Statins such as simvastatin are 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors that inhibit cholesterol synthesis. We presently investigated statin effects on vascular endothelial growth factor (VEGF) expression in osteoblastic cells. Hydrophobic statins including simvastatin, atorvastatin, and cerivastatin-but not a hydrophilic statin, pravastatin-markedly increased VEGF mRNA abundance in nontransformed osteoblastic cells (MC3T3-E1). Simvastatin (10(-6) M) time-dependently augmented VEGF mRNA expression in MC3T3-E1 cells, mouse stromal cells (ST2), and rat osteosarcoma cells (UMR-106). According to heterogeneous nuclear RNA and Northern analyses, 10(-6) M simvastatin stimulated gene expression for VEGF in MC3T3-E1 cells without altering mRNA stability. Transcriptional activation of a VEGF promoter-luciferase construct (-1128 to +827), significantly increased by simvastatin administration. As demonstrated by gel mobility shift assay, simvastatin markedly enhanced the binding of hypoxia-responsive element-protein complexes. These results indicate that the stimulation of the VEGF gene by simvastatin in MC3T3-E1 cells is transcriptional in nature. VEGF secretion into medium was increased in MC3T3-E1 by 10(-6) M simvastatin. Pretreating MC3T3-E1 cells with mevalonate or geranylgeranyl pyrophosphate, a mevalonate metabolite, abolished simvastatin-induced VEGF mRNA expression; manumycin A, a protein prenylation inhibitor, mimicked statin effects on VEGF expression. The effect of simvastatin was blocked by pretreatment with wortmannin and LY294002, specific phosphatidylinositide-3 kinase inhibitors. Simvastatin enhanced mineralized nodule formation in culture, whereas coincubation with mevalonate, geranylgeranyl pyrophosphate, LY294002, or VEGF receptor 2 inhibitor (SU1498) abrogated statin-induced mineralization. Thus, statins stimulate VEGF expression in osteoblasts via reduced protein prenylation and the phosphatidylinositide-3 kinase pathway, promoting osteoblastic differentiation.