NF-κB modulators in osteolytic bone diseases

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Cytokine & Growth Factor Reviews 20 (2009) 7–17 www.elsevier.com/locate/cytogfr

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NF-kB modulators in osteolytic bone diseases Jiake Xu a,*, Hua Fei Wu a, Estabelle S.M. Ang a, Kirk Yip a, Magdalene Woloszyn a, Ming H. Zheng a, Ren Xiang Tan b a

Molecular Orthopaedic Laboratory, Centre for Orthopaedic Research, School of Surgery, University of Western Australia, Nedlands, WA 6009, Australia b Institute of Functional Biomolecules, Medical School, Nanjing University, Nanjing 210093, PR China Available online 28 November 2008

Abstract Osteoclasts are responsible for bone resorption and play a pivotal role in the pathogenesis of osteolytic disorders. NF-kB is a set of nuclear factors that bind to consensus DNA sequences called kB sites, and is essential for osteoclast formation and survival. NF-kB signalling pathways are strictly regulated to maintain bone homeostasis by cytokines such as RANKL, TNF-a and IL-1, which differentially regulate classical and/or alternative NF-kB pathways in osteoclastic cells. These pathways are also modulated by NF-kB mediators, including TRAF6, aPKC, p62/SQSTM1 and deubiquitinating enzyme CYLD that are involved in the ubiquitin–proteasome system during RANK-mediated osteoclastogenesis. Abnormal activation of NF-kB signalling in osteoclasts has been associated with excessive osteoclastic activity, and frequently observed in osteolytic conditions, including periprosthetic osteolysis, arthritis, Paget’s disease of bone, and periodontitis. NF-kB modulators such as parthenolide and NEMO-binding domain peptide demonstrate therapeutic effects on inflammation-induced bone destruction in mouse models. Unravelling the structure and function of NF-kB pathways in osteoclasts and other cell types will be important in developing new strategies for treatments of bone diseases. # 2008 Elsevier Ltd. All rights reserved. Keywords: NF-kB modulator; Osteoclasts; Cytokines; NF-kB signalling pathway; Osteolysis

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NF-kB signalling pathway and function . . . . . . . . . . . . . . . . . . . . . . . . The central role of NF-kB in osteoclast physiology: RANKL, RANK and NF-kB in pathological bone diseases . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. NF-kB in periprosthetic osteolysis . . . . . . . . . . . . . . . . . . . . . . . 4.2. NF-kB in rheumatoid arthritis (RA) and osteoarthritis (OA) . . . . . 4.3. NF-kB in Paget’s disease of bone (PDB). . . . . . . . . . . . . . . . . . . 4.4. NF-kB in periodontitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modulation of NF-kB as a therapeutic intervention . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author at: Molecular Orthopaedics Laboratory, Centre for Orthopaedic Research, School of Surgery, University of Western Australia, QEII Medical Centre, 2nd Floor M Block, Australia. Tel.: +61 8 9346 4051; fax: +61 8 9346 3210. E-mail address: [email protected] (J. Xu). 1359-6101/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.cytogfr.2008.11.007

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1. Introduction Bone remodelling depends on a fine balance between two dynamic processes, bone formation and bone resorption. The cell types responsible for these two opposing processes are the bone-forming osteoblasts and the bone-resorbing osteoclasts, respectively. An imbalance between these two forces has been linked to the development of several human bone disorders, including osteoporosis, osteopetrosis, Paget’s disease of bone, malignant bone diseases, rheumatoid and osteoarthritis, periodontal diseases and bacteriainduced osteolysis [1]. Although normal bone architecture depends on a fine balance between the proper function and regulation of both osteoblasts and osteoclasts, a majority of evidence shows that the primary mechanisms responsible for osteolytic disorders are related to an overproduction or excessive activation of osteoclasts specifically; mediated by tumours, inflammatory cytokines or genetic disorders [1,2]. Osteoclasts are cells of haematopoietic origin and their precursors undergo proliferation and differentiation to become multinucleated bone-resorbing osteoclasts. The process of osteoclastogenesis is modulated by various cytokines such as RANKL, TNF-a and IL-1 [1,2]. In particular, these cytokines have been shown to induce NFkB activation, which is important for osteoclast formation and activation under both physiological and pathological conditions [1,2]. This article focuses on the structure and function of NF-kB in osteoclasts and in particular how selective modulation of this signalling pathway can be used as a therapeutic target for the treatment of many osteoclastrelated osteolytic disorders.

2. NF-kB signalling pathway and function NF-kB is a transcription factor that exists in virtually all eukaryotic cell types and is conserved to operate on divergent genes in many species [3]. It consists of five mammalian reticuloendotheliosis family (REL)/nuclear factor kB (NF-kB) proteins that belong to two groups [4]. The first group consists of RELA (also known as p65), cREL and RELB. These members of the family are produced as transcriptionally active proteins that do not require proteolytic processing for the acquisition of their activities. The second group includes NF-kB1 (also known as p105) and NF-kB2 (also known as p100), which are synthesized as transcriptionally inactive precursor molecules [4]. One of the most fascinating features of NF-kB is the variety and the nature of inducers that lead to its activation. Many of the identified inducers of NF-kB are important regulators for host responses to stress, injury and inflammation and consequently, NF-kB can be activated by signals that are associated with such states [3,4]. Potent inducers for NFkB include cytokines such as RANKL, TNF-a and IL-1; bacterial and viral products such as lipopolysaccharide (LPS), sphingomyelinase, double-stranded RNA and the Tax protein

from human T-cell leukaemia virus 1 (HTLV-1); also proapoptotic and necrotic stimuli such as reactive oxygen intermediates (ROIs), UV light and g-irradiation [3,4]. NF-kB is activated by two separate pathways [3,4]. In the classical pathway, upon activation, IkB proteins become phosphorylated by the macromolecular IkB kinase complex (IKK) that contains two catalytic subunits IKKa, IKKb, and a regulatory subunit IKKg or NEMO. This response in turn triggers the rapid ubiquitination and subsequent degradation of the IkB proteins by the ubiquitin-mediated proteasomal degradation pathway. The degradation of IkB allows NF-kB proteins to translocate from cytoplasm to the nucleus and bind to their cognate DNA binding sites to regulate the transcription of a large number of downstream target genes [5]. The second activation pathway, known as alternative pathway, involves inducible proteolytic processing of NFkB2/p100 REL protein [3]. Activation of the NF-kBinducing kinase (NIK) and IKKa, lead to the phosphorylation of p100 and the processing of p100 to p52 by the proteasome. This pathway principally generates p52/RELB heterodimers, which also translocate into the nucleus and regulate various target genes [3,4].

3. The central role of NF-kB in osteoclast physiology: RANKL, RANK and TRAF6 The receptor activator of NF-kB ligand (RANKL) is essential for osteoclastogenesis, bone resorption and calcium homeostasis [6–8]. Mice deficient in rankl gene exhibit severe osteopetrosis, defective in tooth eruption, and completely lack osteoclasts because of impaired osteoclastogenesis [7]. In addition to its established role in osteoclast physiology, RANKL has been implicated in the migration and metastatic behaviour of cancer cells [9]. The functional receptor of RANKL, RANK is encoded by a tumour necrosis factor receptor (TNFR) superfamily gene TNFGS11A [10]. Mice lacking TNFGS11A gene have a profound defect in bone resorption and in the development of cartilaginous growth plates of endochondral bone [11]. One of the key steps upon activation of the RANK pathway is the binding of TNFR-associated cytoplasmic factors (TRAFs), to specific domains within the cytoplasmic domain of RANK, with different TRAFs isoforms bearing various binding affinity toward RANK [12]. Moreover, truncated RANK receptor, with its TRAF binding Cterminal tail deleted, preserved its capability to stimulate JNK activity but not NF-kB, suggesting that interaction with TRAFs is necessary for NF-kB activation but not necessary for activation of the JNK pathway [13]. The TRAF family proteins are cytoplasmic adapter proteins with multiple domains that are involved in the mediation of several cytokine-signalling pathways [14]. To date, six members of this family have been described, named as TRAF1, 2, 3, 4, 5 and 6. Different members of the family have been reported to activate transcriptional pathways

