Rapamycin inhibits human renal epithelial cell proliferation: effect on cyclin D3 mRNA expression and stability.
BACKGROUND
Recent data have suggested that rapamycin use during the initial period after transplantation is associated with prolonged delayed graft function (DGF). Because of the known effects of rapamycin in other cell types, we speculated that this action may be secondary to human renal epithelial cells (HRECs) inhibition of proliferation.


METHODS
Primary cultures of HRECs were incubated with various concentrations of rapamycin. Cell proliferation was evaluated by cytotoxicity assays. The cell cycle was analyzed by flow cytometry. Protein expression levels were assessed by Western blot. Cyclin D3 mRNA levels were measured by quantitative real-time polymerase chain reaction (PCR). The transcriptional activity of the cyclin D3 gene was evaluated using transient transfection.


RESULTS
Rapamycin exerted a significant concentration-dependent antiproliferative effect on growing HRECs by inhibiting the G(1) to S transition. The p70(S6) kinase pathway leading to cell cycle progression was found to be active, and low concentrations of rapamycin dramatically reduced p70(S6) kinase phosphorylation. Rapamycin completely inhibited the increase in cyclin D3 protein expression and mRNA accumulation induced by fetal calf serum, but did not affect cyclin E or cdk-inhibitor expression levels. This regulation of cyclin D3 protein expression is mainly due to a destabilization of its mRNA. Rapamycin reduced the mRNA half-life by 26% (4.8 +/- 1.3 hours vs. 6.5 +/- 1.0 hours, P < 0.001).


CONCLUSIONS
Rapamycin inhibits the proliferative response of HRECs to mitogenic stimuli, and causes cell cycle arrest in the early G(1) phase, not only by a nonspecific process due to inhibition of the p70(S6k) pathway, but also by a direct effect on cyclin D3 mRNA stability.
Pallet
Nicolas
N
Unité INSERM UMR S490, Molecular Toxicology, Centre Universitaire des Saints-Pères, Université René Descartes, Paris, France.
Thervet
Eric
E
Le Corre
Delphine
D
Knebelmann
Bertrand
B
Nusbaum
Patrick
P
Tomkiewicz
Céline
C
Meria
Paul
P
Flinois
Jean-Pierre
JP
Beaune
Philippe
P
Legendre
Christophe
C
Anglicheau
Dany
D
D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D056743:Cyclin D3; D016213:Cyclins / Q000235:genetics*; D004847:Epithelial Cells / Q000187:drug effects; D016193:G1 Phase / Q000187:drug effects; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D007668:Kidney / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation; D020871:RNA Stability / Q000187:drug effects*; D012333:RNA, Messenger / Q000032:analysis*; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D016196:S Phase / Q000187:drug effects; D020123:Sirolimus / Q000494:pharmacology*
C534696:CCND3 protein, human; D056743:Cyclin D3; D016213:Cyclins; D007166:Immunosuppressive Agents; D012333:RNA, Messenger; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D020123:Sirolimus