PAR2 Knockout Mice Research Articles

Protease activated receptor 2 deficiency retards progression of abdominal aortic aneurysms by modulating phenotypic transformation of vascular smooth muscle cells via ERK signaling

Abdominal aortic aneurysm (AAA) is characterized by localized structural deterioration of the aortic wall, leading to progressive dilatation and rupture. Protease activated receptor 2 (PAR2) dependent signaling has been implicated in the pathophysiology of atherosclerosis through the regulation of smooth muscle cell function. However, its role in AAA remains unclear. This study investigates the function and potential mechanism of PAR2 in AAA progression. Angiotensin II (Ang II) and β-aminopropionitrile (BAPN) were administered to wild type (WT) mice to induce AAA. Increased PAR2 expression was observed in the aneurysmal tissues of these mice and in Ang II-treated vascular smooth muscle cells (VSMCs). We demonstrated that PAR2 deficiency markedly inhibited aorta dilatation and vascular remodeling in the AAA model relative to WT mice. Immunohistochemical staining showed significant upregulation of contractile markers and a reduction in synthetic markers in PAR2 knockout mice. Consistent with in vivo results, PAR2 knockdown diminished the effects of Ang II on VSMCs phenotypic switching, resulting in reduced proliferation and migration. Conversely, a PAR2 agonist (SLIGRL) induced the opposite effect, which was partially mitigated by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor (PD98059). This study suggests that PAR2 deficiency restrains aortic expansion and mitigates adverse vascular remodeling in AAA models, mediated in part by the ERK signaling pathway, indicating that PAR2 could be a potential therapeutic target for mitigating AAA development or progression.

Controlling autoimmune diabetes onset by targeting Protease-Activated Receptor 2

BackgroundType 1 diabetes (T1D) is a challenging autoimmune disease, characterized by an immune system assault on insulin-producing β-cells. As insulin facilitates glucose absorption into cells and tissues, β-cell deficiency leads to elevated blood glucose levels on one hand and target-tissues starvation on the other. Despite efforts to halt β-cell destruction and stimulate recovery, success has been limited. Our recent investigations identified Protease-Activated Receptor 2 (Par2) as a promising target in the battle against autoimmunity. We discovered that Par2 activation’s effects depend on its initial activation site: exacerbating the disease within the immune system but fostering regeneration in affected tissue. MethodsWe utilized tissue-specific Par2 knockout mice strains with targeted Par2 mutations in β-cells, lymphocytes, and the eye retina (as a control) in the NOD autoimmune diabetes model, examining T1D onset and β-cell survival. ResultsWe discovered that Par2 expression within the immune system accelerates autoimmune processes, while its presence in β-cells offers protection against β-cell destruction and T1D onset. This suggests a dual-strategy treatment for T1D: inhibiting Par2 in the immune system while activating it in β-cells, offering a promising strategy for T1D. ConclusionsThis study highlights Par2's potential as a drug target for autoimmune diseases, particularly T1D. Our results pave the way for precision medicine approaches in treating autoimmune conditions through targeted Par2 modulation.

Abstract 684: DT-9045, a novel PAR2 inhibitor with best-in-class properties that reduces resistance to both EGFR-targeting therapies and immunotherapy in oncology models

Abstract Protease-activated receptor 2 (PAR2) is a promising therapeutic target in oncology and immuno-oncology. It is upregulated and associated with poor prognosis in several cancer types. A pan-cancer meta-analysis showed that PAR2 is one of the genes most significantly associated with resistance to immune checkpoint blockade (ICB) and T cells dysfunction in cancer patients. Mechanistically, being expressed by various cells of the tumor microenvironment, PAR2 promotes both survival and proliferation of cancer cells and dampens the anti-cancer immune response. Domain Therapeutics has developed a novel PAR2 inhibitor, DT-9045, with best-in-class properties. Indeed, compared to its most advanced competitors, DT-9045 is a small molecule, orally bioavailable, insurmountable, biased, and active in tumor-like conditions (i.e. acidic pH and high level of activating proteases). This compound is also highly potent and selective on PAR2. Pharmacokinetic properties are suitable with a once-a-day oral administration. In vitro, DT-9045 completely prevented PAR2-mediated resistance to EGFR-targeting drugs. In vivo, its combination with anti-PD1 therapy showed a significant increase in efficacy over monotherapies in preclinical syngeneic mouse cancer models. This result was similar to the one observed in PAR2 knockout mice, indicating that oral administration of DT-9045 induces a complete inhibition of the receptor. We have further demonstrated that treatment with a PAR2 inhibitor enhances dendritic cell-mediated T cell activation and intratumoral infiltration of CD4+ and CD8+ T cells as well as a decrease in M2 macrophage infiltration. In conclusion, Domain Therapeutic has identified a novel PAR2 inhibitor, DT-9045, with clear competitive advantages. This preclinical candidate has shown strong potency in alleviating the resistance to both EGFR-targeting therapies and immunotherapy in preclinical cancer models. IND-enabling studies are currently ongoing to bring this new hope for cancer patients to the clinic. Citation Format: Thibaut Brugat, Maleck Kadiri, Samya Aouad, Anne-Laure Blayo, Baptiste Rugeri, Antoine Mousson, Aurélie Janvier, Edith Steinberg, Luc Baron, Mandy Recolet, Xavier Wirth, Maria Jesus Garcia-Leon, Quentin Ruet, Meriem Semache, Laurent Sabbagh, Orphée Blanchard, Christel Franchet, Stanislas Mayer, John Stagg, Nathalie Lenne, Stephan Schann. DT-9045, a novel PAR2 inhibitor with best-in-class properties that reduces resistance to both EGFR-targeting therapies and immunotherapy in oncology models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 684.