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differently. TRAF2, 5 and 6 has been shown to activate transcription factors NF-kB through IkB kinase (IKK) activation and AP-1 through activation of mitogen-activated protein kinases (MAPKs), including Jun-N-terminal kinase (JNK), p38 and extracellular signal-regulated kinase (ERK) [14]. In addition, Deng et al. [15] reported TRAF6 functions as an ubiquitin ligase, which, in conjunction with a dimeric Ub-conjugating enzyme complex consisting of Ubc13 and Uev1A/Mms2, catalyzes the formation of a unique polyUb chain linked through lysine-63 (K63) of ubiquitin. This unique form of polyubiquitination leads to the activation of IKK and JNK through a proteasome-independent mechanism [15], reinforcing the central regulatory role of TRAF proteins in activation of various signalling pathways. The significance of TRAF6 in the maintenance of normal bone architecture was demonstrated in TRAF6 / mice, which displayed a defect in NF-kB signalling and consequently developed osteopetrosis [16]. Among the signalling pathways activated by RANKL/ RANK/TRAFs, NF-kB is critical for osteoclastogenesis. As shown in Table 1, several molecules in the NF-kB signalling pathway are vital for osteoclastogenesis and a disruption in these proteins resulted in defective bone phenotype. Firstly, p50/p52 double-knockout mice developed osteopetrosis because of a defect in osteoclast differentiation [17], whereas p50 and p52 single knockout mice only resulted in altered immune responses, but had no effect on osteoclast development [17]. While IKKa is required for RANK ligand-induced osteoclast formation in vitro, it is not needed in vivo. By comparison, IKKb is required for osteoclastogenesis both in vitro and in vivo [18]. The role of IKKg subunit (NEMO) in bone development remains unknown as mice lacking in NEMO failed to survive due to liver apoptosis [19]. Similarly, global p65/RelA knockout mice resulted in embryonic lethality and liver degeneration [20]. However, mice lacking RelA/p65 in the hematopoietic compartment exhibited a deficient osteoclastogenic response to RANKL [21]. In addition, in vitro studies have shown that inhibition of p65 nuclear translocation suppresses osteoclastogenesis [22]. RelB is required for full osteoclast differentiation in vitro and involved in inflamma-

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tion and tumour-induced osteolysis in vivo [23]. No abnormalities in bone phenotype were reported in IkB-a and cRel knockout mice [24,25], however, further studies are needed to clarify this. Targeted gene disruption of NIK, which is involved in the activation of alternative NF-kB signalling pathways exhibited a blunted response to RANKL-induced osteoclastogenesis [26] and lack inflammation-induced arthritis [27]. Together, these findings underpin the pivotal role of NF-kB signalling pathways in osteoclast differentiation and most importantly, provide a great insight into formulating new ideas for treating bone disorders. A recent study has found that in mice lacking the NF-kB adaptor molecule p62, the animals exhibited impaired RANKL-induced osteoclastogenesis in vitro and in vivo [28]. p62 appears to recruit atypical protein kinase C (aPKC) to TRAF6 and contributes to IKK activation and NF-kB nuclear translocation [28]. Interestingly, deubiquitinating enzyme CYLD targets TRAF6 via its interaction with the signalling adaptor p62, and thereby negatively regulating TRAF6 ubiquitination and RANK-mediated NFkB signalling and osteoclastogenesis [29]. These data suggest that NF-kB signalling is mediated by the ubiquitin– proteasome pathway, which involves TRAF6, p62, and CYLD. In addition to RANKL, TNF-a and IL-1 have been found to directly regulate NF-kB signalling in osteoclasts. As shown in Fig. 1, RANKL activates both classical and alternative pathways of NF-kB in osteoclastic like cells [26]. By comparison, the activation of NF-kB by TNF-a and IL-1 was restricted to classical pathways [26,30]. The induction of RANKL, TNF-a and IL-1 expression and its resulting NF-kB signalling activation might manifest the outcome of osteoclast-mediated osteolysis in different pathological bone conditions.

