Dasatinib promotes muscle differentiation and disrupts normal muscle regeneration.

Dasatinib is one of the second-generation tyrosine kinase inhibitors used to treat chronic myeloid leukemia and has a broad target spectrum, including KIT, PDGFR, and SRC family kinases. Due to its broad drug spectrum, dasatinib has been reported at the basic research level to improve athletic performance by eliminating senescent cell removal and to have an effect on muscle diseases such as Duchenne muscular dystrophy, but its effect on myoblasts has not been investigated. In this study, we evaluated the effects of dasatinib on skeletal muscle both under normal conditions and in the regenerating state. Dasatinib suppressed the proliferation and promoted the fusion of C2C12 myoblasts. During muscle regeneration, dasatinib increased the gene expressions of myogenic-related genes (Myod, Myog, and Mymx), and caused abnormally thin muscle fibers on the CTX-induced muscle injury mouse model. From these results, dasatinib changes the closely regulated gene expression pattern of myogenic regulatory factors during muscle differentiation and disrupts normal muscle regeneration. Our data suggest that when using dasatinib, its effects on skeletal muscle should be considered, particularly at regenerating stages.

Fuzapladib reduces postsurgical inflammation in the intestinal muscularis externa.

Postoperative ileus (POI) is a surgical complication that induces emesis and anorexia. Fuzapladib (FUZ), an inhibitor of leukocyte-function-associated antigen type 1 (LFA-1) activation, a leukocyte adhesion molecule, exerts anti-inflammatory effects by inhibiting leukocyte migration into the inflammatory site. In this study, we examined the prophylactic impact of FUZ on POI in a mouse model. POI model mice were generated by intestinal manipulation, and the effect of FUZ on intestinal transit and the infiltration of inflammatory cells into the ileal muscularis externa was assessed. The increased number of macrophages was significantly suppressed by FUZ, whereas the infiltration of neutrophils into the ileal muscularis externa was not sufficiently inhibited in the POI model mice. Additionally, FUZ did not ameliorate delayed gastrointestinal transit in POI model mice. In conclusion, our results suggest that FUZ does not improve delayed gastrointestinal transit but partially inhibits inflammation in the ileal muscularis externa in POI model mice. FUZ may be a potential anti-inflammatory agent for the management of post-surgical inflammation.

Toceranib phosphate (Palladia) reverses type 1 diabetes by preserving islet function in mice.

In recent years, strategies targeting ß-cell protection via autoimmune regulation have been suggested as novel and potent immunotherapeutic interventions against type 1 diabetes mellitus (T1D). Here, we investigated the potential of toceranib (TOC), a receptor-type tyrosine kinase (RTK) inhibitor used in veterinary practice, to ameliorate T1D. TOC reversed streptozotocin-induced T1D and improved the abnormalities in muscle and bone metabolism characteristic of T1D. Histopathological examination revealed that TOC significantly suppressed ß-cell depletion and improved glycemic control with restoration of serum insulin levels. However, the effect of TOC on blood glucose levels and insulin secretion capacity is attenuated in chronic T1D, a more ß-cell depleted state. These findings suggest that TOC improves glycemic control by ameliorating the streptozotocin-induced decrease in insulin secretory capacity. Finally, we examined the role of platelet-derived growth factor receptor (PDGFR) inhibition, a target of TOC, and found that inhibition of PDGFR reverses established T1D in mice. Our results show that TOC reverses T1D by preserving islet function via inhibition of RTK. The previously unrecognized pharmacological properties of TOC have been revealed, and these properties could lead to its application in the treatment of T1D in the veterinary field.

Purinergic P2X7 receptor antagonist ameliorates intestinal inflammation in postoperative ileus.

Postoperative ileus (POI) is a postsurgical gastrointestinal motility dysfunction caused by mechanical stress to the intestine during abdominal surgery. POI leads to nausea and vomiting reduced patient quality of life, as well as high medical costs and extended hospitalization. Intestinal inflammation caused by macrophages and neutrophils is thought to be important in the mechanism of POI. Surgery-associated tissue injury and inflammation induce the release of adenosine triphosphate (ATP) from injured cells. Released ATP binds the purinergic P2X7 receptor (P2X7R) expressed on inflammatory cells, inducing the secretion of inflammatory mediators. P2X7R antagonists are thought to be important mediators of the first step in the inflammation process, and studies in chemically induced colitis models confirmed that P2X7R antagonists exhibit anti-inflammatory effects. Therefore, we hypothesized that P2X7R plays an important role in POI. POI models were generated from C57BL/6J mice. Mice were treated with P2X7R antagonist A438079 (34 mg/kg) 30 min before and 2 hr after intestinal manipulation (IM). Inflammatory cell infiltration and gastrointestinal transit were measured. A438079 ameliorated macrophage and neutrophil infiltration in the POI model. Impaired intestinal transit improved following A438079 treatment. P2X7R was expressed on both infiltrating and resident macrophages in the inflamed ileal muscle layer. The P2X7R antagonist A438079 exhibits anti-inflammatory effects via P2X7R expressed on macrophages and therefore could be a target in the treatment of POI.