Involvement of PAR-2 in the Induction of Cell-Specific Matrix Metalloproteinase-2 by Activated Protein C in Cutaneous Wound Healing.

We previously reported that human keratinocytes express protease-activated receptor (PAR)-2 and play an important role in activated protein C (APC)-induced cutaneous wound healing. This study investigated the involvement of PAR-2 in the production of gelatinolytic matrix metalloproteinases (MMP)-2 and -9 by APC during cutaneous wound healing. Full-thickness excisional wounds were made on the dorsum of male C57BL/6 mice. Wounds were treated with APC on days 1, 2, and 3 post-wounding. Cultured neonatal foreskin keratinocytes were treated with APC with or without intact PAR-2 signalling to examine the effects on MMP-2 and MMP-9 production. Murine dermal fibroblasts from PAR-2 knock-out (KO) mice were also assessed. MMP-2 and -9 were measured via gelatin zymography, fluorometric assay, and immunohistochemistry. APC accelerated wound healing in WT mice, but had a negligible effect in PAR-2 KO mice. APC-stimulated murine cutaneous wound healing was associated with the differential and temporal production of MMP-2 and MMP-9, with the latter peaking on day 1 and the former on day 6. Inhibition of PAR-2 in human keratinocytes reduced APC-induced MMP-2 activity by 25~50%, but had little effect on MMP-9. Similarly, APC-induced MMP-2 activation was reduced by 40% in cultured dermal fibroblasts derived from PAR-2 KO mice. This study shows for the first time that PAR-2 is essential for APC-induced MMP-2 production. Considering the important role of MMP-2 in wound healing, this work helps explain the underlying mechanisms of action of APC to promote wound healing through PAR-2.

6-OR: PAR2 Downregulates Adipose LPL through MIF—A Novel Adipose Mechanism Contributing to the Development of Hypertriglyceridemia

Lipoprotein lipase (LPL) is a key enzyme that hydrolyzes circulating triglycerides to release fatty acid (FA). In adipose tissue, LPL is required for lipid storage. However, it is currently unknown whether alteration of LPL in white adipose tissue (WAT) contributes to the development of hypertriglyceridemia. Our present study indicated that WAT isolated from human obese patients have increased expression of PAR2 which is negatively associated with LPL gene. The reduced LPL expression is also negatively correlated with increased plasma TG levels, suggesting that adipose PAR2 may modulate hyperlipidemia through downregulating LPL. In mice, aging and high palmitic oil diet significantly increased PAR2 expression in adipose tissue which was associated with high plasma MIF levels. PAR2 deficiency attenuates the rise of MIF, suggesting a key role of PAR2 in regulating adipose MIF release. MIF reduced LPL expression and activity in adipocytes. In a MIF overexpressed animal model (Mif lung Tg), high circulating MIF levels inhibited adipose LPL which was associated with increased plasma triglyceride but not fatty acid. Following high palmitic oil diet feeding, adipose LPL expression and activity were also reduced, and this reduction was reversed in PAR2 knockout mice. Interestingly, PAR2 mediated LPL in adipose tissue regulates hypertriglyceridemia through controlling adipocyte lipid storage. In Par2-/- mice, recombinant MIF perfusion recovered high plasma MIF levels, which decreased LPL and attenuated adipocyte lipid storage leading to hypertriglyceridemia. These data together suggest that the downregulation of adipose LPL by PAR2/MIF is an important mechanism for the development of hypertriglyceridemia. Disclosure Y.Huang: None. L.Li: None. L.Chen: None. X.Chen: None. P.Gao: None. Y.Qi: None. D.Qi: None. Funding Canadian Institutes of Health Research (PJT156116)

Protease-Activated Receptor 2 (PAR2) Expressed in Sensory Neurons Contributes to Signs of Pain and Neuropathy in Paclitaxel Treated Mice

Chemotherapy-induced peripheral neuropathy (CIPN) is a common, dose-limiting side effect of cancer therapy. Protease-activated receptor 2 (PAR2) is implicated in a variety of pathologies, including CIPN. In this study, we demonstrate the role of PAR2 expressed in sensory neurons in a paclitaxel (PTX)-induced model of CIPN in mice. PAR2 knockout/wildtype (WT) mice and mice with PAR2 ablated in sensory neurons were treated with PTX administered via intraperitoneal injection. In vivo behavioral studies were done in mice using von Frey filaments and the Mouse Grimace Scale. We then examined immunohistochemical staining of dorsal root ganglion (DRG) and hind paw skin samples from CIPN mice to measure satellite cell gliosis and intra-epidermal nerve fiber (IENF) density. The pharmacological reversal of CIPN pain was tested with the PAR2 antagonist C781. Mechanical allodynia caused by PTX treatment was alleviated in PAR2 knockout mice of both sexes. In the PAR2 sensory neuronal conditional knockout (cKO) mice, both mechanical allodynia and facial grimacing were attenuated in mice of both sexes. In the DRG of the PTX-treated PAR2 cKO mice, satellite glial cell activation was reduced compared to control mice. IENF density analysis of the skin showed that the PTX-treated control mice had a reduction in nerve fiber density while the PAR2 cKO mice had a comparable skin innervation as the vehicle-treated animals. Similar results were seen with satellite cell gliosis in the DRG, where gliosis induced by PTX was absent in PAR cKO mice. Finally, C781 was able to transiently reverse established PTX-evoked mechanical allodynia. PerspectiveOur work demonstrates that PAR2 expressed in sensory neurons plays a key role in PTX-induced mechanical allodynia, spontaneous pain, and signs of neuropathy, suggesting PAR2 as a possible therapeutic target in multiple aspects of PTX CIPN.

FSLLRY-NH2, a protease-activated receptor 2 (PAR2) antagonist, activates mas-related G protein-coupled receptor C11 (MrgprC11) to induce scratching behaviors in mice

AimsProtease-activated receptor 2 (PAR2), a type of G protein-coupled receptor (GPCR), plays a significant role in pathophysiological conditions such as inflammation. A synthetic peptide SLIGRL-NH2 (SLIGRL) can activate PAR2, while FSLLRY-NH2 (FSLLRY) is an antagonist. A previous study showed that SLIGRL activates both PAR2 and mas-related G protein-coupled receptor C11 (MrgprC11), a different type of GPCR expressed in sensory neurons. However, the impact of FSLLRY on MrgprC11 and its human ortholog MRGPRX1 was not verified. Hence, the present study aims to verify the effect of FSLLRY on MrgprC11 and MRGPRX1. MethodsThe calcium imaging technique was applied to determine the effect of FSLLRY in HEK293T cells expressing MrgprC11/MRGPRX1 or dorsal root ganglia (DRG) neurons. Scratching behavior was also investigated in wild-type and PAR2 knockout mice after injecting FSLLRY. Key findingsIt was surprisingly discovered that FSLLRY specifically activates MrgprC11 in a dose-dependent manner, but not other MRGPR subtypes. Furthermore, FSLLRY also moderately activated MRGPRX1. FSLLRY stimulates downstream pathways including Gαq/11, phospholipase C, IP3 receptor, and TRPC ion channels to evoke an increase in the intracellular calcium levels. The molecular docking analysis predicted that FSLLRY interacts with the orthosteric binding pocket of MrgprC11 and MRGPRX1. Finally, FSLLRY activated primary cultures of mouse sensory neurons, and induced scratching behaviors in mice. SignificanceThe present study has revealed that FSLLRY is capable of triggering itch sensation through activation of MrgprC11. This finding highlights the importance of considering the unexpected activation of MRGPRs in future therapeutic approaches aimed at the inhibition of PAR2.

ACTIVATED PROTEIN C (APC) PROMOTES A HEALING PHENOTYPE IN CULTURED MURINE TENOCYTES VIA PROTEASE-ACTIVATED RECEPTOR (PAR)-2, BUT NOT PAR-1

Tendinopathy is a tendon pathology often resulting from a failed healing response to tendon injury. Activated protein C (APC) is a natural anti-coagulant with anti-inflammatory and wound healing promoting functions, which are mainly mediated by its receptors, endothelial protein C receptor (EPCR) and protease activated receptors (PARs). This study aimed to determine whether APC stimulates tenocyte healing and if so, to assess the involvement of the receptors.Mouse-tail tenocytes were isolated from 3-week-old wild type (WT), PAR- 1 knockout (KO) and PAR-2 KO mice. The expression of EPCR, PAR-1 and −2 and the effect of APC on tenocytes tendon healing and the underlying mechanisms were investigated by Reverse transcription real time PCR, western blot, 3- (4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, zymography, and scratch wound healing/ migration assay.When compared to WT cells, PAR-1 KO tenocytes showed increased cell proliferation (3.3-fold, p<0.0001), migration (2.7-fold, p<0.0001) and wound healing (3-fold, p<0.0001), whereas PAR-2 KO cells displayed decreased cell proliferation (0.6-fold, p<0.05) and no change in cell migration or wound healing. APC at 1 μg/ml stimulated WT and PAR-1 KO tenocyte proliferation (~1.3, respectively, p<0.05) and wound healing (~1.3-fold, respectively, p<0.05), and additionally promoted PAR1-KO cell migration (1.4-fold, p<0.0001). APC only increased the migration (2-fold, p<0.05) of PAR-2 KO tenocytes. The activation of AKT, extracellular signal-regulated kinase (ERK)-2, and glycogen synthase kinase (GSK)-β3, the intracellular molecules that are associated with cell survival/growth, and matrix metalloproteinase (MMP)-2 that is related to cell migration and wound healing, were increased in all three cell lines in response to APC treatment.These findings show that PAR-1 and PAR-2 act differentially in tenocyte proliferation/migration/wound healing. APC likely promotes tenocyte proliferation/ wound healing via PAR-2, not PAR-1.

PAR2 promotes impaired glucose uptake and insulin resistance in NAFLD through GLUT2 and Akt interference.

Insulin resistance and poor glycemic control are key drivers of the development of NAFLD and have recently been shown to be associated with fibrosis progression in NASH. However, the underlying mechanisms involving dysfunctional glucose metabolism and relationship with NAFLD/NASH progression remain poorly understood. We set out to determine whether protease-activated receptor 2 (PAR2), a sensor of extracellular inflammatory and coagulation proteases, links NAFLD and NASH with liver glucose metabolism. Here, we demonstrate that hepatic expression of PAR2 increases in patients and mice with diabetes and NAFLD/NASH. Mechanistic studies using whole-body and liver-specific PAR2-knockout mice reveal that hepatic PAR2 plays an unexpected role in suppressing glucose internalization, glycogen storage, and insulin signaling through a bifurcating Gq -dependent mechanism. PAR2 activation downregulates the major glucose transporter of liver, GLUT2, through Gq -MAPK-FoxA3 and inhibits insulin-Akt signaling through Gq -calcium-CaMKK2 pathways. Therapeutic dosing with a liver-homing pepducin, PZ-235, blocked PAR2-Gq signaling and afforded significant improvements in glycemic indices and HbA1c levels in severely diabetic mice. This work provides evidence that PAR2 is a major regulator of liver glucose homeostasis and a potential target for the treatment of diabetes and NASH.

Mast cells instruct keratinocytes to produce thymic stromal lymphopoietin: Relevance of the tryptase/protease-activated receptor 2 axis

Thymic stromal lymphopoietin (TSLP) promotes TH2 inflammation and is deeply intertwined with inflammatory dermatoses like atopic dermatitis. The mechanisms regulating TSLP are poorly defined. We investigated whether and by what mechanisms mast cells (MCs) foster TSLP responses in the cutaneous environment. Exvivo and invivo skin MC degranulation was induced by compound 48/80 in wild-type protease-activated receptor 2 (PAR-2)- and MC-deficient mice in the presence or absence of neutralizing antibodies, antagonists, or exogenous mouse MC protease 6 (mMCP6). Primary human keratinocytes and murine skin explants were stimulated with lysates/supernatants of human skin MCs, purified tryptase, or MC lysate diminished of tryptase. Chymase and histamine were also used. TSLP was quantified by ELISA, real-time quantitative PCR, and immunofluorescence staining. Mas-related G protein-coupled receptor X2 (Mrgprb2) activation elicited TSLP in intact skin, mainly in the epidermis. Responses were strictly MC dependent and relied on PAR-2. Complementarily, TSLP was elicited by tryptase in murine skin explants. Exogenous mMCP6 could fully restore responsiveness in MC-deficient murine skin explants. Conversely, PAR-2 knockout mice were unresponsive to mMCP6 while displaying increased responsiveness to other inflammatory pathways, such as IL-1α. Indeed, IL-1α acted in concert with tryptase. In primary human keratinocytes, MC-elicited TSLP generation was likewise abolished by tryptase inhibition or elimination. Chymase and histamine did not affect TSLP production, but histamine triggered IL-6, IL-8, and stem cell factor. MCs communicate with kerationocytes more broadly than hitherto suspected. The tryptase/PAR-2 axis is a crucial component of this cross talk, underlying MC-dependent stimulation of TSLP in neighboring kerationocytes. Interference specifically with MC tryptase may offer a treatment option for disorders initiated or perpetuated by aberrant TSLP, such as atopic dermatitis.

