Sesquiterpene lactone parthenolide blocks lipopolysaccharide-induced osteolysis through the suppression of NF-kappaB activity.
Effective treatment for bacteria-induced bone lytic diseases is not yet available. In this study, we showed that PAR, an NF-kappaB inhibitor found in medicinal herbs, can block LPS-induced osteolysis. PAR does this by inhibiting osteoclastogenesis and bone resorption and promoting apoptosis of osteoclasts through the suppression of NF-kappaB activity.


BACKGROUND
Osteolysis induced by chronic gram-negative bacterial infection underlies many bone diseases such as osteomyelitis, septic arthritis, and periodontitis. Drugs that inhibit lipopolysaccharide (LPS)-induced osteolysis are critically needed for the prevention of bone destruction in infective bone diseases. In this study, we investigated the effect of parthenolide (PAR) on LPS-induced osteolysis in vivo and studied its role in osteoclastogenesis, bone resorption, apoptosis, and NF-kappaB activity.


METHODS
The LPS-induced osteolysis in the mouse calvarium model was used to examine the effect of PAR in vivo. RANKL-induced osteoclast differentiation from RAW264.7 cells and bone resorption assays were used to assess the effect of PAR in vitro. Assays for NF-kappaB activation, p65 translocation, and IkappaB-alpha degradation were used to determine the mechanism of action of PAR in osteoclasts and their precursors. Flow cytometry and confocal microscopic analysis were used to examine cell apoptosis. Semiquantitative RT-PCR was performed to examine the effect of PAR on gene expression of RANK and TRAF6.


RESULTS
We found that PAR (0.5 and 1 mg/kg), injected simultaneously with LPS (25 mg/kg) or 3 days later, blocked the LPS-induced osteolysis in the mouse calvarium model. In vitro studies showed that low concentrations of PAR (<1 microM) inhibited in vitro osteoclastogenesis and osteoclastic bone resorption, whereas higher concentrations (>5 microM) triggered apoptotic cell death of osteoclasts and their precursor cells in a dose-dependent manner. Furthermore, PAR inhibited LPS-induced NF-kappaB activation, p65 translocation, and IkappaB-alpha degradation both in mature osteoclasts and their precursors in a time- and dose-dependent manner. In addition, PAR inhibited NF-kappaB activation induced by osteoclastogenic factors RANKL, interleukin (IL)-1beta, or TNF-alpha to varying degrees and reduced the gene expression of RANK and TRAF6.


CONCLUSIONS
The NF-kappaB pathway is known to mediate both osteoclast differentiation and survival. These findings indicate that PAR blocks LPS-induced osteolysis through the suppression of NF-kappaB activity and suggest that it might have therapeutic value in bacteria-induced bone destruction.
Molecular Orthopaedic Laboratory, School of Surgery and Pathology, University of Western Australia, Nedlands, Western Australia, Australia.
Kirk Ho Man
KH
Yip
Ming H
MH
Zheng
Hao Tian
HT
Feng
James H
JH
Steer
David A
DA
Joyce
Jiake
J
Xu
D021581:Active Transport, Cell Nucleus; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D017209:Apoptosis; D015153:Blotting, Western; D001862:Bone Resorption; D001842:Bone and Bones / Q000378:metabolism; D002454:Cell Differentiation; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006023:Glycoproteins / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007375:Interleukin-1 / Q000378:metabolism; D007783:Lactones / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000378:metabolism / Q000494:pharmacology*; D008156:Luciferases / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000378:metabolism*; D010010:Osteoclasts / Q000378:metabolism; D010014:Osteolysis; D053244:Osteoprotegerin; D021381:Protein Transport; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012717:Sesquiterpenes / Q000494:pharmacology*; D048029:TNF Receptor-Associated Factor 6 / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000894:Anti-Inflammatory Agents, Non-Steroidal; D006023:Glycoproteins; D007375:Interleukin-1; D007783:Lactones; D008070:Lipopolysaccharides; D016328:NF-kappa B; D053244:Osteoprotegerin; D018160:Receptors, Cytoplasmic and Nuclear; D018124:Receptors, Tumor Necrosis Factor; D012717:Sesquiterpenes; D048029:TNF Receptor-Associated Factor 6; C506031:Tnfrsf11b protein, mouse; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; C002669:parthenolide; D008156:Luciferases