title: Complement (C5b-9) induces glomerular epithelial cell DNA synthesis but not proliferation in vitro.
abstract: BACKGROUND
  The C5b-9 membrane attack complex of complement is the principal mediator of injury induced experimentally by antibodies directed at glomerular cell membranes. In experimental membranous nephropathy, C5b-9 induced injury to the glomerular visceral epithelial cell (VEC) is associated with DNA synthesis, but not cytokinesis. In the current study we determined if C5b-9 increases DNA synthesis in VEC in vitro, and defined the mechanisms involved.
  
  
  METHODS
  Rat VEC in vitro were divided into three groups: (1) sensitized with anti-VEC antibody and exposed to sublytic concentrations of C +/PVG serum (normal complement components); (2) anti-VEC antibody and control C-/PVG serum (C6 deficient); (3) no anti-VEC antibody. DNA synthesis (BrdU staining), mitosis (mitotic figures) and cytokinesis (cell counts) were measured at 24 and 48 hours. To examine the expression of specific S-phase and M-phase cell cycle regulatory proteins and their inhibitors, immunostaining and Western blot analysis was performed for cyclin A, CDK2, p21 and p27, cyclin B and cdc2.
  
  
  RESULTS
  In the absence of growth factors, sublytic C5b-9 attack did not increase proliferation. In contrast, sublytic C5b-9 attack (group 1) augmented growth factor induced DNA synthesis by 50% compared to controls (groups 2 and 3; P < 0.001), and was accompanied by increased levels of cyclin A and CDK2, and a decrease in the cyclin kinase inhibitor p27 (but not p21). Sublytic C5b-9 attack reduced the expression of the M phase cell cycle proteins, cyclin B and cdc2, accompanied by reduced mitosis (mitotic figures) and cytokinesis (cell number).
  
  
  CONCLUSIONS
  Our results show that the C5b-9 augmented growth factor entry into the S phase in VEC is regulated by changes in specific cell cycle regulatory proteins. However, antibody and complement decreased the M phase cell cycle proteins, and prevented VEC mitosis and cytokinesis, suggesting a delay or arrest at the G2/M phase.
authors.affiliation: Department of Medicine, University of Washington, Seattle 98195, USA. stuartjs@u.washington.edu
authors.forename: S J
authors.initials: SJ
authors.lastname: Shankland
authors.forename: J W
authors.initials: JW
authors.lastname: Pippin
authors.forename: W G
authors.initials: WG
authors.lastname: Couser
mesh_terms: D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D015938:Complement Membrane Attack Complex / Q000378:metabolism / Q000494:pharmacology*; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology; D016222:Fibroblast Growth Factor 2 / Q000276:immunology / Q000494:pharmacology; D016195:G2 Phase / Q000187:drug effects; D066298:In Vitro Techniques; D007678:Kidney Glomerulus / Q000166:cytology*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008934:Mitogens / Q000378:metabolism; D008938:Mitosis / Q000187:drug effects; D009500:Neutralization Tests; D051381:Rats; D016196:S Phase / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000494:pharmacology
chemical_list: D000906:Antibodies; D015938:Complement Membrane Attack Complex; D008934:Mitogens; D016212:Transforming Growth Factor beta; D016222:Fibroblast Growth Factor 2; D004247:DNA; D007770:L-Lactate Dehydrogenase; D018844:Cyclin-Dependent Kinases