Renal tubular PAR2 promotes interstitial fibrosis by increasing inflammatory responses and EMT process.

Renal fibrosis is defined by excessive extracellular matrix (ECM) accumulation and is associated with a decreased kidney function. Increased inflammation and infiltration of inflammatory cells are the key features of renal fibrosis development; however, the mechanism of how inflammation starts is still un-known. Here, we show that the activation of epithelial Protease-activating receptor 2 (PAR2) signaling plays an important role in the initiation of inflammation via increased chemokine expression and inflammatory cell induction. In the adenine diet-induced renal fibrosis mouse model, PAR2 expression was significantly increased in the renal tubule region. Kidneys from PAR2-knockout mice were protected from adenine diet-induced renal fibrosis, kidney dysfunction, and inflammation. Using NRK52E kidney epithelial cells, we further elucidated the mechanisms underlying these processes. Activation of PAR2 signaling pathway by PAR2 agonist specifically increased the levels of chemokines, including MCP1 and MCP3, via the MAPK-NF-κB signaling pathway. Inhibition of the MAPK signaling pathway attenuated PAR2 agonist-induced NF-κB activation, chemokine expression, and macrophage cell induction. Furthermore, PAR2 activation directly increased mesenchymal cell markers in epithelial cells. Taken together, we found that increased PAR2 expression and the PAR2/MAPK signaling pathway promote renal fibrosis by increasing the inflammatory responses and promoting EMT process.

Protease-activated receptor 2 induces ROS-mediated inflammation through Akt-mediated NF-κB and FoxO6 modulation during skin photoaging

Long-term exposure to ultraviolet irradiation to skin leads to deleterious intracellular effects, including reactive oxygen species (ROS) production and inflammatory responses, causing accelerated skin aging. Previous studies have demonstrated that increased expression and activation of protease-activated receptor 2 (PAR2) and Akt is observed in keratinocyte proliferation, suggesting their potential regulatory role in skin photoaging. However, the specific underlying molecular mechanism of PAR2 and the Akt/NF-κB/FoxO6-mediated signaling pathway is not clearly defined. In this study, we first used the UVB-irradiated photoaged skin of hairless mice and observed an increase in PAR2 and Gαq expression and PI3-kinase/Akt, NF-κB, and suppressed FoxO6. Consequently, increased levels of proinflammatory cytokines and decreased levels of antioxidant MnSOD was observed. Next, to investigate PAR2-specific roles in inflammation and oxidative stress, we used photoaged hairless mice topically applied with PAR2 antagonist GB83 and photoaged PAR2 knockout mice. PAR2 inhibition and deletion significantly suppressed inflammatory and oxidative stress levels, which were associated with decreased IL-6 and IL-1β levels and increased MnSOD levels, respectively. Furthermore, NF-κB phosphorylation and decreased FoxO6 was reduced by PAR2 inhibition and deletion in vivo. To confirm the in vivo results, we conducted PAR2 knockdown and overexpression in UVB-irradiated HaCaT cells. In PAR2 knockdown cells by si-PAR2 treatment, it suppressed Akt/NF-κB and increased FoxO6, whereas PAR2 overexpression reversed these effects and subsequently modulated proinflammatory target genes. Collectively, our data define that PAR2 induces oxidative stress and inflammation through Akt-mediated phosphorylation of NF-κB (Ser536) and FoxO6 (Ser184), which could be a critical upstream regulatory mechanism in ROS-mediated inflammatory response.

PAR2 promotes high-fat diet-induced hepatic steatosis by inhibiting AMPK-mediated autophagy

