Medicarpin inhibits osteoclastogenesis and has nonestrogenic bone conserving effect in ovariectomized mice

https://doi.org/10.1016/j.mce.2010.05.016Get rights and content

Abstract

Medicarpin, a pterocarpan class of naturally occurring benzopyran furanobenzene compound was synthesized in gram scale to investigate its effects on murine bone cells and in ovariectomized (OVx) mice. Medicarpin, at as low as 10−10 M suppressed osteoclastogenesis in bone marrow cells (BMCs). Medicarpin-induced apoptosis of mature osteoclasts isolated from long bones. Effects of medicarpin in osteoclasts appear to be independent of estrogen receptor (ER) activation as ICI 180,782 failed to abrogate its effects on osteoclasts. In calvarial osteoblasts, medicarpin (10−10 M) blocked nuclear factor kappaB (NF-κB) signaling assessed by tumor necrosis factor alpha (TNFα)-stimulated nuclear translocation of p65 subunit of NF-κB. Medicarpin also inhibited the expression of TNFα in mouse calvarial osteoblasts. This effect was ER dependent as ICI 180,782 reversed the suppressive effect of medicarpin on TNFα mRNA levels in osteoblasts. In addition, like 17β-estradiol, presence of medicarpin inhibited TNFα-induced upregulation of interleukin-1, and -6 mRNA levels in osteoblasts. In co-cultures consisting of calvarial osteoblasts and BMCs, presence of medicarpin increased osteoprotegerin (OPG)/receptor activator of NF-κB ligand (RANKL) ratio and reduced mRNA levels of osteoclast markers including tartrate-resistant acid phosphatase and RANK. OVx mice administered medicarpin (10.0 mg kg−1 day−1) orally for 30 days had reduced formation of osteoclasts but increased formation of osteoprogenitor cells in BMCs compared with OVx + vehicle group. Medicarpin treatment to OVx mice maintained parameters of trabecular microarchitecure. Medicarpin exhibited no uterine estrogenicity. Our findings point towards direct and indirect inhibitory effects of medicarpin on osteoclastogenesis in vitro that contribute to its bone sparing effect in OVx mice.

Introduction

The increase in bone resorption caused by estrogen deficiency is the major cause of postmenopausal bone loss and osteoporosis. This increase in bone resorption is considered to be mediated largely by changes in cytokine levels in the bone microenvironment (Pfeilschifter et al., 2002). Several proinflammatory cytokines have been suggested as mediators of bone resorption including TNFα, interleukin (IL)-1β, IL-6, IL-7 and macrophage-colony stimulating factor (M-CSF) (Baek et al., 2006, Bandyopadhyay et al., 2006, Pfeilschifter et al., 2002). Among these, TNFα and IL-1β have been found to be the most consistent mediators of estrogen action (Charatcharoenwitthaya et al., 2007). Bone loss does not occur after ovariectomy in mice deficient in receptors for IL-1 or transgenic mice overexpressing soluble TNFα decoy receptor (Roggia et al., 2001, Vargas et al., 1996). Moreover, treatment with IL-1 receptor antagonist or TNF binding protein decrease osteoclast formation and bone resorption in ovariectomized (OVx) mice (Kitazawa et al., 1994).

TNF also inhibits osteoblastic bone formation (Abbas et al., 2003, Gilbert et al., 2000). TNF inhibits recruitment of osteoblast progenitors, reduces expression of genes produced by mature osteoblasts, and promotes osteoblast apoptosis through nuclear factor-κB (NF-κB) signaling pathway (Nanes, 2003, Zhou et al., 2006). Together, TNFα is a potent proinflammatory cytokine that has overall detrimental effects on bone. Agents that can disrupt TNF receptor signaling in bone have the potential to prevent bone loss.

Osteoblasts express receptor activator of NF-κB ligand (RANKL, a TNF family ligand) and its decoy receptor, osteoprotegerin (OPG, a TNF family receptor) (Caetano-Lopes et al., 2007). RANKL and OPG are fundamental factors that control osteoclast formation and activation (Kearns et al., 2008, Kobayashi et al., 2009). Bone remodeling appears to be mainly controlled by the balance of RANKL and OPG (Boyce and Xing, 2007). Postmenopausal osteoporosis is associated with an imbalance in the RANKL/OPG system in favor of RANKL (Giner et al., 2009). RANKL stimulates and OPG inhibits osteoclast formation and activation. A single, subcutaneous dose of OPG resulted in a profound and sustained suppression of bone resorption in postmenopausal women (Marini et al., 2008). Estrogen contributes to bone protection since it increases OPG production in osteoblasts and decreases the response of osteoclasts to RANKL and induces osteoclast apoptosis (Han et al., 2005).

Flavonoids as natural selective estrogen receptor modulators (SERM) have gained preference as prophylactic agents against postmenopausal bone loss due to their lack of adverse effects that are commonly associated with estrogen replacement therapy (ERT) (Bhargavan et al., 2009a, Bhargavan et al., 2009b, Maggiolini et al., 2005, Sharan et al., 2009). Medicarpin, a phytoalexin having chemical resemblance with isoflavonoids, is found in a wide range of leguminous species (Ingham and Harborne, 1976) including chickpea and in the extracts of Butea monosperma. Recently, we have shown that an acetone soluble fraction is more potent than total ethanolic extract made from the stem-bark of Butea monosperma in mitigating OVx-induced bone loss in rats (Pandey et al., 2010) Principal component analysis revealed ∼2.0-fold increase in medicarpin concentration in acetone soluble fraction compared with total ethanolic extract of Butea monosperma. Based on our observations with Butea monosperma, we hypothesized that medicarpin could inhibit osteoclast function and in the process protect bone against estrogen deficiency-induced bone loss. We also sought to characterize the modulatory roles of medicarpin in proinflammatory signaling in bone cells.