Atlantoaxial Rotatory Fixation after Microtia Reconstruction Surgery.

Nontraumatic atlantoaxial rotatory fixation after microtia reconstruction surgery is a rare complication. Intraoperative cervical hyperextension and/or excessive rotation and postoperative inflammation have been reported as causes of atlantoaxial rotatory fixation. We herein describe cases of atlantoaxial rotatory fixation after microtia reconstruction surgery. METHODS: This was a retrospective study of 80 patients (165 surgeries) who underwent microtia reconstruction surgery in Dokkyo Medical University Hospital between April 2006 and December 2012. The patient- and operation-related variables were obtained from medical charts. Neck radiographs and computed tomography scans of patients with atlantoaxial rotatory fixation were evaluated to check for cervical spine abnormalities. RESULTS: Five cases of atlantoaxial rotatory fixation after microtia reconstruction surgery were recorded. Three of these five cases were diagnosed with Klippel-Feil syndrome after the onset of atlantoaxial rotatory fixation. No significant difference was found in the operative duration and other variables between patients with atlantoaxial rotatory fixation and those without. All patients immediately underwent conservative treatment and showed complete recovery and no recurrences. CONCLUSIONS: Although atlantoaxial rotatory fixation is a rare complication, surgeons should consider it in patients with neck problems following microtia reconstruction surgery. A patient with microtia may have unrecognized Klippel-Feil syndrome. Patients with Klippel-Feil syndrome are more likely to develop atlantoaxial rotatory fixation, which may have severe consequences. Thus, it is crucial to preoperatively identify Klippel-Feil syndrome with neck radiography and to detect atlantoaxial rotatory fixation at the earliest.

Liver fibrosis-induced muscle atrophy is mediated by elevated levels of circulating TNFα.

Liver cirrhosis is a critical health problem associated with several complications, including skeletal muscle atrophy, which adversely affects the clinical outcome of patients independent of their liver functions. However, the precise mechanism underlying liver cirrhosis-induced muscle atrophy has not been elucidated. Here we show that serum factor induced by liver fibrosis leads to skeletal muscle atrophy. Using bile duct ligation (BDL) model of liver injury, we induced liver fibrosis in mice and observed subsequent muscle atrophy and weakness. We developed culture system of human primary myotubes that enables an evaluation of the effects of soluble factors on muscle atrophy and found that serum from BDL mice contains atrophy-inducing factors. This atrophy-inducing effect of BDL mouse serum was mitigated upon inhibition of TNFα signalling but not inhibition of myostatin/activin signalling. The BDL mice exhibited significantly up-regulated serum levels of TNFα when compared with the control mice. Furthermore, the mRNA expression levels of Tnf were markedly up-regulated in the fibrotic liver but not in the skeletal muscles of BDL mice. The gene expression analysis of isolated nuclei revealed that Tnf is exclusively expressed in the non-fibrogenic diploid cell population of the fibrotic liver. These findings reveal the mechanism through which circulating TNFα produced in the damaged liver mediates skeletal muscle atrophy. Additionally, this study demonstrated the importance of inter-organ communication that underlies the pathogenesis of liver cirrhosis.

Protease-Activated Receptor-2 Is Associated With Adverse Outcomes in Canine Mammary Carcinoma.