Protease-activated receptor 2 (PAR2) is a member of G protein-coupled receptors. There are two types of PAR2 signaling pathways: Canonical G-protein signaling and β-arrestin signaling. Although PAR2 signaling has been reported to aggravate hepatic steatosis, the exact mechanism is still unclear, and the role of PAR2 in autophagy remains unknown. In this study, we investigated the regulatory role of PAR2 in autophagy during high-fat diet (HFD)-induced hepatic steatosis in mice. Increased protein levels of PAR2 and β-arrestin-2 and their interactions were detected after four months of HFD. To further investigate the role of PAR2, male and female wild-type (WT) and PAR2-knockout (PAR2 KO) mice were fed HFD. PAR2 deficiency protected HFD-induced hepatic steatosis in male mice, but not in female mice. Interestingly, PAR2-deficient liver showed increased AMP-activated protein kinase (AMPK) activation with decreased interaction between Ca2+/calmodulin-dependent protein kinase kinase β (CAMKKβ) and β-arrestin-2. In addition, PAR2 deficiency up-regulated autophagy in the liver. To elucidate whether PAR2 plays a role in the regulation of autophagy and lipid accumulation in vitro, PAR2 was overexpressed in HepG2 cells. Overexpression of PAR2 decreased AMPK activation with increased interaction of CAMKKβ with β-arrestin-2 and significantly inhibited autophagic responses in HepG2 cells. Inhibition of autophagy by PAR2 overexpression further exacerbated palmitate-induced lipid accumulation in HepG2 cells. Collectively, these findings suggest that the increase in the PAR2-β-arrestin-2-CAMKKβ complex by HFD inhibits AMPK-mediated autophagy, leading to the alleviation of hepatic steatosis.

The thrombin receptor modulates astroglia-neuron trophic coupling and neural repair after spinal cord injury.

Excessive activation of the thrombin receptor, protease activated receptor 1 (PAR1) is implicated in diverse neuropathologies from neurodegenerative conditions to neurotrauma. PAR1 knockout mice show improved outcomes after experimental spinal cord injury (SCI), however information regarding the underpinning cellular and molecular mechanisms is lacking. Here we demonstrate that genetic blockade of PAR1 in female mice results in improvements in sensorimotor co-ordination after thoracic spinal cord lateral compression injury. We document improved neuron preservation with increases in Synapsin-1 presynaptic proteins and GAP43, a growth cone marker, after a 30 days recovery period. These improvements were coupled to signs of enhanced myelin resiliency and repair, including increases in the number of mature oligodendrocytes, their progenitors and the abundance of myelin basic protein. These significant increases in substrates for neural recovery were accompanied by reduced astrocyte (Serp1) and microglial/monocyte (CD68 and iNOS) pro-inflammatory markers, with coordinate increases in astrocyte (S100A10 and Emp1) and microglial (Arg1) markers reflective of pro-repair activities. Complementary astrocyte-neuron co-culture bioassays suggest astrocytes with PAR1 loss-of-function promote both neuron survival and neurite outgrowth. Additionally, the pro-neurite outgrowth effects of switching off astrocyte PAR1 were blocked by inhibiting TrkB, the high affinity receptor for brain derived neurotrophic factor. Altogether, these studies demonstrate unique modulatory roles for PAR1 in regulating glial-neuron interactions, including the capacity for neurotrophic factor signaling, and underscore its position at neurobiological intersections critical for the response of the CNS to injury and the capacity for regenerative repair and restoration of function.

A50 MODULATION OF GOBLET CELL ACTIVITY DURING GIARDIA DUODENALIS INFECTION: A ROLE FOR PAR2

Abstract Background Giardia duodenalis has been shown to alter the structure of the intestinal mucus layers during infection via obscure mechanisms. We hypothesize that goblet cell activity may be disrupted in part due to proteolytic activation of protease-activated receptor 2 (PAR2) by Giardia proteases, resulting in disruption of mucus production and secretion by intestinal goblet cells. Aims Characterize alterations in goblet cell activity during Giardia infection, focusing on the roles of Giardia protease activity and PAR2. Methods Chinese hamster ovary cells transfected with nano-luciferase tagged PAR2 were incubated with Giardia NF or GSM trophozoites. Cleavage within the activation domain results in release of enzymes into the supernatant. Luminescence in the supernatant was measured as an indication of PAR cleavage by Giardia. LS174T, a human colonic mucus-producing cell line, was infected with Giardia trophozoites (isolates NF, WB, S2, and GSM). Prior to infection, trophozoites were treated with E64, a broad-spectrum cysteine protease inhibitor, and LS174T were treated with a PAR2 antagonist, a calcium chelator, or an ERK1/2 inhibitor. Quantitative PCR (qPCR) was performed for the MUC2 mucin gene. Wild-type (WT) and PAR2 knockout (KO) mice were infected with Giardia. Colonic mucus was stained using fluorescein-coupled wheat-germ agglutinin (WGA), and qPCR was performed for Muc2 and Muc5ac. Results Giardia trophozoites cleaved PAR2 within the N-terminal activation domain in a cysteine protease-dependent manner. Cleavage was isolate dependent, with isolates that show higher protease activity cleaving at a higher rate. High protease activity Giardia isolates increased MUC2 gene expression in LS714T. This increase was attenuated by inhibition of Giardia cysteine protease activity, and by antagonism of PAR2, inhibition of calcium release, or inhibition of ERK1/2 activity in LS174T cells. Both Muc2 and Muc5ac expression were upregulated in the colons of WT mice in response to Giardia infection, while in the jejunum Muc2 expression decreased and Muc5ac expression increased. In KO, no changes in gene expression were seen in the colon in response to Giardia infection, while in the jejunum, Muc2 expression was unchanged and Muc5ac expression decreased. Both WT infected and KO noninfected mice showed thinning of the colonic mucus layer compared to WT controls. There was some recovery in thickness in KO infected mice. Conclusions PAR2 plays a significant role in the regulation of mucin gene expression in mice and in a human colonic cell line. Results suggest that Giardia cysteine proteases cleave and activate PAR2, leading to calcium release and activation of the MAPK pathway in goblet cells, ultimately leading to altered mucin gene expression. Findings identify a novel regulatory pathway for mucus production by intestinal goblet cells. Funding Agencies CAG, CCC