Section snippets

Reagents and chemicals

Cell culture media and supplements were purchased from Invitrogen (Carlsbad, CA). All fine chemicals were purchased from Sigma–Aldrich (St. Louis, MO). ECL kit was purchased from Amersham Pharmacia, USA. Antibodies for western blot analysis were obtained from Santacruz, USA. Mouse cytokine array was purchased from Ray Biotech, USA.

Synthesis of medicarpin

The substituted deoxybenzoin, 1-(2,4-dihydroxy-phenyl)-2-(2,4-dimethoxy-phenyl)-ethanone (3) was synthesized to 76% yield by heating a mixture of resorcinol (1),

Medicarpin inhibits differentiation of osteoclasts from BMCs

RANKL, a member of the TNFα family, is expressed by osteoblasts and stimulates formation of multinucleated TRAP-positive osteoclasts (Pacifici, 2007). Using murine BMCs, the effect of medicarpin on osteoclast formation by RANKL and M-CSF was determined. Fig. 1A shows the representative image of TRAP positive cells in BMC cultures treated with or without medicarpin. Medicarpin at 10−10 M and 10−8 M exhibited concentration-dependent inhibition of multinucleated cells by ∼38% and 54%, respectively (P

Discussion

Bone remodeling requires a balance in the activity of the osteoblasts and the osteoclasts. Effect of medicarpin was characterized in osteoclasts and osteoblasts, the cells involved in bone remodeling. Our data show that medicarpin modulates function of these cells in ways that appear to apply osteoprotective effect.

Medicarpin is present in leguminous species including chickpea and in the extracts of Butea monosperma. The latter is known to have osteoprotective action in OVx rats (Pandey et al.,

Conflict of interest

Authors have no conflict of interest.

Acknowledgement

Funding from the Ministry of Health and Family Welfare, Department of Biotechnology and Department of Science and Technology, Government of India. RM thanks Le Conseil Régional de Picardie for funding. Fellowship grants from the Council of Scientific and Industrial Research (BB, AK, DKY), University Grants Commission (AKG), Government of India.

References (53)

  • J.L. Pang et al.

    Differential activity of kaempferol and quercetin in attenuating tumor necrosis factor receptor family signaling in bone cells

    Biochem. Pharmacol.

    (2006)
  • R. Trivedi et al.

    Kaempferol has osteogenic effect in ovariectomized adult Sprague–Dawley rats

    Mol. Cell. Endocrinol.

    (2008)
  • R. Trivedi et al.

    Effects of Egb, 761 on bone mineral density, bone microstructure, and osteoblast function: possible roles of quercetin and kaempferol

    Mol. Cell. Endocrinol.

    (2009)
  • W. Ariyoshi et al.

    Heparin inhibits osteoclastic differentiation and function

    J. Cell. Biochem.

    (2008)
  • J.E. Aubin et al.

    Osteoprotegerin and its ligand: a new paradigm for regulation of osteoclastogenesis and bone resorption

    Osteoporos. Int.

    (2000)
  • U. Beck et al.

    Phytoestrogens derived from red clover: An alternative to estrogen replacement therapy?

    Journal of steroid biochemistry and Molecular Biology

    (2005)
  • B. Bhargavan et al.

    Methoxylated isoflavones, cajanin and isoformononetin, have non-estrogenic bone forming effect via differential mitogen activated protein kinase (MAPK) signaling

    J. Cell. Biochem.

    (2009)
  • B. Bhargavan et al.

    Methoxylated isoflavones, cajanin and isoformononetin, have non-estrogenic bone forming effect via differential mitogen activated protein kinase (MAPK) signaling

    J. Cell. Biochem.

    (2009)
  • B.F. Boyce et al.

    Biology of RANK, RANKL, and osteoprotegerin

    Arthritis Res. Ther.

    (2007)
  • J. Caetano-Lopes et al.

    Osteoblasts and bone formation

    Acta Reumatol. Port.

    (2007)
  • N. Charatcharoenwitthaya et al.

    Effect of blockade of TNF-alpha and interleukin-1 action on bone resorption in early postmenopausal women

    J. Bone Miner. Res.

    (2007)
  • W. Cocker et al.

    A synthesis of demethylhomopterocarpin

    J. Chem. Soc.

    (1965)
  • Y.H. Gao et al.

    Suppressive effect of genistein on rat bone osteoclasts: involvement of protein kinase inhibition and protein tyrosine phosphatase activation

    Int. J. Mol. Med.

    (2000)
  • L. Gilbert et al.

    Inhibition of osteoblast differentiation by tumor necrosis factor-alpha

    Endocrinology

    (2000)
  • Y. Goda et al.

    Inhibitor of prostaglandin biosynthesis from Dalbergia odorifera

    Chem. Pharm. Bull.

    (1992)
  • F. Grassi et al.

    Oxidative stress causes bone loss in estrogen-deficient mice through enhanced bone marrow dendritic cell activation

    Proc. Natl. Acad. Sci. U.S.A.

    (2007)
  • Cited by (0)

    Supporting grants: Ministry of Heath and Family Welfare, Council of Scientific and Industrial Research, University Grants Commission, Government of India, Department of Biotechnology (DBT), Department of Science and Technology (DST).

    1

    Authors have contributed equally.

    View full text