Defective Osteoclastogenesis by IKKβ-null Precursors Is a Result of Receptor Activator of NF-κB Ligand (RANKL)-induced JNK-dependent Apoptosis and Impaired Differentiation

It has been reported previously that inhibitory κB kinase (IKK) supports osteoclastogenesis through NF-κB-mediated prevention of apoptosis. This finding suggests that the ligand for receptor activator of NF-κB (RANKL), the master osteoclastogenic cytokine, induces apoptosis of osteoclast precursors (OCPs) in the absence of IKKβ/NF-κB competency. To validate this hypothesis, we sought to determine the pro-apoptotic signaling factors induced by RANKL in IKKβ-null osteoclast OCPs and to rescue osteoclast differentiation in the absence of IKKβ through their inhibition. To accomplish this, we generated mice that lack IKKβ in multiple hematopoietic lineages, including OCPs. We found that these mice possess both in vitro and in vivo defects in osteoclast generation, in concurrence with previous reports, and that this defect is a result of susceptibility to RANKL-mediated apoptosis as a result of gain-of-function of JNK activation. We demonstrate that differentiation of OCPs depends on IKKβ because reduced IKKβ mRNA expression correlates with impaired induction of osteoclast differentiation markers in response to RANKL stimulation. We further show that fine-tuned inhibition of JNK activation in these cells inhibits RANKL-induced apoptosis and restores the ability of IKKβ-null OCPs to become mature osteoclasts. Our data highlight the pro-osteoclastogenic and anti-apoptotic roles of IKKβ in OCPs and identify a pro-apoptotic mechanism activated within the RANK signalosome. It has been reported previously that inhibitory κB kinase (IKK) supports osteoclastogenesis through NF-κB-mediated prevention of apoptosis. This finding suggests that the ligand for receptor activator of NF-κB (RANKL), the master osteoclastogenic cytokine, induces apoptosis of osteoclast precursors (OCPs) in the absence of IKKβ/NF-κB competency. To validate this hypothesis, we sought to determine the pro-apoptotic signaling factors induced by RANKL in IKKβ-null osteoclast OCPs and to rescue osteoclast differentiation in the absence of IKKβ through their inhibition. To accomplish this, we generated mice that lack IKKβ in multiple hematopoietic lineages, including OCPs. We found that these mice possess both in vitro and in vivo defects in osteoclast generation, in concurrence with previous reports, and that this defect is a result of susceptibility to RANKL-mediated apoptosis as a result of gain-of-function of JNK activation. We demonstrate that differentiation of OCPs depends on IKKβ because reduced IKKβ mRNA expression correlates with impaired induction of osteoclast differentiation markers in response to RANKL stimulation. We further show that fine-tuned inhibition of JNK activation in these cells inhibits RANKL-induced apoptosis and restores the ability of IKKβ-null OCPs to become mature osteoclasts. Our data highlight the pro-osteoclastogenic and anti-apoptotic roles of IKKβ in OCPs and identify a pro-apoptotic mechanism activated within the RANK signalosome. Osteoclasts develop from bone marrow macrophage precursors under the control of two cytokines, receptor activator of NF-κB ligand (RANKL) 2The abbreviations used are:RANKLreceptor activator of NF-κB ligandm-CSFmacrophage colony-stimulating factorOCPosteoclast precursorWTwild typeTRAPtartrate-resistant acid phosphataseNFATnuclear factor and activator of T-cellsMMPmatrix metalloproteinasecIAPcellular inhibitor of apoptosisXIAPX-linked inhibitor of apoptosisMKPMAPK phosphatasePARPpoly(ADP-ribose) polymeraseTUNELTdT-mediated dUTP nick end labelingJIPJNK-interacting proteinOCPosteoclast precursorTNFtumor necrosis factorGAPDHglyceraldehyde-3-phosphate dehydrogenaseMAPmitogen-activated proteinLPSlipopolysaccharideIKKinhibitory κB kinase. (1Kong Y.Y. Yoshida H. Sarosi I. Tan H.L. Timms E. Capparelli C. Morony S. Oliveira-dos-Santos A.J. Van G. Itie A. Khoo W. Wakeham A. Dunstan C.R. Lacey D.L. Mak T.W. Boyle W.J. Penninger J.M. Nature. 