title: Activation of P2X7R and downstream effects in bleomycin treated lung epithelial cells.
abstract: Changes in intracellular calcium concentration [Ca(2+)](i) are believed to influence the proliferation and differentiation of airway epithelial cells both in vivo and in vitro. In the present study, using mouse alveolar epithelial E10 cells, we demonstrated that the treatment of lung epithelial cells with BLM resulted in elevated intracellular Ca(2+) levels. BLM further increased P2rx7 mRNA expression and P2X7R protein levels, paralleled by increased PKC-β1 levels. BLM treatment or stimulation of the P2X7R with the P2X7R agonist BzATP induced translocation of PKC-β1 from the cytoplasm to the membrane. The expression of PKC-β1 was repressed by the P2X7R inhibitor oxATP, suggesting that PKC-β1 is downstream of P2X7R activation. Furthermore, cells exposed to BLM contained increased amounts of P2X7R and PKC-β1 in Cav-1 containing lipid raft fractions. The comparison of lung tissues from wild-type and P2rx7(-/-) mice revealed decreased protein and mRNA levels of PKC-β1 and CaM as well as decreased immunoreactivity for PKC-β1. The knockdown of P2X7R in alveolar epithelial cells resulted also in a loss of PKC-β1. These data suggest that the effect of P2X7R on expression of PKC-β1 detected in alveolar epithelial cells is also functioning in the animal model. Immunohistochemical evaluation of fibrotic lungs derived from a BLM-induced mouse model revealed a strong increase in PKC-β1 immunoreactivity. The present experiments demonstrated that the increased expression of P2X7R influences PKC-β1. We predict that increased Ca(2+) concentration stimulates PKC-β1, whereas the prerequisite for activating PKC-β1 after P2X7R increase remained to be determined. Our findings suggest that PKC-β1 is important in the pathogenesis of pulmonary fibrosis.
authors.affiliation: University of Technology Dresden, Department of Anatomy, Medical Clinic, Fetscherstr. 76, 01307 Dresden, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Bläsche
authors.forename: Georg
authors.initials: G
authors.lastname: Ebeling
authors.forename: Srikanth
authors.initials: S
authors.lastname: Perike
authors.forename: Karina
authors.initials: K
authors.lastname: Weinhold
authors.forename: Michael
authors.initials: M
authors.lastname: Kasper
authors.forename: Kathrin
authors.initials: K
authors.lastname: Barth
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001761:Bleomycin / Q000494:pharmacology*; D020013:Calcium Signaling; D002460:Cell Line; D004195:Disease Models, Animal; D004847:Epithelial Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008168:Lung / Q000473:pathology; D051379:Mice; D018345:Mice, Knockout; D011493:Protein Kinase C / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D064546:Protein Kinase C beta; D021381:Protein Transport / Q000187:drug effects; D011658:Pulmonary Fibrosis / Q000139:chemically induced / Q000378:metabolism*; D058911:Purinergic P2X Receptor Agonists / Q000494:pharmacology*; D058486:Receptors, Purinergic P2X7 / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D058911:Purinergic P2X Receptor Agonists; D058486:Receptors, Purinergic P2X7; D001761:Bleomycin; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D064546:Protein Kinase C beta