Protease-activated receptor-2 (PAR2) is a G protein-coupled receptor that is activated by serine proteases. In humans, PAR2 is highly expressed in various cancers, including breast cancer, and is associated with cancer progression and metastasis. However, the expression and roles of PAR2 in canine mammary carcinoma remain unclear. The purpose of this study was to examine the expression of PAR2 in canine mammary carcinoma, the association between PAR2 expression and clinical characteristics, and the role of PAR2 in the metastatic phenotypes of tumor cells. Mammary carcinoma from 31 dogs and 10 normal mammary glands were included in this study, and used for immunohistochemical analysis of PAR2 expression. Normal mammary glands did not express PAR2. In contrast, mammary carcinomas showed PAR2 immunoreactivity in the cytoplasm, and its expression level varied between specimens from negative to strongly positive. The overall survival of dogs with high PAR2 expression was shorter than that of dogs with low PAR2 expression. Moreover, PAR2 expression level was associated with the presence of lymph node involvement, advanced clinical stage, and high histopathological grade. In vitro analyses revealed that a PAR2 agonist accelerated cell migration and invasion in a canine mammary carcinoma cell line. In addition, the PAR2 agonist induced epithelial-mesenchymal transition and actin polymerization. These results suggest that PAR2 expression plays a role in tumor progression and clinical outcomes in canine mammary carcinoma.

A new zinc chelator, IPZ-010 ameliorates postoperative ileus.

Zinc was discovered to be a novel second messenger in immunoreactive cells. We synthesized a novel free zinc chelator, IPZ-010. Here, we investigated the effects of IPZ-010 in a mouse postoperative ileus model and determined the effects of zinc signal inhibition as a new therapeutic strategy against postoperative ileus. Zinc waves were measured in bone marrow-derived mast cells (BMMCs) loaded with a zinc indicator, Newport green. Degranulation and cytokine expression were measured in BMMCs and bone marrow-derived macrophages (BMDMs). Postoperative ileus model mice were established with intestinal manipulation. Mice were treated with IPZ-010 (30 mg/kg, s.c. or p.o.) 1 h before and 2 h and 4 h after intestinal manipulation. Gastrointestinal transit, inflammatory cell infiltration, and expression of inflammatory mediators were measured. Free zinc waves occurred following antigen stimulation in BMMCs and were blocked by IPZ-010. IPZ-010 inhibited interleukin-6 secretion and degranulation in BMMCs. IPZ-010 inhibited tumor necrosis factor-α mRNA expression in BMMCs stimulated with lipopolysaccharide or adenosine triphosphate, whereas IPZ-010 had no effects on tumor necrosis factor-α mRNA expression in BMDMs stimulated with lipopolysaccharide or adenosine triphosphate. In postoperative ileus model mice, IPZ-010 inhibited leukocyte infiltration and cytokine expression, which ameliorated gastrointestinal transit. Furthermore, ketotifen (1 mg/kg) induced similar effects as IPZ-010. These effects were not amplified by co-administration of IPZ-010 and ketotifen. IPZ-010 inhibited zinc waves, resulting in inhibition of inflammatory responses in activated BMMCs in vitro. Targeting zinc waves in inflammatory cells may be a novel therapeutic strategy for treating postoperative ileus.

Hyperglycemia in the early stages of type 1 diabetes accelerates gastric emptying through increased networks of interstitial cells of Cajal.

Gastric emptying (GE) can be either delayed or accelerated in diabetes mellitus (DM). However, most research has focused on delayed GE mediated by a chronic hyperglycemic condition in DM. As such, the function of GE in the early stages of DM is not well understood. Interstitial cells of Cajal (ICC) are pacemaker cells in the gastrointestinal tract. In the present study, we investigated changes in GE and ICC networks in the early stages of DM using a streptozotocin-induced type 1 diabetic mouse model. The changes in GE were measured by the 13C-octanoic acid breath test. ICC networks were immunohistochemically detected by an antibody for c-Kit, a specific marker for ICC. Our results showed that GE in type 1 DM was significantly accelerated in the early stages of DM (2-4 weeks after onset). In addition, acute normalization of blood glucose levels by a single administration of insulin did not recover normal GE. ICC networks of the gastric antrum were significantly increased in DM and were not affected by the acute normalization of blood glucose. In conclusion, our results suggest that GE is accelerated in the early stages of DM, and it is associated with increased ICC networks. This mechanism may help to clarify a link between the onset of DM and GE disorders.

Neural anti-inflammatory action mediated by two types of acetylcholine receptors in the small intestine.

Gastrointestinal prokinetic agents function as serotonin-4 receptor (5-HT4R) agonists to activate myenteric plexus neurons to release acetylcholine (ACh), which then induce anti-inflammatory action. Details of this pathway, however, remain unknown. The aim of this study is to clarify the anti-inflammatory mechanism underlying the 5-HT4R agonist, mosapride citrate (MOS)-induced anti-inflammatory action on postoperative ileus (POI). POI models were generated from wild-type C57BL6/J (WT), 5-HT4R knock-out (S4R KO), α7 nicotinic AChR KO (α7 R KO), and M2 muscarinic ACh receptor KO (M2R KO) mice. MOS attenuated leukocyte infiltration in WT. MOS-induced anti-inflammatory action was completely abolished in both S4R KO and S4R KO mice upon wild-type bone marrow transplantation. MOS-induced anti-inflammatory action against macrophage infiltration, but not neutrophil infiltration, was attenuated in α7 R KO mice. Selective α7nAChR agonists (PNU-282987 and AR-R17779) also inhibited only macrophage infiltration in POI. MOS-mediated inhibition of neutrophil infiltration was diminished by atropine, M2AChR antagonist, methoctramine, and in M2R KO mice. Stimulation with 5-HT4R inhibits leukocyte infiltration in POI, possibly through myenteric plexus activation. Released ACh inhibited macrophage and neutrophil infiltration likely by activation of α7nAChR on macrophages and M2AChR. Thus, macrophage and neutrophil recruitment into inflamed sites is regulated by different types of AChR in the small intestine.

Serotonin 3 receptor signaling regulates 5-fluorouracil-mediated apoptosis indirectly via TNF-α production by enhancing serotonin release from enterochromaffin cells.

Antagonists of the 5-hydroxytryptamine (serotonin) 3 receptor (5-HT3R) have anti-inflammatory and anti-apoptotic activities, but the detailed, underlying mechanisms are not well understood. We focused on anti-apoptotic activities via 5-HT3R signaling to clarify the underlying mechanisms. Mice were administered 5-fluorouracil (5-FU), which induced apoptosis in intestinal epithelial cells. Coadministration with 5-HT3R antagonists or agonists tended to decrease or increase the number of apoptotic cells, respectively. In serotonin 3A receptor (5-HT3AR) null (HTR3A-/-) mice, the number of apoptotic cells induced by 5-FU was decreased compared with that in wild-type (WT) mice. Bone marrow (BM) transplantation was performed to determine if BM-derived immune cells regulated 5-FU-induced apoptosis, but they were found to be unrelated to this process. Data from 5-HT3AR/enhanced green fluorescent protein reporter mice revealed that 50% of enterochromaffin (EC) cells expressed 5-HT3AR, but the number of apoptotic cells induced by 5-FU in the intestinal crypt organoids of HTR3A-/- mice was not altered compared with WT mice. In contrast, plasma 5-HT concentrations in WT mice but not in HTR3A-/- mice administered 5-FU were increased significantly. In conclusion, 5-HT3R signaling may enhance 5-HT release, possibly from EC cells intravascularly, or paracrine, resulting in increases in plasma 5-HT concentration, which in turn, enhances apoptotic activities induced by 5-FU.-Mikawa, S., Kondo, M., Kaji, N., Mihara, T., Yoshitake, R., Nakagawa, T., Takamoto, M., Nishimura, R., Shimada, S., Ozaki, H., Hori, M. Serotonin 3 receptor signaling regulates 5-fluorouracil-mediated apoptosis indirectly via TNF-α production by enhancing serotonin release from enterochromaffin cells.

Induction of myosin light chain kinase and CPI-17 by TGF-ß accelerates contractile activity in intestinal epithelial cells.

Epithelial-mesenchymal transition (EMT) is an orchestral and functional change in epithelial cells. Many signaling pathways are involved in EMT, and transforming growth factor-beta (TGF-ß) is considered to be one of the most important factors in induction of EMT. In this study, we treated the rat intestinal epithelial cell line (IEC-6) with TGF-ß1 as a signaling stimulant. Gross analysis of IEC-6 cells showed typical characteristics of epithelial cells such as cuboidal morphology and cell-cell contact, whereas treatment with TGF-ß1 (10 ng/ml-1) for 7 days produced robust, spindle-shaped morphology. Immunocytochemistry analysis showed distinct E-cadherin staining in IEC-6 cells, but weak and faint in EMT cells. EMT cells showed positive expression of α-SMA and tenascin-C but IEC-6 cells did not. Quantitative real-time PCR analysis showed that myosin light chain kinase and C-kinase potentiated protein phosphatase-1 inhibitor (CPI-17) mRNAs were significantly upregulated in EMT cells. Immunocytochemistry analysis also showed that EMT cells strongly expressed CPI-17 but IEC-6 cells did not. A collagen gel contraction assay revealed that EMT cells had greatly increased contraction compared with control cells. These results suggest that the increased contractile activity induced by TGF-ß in EMT cells may be attributable to the upregulation of molecules responsible for myosin phosphorylation/de-phosphorylation.

