Inositol Hexakisphosphate Inhibits Osteoclastogenesis on RAW 264.7 Cells and Human Primary Osteoclasts

Monjo M (2012) Inositol Hexakisphosphate Inhibits Osteoclastogenesis on RAW 264.7 Cells and Human Primary Osteoclasts. PLoS ONE 7(8): e43187. doi:10.1371/journal.pone.0043187 Inositol Hexakisphosphate Inhibits Osteoclastogenesis on RAW 264.7 Cells and Human Primary Osteoclasts Mara del Mar Arriero 0 Joana M. Ramis 0 Joan Perello 0 Marta Monjo 0 Dominique Heymann, Faculte de medecine de Nantes, France 0 1 Department of Fundamental Biology and Health Sciences, Research Institute on Health Sciences (IUNICS), University of Balearic Islands , Palma de Mallorca, Spain, 2 Laboratoris Sanifit, ParcBIT, Palma de Mallorca , Spain Background: Inoxitol hexakisphosphate (IP6) has been found to have an important role in biomineralization and a direct effect inhibiting mineralization of osteoblasts in vitro without impairing extracellular matrix production and expression of alkaline phosphatase. IP6 has been proposed to exhibit similar effects to those of bisphosphonates on bone resorption, however, its direct effect on osteoclasts (OCL) is presently unknown. Methodology/Principal Findings: The aim of the present study was to investigate the effect of IP6 on the RAW 264.7 monocyte/macrophage mouse cell line and on human primary osteoclasts. On one hand, we show that IP6 decreases the osteoclastogenesis in RAW 264.7 cells induced by RANKL, without affecting cell proliferation or cell viability. The number of TRAP positive cells and mRNA levels of osteoclast markers such as TRAP, calcitonin receptor, cathepsin K and MMP-9 was decreased by IP6 on RANKL-treated cells. On the contrary, when giving IP6 to mature osteoclasts after RANKL treatment, a significant increase of bone resorption activity and TRAP mRNA levels was found. On the other hand, we show that 1 mM of IP6 inhibits osteoclastogenesis of human peripheral blood mononuclear cells (PBMNC) and their resorption activity both, when given to undifferentiated and to mature osteoclasts. Conclusions/Significance: Our results demonstrate that IP6 inhibits osteoclastogenesis on human PBMNC and on the RAW264.7 cell line. Thus, IP6 may represent a novel type of selective inhibitor of osteoclasts and prove useful for the treatment of osteoporosis. - Funding: This work was supported by INVEST IN SPAIN (C2_10_32), European Regional Development Fund (ERDF) from the European Union, Eureka-Eurostars Project Application E!5069 NewBone, Interempresas Internacional Program (CIIP20101024) from the Centre for the Development of Industrial Technology (CDTI) and the Ministry of Science and Innovation of Spain (Torres Quevedo contract to J.M. Ramis, and Ramo n y Cajal contract to M. Monjo). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Joan Perello is employed by Laboratoris Sanifit. All authors are inventors of a pending patent application based on some aspects of this work (EP11382404.9, priority date 26.12.2011, Biocompatible implant). There are no other patents, products in development or marketed products to declare. This does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials. Inositol hexakisphosphate (IP6, phytic acid) is found in high amounts in plant seeds, being their major phosphate store [1,2]. Afterwards, it has also been shown to be widely distributed in animal cells and tissues [36]. A large body of evidence has implicated IP6 in a variety of cellular functions such as cell proliferation [7], cell differentiation [8], signal transduction [9], cation transport [10,11], exocytosis [9], neurotransmission [12], antioxidant [12], efficient transport of mRNA [13] and DNA repair [14]. As regards to biomineralization, different in vitro and in vivo studies have demonstrated that IP6 is a potent inhibitor of crystallization of calcium salts (oxalate and phosphate salts) [15 18]. It has been demonstrated that IP6 inhibits pericardial [19], vascular [20], tooth enamel [21] and renal calcification [22,23], in addition to inhibiting dental tartar formation [24]. Some results suggest that the mechanism of IP6 in the inhibition of soft tissue calcification is by a reduced hydroxyapatite crystal formation in the first steps, i.e. IP6 would adsorb onto growing crystal faces or avoid nascent crystal nuclei formation, thus impeding further apposition of mineral ions to the crystal [2527]. At the same time, the adsorption of IP6 on critical points of the crystal surface, when already formed, would contribute to its stabilization, thus preventing its dissolution [28]. Therefore, IP6 acts both, preventing the process of formation of calcium salts, but also stabilizing already formed calcium salts, avoiding its subsequent growth and dissolution. The effect of IP6 on the inhibition of the dissolution of already formed calcium salts is of importance in the prevention of osteoporosis. In agreement with this effect, higher IP6 consumption has been shown to correlate with an increase on bone mineral density (BMD) [29,30] and with a reduced BMD loss due to estrogen deficiency in an osteoporosis animal model [28]. In fact, IP6 has been proposed to exhibit similar effects to those of nonnitrogen containing bisphosphonates (BP) on bone resorption and to be of use in the primary prevention of osteoporosis [28]. The simplest ones, nonnitrogen-containing BP (such as clodronate and etidronate), can be metabolically incorporated into nonhydrolyzable analogs of ATP that may inhibit ATP-dependent intracellular enzymes resulting in induction of osteoclast apoptosis. The most potent ones, nitrogen-containing bisphosphonates (such as pamidronate, alendronate, risedronate, ibandronate, and zoledronate), can inhibit a key enzyme, farnesyl pyrophosphate synthase, in the mevalonate pathway, thereby preventing the biosynthesis of isoprenoid compounds that are essential for the posttranslational modification of small GTP-binding proteins (GTPases), resulting in the loss of osteoclast activity. Since osteoporosis results from an imbalance between osteoblast and osteoclast (OCL) activity, it is of interest to study the direct effect of IP6 on both types of cells. A recent study by Addison et al [31] showed that IP6 inhibits mineralization of MC3T3-E1 osteoblast cultures by binding to growing crystals, increases gene expression of the mineralization inhibitor osteopontin, but does not impair the ability of osteoblasts to synthesize a collagenous matrix, express alkaline phosphatase or differentiate to produce specific bone matrix proteins. However, further investigation is needed to fully understand these effects on osteoblasts and the net effect of IP6 on bone formation, since both in vivo animal and clinical data indicate a positive correlation between IP6 physiological levels and BMD. The aim of the present study was to investigate the effect of IP6 on the RAW 264.7 monocyte/macrophage mouse cell line and human peripheral blood mononu (...truncated)