1999; 397: 315-323Crossref PubMed Scopus (2925) Google Scholar) and m-CSF (2Yoshida H. Hayashi S.-I. Kunisada T. Ogawa M. Nishikawa S. Okamura H. Sudo T. Shultz L.D. Nishikawa S.-I. Nature. 1990; 345: 442-443Crossref PubMed Scopus (1546) Google Scholar). RANKL induces osteoclast commitment and development by signaling downstream to several transcription factors, the most important of which is NF-κB (3Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. DuBose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1978) Google Scholar). NF-κB is a family of transcription factors whose activity coordinates a major component of the cellular inflammatory program, and its function is essential for osteoclastogenesis (4Iotsova V. Caamano J. Loy J. Young Y. Lewin A. Bravo R. Nat. Med. 1997; 3: 1285-1289Crossref PubMed Scopus (895) Google Scholar, 5Yamashita T. Yao Z. Li F. Zhang Q. Badell I.R. Schwarz E.M. Takeshita S. Wagner E.F. Noda M. Matsuo K. Xing L. Boyce B.F. J. Biol. Chem. 2007; 282: 18245-18253Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). NF-κB signaling involves two distinct but cooperating pathways, one canonical and one alternative pathway (6Senftleben U. Cao Y. Xiao G. Greten F.R. Krahn G. Bonizzi G. Chen Y. Hu Y. Fong A. Sun S.C. Karin M. Science. 2001; 293: 1495-1499Crossref PubMed Scopus (1161) Google Scholar), which has recently been shown to be critical in osteoclast biology (7Vaira S. Johnson T. Hirbe A.C. Alhawagri M. Anwisye I. Sammut B. O'Neal J. Zou W. Weilbaecher K.N. Faccio R. Novack D.V. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 3897-3902Crossref PubMed Scopus (125) Google Scholar). receptor activator of NF-κB ligand macrophage colony-stimulating factor osteoclast precursor wild type tartrate-resistant acid phosphatase nuclear factor and activator of T-cells matrix metalloproteinase cellular inhibitor of apoptosis X-linked inhibitor of apoptosis MAPK phosphatase poly(ADP-ribose) polymerase TdT-mediated dUTP nick end labeling JNK-interacting protein osteoclast precursor tumor necrosis factor glyceraldehyde-3-phosphate dehydrogenase mitogen-activated protein lipopolysaccharide inhibitory κB kinase. NF-κB is activated by the inhibitory κB kinase (IKK) complex, which is crucial for osteoclastogenesis. The IKK complex is composed of two catalytically active members, IKKα and IKKβ, and a regulatory subunit IKKγ/NEMO. IKKα mediates activation of the alternative pathway by phosphorylation of NF-κB2/p100 (6Senftleben U. Cao Y. Xiao G. Greten F.R. Krahn G. Bonizzi G. Chen Y. Hu Y. Fong A. Sun S.C. Karin M. Science. 2001; 293: 1495-1499Crossref PubMed Scopus (1161) Google Scholar), whereas IKKβ is important for activation of the canonical pathway through phosphorylation of IκB (8Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1608) Google Scholar). The importance of the signaling activity of the IKK complex in osteoclasts is demonstrated by the defect in osteoclastogenesis noted in mice lacking IKKα (9Chaisson M.L. Branstetter D.G. Derry J.M. Armstrong A.P. Tometsko M.E. Takeda K. Akira S. Dougall W.C. J. Biol. Chem. 2004; 279: 54841-54848Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) or IKKβ (10Ruocco M.G. Maeda S. Park J.M. Lawrence T. Hsu L.