Mechanism of Lipid Accumulation through PAR2 Signaling in Diabetic Male Mice.

BackgroundProtease-activated protein-2 (PAR2) has been reported to regulate hepatic insulin resistance condition in type 2 diabetes mice. However, the mechanism of lipid metabolism through PAR2 in obesity mice have not yet been examined. In liver, Forkhead box O1 (FoxO1) activity induces peroxisome proliferator-activated receptor γ (PPARγ), leading to accumulation of lipids and hyperlipidemia. Hyperlipidemia significantly influence hepatic steatoses, but the mechanisms underlying PAR2 signaling are complex and have not yet been elucidated.MethodsTo examine the modulatory action of FoxO1 and its altered interaction with PPARγ, we utilized db/db mice and PAR2-knockout (KO) mice administered with high-fat diet (HFD).ResultsHere, we demonstrated that PAR2 was overexpressed and regulated downstream gene expressions in db/db but not in db+ mice. The interaction between PAR2/β-arrestin and Akt was also greater in db/db mice. The Akt inhibition increased FoxO1 activity and subsequently PPARγ gene in the livers that led to hepatic lipid accumulation. Our data showed that FoxO1 was negatively controlled by Akt signaling and consequently, the activity of a major lipogenesis-associated transcription factors such as PPARγ increased, leading to hepatic lipid accumulation through the PAR2 pathway under hyperglycemic conditions in mice. Furthermore, the association between PPARγ and FoxO1 was increased in hepatic steatosis condition in db/db mice. However, HFD-fed PAR2-KO mice showed suppressed FoxO1-induced hepatic lipid accumulation compared with HFD-fed control groups.ConclusionCollectively, our results provide evidence that the interaction of FoxO1 with PPARγ promotes hepatic steatosis in mice. This might be due to defects in PAR2/β-arrestin-mediated Akt signaling in diabetic and HFD-fed mice.

Protease-activated receptor 2 contributes to placental development and fetal growth in mice

BackgroundProtease-activated receptor 2 (PAR2) is activated by serine proteases such as coagulation tissue factor/VIIa complex, factor Xa or trypsin and is pro-angiogenic in several disease models. Impaired angiogenesis in placenta causes placental dysfunction and fetal growth restriction. PAR2 is expressed in the placenta trophoblast. However, the role of PAR2 in pregnancy remains unknown. ObjectiveThe present study aimed to examine the role of PAR2 in placental development and fetal growth using a murine model. MethodsPAR2−/− or PAR2+/+ mice in the ICR background were used. Female PAR2−/− mice were mated with male PAR2−/− mice, and female PAR2+/+ mice were mated with male PAR2+/+ mice to obtain PAR2−/− and PAR2+/+ fetuses, respectively. The day a virginal plug was observed in the morning was determined as 0.5-day post-coitum (dpc). Pregnant mice were sacrificed on 13.5 or 18.5 dpc to collect samples. ResultsA deficiency of PAR2 significantly reduced the fetal and placental weight and impaired placental labyrinth development in mice on 18.5 dpc. Collagen IV expression in placenta labyrinth was smaller in PAR2 knockout mice compared to that of wild-type mice. A deficiency of PAR2 also reduced the expression levels of genes related to angiogenesis and coagulation in placenta. ConclusionOur data suggest that PAR2 is required for fetal growth and angiogenesis in the placenta and is thus important for a normal pregnancy.

