No abstract

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.

Leptin is the LEP (ob) gene product secreted by adipocytes. We previously reported that leptin decreases renal expression of the 25-hydroxyvitamin D 3 1a-hydroxylase (CYP27B1) gene through the leptin receptor (ObRb) by indirectly acting on the proximal tubules. This study focused on bone-derived fibroblast growth factor 23 (FGF-23) as a mediator of the influence of leptin on renal 1a-hydroxylase mRNA expression in leptin-deficient ob/ob mice. Exposure to leptin (200 ng/mL) for 24 hours stimulated FGF-23 expression by primary cultured rat osteoblasts. Administration of leptin (4 mg/kg i.p. at 12-hour intervals for 2 days) to ob/ob mice markedly increased the serum FGF-23 concentration while significantly reducing the serum levels of calcium, phosphate, and 1a,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ]. Administration of FGF-23 (5 mg i.p. at 12-hour intervals for 2 days) to ob/ob mice suppressed renal 1a-hydroxylase mRNA expression. The main site of FGF-23 mRNA expression was the bone, and leptin markedly increased the FGF-23 mRNA level in ob/ob mice. In addition, leptin significantly reduced 1a-hydroxylase and sodium-phosphate cotransporters (NaP i -IIa and NaP i -IIc) mRNA levels but did not affect Klotho mRNA expression in the kidneys of ob/ob mice. Furthermore, the serum FGF-23 level and renal expression of 1a-hydroxylase mRNA were not influenced by administration of leptin to leptin receptor-deficient (db/db) mice. These results indicate that leptin directly stimulates FGF-23 synthesis by bone cells in ob/ob mice, suggesting that inhibition of renal 1,25(OH) 2 D 3 synthesis in these mice is at least partly due to elevated bone production of FGF-23. ß

Interstitial collagenases, including matrix metalloproteinase-1 (MMP-1) and -8 (MMP-8), serve as initiators of extracellular matrix destruction in periodontal disease. Collagenase activities are mainly regulated by tissue inhibitors of metalloproteinases (TIMPs). We tested the effects of inflammation on MMP-1 and MMP-8 gene expression in periodontal disease. To determine the relative abundance of these mRNAs in gingiva, we used a reverse transcription-polymerase chain reaction (RT-PCR) assay. Gingival biopsies were divided into 2 groups; a control group and an inflamed group with severe gingivitis or periodontitis. The MMP-1 mRNA levels were significantly elevated in inflamed gingiva, while the levels of the MMP-8 transcript were not different in the 2 groups and barely detectable by RT-PCR assay. The expression of the TIMP-1 gene was not altered, and remained higher than any of these other genes in both control and diseased gingivae. These results suggest that MMP-1 rather than MMP-8 may play an important role in the initiation of collagen degradation in periodontal disease. However, the possibility remains that MMP-8 plays an important role in periodontal tissue destruction, since the mRNA abundance and not the enzyme activity was assessed.

Runx2 and Sp7 are essential transcription factors for osteoblast differentiation. However, the molecular mechanisms responsible for the proliferation of osteoblast progenitors remain unclear. The early onset of Runx2 expression caused limb defects through the Fgfr1–3 regulation by Runx2. To investigate the physiological role of Runx2 in the regulation of Fgfr1–3, we compared osteoblast progenitors in Sp7−/− and Runx2−/− mice. Osteoblast progenitors accumulated and actively proliferated in calvariae and mandibles of Sp7−/− but not of Runx2−/− mice, and the number of osteoblast progenitors and their proliferation were dependent on the gene dosage of Runx2 in Sp7−/− background. The expression of Fgfr2 and Fgfr3, which were responsible for the proliferation of osteoblast progenitors, was severely reduced in Runx2−/− but not in Sp7−/− calvariae. Runx2 directly regulated Fgfr2 and Fgfr3, increased the proliferation of osteoblast progenitors, and augmented the FGF2-induced proliferation. The proliferation of Sp7−/− osteoblast progenitors was enhanced and strongly augmented by FGF2, and Runx2 knockdown reduced the FGF2-induced proliferation. Fgfr inhibitor AZD4547 abrogated all of the enhanced proliferation. These results indicate that Runx2 is required for the proliferation of osteoblast progenitors and induces proliferation, at least partly, by regulating Fgfr2 and Fgfr3 expression.