Elsevier

Nutrition Research

Volume 30, Issue 3, March 2010, Pages 191-199
Nutrition Research

Simvastatin promotes osteoblast viability and differentiation via Ras/Smad/Erk/BMP-2 signaling pathway

https://doi.org/10.1016/j.nutres.2010.03.004Get rights and content

Abstract

Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A reductase, which catalyzes the conversion of 3-hydroxy-3-methylglutaryl-coenzyme A to mevalonate, a rate-limiting step in cholesterol synthesis. Statins are able to reduce cardiovascular risk in hypercholesterolemic patients. In recent years, the possible effect of statins on bone tissue has received particular attention. The present study was undertaken to understand the events of osteoblast differentiation induced by statins. Our hypothesis is that simvastatin promotes osteoblast viability and differentiation via Ras/Smad/Erk/bone morphogenic protein (BMP)-2 signaling pathway. The viability and differentiation of osteoblasts were examined by mitochondrial activity assay, alkaline phosphatase (ALP) activity, and gene expression. The associated signaling pathways were analyzed by cytoplasmic and membrane proteins manifestation. After administration of 10−6 M simvastatin, the ALP activity was significantly enhanced, and the expression of BMP-2, ALP, sialoprotein, and type I collagen genes were up-regulated. After simvastatin treatment, both the RasGRF1 and phospho-RasGRF1 in the cytoplasm decreased significantly, whereas those on the plasma membrane increased. A marked increase in membranous GAP-associated protein (P190) and the activated form of both phospho–extracellular signal-regulated kinase1/2 and phospho-Smad1 were also noted. In conclusion, this study shows that statins pose a positive effect on the metabolism of osteoblasts. Simvastatin can promote osteoblast viability and differentiation via membrane-bound Ras/Smad/Erk/BMP-2 pathway. Statins stimulate osteoblast differentiation in vitro and may be a promising drug for the treatment of osteoporosis in the future.

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)

  • RajalingamK. et al.

    Ras oncogenes and their downstream targets

    Biochimica et Biophysica Acta

    (2007)
  • CoutantK.D. et al.

    Fluvastatin enhances receptor-stimulated intracellular Ca2+ release in human keratinocytes

    Biochem Biophys Res Commun

    (1998)
  • RobinsonK.N. et al.

    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

    (2005)
  • ClaphamD.E.

    Calcium signaling

    Cell

    (2007)
  • DownsJ.R. et al.

    Primary prevention of acute coronary events with lovasatin in men and women with average cholesterol levels: results of AFCAPS/TexCAPS

    JAMA

    (1998)
  • CelesteA.J. et al.

    Identification of transforming growth factor beta family members present in boneinductive protein purified from bovine bone

    Proc Natl Acad Sci U S A

    (1990)
  • GerberH.P. et al.

    VEGF couples hypertrophic cartilage remodeling, ossification, and angiogenesis during endochondral bone formation

    Nat Med

    (1999)
  • GoadD.L. et al.

    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

    (1996)
  • HughesF.J. et al.

    The effects of bone morphogenetic protein-2, -4, and -6 on differentiation of rat osteoblast cells in vitro

    Endocrinology

    (1995)
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