4. NF-kB in pathological bone diseases NF-kB has been shown to play an important role in pathological processes of infection, inflammation and

Table 1 NF-kB subunits and signalling molecules in bone. Protein IKKa IKKb IKKg IkB-a p50 p52 p50/p52 p65 RelB C-Rel

Other name

NEMO NF-kB1 p105 NF-kB2 p100 NF-kB1/NF-kB2 RelA

Bone phenotype

References

Required for RANKL-induced osteoclast formation in vitro but not in vivo Required for RANKL-induced osteoclast formation in vitro and in vivo Mutant embryos die at E12.5-E13.0 from severe liver damage due to apoptosis Severe widespread dermatitis in mice Altered immune responses, normal development Normal development Osteopetrosis; defect in osteoclast differentiation Embryonic lethality and liver degeneration in mice Required for RANKL-induced osteoclastogenesis and involved in arthritis-induced osteolysis Required for full osteoclast differentiation in vitro and involved in inflammation and tumour-induced osteolysis in vivo Defective in mitogenic activation of B and T lymphocytes and display impaired humoral immunity

[18] [18] [19] [25] [17] [17] [17] [20] [21] [23] [24]

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Fig. 1. Pathways leading to the activation of NF-kB (3, 4) in the osteoclast lineage regulated by RANKL, IL-1 and TNF-a. RANKL activates both classical and alternative pathways of NF-kB in osteoclastic like cells. By comparison, the activation of NF-kB by TNF-a and IL-1 was restricted to the classical pathway. The role of individual NF-kB subunits in osteoclast biology is listed in Table 1. Note that the binding of RANKL to RANK induces TRAF6 ubiquitination and activation of down stream signalling molecules. p62 mediates NF-kB signalling via interacting with aPKC or CYLD which confer positive or negative effect on NF-kB activation, respectively. The ubiquitin–proteasome pathway is involved in NF-kB signalling via the regulation of IkBa, TRAF6, p62, and CYLD.

cancer [3]. Given the central role of NF-kB in osteoclast differentiation and singling pathways, in the following section, the involvement of NF-kB in the pathogenesis of several osteolytic disorders will be discussed. 4.1. NF-kB in periprosthetic osteolysis Currently, over one million of total joint arthroplasties are performed each year because of joint destruction from various pathological conditions, including rheumatoid arthritis and osteoarthritis. Studies have shown that orthopaedic prostheses implant developed radiographic evidence of aseptic loosening within 10 years and high occurrence of implant failure, in particular, among the younger patients [31].

Periprosthetic failure arises from multiple factors, including bacterial, biomechanical, and host responses. Numbers of studies have defined a correlation between the amount of debris produced by wearing of the joint and the occurrence of aseptic loosening, and wear-debris-induced osteolysis as the main cause of the implant failure has been suggested [32]. Wear particles such as polymethylmethacrylate (PMMA) [33], a major component of the implant supporting cement generated from the prosthesis are phagocytosed by macrophages and initiate an inflammatory response, which subsequently leads to the recruitment of activated osteoclasts to the bone–implant interface. There are lines of evidence supporting the role of host immune responses in this scenario. For instance, results have shown that several potent osteoclastogenic cytokines such as IL-1