The anti-inflammatory pathway regulated via nicotinic acetylcholine receptors in rat intestinal mesothelial cells.

Regulation of inflammation in intestinal mesothelial cells in the abdominal cavity is important for the pathogeny of clinical conditions, such as postoperative ileus, peritonitis and encapsulating peritoneal sclerosis. Here we have examined the inflammatory effect of lipopolysaccharide (LPS) and the anti-inflammatory effect of nicotinic acetylcholine receptor stimulation in rat intestinal mesothelial cells. LPS upregulated mRNA expression of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1) and inducible nitric oxide synthase (iNOS). The α7, α9 and α10 subunits of nicotinic acetylcholine receptor were detected in intestinal mesothelial cells. Nicotine (10 nM) significantly inhibited LPS-induced mRNA expression of IL-1ß and iNOS, but not TNF-α and MCP-1. In addition, the α7 nicotinic acetylcholine receptor selective agonist, PNU-282987 (10 nM), significantly inhibited LPS-induced mRNA expression of IL-1ß but not TNF-α, iNOS and MCP-1. Finally, we found that enteric nerves adhered to intestinal mesothelial cells located under the ileal serosa. In conclusion, intestinal mesothelial cells react to LPS to induce the production of nitric oxide from iNOS. The anti-inflammatory action of intestinal mesothelial cells expressing α7nAChR may be mediated via their connectivity with enteric nerves.

Therapeutic Action of Honokiol on Postoperative Ileus via Downregulation of iNOS Gene Expression.

Postoperative ileus is a common complication after intra-abdominal surgery. Nitric oxide produced by macrophages in the inflamed gastrointestinal tract plays a crucial role in the pathogeny of postoperative ileus. Honokiol, extracted from the bark of Magnolia spp., is a natural compound with a biphenolic structure. In the present study, we examined the effect of honokiol on postoperative ileus and discussed its site of action. Postoperative ileus model mice were generated by surgical intestinal manipulation. Mice were administered honokiol (10 mg kg-1, per os) 1 h before and after intestinal manipulation. Gastrointestinal transit, leukocyte infiltration, and messenger RNA (mRNA) expression of inflammatory mediators were measured in postoperative ileus model mice with or without honokiol. We also investigated the inflammatory effect of honokiol in lipopolysaccharide-stimulated peritoneal macrophages. Gastrointestinal transit was delayed in postoperative ileus model mice and honokiol recovered the impaired transit. Honokiol significantly inhibited leukocyte infiltration and upregulation of proinflammatory cytokines (tumor necrosis factor-α, interleukin-1ß, and interleukin-6) and inducible nitric oxide synthase in the ileal muscle layer of postoperative ileus model mice. In peritoneal macrophages activated by lipopolysaccharide, honokiol significantly inhibited the upregulated mRNA expression of proinflammatory cytokines and inducible nitric oxide synthase. Honokiol significantly recovered gastrointestinal dysmotility and inhibited intestinal inflammation in postoperative ileus. Moreover, honokiol was suggested to have effects on macrophages, namely, inhibiting mRNA expression of proinflammatory cytokines and inducible nitric oxide synthase. Taken together, honokiol represents a potential novel therapeutic agent for postoperative ileus.

Nitric oxide-induced oxidative stress impairs pacemaker function of murine interstitial cells of Cajal during inflammation.

The pacemaker function of interstitial cells of Cajal (ICC) is impaired during intestinal inflammation. The aim of this study is to clarify the pathophysiological mechanisms of ICC dysfunction during inflammatory condition by using intestinal cell clusters. Cell clusters were prepared from smooth muscle layer of murine jejunum and treated with interferon-gamma and lipopolysaccharide (IFN-γ+LPS) for 24h to induce inflammation. Pacemaker function of ICC was monitored by measuring cytosolic Ca(2+) oscillation in the presence of nifedipine. Treatment with IFN-γ+LPS impaired the pacemaker activity of ICC with increasing mRNA level of interleukin-1 beta, tumor necrosis factor-alpha and interleukin-6 in cell clusters; however, treatment with these cytokines individually had little effect on pacemaker activity of ICC. Treatment with IFN-γ+LPS also induced the expression of inducible nitric oxide synthase (iNOS) in smooth muscle cells and resident macrophages, but not in ICC. Pretreatment with NOS inhibitor, L-NAME or iNOS inhibitor, 1400W ameliorated IFN-γ+LPS-induced pacemaker dysfunction of ICC. Pretreatment with guanylate cyclase inhibitor, ODQ did not, but antioxidant, apocynin, to suppress NO-induced oxidative stress, significantly suppressed the impairment of ICC function induced by IFN-γ+LPS. Treatment with IFN-γ+LPS also decreased c-Kit-positive ICC, which was prevented by pretreatment with L-NAME. However, apoptotic ICC were not detected in IFN-γ+LPS-treated clusters, suggesting IFN-γ+LPS stimulation just changed the phenotype of ICC but not induced cell death. Moreover, ultrastructure of ICC was not disturbed by IFN-γ+LPS. In conclusion, ICC dysfunction during inflammation is induced by NO-induced oxidative stress rather than NO/cGMP signaling. NO-induced oxidative stress might be the main factor to induce phenotypic changes of ICC.

