Simvastatin promotes osteoblast viability and differentiation via Ras/Smad/Erk/BMP-2 signaling pathway
Introduction
Statins, the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors are well-established cholesterol-lowering drugs able to inhibit cholesterol synthesis in humans and animals and reduce cardiovascular risk in hypercholesterolemic patients [1], [2]. Statins, including simvastatin, lovastatin, and cerivastatin, have been widely used for the treatment of hypercholesterolemia in humans [3]. By inhibiting the initial part of the cholesterol synthesis pathway, statins decrease availability of several important lipid intermediate compounds including isoprenoids such as geranylgeranyl pyrophosphate; these are attached as posttranslational modification to certain proteins, such as small G proteins including Ras and Ras-like proteins (Rho, Rap, Rab, and Ral) [4].
Osteoblasts, which arise from mesenchymal stem cell precursors, undergo differentiation in response to a number of factors including bone morphogenetic proteins (BMPs), transforming growth factor, insulin-like growth factor I, vascular endothelial growth factor, and glucocorticoids [5], [6], [7], [8], [9], [10], [11], [12]. In addition, several different molecules are associated with deposition and maintenance of mineralized skeletal elements. Once matrix synthesis begins in osteoblast culture models such as primary osteoblast cultures, the cells differentiate as genes encoding osteoblastic markers such as alkaline phosphatase (ALP), collagen type I (Col 1), and osteocalcin are activated. Finally, osteoblasts become embedded in the extracellular matrix consisting mainly of collagen fibrils, and matrix mineralization begins as mineral deposits extend along and within collagen fibrils [13].
In recent years, there has been a growing interest in the potential effects of statins which appear to be different to those better-known on serum cholesterol. Among these, the possible effect of statins on bone tissue has received particular attention [14]. Mundy et al. [15] first reported that statins stimulated in vivo bone formation in rodents and increased new bone volume in cultures from mouse calvaria. Recently, several reports about the positive effect of statins on bone tissue have been confirmed both in vitro and in vivo [16], [17], [18], [19]. In a study to assess the effects of simvastatin on osteoblastic differentiation, it was found that simvastatin has anabolic effects on bone through the promotion of osteoblastic differentiation [16]. Later studies demonstrated that low concentration simvastatin exhibits a positive effect on osteoblastic proliferation and differentiation may be caused by the inhibition of the mevalonate pathway [20]. Transient exposure of bone cultures to lipophilic statins is sufficient to initiate the cascade resulting in bone formation, most probably because of the local production of BMP-2 [15]. In vivo, simvastatin treatment can enhance the production of BMP-2, Col 1 and osteocalcin in vertebral bones, and this effect may contribute to the prevention of bone loss in ovariectomized rats [21].
In an in vitro MC3T3-E1 cell culture model, simvastatin-induced osteoblast differentiation was shown to be accompanied by an increase in mRNA expression of BMP-2, vascular endothelial growth factor, alkaline phosphatase, Col 1, bone sialoprotein, and osteocalcin, but the expression of Runx2/Cbfa1 was found to be unchanged by simvastatin treatment [22]. This implies that little is known about effects of statins on regulation of osteoblast function. The present study was undertaken to investigate the events of osteoblast differentiation induced by statins. The purpose of this study was to first document whether simvastatin has an osteoinductive effect on osteoblast cells; second, clarify whether the osteoinductive effect of simvastatin occurs via the BMP pathway; and third, determine the signaling pathways during the simvastatin-BMP osteoinductive processes. Our hypothesis is that simvastatin promotes osteoblast viability and differentiation via Ras/Smad/Erk/BMP-2 signaling pathway. There were 3 primary research objectives in this study: (1) to determine osteoblast viability and differentiation after simvastatin treatment; (2) to characterize the osteoblasts' early genes (BMP-2, BMP-4, ALP, and Runx2) and late genes (bone sialoprotein, Col 1 and osteocalcin) expression after simvastatin treatment; and (3) to evaluate the possible roles about the signaling pathways of guanine nucleotide exchange factors (GEFs), Ras GTPase-activating protein (RasGAP), and Smad/Erk for the osteoblast differentiation after simvastatin treatment.