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and TNF-a, produced by macrophages were found in the fluid and tissue surrounding the loose implants [34]. In addition, recent findings have shown an abnormally high level of expression of RANKL in peri-implant tissues from patients with prosthetic loosening, giant cell tumour of bone and bone allograft non-union [35–37]. On the other hand, activation of the NF-kB signalling pathway has been directly linked to the development of prosthetic osteolysis in vitro. Titanium-alloy debris particles have been shown to activate NF-kB and induce the production of TNF-a and IL-6 from healthy human peripheral blood monocytes [38]. In addition, NF-kB is rapidly induced by titanium particles in ANA-1 cells via the p105 degradation pathway, and it has been suggested that the TNF-a induction is mediated by, at least in part, through NF-kB binding to the kB2a site of the TNFa promoter [39]. Moreover, anti-TNF therapy has been suggested for the treatment of implant loosening [40]. Taken together, modification of NF-kB signalling may represent a new approach to retard wear debris-induced prosthetic loosening. 4.2. NF-kB in rheumatoid arthritis (RA) and osteoarthritis (OA) Rheumatoid arthritis (RA) and osteoarthritis (OA) are the two most common human articulating diseases. The debilitating joint destruction associated with RA diseases has long been attributed to the ongoing chronic inflammation of the synovial lining. The infiltration of immunocompetent cells and proliferation of synovial fibroblasts in synovial lining leads to the formation of pannus tissue, which invades and degrades the articular cartilage and subchondral bone. In comparison, the major pathological feature of OA is the gradual destruction and loss of the articular cartilage, which has been linked to a combination of mechanical and biochemical factors. Synovial inflammation in OA is believed to be a secondary feature driven by the degradation of articular cartilage and the release of potentially immunogenic molecules during this process [41]. Although RA and OA display obvious pathological differences, the pleiotropic proinflammatory cytokines IL1b and TNF-a; extracellular matrix metalloproteinase (MMPs); prostaglandins and nitric oxide, in particular, IL-1b and TNF-a, have all been shown to play pivotal roles in the pathogenesis of RA both in preclinical [42] and clinical studies using biological agents such as etanercept (Enbrel1; Immunex Corporation, Seattle, WA, USA) and infliximab (Remicade1; Centocor Inc., Malvern, PA, USA) [43,44]. In addition, there is compelling evidence to suggest that the involvement of these factors also occurs in the progressive destruction of the articular cartilage in OA [45]. The activation of NF-kB is known to be central for the regulation of the synthesis and activity of inflammatory cytokines, including TNF-a and IL-1b, and also several other mediators also involved in the pathogenesis of OA and

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RA. These include cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and MMP-1 [46] and the link between NF-kB signalling and inflammatory joint diseases, to this date has been well documented. Constitutive activation of NF-kB has been reported in both RA and OA, as immunohistochemical staining for active NF-kB has been demonstrated in the nuclei of cells in the synovial lining of RA patients [47] and in OA patients, albeit to a lesser extent [48]. NF-kB is essential for TNF-induced synovial cell activation and proliferation as several studies showed that treatment of synovial cells with N-acetyl-Lcysteine (NAC), an antioxidant agent, inhibited TNF-ainduced NF-kB activation and transcription, and NAC subsequently inhibited synovial cell proliferation induced by TNF-a [49]. Moreover, nuclear extracts from IL-1b stimulated human synovial fibroblasts contained p65 DNA-binding NF-kB complexes and both the NF-kB classical oligonucleotide decoy and antisense oligonucleotide specific to p65 and they produced a concentrationdependent decrease in IL-1-stimulated PGE2 production [46]. Additionally, NF-kB activator, IL-18 can indirectly stimulate osteoclast formation through up-regulation of RANKL production from T cells in RA synovitis; as IL-18 is as effective as IL-1b, but less potent than TNF-a [50]. Blocking of IKKb in vitro with a dominant negative adenoviral construct was shown to inhibit the induction of IL-6, IL-8, and intercellular adhesion molecule-1 (ICAM-1) after stimulation with IL-1 or TNF-a [51]. The significance of NF-kB in inflammatory joint disease is further validated by numbers of in vivo arthritis models. In animal models such as carrageenan-induced paw edema, collagen-induced arthritis and adjuvant-induced arthritis, the time course of NF-kB activation appears to precede the onset of disease, and the blockade of NF-kB by different means all decreases mediator production and disease severity [52,53]. Intraarticular gene transfer of IKKb-wild type into the joints of normal rats resulted in significant paw swelling and accompanied histological evidence of synovial inflammation. Increased IKK activity was detectable in the IKKb-wt-injected ankle joints, coincident with enhanced NF-kB DNA binding activity. Intraarticular gene transfer of IKKb-dominant negative significantly ameliorated the severity of adjuvant arthritis, accompanied by a significant decrease in NF-kB DNA expression in the joints of adenoviral IKKb-dominant negative-treated animals [54]. In addition, studies using the selective IKKb inhibitor SPC 839 showed efficiency in arthritis models [51]. Recently, Jimi et al. [55] demonstrated that a cell-permeable peptide inhibitor of IkB-kinase complex can suppress RANKLstimulated NF-kB activation and osteoclastogenesis both in vitro and in vivo. Additionally, this peptide significantly reduced the severity of collagen-induced arthritis in mice by reducing levels of TNF-a and IL-1b, abrogating joint swelling and reducing destruction of bone and cartilage [55]. Therefore, in light of the central role NF-kB has in mediating inflammation, selective inhibition of NF-kB

