the specific substrate BApNA. Results: PAR2 expression was significantly higher (p<0.001) in periodontitis compared to healthy individuals, independently of the diseases severity. In addition, in periodontitis group, there was a significantly increased proteolytic activity (p<0.001) compared with controls. Conclusions: we conclude that in chronic periodontitis there is an increased expression of PAR2 and an increased proteolytic activity at the crevicular fluid. Thus, it is suggested that PAR2 might be involved in human periodontal inflammation.

Neste trabalho investigamos o efeito de doadores de H2S no prurido agudo mediado por PAR-2 em camundongos. A injeção i.d. do agonista PAR-2 SLIGRL-NH2, induziu prurido que não foi afetado pelo pré-tratamento com o antagonista H1 pirilamina. A coinjeção dos doadores de H2S GYY4137 (lento) ou NaHS (espontâneo) com SLIGRL-NH2 reduziu significativamente o prurido (P<0,05). A glibenclamida (bloqueador de canais KATP) e o SNP (doador de NO), mas não o ODQ (inibidor da sGC), evitaram estes efeitos. O antagonista TRPA1 HC-030031 reduziu significativamente o prurido induzido pelo SLIGRL-NH2 (P<0,05), mas o prurido induzido pelo agonista TPRA1 AITC não foi afetado por NaHS. Ensaios de Western blot mostraram que ambos PAR-2 e TRPA1 são expressos constitutivamente na pele de camundongos. Nossos dados mostram que o prurido secundário à ativação do PAR-2 pode ser reduzido por H2S, atuando via a abertura dos canais KATP e ativação da via NO-GMPc. Ademais, o receptor TRPA1 pode mediar o prurido induzido por SLIGRL-NH2, mas o H2S não interfere nesta via. / In this study we investigated the effect of H2S donors in PAR-2-mediated acute pruritus in mice. The i.d. injection of the PAR-2 agonist SLIGRL-NH2 induced itching that was unaffected by pre-treatment with the H1 antagonist pyrilamine. Co-injection of the H2S donors GYY4137 (slow) or NaHS (spontaneous) with SLIGRL-NH2 significantly reduced pruritis (P <0.05). Glibenclamide (a KATP channel blocker) and SNP (a NO donor), but not ODQ (a sGC inhibitor) prevented these effects. The TRPA1 antagonist HC-030031 significantly reduced SLIGRL-NH2-induced pruritus (P<0.05), but the pruritus induced by the TPRA1 agonist AITC was unaffected by NaHS. Western blot assays showed that both TRPA1 and PAR-2 are constitutively expressed in the mouse skin. Our data show that itching secondary to PAR-2 activation can be reduced by H2S which acts via the opening of KATP channels and activation of the NO-cGMP pathway. Furthermore, TRPA1 receptors may mediate SLIGRL-NH2-induced pruritus, however, H2S does not interfere with this pathway.

O receptor ativado por protease do tipo 2 (PAR2) está envolvido na patogênese de doenças inflamatórias crônicas, incluindo a periodontite. O PAR2 pode ser ativado pela gingipaína, produzida pela Porphyromonas gingivalis (P. gingivalis) e proteinase 3 (P3) do neutrófilo. A ativação do PAR2 desempenha um papel relevante nos processos inflamatórios ao induzir a liberação de importantes mediadores pró-inflamatórios associados à destruição periodontal. No presente estudo, o efeito do tratamento periodontal não-cirúrgico na expressão do PAR2 por células do fluido gengival, bem como a sua associação com os níveis de mediadores pró-inflamatórios e proteases ativadoras foram investigados em pacientes com periodontite. Além disso, avaliou-se in vitro o envolvimento da via de sinalização MAPK-p38 na ativação do PAR2 e a influência deste na produção de superóxido (O2-) e na capacidade de fagocitose em neutrófilos do sangue periférico humano infectados pela P. gingivalis. Um aumento significativo (P < 0,05) da expressão do RNAm e proteica do PAR2 por células do fluido gengival foi positivamente associado aos parâmetros clínicos inflamatórios e aos níveis de interleucina (IL)-6, IL-8, fator de necrose tumoral-, metaloproteinases da matriz (MMP)-2, MMP-8, fator de crescimento de hepatócitos e fator de crescimento endotelial vascular, avaliados por Bioplex. Níveis elevados de RNAm da gingipaína e P3, expressão reduzida do RNAm da dentilisina (P < 0,05), bem como redução significativa (P < 0,05) dos níveis de inibidores de protease secretados por leucócitos e elafina (avaliados por ELISA), também foram associados à superexpressão do PAR2. Sítios periodontalmente saudáveis de indivíduos com periodontite crônica apresentaram uma menor expressão do RNAm e proteica do PAR2 (P < 0,05). O tratamento periodontal resultou na diminuição significativa da expressão de PAR2 (P < 0,05), correlacionada com a diminuição da expressão de mediadores inflamatórios (P < 0,05), bem como com suas proteases ativadoras (P < 0,05). Além disso, o uso do antagonista do PAR2 suprimiu significativamente (P < 0,05) a produção de superóxido em neutrófilos do sangue periférico humano, denotando