Protease-activated receptor 2 deficiency in hematopoietic lineage protects against myocardial infarction through attenuated inflammatory response and fibrosis

Ischaemic heart disease is one of the leading causes of death. Protease-activated receptor 2 (PAR2) is widely expressed within the cardiovascular system and is known to mediate inflammatory processes in various immunocytes, such as macrophages, mastocytes and neutrophils. Here, we investigated whether activating macrophage PAR2 modulates cardiac remodelling in a murine model of myocardial infarction. Myocardial infarction was produced by the permanent ligation of the left anterior descending coronary artery (LAD) in C57BL/6J background wild-type (WT) mice transplanted with bone marrow from WT or PAR2 knockout (PAR2 KO) mice. Hematopoietic deficiency of PAR2 had improvement of left ventricular systolic dysfunction and dilatation and decreased fibrosis deposition in remote zone at 1 week after LAD ligation. Inactivation of PAR2 also led to less recruitment of macrophages in myocardium, which was accompanied by decreased expression of pro-inflammatory cytokines. Furthermore, cultured cardiac fibroblasts (CFs) were activated and showed a fibrotic phenotype after being co-cultured in medium containing PAR2-activating macrophage, which enhances interferon-beta (INF-β) expression. The beneficial effects of macrophages with INF-β neutralisation or PAR2-deletion ameliorates the JAK/STAT3 pathway in CFs, which might be attributed to CF activation. These data suggest that macrophage-derived IFN-β plays a crucial role in adverse cardiac remodelling after myocardial infarction, at least in part, through a PAR2-dependent mechanism.

Blocking the Thrombin Receptor Promotes Repair of Demyelinated Lesions in the Adult Brain.

Myelin loss limits neurological recovery and myelin regeneration and is critical for restoration of function. We recently discovered that global knock-out of the thrombin receptor, also known as Protease Activated Receptor 1 (PAR1), accelerates myelin development. Here we demonstrate that knocking out PAR1 also promotes myelin regeneration. Outcomes in two unique models of myelin injury and repair, that is lysolecithin or cuprizone-mediated demyelination, showed that PAR1 knock-out in male mice improves replenishment of myelinating cells and remyelinated nerve fibers and slows early axon damage. Improvements in myelin regeneration in PAR1 knock-out mice occurred in tandem with a skewing of reactive astrocyte signatures toward a prorepair phenotype. In cell culture, the promyelinating effects of PAR1 loss of function are consistent with possible direct effects on the myelinating potential of oligodendrocyte progenitor cells (OPCs), in addition to OPC-indirect effects involving enhanced astrocyte expression of promyelinating factors, such as BDNF. These findings highlight previously unrecognized roles of PAR1 in myelin regeneration, including integrated actions across the oligodendrocyte and astroglial compartments that are at least partially mechanistically linked to the powerful BDNF-TrkB neurotrophic signaling system. Altogether, findings suggest PAR1 may be a therapeutically tractable target for demyelinating disorders of the CNS.SIGNIFICANCE STATEMENT Replacement of oligodendroglia and myelin regeneration holds tremendous potential to improve function across neurological conditions. Here we demonstrate Protease Activated Receptor 1 (PAR1) is an important regulator of the capacity for myelin regeneration across two experimental murine models of myelin injury. PAR1 is a G-protein-coupled receptor densely expressed in the CNS, however there is limited information regarding its physiological roles in health and disease. Using a combination of PAR1 knock-out mice, oligodendrocyte monocultures and oligodendrocyte-astrocyte cocultures, we demonstrate blocking PAR1 improves myelin production by a mechanism related to effects across glial compartments and linked in part to regulatory actions toward growth factors such as BDNF. These findings set the stage for development of new clinically relevant myelin regeneration strategies.

Alternaria induces airway epithelial cytokine expression independent of protease‐activated receptor

A novel fungal allergen, Alternaria (Alt), has been previously shown to associate with the pathogenesis of allergic rhinitis and bronchial asthma, particularly in arid and semi-arid regions. Airway epithelial cells are among the first to encounter Alt, and epithelial cytokine production and subsequent airway inflammation are early events in the response to Alt exposure. However, the underlying mechanism is unclear. As protease-activated receptor 2 (PAR2) has been implicated in most of the Alt-induced biological events, we investigated the regulation of airway inflammation and epithelial cytokine expression by PAR2. Wild-type (WT) and Par2 knockout (Par2-KO) mice were used to evaluate the in vivo role of PAR2. Primary human and mouse airway epithelial cells were used to examine the mechanistic basis of epithelial cytokine regulation in vitro. Surprisingly, Par2 deficiency had no negative impact on the change of lung function, inflammation and cytokine production in the mouse model of Alt-induced asthma. Alt-induced cytokine production in murine airway epithelial cells from Par2-KO mice was not significantly different from the WT cells. Consistently, PAR2 knockdown in human cells also had no effect on cytokine expression. In contrast, the cytokine expressions induced by synthetic PAR2 agonist or other asthma-related allergens (e.g. cockroach extracts) were indeed mediated via a PAR2-dependent mechanism. Finally, we found that EGFR pathway was responsible for Alt-induced epithelial cytokine expression. The activation of EGFR, but not PAR2, was likely to drive the airway inflammation and epithelial cytokine production induced by Alt.