Novel germacrane sesquiterpene esters inhibit NF-κB activity and RANKL-induced osteoclast formation and bone resorption

Bone 44 (2009) S131–S141

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Bone j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b o n e

Abstracts

Category 5. Bone resorption and its regulation 371 Osteocyte cell density is reduced during vitamin D depletion and predicts osteoclast surface and bone mineral volume P.H. Andersona, R.K. Sawyera, A.J. Moorea, G.J. Atkinsb, P.D. O'Loughlinc, H.A. Morrisb a Hanson Institute, IMVS, Adelaide, SA, Australia b Department of Orthopaedics and Trauma, University of Adelaide, Adelaide, SA, Australia c Chemical Pathology, IMVS, Adelaide, SA, Australia

372 Novel germacrane sesquiterpene esters inhibit NF-κB activity and RANKL-induced osteoclast formation and bone resorption E. Ang, M. Zheng, J. Xu Molecular Orthopaedic Laboratory, Centre for Orthopaedics Research, School of Su, The University of Western Australia, Nedlands, WA, Australia

Although the association between hip fracture risk and vitamin D deficiency is well established, understanding of the cellular mechanisms is less clear. Previously, we have reported maintaining adequate levels of serum 25-hydroxyvitamin D (25D) prevent bone loss by reducing RANKL-mediated osteoclastic bone resorption in an animal model. Interestingly, it has been demonstrated that increased bone resorption is mediated by osteocytes (OCY) either via apoptosis or trans-cellular signalling. Hence, we have used our vitamin D-deplete animal model to investigate the relationship between trabecular bone OCY cell density and other bone variables. Briefly, vitamin D-depleted animals were fed one of several levels of vitamin D3 (2 IU–20 IU/rat/d) with 0.4% dietary Ca from 3 m of age. At 7 m, the mean 25D level ranged from 20 nmol/L to 115 nmol/L which was demonstrated to be stable for at least 12 weeks. In addition to serum biochemical analyses, the distal femoral metaphysis from each animal was analysed on normal and decalcified 5 μm sagittal sections for standard histomorphometric variables, such as osteoclast surface (OcS, %), plus osteocyte numbers relative to bone mineral area (OCY/BMA, #/mm2). As previously reported, trabecular bone volume (BV/TV) was positively related to circulating 25D (R2 = 0.51, P < 0.001), but not to serum 1,25(OH)2D3, serum calcium nor parathyroid hormone (PTH) levels. The reduction in BV/TV as result of reduced serum 25D levels was negatively associated with increases in both RANKL:OPG mRNA ratio (R2 = 0.35, P < 0.05) and OcS (R2 = 0.36, P < 0.05). The OCY/ BMA in trabecular bone was lowest in animals with 20 nmol/L 25D (P < 0.01) when compared to all other groups and positively correlated with serum 25D levels (R2 = 0.46, P < 0.01). Furthermore, of all the measures of bone formation and resorption, only OcS was negatively associated with OCY/BMA (R2 = 0.3, P < 0.05). OCY/BMA was also correlated with BV/TV (R2 = 0.23, P < 0.05), suggesting that a low osteocyte cell density in trabecular bone may play a role in determining osteoclastic bone resorption and ultimately bone mineral volume. Measures of osteocyte survival/activity in this vitamin D-depletion animal model, will confirm whether osteoclast activity during vitamin D depletion is due partly to osteocyte signalling.

Osteoclasts are responsible for bone resorption and play a pivotal role in the pathogenesis of osteolytic disorders. NF-κB is a set of nuclear factors essential for osteoclast formation and survival. NF-κB signalling pathways are strictly regulated to maintain bone homeostasis by cytokines such as RANKL, TNF-α and IL-1. Abnormal activation of NF-κB in osteoclasts has been frequently observed in osteolytic bone diseases such as periprothetic osteolysis and arthritis, and this raises an intriguing possibility that NF-κB might serve as a therapeutic target for the treatment of osteolytic bone diseases. We have previously shown that sesquiterpene lactone parthenolide attenuates osteoclast formation and function through the inhibition of NF-κB activity. Further screening of sesquiterpene structural analogues from a natural compound library identified 3 novel germacrane sesquiterpene bearing the same core carbon structure as parthenolide. These compounds were isolated from the whole plant of Salvia roborowskii. Using primary bone marrow monocytes (BMMs) osteoclastogenic culture system, we examined the effects of these compounds on osteoclast formation. Bone resorption assay was employed to examine their inhibitory effects on the function of osteoclasts. Immunoblot analysis, together with luciferase reporter gene assay was used to dissect the molecular mechanism(s) underlying the observed effects of the compounds on RANKL-induced NFκB and p-ERK signalling. We demonstrated that structural analogs of parthenolide dose-dependently inhibit RANKL-induced osteoclastogenesis in BMM cells as well as disrupting the ability of the osteoclasts to resorb bone. Compound A was identified to be the most potent amongst the 3 analogues. Luciferase reporter gene assay revealed that these compounds decrease RANKL-induced NF-κB activation. Furthermore, western blotting analysis also demonstrated that the compounds inhibit IκB-α degradation, but had little effect on RANKL-induced ERK phosphorylation. Taken together, these results indicate that structural analogues of parthenolide effectively inhibit osteoclast formation and function through the inhibition of NF-κB. We propose that novel germacrane sesquiterpene esters might provide therapeutic candidates for the treatment of osteolytic bone diseases associated with enhanced osteoclastic activity.

doi:10.1016/j.bone.2009.01.449

doi:10.1016/j.bone.2009.01.286

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