Rapamycin inhibits expression of minichromosome maintenance proteins in vascular smooth muscle cells

Abstract

Background: Rapamycin inhibits proliferation of vascular smooth muscle cells (VSMC) and local delivery of rapamycin from polymer-coated stents represents a novel therapeutic strategy for preventing postangioplasty restonsis. The molecular mechanims by which rapamycin exerts its antiproliferative activity involves an inhibition of Gi +S phase transition. However, the effects of rapamycin on S phase and DNA replication remain to be investigated. In the present study the effect of rapamycin on minichromosome maintenance (MCM) proteins 6 and 7, two proteins essential for initiation of DNA’replication. was investigated in rat aortic VSMC. Methods and results: Rapamycin substantially inhibited mitogen-induced MCM6 and MCM7 mRNA (68.2 f 5.6 %. 71.2 f 6.9 % inhibition vs. IO % FBS after 12 hours treatment with 100 nM rapamycin, n=3, ~~0.05) and protein (61 .l * 8.3 %. 52.7 f 6.0 % inhibition vs. 10 % FBS after 24 hours treatment with 100 nM rapamycin, n=3, ~~0.05) expression in a dose-dependent fashion. Transient transfection experiments revealed that rapamycin inhibited MCM6 and MCM7 promotor activity (58.3 f 6.8 %, 67.1 f 8.4 % inhibition vs. 10% FBS after 48 hours treatment with 100 nM rapamycin, n=3, ~~0.05) implicating a transcriptional mechanism. MCM6 and MCM7 transcriptional activation is regulated by E2F and activity of a luciferase reporter plasmid driven by four E2F elements was also strongly inhibited by rapamycin (63.1 f 7.3 % inhibition after 48 hours, n=3. p<O.O5). The inhibitory effect of rapamycin on MCM8 and MCM7 was reversed by adenoviral mediated overexpression of E2F, indicating that their downregulation by rapamycin involves an E2F-dependent mechanism. Conclusion: These observation suggest that rapamycin inhibits MCMG and MCM7 expression by blocking EZF function which may contribute importantly to the inhibition of VSMC DNA synthesis by rapamycin.