o papel do PAR2 nessa importante função celular. Dessa forma, concluímos que o tratamento periodontal, o qual resulta na diminuição dos níveis de proteases e mediadores pró-inflamatórios, está associado com uma diminuição da expressão do PAR2, portanto, sugerindo que a expressão de PAR2 é influenciada pela presença da infecção periodontal, e não uma característica constitutiva favorecendo a inflamação periodontal. / Protease-activated receptor-2 (PAR2) is implicated in the pathogenesis of chronic inflammatory diseases including periodontitis; it can be activated by gingipain, produced by Porphyromonas gingivalis (P. gingivalis), and by neutrophil-protease 3 (P3). PAR2 activation plays a relevant role in inflammatory processes by inducing the release of important inflammatory mediators associated with periodontal breakdown. In the present study, the effect of non-surgical periodontal treatment on PAR2 expression and its association with levels of pro-inflammatory mediators, and activating proteases were investigated in chronic periodontitis patients. Moreover it was evaluated in vitro the role of MAPK-p38 signaling pathway during PAR2 activation and its influence on superoxide production and phagocytosis capacity in peripheral human neutrophils infected with P. gingivalis. A significant mRNA and protein over-expression of PAR2 (P < 0.05) in gingival crevicular fluid cells was positively associated to inflammatory clinical parameters, and to the levels of interleukin (IL)-6, IL-8, tumor necrosis factor-alpha, matrix metalloproteases (MMP)-2, MMP-8, hepatocyte growth factor, and vascular endothelial growth factor (evaluated by Bioplex). Elevated levels of gingipain and P3 (P < 0.05), and decreased levels of dentilisin mRNA, and protease inhibitors, secretory leucocyte protease inhibitor and elafina (P < 0.05) (evaluated by ELISA), were also associated to PAR2 overexpression. Periodontally healthy sites from chronic periodontitis individuals showed a diminished expression of PAR2 mRNA and PAR2 protein level (P < 0.05). Furthermore, periodontal treatment resulted in decreased PAR2 expression (P < 0.05), correlated with decreased expression of inflammatory mediators (P < 0.05) and activating proteases (P < 0.05). Moreover, PAR2 antagonist reduced significantly superoxide production in human neutrophils (P < 0.05) showing the role of PAR2 on this important cellular function. We concluded that periodontal treatment resulted on decreased levels of proteases, and pro-inflammatory mediators is associated to decreased PAR2 expression, therefore suggesting that PAR2 expression is influenced by the presence of periodontal infection, and not a constitutive characteristic favoring periodontal inflammation.

Neste trabalho investigamos o efeito de doadores de H2S no prurido agudo mediado por PAR-2 em camundongos. A injeção i.d. do agonista PAR-2 SLIGRL-NH2, induziu prurido que não foi afetado pelo pré-tratamento com o antagonista H1 pirilamina. A coinjeção dos doadores de H2S GYY4137 (lento) ou NaHS (espontâneo) com SLIGRL-NH2 reduziu significativamente o prurido (P<0,05). A glibenclamida (bloqueador de canais KATP) e o SNP (doador de NO), mas não o ODQ (inibidor da sGC), evitaram estes efeitos. O antagonista TRPA1 HC-030031 reduziu significativamente o prurido induzido pelo SLIGRL-NH2 (P<0,05), mas o prurido induzido pelo agonista TPRA1 AITC não foi afetado por NaHS. Ensaios de Western blot mostraram que ambos PAR-2 e TRPA1 são expressos constitutivamente na pele de camundongos. Nossos dados mostram que o prurido secundário à ativação do PAR-2 pode ser reduzido por H2S, atuando via a abertura dos canais KATP e ativação da via NO-GMPc. Ademais, o receptor TRPA1 pode mediar o prurido induzido por SLIGRL-NH2, mas o H2S não interfere nesta via. / In this study we investigated the effect of H2S donors in PAR-2-mediated acute pruritus in mice. The i.d. injection of the PAR-2 agonist SLIGRL-NH2 induced itching that was unaffected by pre-treatment with the H1 antagonist pyrilamine. Co-injection of the H2S donors GYY4137 (slow) or NaHS (spontaneous) with SLIGRL-NH2 significantly reduced pruritis (P <0.05). Glibenclamide (a KATP channel blocker) and SNP (a NO donor), but not ODQ (a sGC inhibitor) prevented these effects. The TRPA1 antagonist HC-030031 significantly reduced SLIGRL-NH2-induced pruritus (P<0.05), but the pruritus induced by the TPRA1 agonist AITC was unaffected by NaHS. Western blot assays showed that both TRPA1 and PAR-2 are constitutively expressed in the mouse skin. Our data show that itching secondary to PAR-2 activation can be reduced by H2S which acts via the opening of KATP channels and activation of the NO-cGMP pathway. Furthermore, TRPA1 receptors may mediate SLIGRL-NH2-induced pruritus, however, H2S does not interfere with this pathway.