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activation offers an effective therapeutic approach for inhibiting the increased proinflammatory mediator production and the infiltration of immunocompetent cells observed in inflammatory joint disease. 4.3. NF-kB in Paget’s disease of bone (PDB) PDB is a common disease of the elderly that affects up to 3% of individuals above the age of 40 years, and is also the second commonest bone disorders after osteoporosis. PDB has a localized and asymmetric nature; it may be monostotic, affecting only a single bone or portion of a bone, or may be polyostotic, involving two or more bones. Osteoclasts in Paget’s patients’ lesions are found to be increased in number, nuclei number and size. These osteoclastic abnormalities, coupled with increased and disorganized new bone formation, lead to debilitating symptoms, including bone pain, deformity and susceptibility to pathological fractures in and up to a third of suffers, and an increased incidence of osteosarcoma [56]. Although the etiology of PDB is yet uncertain, there is a genetic predisposition for the disease, and recent genetic linkage studies have shown that chromosome 5-linked forms of PDB are caused by mutations in a gene on 5q35-QTER, which encodes the ubiquitin-binding protein, sequestasome1(SQSTM1)/p62 [57]. Over the past few years, over 10 separate mutations have been identified, all of which affects the UBA domain of p62, a region of the protein that interacts with ubiquitin [58,59]. The mutations identified showed different penetrance to patients with the mutation results in a proline to leucine substitution at amino acid-392 of the gene displaying the highest penetrance. P932L mutation was detected in 11 of 24 French-Canadian families with Paget’s disease and 18 unrelated Paget’s disease patients, whereas

the mutation was not found in 291 controls [57]. Collectively, 30% of studied patients with familial Paget’s disease have mutations in the p62 gene. p62 is a scaffold adaptor molecule involved in number of signalling pathways, including the RANK-TRAF6-NF-kB pathway. Remarkably, recent studies have shown that in vitro p62 mutants enhanced RANKL-mediated NF-kB activity, osteoclast formation and activity [60,61]. The activation of NF-kB mediated by p62 mutants provides mechanistic explanations for the linkage between p62 and the progression of PDB. Thus, inhibition of NF-kB might represent an important approach to treat PDB. 4.4. NF-kB in periodontitis Periodontitis is a complex, multifactorial process involved bacterial plaque-components and host defense mechanisms. Inflammation of the periodontitium could result in the destruction of the underlying ligament and alveolar bone. Up regulation of RANKL mRNA in both inflammatory cells and epithelium has been associated with osteoclastic bone destruction in periodontitis [62]. In gingival crevicular fluid (GCF) samples from adult patients with untreated chronic periodontitis and in healthy controls, total amount of RANKL is significantly increased in periodontal disease, supporting its role in the alveolar bone loss [63]. Activation of NF-kB was observed on the oral epithelial cells when exposed to periodontopathogens Fusobacterium nucleatum and Porphyromonas gingivalis [64]. Porphyromonas gingivalis LPS induction of gene expression was abolished under the inactivation of IKK/NFkB [65]. Inhibition of NF-kB activation also reduced the production of pro-inflammatory mediators (IL-6, IL-8 and MCP-1) induced by Porphyromonas gingivalis LPS in

Table 2 Impact of NF-kB modulators/inhibitors on bone cells. NF-kb inhibitor

Target site

Impact on bone cells

Reference

AKBA

Phosphorylation/degradation of IKK; nuclear translocation of p65 Phosphorylation, IKKb DNA binding; phosphorylation of IkBa and p65 NF-kB activation