Anti-emetic drug maropitant induces intestinal motility disorder but not anti-inflammatory action in mice.

Maropitant is a neurokinin 1 receptor (NK1R) antagonist that is clinically used as a new anti-emetic drug for dogs. Substance P (SP) and its receptor NK1R are considered to modulate gastrointestinal peristalsis. In addition, SP works as an inflammatory mediator in gastrointestinal diseases. Aim of this study is to clarify the effects of maropitant on intestinal motility and inflammation in mice. Ex vivo examination of luminal pressure-induced intestinal motility of whole intestine revealed that maropitant (0.1-10 µM) increased frequency of contraction, decreased amplitude of contraction and totally inhibited motility index in a concentration-dependent manner. We measured intestinal transit in vivo by measuring transportation of orally administered luminal content labeled with phenol red. Our results demonstrated that maropitant (10 mg/kg, SC) delayed intestinal transit. Geometric center value was significantly decreased in maropitant-treated mice. Anti-inflammatory effects of maropitant against leukocytes infiltration into the intestinal smooth muscle layer in post-operative ileus (POI) model mice were measured by immunohistochemistry. In POI model mice, a great number of CD68-positive macrophages or MPO-stained neutrophils infiltrated into the inflamed muscle region of the intestine. However, in the maropitant treated mice, the infiltration of leukocytes was not inhibited. The results indicated that maropitant has ability to induce disorder of intestinal motility in mice, but has no anti-inflammatory action in the mouse of a POI model. In conclusion, in mice, maropitant induces disorder of intestinal motility in vivo.

Protease-activated receptor-2 induces proinflammatory cytokine and chemokine gene expression in canine keratinocytes.

Although the molecular basis of the allergenicity remains to be fully elucidated, the ability of allergens to elicit allergic responses is at least partly attributed to their proteolytic activity. Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is activated by site-specific proteolysis by serine proteases and is known to mediate inflammatory processes in various tissues. In this study, we investigated the effects of trypsin, a major serine protease, and a human PAR-2 agonist peptide (SLIGKV-NH2) on proinflammatory cytokine and chemokine gene expression in the canine keratinocyte cell line CPEK. The expression of PAR-2 mRNA and protein in CPEK cells was detected by RT-PCR and Western blotting, respectively. The localization of PAR-2 in CPEK was examined by immunofluorescence. The mRNA expression levels of proinflammatory cytokines and chemokines were quantified by real-time RT-PCR. The free intracellular Ca(2+) concentration was measured using the Ca(2+)-sensitive fluorescent dye. CPEK cells constitutively expressed PAR-2 mRNA and protein. Stimulation of CPEK cells with trypsin induced significant upregulation of the mRNA expression levels of tumor necrosis factor alpha (TNF-α, P<0.05), granulocyte-macrophage colony-stimulating factor (GM-CSF, P<0.01), thymus and activation regulated chemokine (TARC/CCL17, P<0.01), and interleukin 8 (IL-8/CXCL8, P<0.01). Similarly, the PAR-2 agonist peptide increased the mRNA expression levels of TNF-α (P<0.05), GM-CSF (P<0.05), TARC/CCL17 (P<0.05), and IL-8/CXCL8 (P<0.05) in CPEK cells. Both trypsin and the PAR-2 agonist peptide increased the intracellular Ca(2+) concentration and PAR-2 internalization. These results suggest that PAR-2 activation can augment inflammatory cytokine and chemokine expression in canine keratinocytes, and it may initiate allergic inflammation through the proteolytic activity of allergens in canine atopic dermatitis.