Effects of lovastatin on expression of cell cycle regulatory proteins in vascular smooth muscle cells.
BACKGROUND
The sequential appearance of cyclins D and E is thought to initiate subsequent DNA synthesis in proliferating cells. Previous studies have reported that DNA synthesis in cultured rat vascular smooth muscle cells (VSMCs) was suppressed by the HMG-CoA reductase inhibitor lovastatin. The effects of lovastatin on cell cycle regulatory proteins in proliferating VSMCs, however, are largely unknown. Thus, we investigated the sequential expression of cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 4, CDK2, and p27Kip1 in cultured rat VSMCs stimulated by platelet-derived growth factor (PDGF)-BB in the presence or absence of lovastatin.


METHODS
Quiescent VSMCs, with and without lovastatin (20 microM) pretreatment for nine hours, were stimulated by PDGF-BB (25 ng/ml). The incorporation of tritiated thymidine was done to assess DNA synthesis. VSMC lysates were obtained every 6 hours for up to 36 hours after stimulation and were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis using relevant polyclonal antibodies. Autoradiograms were analyzed using a densitometer.


RESULTS
The peak expression of cyclins D1 and E occurred at 18 and 30 hours of PDGF stimulation, respectively. Concomitant expression of CDK4 and CDK2 was also observed. The expression of p27Kip1, by contrast, was reduced in association with DNA synthesis. Lovastatin suppressed DNA synthesis and reduced the expression of cyclin D1 and cyclin E, whereas p27Kip1 expression was strongly induced by lovastatin pretreatment. CDK4 and CDK2 expression was unaffected by lovastatin treatment.


CONCLUSIONS
PDGF-BB induces cyclins D1 and E prior to the onset of DNA synthesis in VSMCs. Lovastatin may suppress DNA synthesis in VSMCs by inducing p27Kip1 and reducing expression of cyclins D1 and E.
Department of Medicine, Hennepin County Medical Center, Minneapolis, Minnesota, USA.
H
H
Oda
B L
BL
Kasiske
M P
MP
O'Donnell
W F
WF
Keane
D000818:Animals; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000187:drug effects*; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000096:biosynthesis / Q000187:drug effects; D019927:Cyclin E / Q000096:biosynthesis / Q000187:drug effects; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000096:biosynthesis / Q000187:drug effects; D004247:DNA / Q000096:biosynthesis / Q000187:drug effects; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology*; D008148:Lovastatin / Q000494:pharmacology*; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000187:drug effects; D011518:Proto-Oncogene Proteins; D051381:Rats; D025521:Tumor Suppressor Proteins
C493962:Cdkn1b protein, rat; D018797:Cell Cycle Proteins; D019927:Cyclin E; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D008869:Microtubule-Associated Proteins; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D025521:Tumor Suppressor Proteins; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004247:DNA; D008148:Lovastatin; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495892:Cdk2 protein, rat; C495903:Cdk4 protein, rat; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases