title: Inflammatory mediators modulate thrombin and cathepsin-G signaling in human bronchial fibroblasts by inducing expression of proteinase-activated receptor-4.
abstract: Human lung fibroblasts express proteinase-activated receptor-1 (PAR1), PAR2 and PAR3, but not PAR4. Because PAR2 has inflammatory effects on human primary bronchial fibroblasts (HPBF), we asked 1) whether the inflammatory mediators TNF-alpha and LPS could modify HPBF PAR expression and 2) whether modified PAR expression altered HPBF responsiveness to PAR agonists in terms of calcium signaling and cell growth. TNF-alpha and LPS induced PAR4 mRNA expression (RT-PCR) at 6 h and 24 h, respectively. TNF-alpha and LPS also upregulated PAR2 mRNA expression with similar kinetics but had negligible effect on PAR1 and PAR3. Flow cytometry for PAR1, PAR2, and PAR3 also demonstrated selective PAR2 upregulation in response to TNF-alpha and LPS. Intracellular calcium signaling to SLIGKV-NH2 (a selective PAR2-activating peptide; PAR2-AP) and AYPGQV-NH2 (PAR4-AP) revealed that TNF-alpha and LPS induced maximal responses to these PAR agonists at 24 h and 48 h, respectively. Upregulation of PAR2 by TNF-alpha heightened HPBF responses to trypsin, while PAR4 induction enabled cathepsin-G-mediated calcium signaling. Cathepsin-G also disarmed PAR1 and PAR2 in HPBF, while tryptase disarmed PAR2. Induction of PAR4 also enabled thrombin to elicit a calcium signal through both PAR1 and PAR4, as determined by a desensitization assay. In cell growth assays the PAR4 agonists cathepsin-G and AYPGQV-NH2 reduced HPBF cell number only in TNF-alpha-treated HPBF. Moreover, the mitogenic effect of thrombin (a PAR1/PAR4 agonist) but not the PAR1-AP TFLLR-NH2, was ablated in TNF-alpha-treated HPBF. These findings point to an important mechanism, whereby cellular responses to thrombin and cathepsin-G can be modified during an inflammatory response.
authors.affiliation: Division of Cardiovascular and Respiratory Studies, Postgraduate Medical Institute of the University of Hull, Hull York Medical Schoo, East Yorkshire, UK.
authors.forename: Rithwik
authors.initials: R
authors.lastname: Ramachandran
authors.forename: Laura R
authors.initials: LR
authors.lastname: Sadofsky
authors.forename: Yupei
authors.initials: Y
authors.lastname: Xiao
authors.forename: Andrew
authors.initials: A
authors.lastname: Botham
authors.forename: Michael
authors.initials: M
authors.lastname: Cowen
authors.forename: Alyn H
authors.initials: AH
authors.lastname: Morice
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Compton
mesh_terms: D001980:Bronchi / Q000166:cytology / Q000378:metabolism*; D056649:Cathepsin G; D002403:Cathepsins / Q000378:metabolism*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D044463:Receptor, PAR-1 / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D044464:Receptor, PAR-2 / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D018179:Receptors, Thrombin / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000378:metabolism*; D015398:Signal Transduction; D013917:Thrombin / Q000378:metabolism*; D013918:Thrombin Time; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D008070:Lipopolysaccharides; D012333:RNA, Messenger; D044463:Receptor, PAR-1; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin; D014409:Tumor Necrosis Factor-alpha; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D013917:Thrombin; C112908:protease-activated receptor 4