Section snippets
Osteoblasts cell culture
For the cell culture, newborn Imprinting Control Region mice were killed by CO2 asphyxia, then the femur and tibia bones were aseptically dissected. The osteoblast-like cells were isolated from sequential digestion of calvaria bone. The harvested cells were cultured in α-minimum essential medium (GibcoBRL; Grand Island, NY) supplemented with 10% fetal bovine serum (HyClone, Logan, Utah), antibiotics (100 U/mL of penicillin G and streptomycin 100 μg/mL, GibcoBRL), ascorbic acid (50 mg/ml; Sigma
Effect of simvastatin on osteoblast viability and differentiation
In the pilot study, the viability of osteoblasts was tested with different concentrations of simvastatin (control, 10−9 to 10−3 M) at 3 days of culture. The results showed that the viability of osteoblasts was not affected when simvastatin was lower than 10−7 mol/L, whereas higher concentrations (10−4 M and 10−3 M) resulted in a decrease in cell viability (Fig. 1). In this study, 10−6 mol/L simvastatin was selected for further study since this was the concentration that attained the highest
Discussion
Mundy et al. [15] reported that pharmacologic doses of statins such as simvastatin stimulated in vitro and in vivo bone formation. Many other studies have confirmed the in vitro osteogenic effect and increase in expression of BMP-2 gene [16], [17], [24], but the detail mechanism about how statins induce the BMP-2 expression is still not well understood. In this study, we sought to unravel the intracellular signaling mechanism by which statins induce BMP-2 expression and osteoblast
Acknowledgment
This work was supported by Grants-in-Aid for Scientific Research from the Department of Health, Taipei City Government (Taiwan, ROC) (Grant No: 95003-62-141).
References (38)
- et al.
The relationship between reduction in low-density lipoprotein cholesterol by statins and reduction in risk of cardiovascular outcomes: an updated meta-analysis
Clin Ther
(2009) - et al.
Hydrophilicity/lipophilicity: relevance for the pharmacology and clinical effects of HMG-CoA reductase inhibitors
Trends Pharmacol Sci
(1998) - et al.
Protein prenyltransferases
J Biol Chem
(1996) - et al.
Vasculotropin/vascular endothelial growth factor induces differentiation in cultured osteoblasts
Biochem Biophys Res Commun
(1994) - et al.
Simvastatin promotes osteoblast differentiation and mineralization in MC3T3-E1 cells
Biochem Biophys Res Commun
(2001) - et al.
Pitavastatin enhanced BMP-2 and osteocalcin expression by inhibition of Rho-associated kinase in human osteoblasts
Biochem Biophys Res Commun
(2001) - et al.
Effect of simvastatin treatment on bone mineral density and bone turnover in hypercholesterolemic postmenopausal women: a 1-year longitudinal study
Bone
(2003) - et al.
Effects of statins on bone mineral density: a meta-analysis of clinical studies
Bone
(2007) - et al.
Compactin and simvastatina, but not pravastatin, induce bone morphogenetic protein 2 in human osteosarcoma cells
Biochem Biophys Res Commun
(2000) - et al.
Statin-induced Ras activation integrates the phosphatidylinositol 3-kinase signal to Akt and MAPK for bone morphogenetic protein-2 expression in osteoblast differentiation
J Biol Chem
(2007)
Ras oncogenes and their downstream targets
Biochimica et Biophysica Acta
Fluvastatin enhances receptor-stimulated intracellular Ca2+ release in human keratinocytes
Biochem Biophys Res Commun
Neurotrophin-dependent tyrosine phosphorylation of Ras guanine-releasing factor 1 and associated neurite outgrowth is dependent on the HIKE domain of TrkA
J Biological Chem 2005
Calcium signaling
Cell
Primary prevention of acute coronary events with lovasatin in men and women with average cholesterol levels: results of AFCAPS/TexCAPS
JAMA
Identification of transforming growth factor beta family members present in boneinductive protein purified from bovine bone
Proc Natl Acad Sci U S A
VEGF couples hypertrophic cartilage remodeling, ossification, and angiogenesis during endochondral bone formation
Nat Med
Enhanced expression of vascular endothelial growth factor in human SaOS-2 osteoblast-like cells and murine osteoblasts induced by insulin-like growth factor I
Endocrinology
The effects of bone morphogenetic protein-2, -4, and -6 on differentiation of rat osteoblast cells in vitro
Endocrinology
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