Suppresses osteoclastogenesis

[70]

Associated with an increase in bone mineral density Inhibits osteoclastogenesis Suppresses osteoclastogenesis

[78] [68] [73]

DNA binding; degradation of IkBa NF-kB activation Nuclear translocation Degradation of IKK IkBa degradation, nuclear translocation of p65 NF-kB activation

Inhibits TNF-a expression Suppresses osteoclastogenesis Inhibits LPS-induced osteolysis Suppresses osteoclastogenesis Suppresses osteoclastogenesis Suppresses osteoclastogenesis

[67] [74] [22] [71] [75] [76]

Phosphorylation/degradation of IKK; nuclear translocation of p65 Nuclear translocation of p65 IKK complex

Suppresses osteoclastogenesis

[72]

Inhibit osteoclast survival Inhibits osteoclasts in inflammatory Arthritis

[69] [55] [50]

Aspirin Curcumin Docosahexaenoic acid Gliotoxin Honokiol Parthenolide Retinamide Salinosporamide A Suberoylanilide hydroxamic acid Withanolide SN50 NEMO binding domain (NBD)

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human gingival fibroblasts [66]. Taken together, these studies indicate a role of NF-kB in periodontal diseases with an association of osteolysis.

5. Modulation of NF-kB as a therapeutic intervention An accumulating amount of evidence has shown that modulators of NF-kB signalling pathways have a great therapeutic potential, particularly in patients with cancer, infection and inflammatory diseases [3]. Therefore, the generation of NF-kB inhibitors/modulators with a higher specificity and effectiveness to treat human diseases is of great interest. Over the past few years, large numbers of

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natural or synthetic NF-kB inhibitors have been identified and produced. Interestingly, these NF-kB inhibitors interfere and exert their suppressive effects on NF-kB activities at different target sites. Moreover, a great number of these inhibitors have successfully demonstrated their effectiveness in inhibiting osteoclastogenesis and bone resorption, and preventing osteolytic-related bone disorders in vivo. Table 2 lists a number of NF-kB inhibitors and their corresponding possible target sites and impact on bone cells. For example, gliotoxin is a fungal metabolite known to inhibit NF-kB activation by suppressing NF-kB DNAbinding activity, thereby blocking RANKL-induced TNF-a expression [67]. Curcumin, a derivative from the Indian spice turmeric, is an inhibitor that is known to have potent anti-inflammatory and anti-oxidant properties. Recently,

Fig. 2. Chemical structure of NF-kB modulators and inhibitors that affect bone cells. Note that all compounds that act as NF-kB inhibitors or modulators bear no structural similarity, and exhibit various modes of action on NF-kB pathways (see Table 2).