O receptor ativado por protease do tipo 2 (PAR2) está envolvido na patogênese de doenças inflamatórias crônicas, incluindo a periodontite. O PAR2 pode ser ativado pela gingipaína, produzida pela Porphyromonas gingivalis (P. gingivalis) e proteinase 3 (P3) do neutrófilo. A ativação do PAR2 desempenha um papel relevante nos processos inflamatórios ao induzir a liberação de importantes mediadores pró-inflamatórios associados à destruição periodontal. No presente estudo, o efeito do tratamento periodontal não-cirúrgico na expressão do PAR2 por células do fluido gengival, bem como a sua associação com os níveis de mediadores pró-inflamatórios e proteases ativadoras foram investigados em pacientes com periodontite. Além disso, avaliou-se in vitro o envolvimento da via de sinalização MAPK-p38 na ativação do PAR2 e a influência deste na produção de superóxido (O2-) e na capacidade de fagocitose em neutrófilos do sangue periférico humano infectados pela P. gingivalis. Um aumento significativo (P < 0,05) da expressão do RNAm e proteica do PAR2 por células do fluido gengival foi positivamente associado aos parâmetros clínicos inflamatórios e aos níveis de interleucina (IL)-6, IL-8, fator de necrose tumoral-, metaloproteinases da matriz (MMP)-2, MMP-8, fator de crescimento de hepatócitos e fator de crescimento endotelial vascular, avaliados por Bioplex. Níveis elevados de RNAm da gingipaína e P3, expressão reduzida do RNAm da dentilisina (P < 0,05), bem como redução significativa (P < 0,05) dos níveis de inibidores de protease secretados por leucócitos e elafina (avaliados por ELISA), também foram associados à superexpressão do PAR2. Sítios periodontalmente saudáveis de indivíduos com periodontite crônica apresentaram uma menor expressão do RNAm e proteica do PAR2 (P < 0,05). O tratamento periodontal resultou na diminuição significativa da expressão de PAR2 (P < 0,05), correlacionada com a diminuição da expressão de mediadores inflamatórios (P < 0,05), bem como com suas proteases ativadoras (P < 0,05). Além disso, o uso do antagonista do PAR2 suprimiu significativamente (P < 0,05) a produção de superóxido em neutrófilos do sangue periférico humano, denotando o papel do PAR2 nessa importante função celular. Dessa forma, concluímos que o tratamento periodontal, o qual resulta na diminuição dos níveis de proteases e mediadores pró-inflamatórios, está associado com uma diminuição da expressão do PAR2, portanto, sugerindo que a expressão de PAR2 é influenciada pela presença da infecção periodontal, e não uma característica constitutiva favorecendo a inflamação periodontal. / Protease-activated receptor-2 (PAR2) is implicated in the pathogenesis of chronic inflammatory diseases including periodontitis; it can be activated by gingipain, produced by Porphyromonas gingivalis (P. gingivalis), and by neutrophil-protease 3 (P3). PAR2 activation plays a relevant role in inflammatory processes by inducing the release of important inflammatory mediators associated with periodontal breakdown. In the present study, the effect of non-surgical periodontal treatment on PAR2 expression and its association with levels of pro-inflammatory mediators, and activating proteases were investigated in chronic periodontitis patients. Moreover it was evaluated in vitro the role of MAPK-p38 signaling pathway during PAR2 activation and its influence on superoxide production and phagocytosis capacity in peripheral human neutrophils infected with P. gingivalis. A significant mRNA and protein over-expression of PAR2 (P < 0.05) in gingival crevicular fluid cells was positively associated to inflammatory clinical parameters, and to the levels of interleukin (IL)-6, IL-8, tumor necrosis factor-alpha, matrix metalloproteases (MMP)-2, MMP-8, hepatocyte growth factor, and vascular endothelial growth factor (evaluated by Bioplex). Elevated levels of gingipain and P3 (P < 0.05), and decreased levels of dentilisin mRNA, and protease inhibitors, secretory leucocyte protease inhibitor and elafina (P < 0.05) (evaluated by ELISA), were also associated to PAR2 overexpression. Periodontally healthy sites from chronic periodontitis individuals showed a diminished expression of PAR2 mRNA and PAR2 protein level (P < 0.05). Furthermore, periodontal treatment resulted in decreased PAR2 expression (P < 0.05), correlated with decreased expression of inflammatory mediators (P < 0.05) and activating proteases (P < 0.05). Moreover, PAR2 antagonist reduced significantly superoxide production in human neutrophils (P < 0.05) showing the role of PAR2 on this important cellular function. We concluded that periodontal treatment resulted on decreased levels of proteases, and pro-inflammatory mediators is associated to decreased PAR2 expression, therefore suggesting that PAR2 expression is influenced by the presence of periodontal infection, and not a constitutive characteristic favoring periodontal inflammation.

