title: Per a 10 activates human derived epithelial cell line in a protease dependent manner via PAR-2.
abstract: BACKGROUND
  Protease activity of Per a 10 has been shown to modulate dendritic cells toward Th-2 polarization and to induce airway inflammation.
  
  
  OBJECTIVE
  To elucidate the role of serine protease activity of Per a 10 in inducing biochemical responses in epithelial cells.
  
  
  METHODS
  Per a 10 was inactivated by heat treatment (ΔPer a 10) or AEBSF (iPer a 10). A549 cells were exposed to either enzymatically active/inactive Per a 10. The supernatant was analyzed for the secretion of proinflammatory cytokines by ELISA. Ca(2+) mobilization was analyzed by flow cytometry. A PAR-2 derived synthetic peptide 28GTNRSSKGRSLIGKVDGTSHVTGKGVTC54 was incubated with Per a 10 and the resultant cleaved products were analyzed by LC-MS. PAR-2 activation was inhibited by PAR-2 cleavage inhibiting antibody.
  
  
  RESULTS
  ΔPer a 10 was completely inactivated whereas iPer a 10 showed some residual activity. nPer a 10 having protease activity increased the secretion of IL-6, IL-8 and GMCSF from A549 in a dose and time dependent manner whereas iPer a 10 has reduced cytokine secretion. ΔPer a 10 and rPer a 10 were unable to activate the cells. nPer a 10 mobilized intracellular Ca(2+). nPer a 10 cleaved the PAR-2 derived peptide between arginine and serine residues (36R-S37) to expose PAR-2 ligand SLIGKV, as determined by LC-MS. Incubating with anti-PAR-2 cleavage antibody showed diminished cytokine secretion when treated with nPer a 10.
  
  
  CONCLUSIONS
  Serine protease activity of Per a 10 activates A549 cells to secrete proinflammatory cytokines by PAR-2 activation and Ca(2+)mobilization and can be exploited therapeutically.
authors.affiliation: Allergy and Immunology Section, CSIR-Institute of Genomics and Integrative Biology, Delhi University Campus, Mall Road, Delhi 110007, India; Department of Biotechnology, University of Pune, Ganeshkhind, Pune 411007, India.
authors.forename: Sagar L
authors.initials: SL
authors.lastname: Kale
authors.affiliation: Allergy and Immunology Section, CSIR-Institute of Genomics and Integrative Biology, Delhi University Campus, Mall Road, Delhi 110007, India. Electronic address: naveen@igib.res.in.
authors.forename: Naveen
authors.initials: N
authors.lastname: Arora
mesh_terms: D000485:Allergens / Q000276:immunology*; D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D016207:Cytokines / Q000096:biosynthesis; D004847:Epithelial Cells / Q000276:immunology* / Q000378:metabolism*; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D019476:Insect Proteins / Q000276:immunology*; D042541:Intracellular Space / Q000378:metabolism; D059748:Proteolysis; D044464:Receptor, PAR-2 / Q000737:chemistry / Q000378:metabolism*; D057057:Serine Proteases / Q000276:immunology*
chemical_list: D000485:Allergens; D016207:Cytokines; D018836:Inflammation Mediators; D019476:Insect Proteins; D044464:Receptor, PAR-2; D057057:Serine Proteases; D002118:Calcium
keywords: Airway epithelium; Ca(2+) mobilization; PAR-2; Proinflammatory cytokines; Serine proteases