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Curcumin has been shown to inhibit both NF-kB activation and osteoclastogenesis induced by RANKL [68]. Administration of the NEMO (NF-kB essential modulator)-binding domain (NBD) peptide in rheumatoid arthritis and collageninduced arthritis mouse model demonstrated that NF-kB activity was inhibited, which delayed the onset, lowered the incidence and decreased the severity of the arthritis [50]. SN50 (cell-permeable peptide inhibitor of NF-kB) suppressed P2Y6 receptor-mediated NF-kB signalling and osteoclasts survival [69]. The effects of selective inhibition of RANKL-induced NF-kB activation for the treatment of bone disorders have also been demonstrated. Recently, Yip et al. demonstrated that parthenolide, an active ingredient of medicinal plant Feverfew, blocks bacterial induced osteolysis in mice via inhibition of LPS induced NF-kB activation [22]. Additionally, acetyl-11-keto-beta-boswellic acid (AKBA), a component of an Ayurvedic therapeutic plant Boswellia serrata, which has been used for a large number of inflammatory diseases, has also found to enhance apoptosis induced by cytokines and chemotherapeutic agents, inhibit invasion, and suppress osteoclastogenesis through inhibition of NF-kB-regulated gene expression [70]. Other NF-kB inhibitors, including retinamide and some withanolides such as Indian ginseng have been widely used to treat tumours, inflammation, arthritis and other disorders. They also have been shown to block NF-kB activation in a non-specific manner by potentiating apoptosis, inhibiting invasion and osteoclastogenesis [71,72]. Recently, Salinosporamide A, Honokiol, Docosahexaenoic acid and Suberoylanilide hydroxamic acid have been shown to inhibit osteoclastogenesis via the suppression of NF-kB [73–76]. Finally, epidemiological studies suggest that aspirin, a NF-kB inhibitor [77] has been associated with an increase in bone mineral density of the hip and lumbar spine [78]. As shown in Fig. 2, all compounds or peptides that act as NF-kB inhibitors or modulators are largely different in their chemical structures, and exhibit various modes of action. More studies will be required to demonstrate direct or indirect binding targets of these compounds in the NF-kB pathway. It has been reported that inhibition of NF-kB may have adverse effects, given the physiological role of NF-kB in immune system [79]. Modulation of the NF-kB pathway, rather than inhibition, could be an alternate therapeutic approach for the treatment of osteolytic bone diseases. For example, modulation of NK-kB signalling pathways by calcium signaling, reactive oxygen species [80,81] and PKC activity [82] have been shown to alter osteoclast formation and survival. Understanding the genetic switch or microenvironmental factors that regulate NF-kB could lead to the development of drugs that can convert the function of NF-kB from one role to another. Therefore, the various NF-kB inhibitors might exert their effects at different target sites and affect different cell types accordingly (Table 2). By elucidating the molecular mechanisms underlying the action of these inhibitors,

novel treatments can be designed specifically for osteolytic bone disorders.

6. Conclusion Over the past few years, the NF-kB signalling pathways have been intensively studied by various in vitro and in vivo approaches and the knowledge gained from these studies has not only given insight into the molecular pathways, but more importantly helped to understand the pathogenesis of human diseases. This review highlights a number of current discoveries in NF-kB signalling pathways with particular emphasis on its association with osteoclast biology and osteolytic bone diseases. NF-kB pathways appear to have indispensable physiological role in various systems, particularly in osteoclast development. Significantly, NFkB is involved in bone diseases associated with over production and activation of osteoclasts, and has become an attractive therapeutic target for the treatment of osteolytic disorders through the modulation of its signalling pathways. Further understanding of the NF-kB pathways in osteoclasts may facilitate the design of novel treatments for osteoclast related bone disorders.

Acknowledgement This work was supported by the National Health and Medical Research Council of Australia.

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Jiake Xu, MD, PhD, studied Medicine in Guangzhou Medical College, China (1980– 1985), and received his PhD at the University of Western Australia (1991–1994) and postdoctoral fellowship at Stanford University (1994– 1998). Currently, he is Associate Professor and Head of Molecular Orthopaedic Laboratory, Centre for Orthopaedic Research, the University of Western Australia. His research focuses on gene discovery and molecular signalling of osteo-

J. Xu et al. / Cytokine & Growth Factor Reviews 20 (2009) 7–17 clasts, the intercellular communication of osteoclasts and osteoblasts, and pathogenesis of bone and joint diseases. Ming Hao Zheng, PhD, DM, FRCPath, graduated from Shantou University, China and obtained his PhD and Doctor of Medicine (DM) from the Faculty of Medicine at the University of Western Australia, Australia, and Fellow of Royal College of Pathologists in the United Kingdom. He has been the director of Research at the Centre for Orthopaedic Research since 1993. His current work focus on pathology of bone loss, cellular therapies and regenerative medicine for bone, cartilage and tendon.

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Ren Xiang Tan, China Pharmaceutical University (B 1983; M 1986), Lanzhou University (PhD 1990, Prof. Z.J. Jia), Technical University of Berlin (Visiting PhD candidate, 1989–1990, Prof. F. Bohlmann), University of Lausanne (Visiting scholar, 1995 and 1997, Prof. K. Hostettmann), University of California (Visiting scholar, 2001 and 2003, Prof. W. Fenical), Nanjing University (Associate Professor, 1992; Professor, 1994–present). International Society for the Development of Natural Products (President, 2005–2007). Research filed: natural product chemistry and microbe–host chemical communication/interaction.