La Fibrose Pulmonaire Idiopathique (FPI) est une pathologie pulmonaire chronique, progressive, irréversible et mortelle, dont les thérapeutiques sont insuffisantes à ce jour. L'activation de la cascade de la coagulation et des protéases à sérine, délétère dans la progression des maladies pulmonaires chroniques, est une caractéristique de la pathologie. Récemment, un lien a été démontré entre Protease-Activated Receptor-2 (PAR-2), un récepteur cellulaire ubiquitaire, et la progression de la fibrose pulmonaire chez l'homme et la souris. Outre certains facteurs de la coagulation, PAR-2 semble aussi pouvoir être activé par des protéases appartenant à la famille récemment identifiée des Type II Transmembrane Serine Proteases (TTSPs), dont la matriptase et la Human Airway Trypsin-like protease (HAT). Leur rôle dans la fibrogénèse pulmonaire humaine et expérimentale est cependant encore inconnu.Nos travaux montrent pour la première fois qu'il existe une dérégulation de l'expression et de l'activité de ces protéases de la famille des TTSPs chez le patient FPI. In vitro, la matriptase induit des réponses pro-fibrosantes dans les fibroblastes pulmonaires primaires humains via l'activation de PAR-2, tandis que la HAT induit des réponses anti-fibrosantes dans ces cellules et une activation de la voie de la prostaglandine E2. Ces deux TTSPs sont ainsi différemment impliquées dans la fibrogénèse pulmonaire : in vivo, l'inhibition génétique et pharmacologique de la matriptase atténue la fibrose dans le modèle murin de fibrose pulmonaire induite par la bléomycine, et des résultats similaires sont observés suite à la surexpression de la HAT médiée par adénovirus dans ce modèle animal. L'ensemble des résultats obtenus dans ce travail de thèse permet de documenter l'implication de deux protéases à sérine, la matriptase et la HAT, dans la pathogénèse de la FPI et de définir des axes de recherche thérapeutique potentiels / Idiopathie Pulmonary Fibrosis (IPF) is a chronic, progressive, irreversible and mortal disease. Therapeutics options that improve the clinical outcome of IPF are limited. Coagulation proteinases and coagulation signaling deregulation, which influences several key inflammatory and fibroproliferative responses, is essential in IPF. Recently, Protease-Activated Receptor-2 (PAR-2) was shown to be involved in pulmonary fibrogenesis, both in Human and in mice. In addition to coagulation factors, PAR-2 can be activated by serine proteases of the emerging Type II Transmembrane Serine Proteases (TTSPs) family, including matriptase and the Human Airway Trypsin-like protease (HAT). Herein we explored the role of matriptase and HAT in the progression of human and experimental pulmonary fibrosis.Our data show that TTSPs matriptase and HAT pulmonary expression and activity are deregulated in patients with IPF. In vitro, matriptase induces PAR-2 dependent pro-fibrotic responses in primary human lung fibroblasts, whereas HAT induces anti-fibrotic effects in these cells, through the activation of prostaglandin E2 pathway. These TTSPs are differently involved in pulmonary fibrogenesis: in vivo, genetic and pharmacological inhibition of matriptase reduces fibrosis in the bleomycin induced lung fibrosis model, while an adenovirus-mediated HAT overexpression in the murine model leads also to a limited lung fibrosis. Here, we highlight the involvement of matriptase and HAT in the pathogenesis of IPF and explore potential therapeutics for lung fibrosis