Sj. Shankland et al., Complement (C5b-9) induces glomerular epithelial cell DNA synthesis but not proliferation in vitro, KIDNEY INT, 56(2), 1999, pp. 538-548
Background. The C5b-9 membrane attack complex of complement is the principa
l mediator of injury induced experimentally by antibodies directed at glome
rular 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 i
nvolved.
Methods. Rat VEC in vitro were divided into three groups: (1) sensitized wi
th anti-VEC antibody and exposed to sublytic concentrations of C +/PVG seru
m (normal complement components); (2) anti-VEC antibody and control C-/PVG
serum (C6 deficient); (3) no anti-VEC antibody. DNA synthesis (BrdU stainin
g), mitosis (mitotic figures) and cytokinesis (cell counts) were measured a
t 24 and 48 hours. To examine the expression of specific S-phase and M-phas
e cell cycle regulatory proteins and their inhibitors, immunostaining and W
estern 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 in
crease proliferation. In contrast, sublytic C5b-9 attack (group 1) augmente
d 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). Sub
lytic C5b-9 attack reduced the expression of the M phase cell cycle protein
s, 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 regu
latory proteins. However, antibody and complement decreased the M phase cel
l cycle proteins, and prevented VEC mitosis and cytokinesis, suggesting a d
elay or arrest at the G(2)/M phase.