title: Protease-activated receptor-2 induces proinflammatory cytokine and chemokine gene expression in canine keratinocytes.
abstract: 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.
authors: Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors: Shingo
authors: S
authors: Maeda
authors: Sadatoshi
authors: S
authors: Maeda
authors: Koichi
authors: K
authors: Ohno
authors: Noriyuki
authors: N
authors: Kaji
authors: Masatoshi
authors: M
authors: Hori
authors: Yasuhito
authors: Y
authors: Fujino
authors: Hajime
authors: H
authors: Tsujimoto
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D018925:Chemokines / Q000235:genetics; D003429:Cross Reactions; D016207:Cytokines / Q000235:genetics; D003876:Dermatitis, Atopic / Q000209:etiology; D004285:Dogs; D057809:HEK293 Cells; D006801:Humans; D015603:Keratinocytes / Q000276:immunology*; D012333:RNA, Messenger / Q000032:analysis; D044464:Receptor, PAR-2 / Q000819:agonists / Q000502:physiology*; D014357:Trypsin / Q000494:pharmacology
chemical_list: D018925:Chemokines; D016207:Cytokines; D012333:RNA, Messenger; D044464:Receptor, PAR-2; D014357:Trypsin; D002118:Calcium