-C. Cao Y. Schett G. Wagner E.F. Karin M. J. Exp. Med. 2005; 201: 1677-1687Crossref PubMed Scopus (213) Google Scholar). Despite the sequence homology of these two kinases, their relative importance in osteoclastogenesis is strikingly different. For example, osteoclasts devoid of active IKKα only demonstrate an in vitro defect in osteoclastogenesis, whereas the bone phenotype of the mouse is remarkably normal. On the other hand, mice with an inducible osteoclast precursor-specific deletion of IKKβ demonstrate both in vitro and in vivo defects in osteoclastogenesis and are resistant to inflammatory osteolysis (10Ruocco M.G. Maeda S. Park J.M. Lawrence T. Hsu L.-C. Cao Y. Schett G. Wagner E.F. Karin M. J. Exp. Med. 2005; 201: 1677-1687Crossref PubMed Scopus (213) Google Scholar). Given these findings, it is evident that investigating the mechanism by which IKK2 supports osteoclastogenesis will improve our understanding of osteoclast biology and diseases attributable to overactive osteoclasts. We and others have shown that diverse methods of IKK blockade arrest osteoclastogenesis by induction of apoptosis (10Ruocco M.G. Maeda S. Park J.M. Lawrence T. Hsu L.-C. Cao Y. Schett G. Wagner E.F. Karin M. J. Exp. Med. 2005; 201: 1677-1687Crossref PubMed Scopus (213) Google Scholar, 11Abbas S. Abu-Amer Y. J. Biol. Chem. 2003; 278: 20077-20082Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 12Dai S. Hirayama T. Abbas S. Abu-Amer Y. J. Biol. Chem. 2004; 279: 37219-37222Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). We were interested in the pro-apoptotic signals downstream of RANKL in the absence of IKKβ, and we hypothesized that inhibition of these signals would be sufficient to rescue the osteoclast defect of cells lacking IKKβ. Our findings reveal that loss of IKKβ in osteoclast precursors (OCPs) results in a gain-of-function of JNK activation in response to RANKL that results in apoptosis. Furthermore, fine-tuned inhibition of this gain-of-function in JNK activation is sufficient to rescue osteoclastogenesis in OCPs lacking IKKβ. This finding demonstrates that the necessity of IKKβ for osteoclastogenesis may be evaded by inhibiting the pro-apoptotic effects of RANKL and designates JNK activation in the osteoclast as a potential means to induce cell death in OCPs. Reagents—Antibodies against IKKβ, IKKα, NEMO, actin, JNK, p38, Akt, MKP1, phospho-c-Jun, and c-Jun as well as horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against phospho-JNK, phospho-p38, phospho-Akt, and PARP were purchased from Cell Signaling Technologies, Inc. (Danvers, MA). Antibody against MKP5 was purchased from Abcam (Cambridge, MA). Cytokines were purchased from R & D Systems (Minneapolis, MN). TAT-TI-JIP was purchased from EMD Biosciences, Inc. (La Jolla, CA). Enhanced chemiluminescence kit was purchased from Pierce. All other chemicals were purchased from Sigma unless otherwise indicated. Animals—CD11b Cre Y-chromosomal transgenic and floxed IKKβ mice on a C57BL/6 background were reported previously (12Dai S. Hirayama T. Abbas S. Abu-Amer Y. J. Biol. Chem. 2004; 279: 37219-37222Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 13Ferron M. Vacher J. Genesis. 2005; 41: 138-145Crossref PubMed Scopus (93) Google Scholar). Male Cre+ Floxed IKKβ homozygotes were generated by crossing the above mice. Cell Culture—Osteoclast precursors were enriched from bone marrow of 2–3-week-old mice. Briefly, whole marrow was flushed from long bones into α-minimum Eagle's medium and was centrifuged at 453 relative centrifugal force. Marrow pellets were resuspended in whole media (α-minimum Eagle's medium with penicillin/streptomycin, 10% heat-inactivated fetal bovine serum) supplemented with 10 ng/ml m-CSF. Cell suspensions were plated onto Petri dishes at 37 °C in 5% CO2 for 5 days and then were plated according to experimental conditions. Osteoclast Formation Assay—Osteoclast precursors were plated in triplicate at a density of 3.0 × 104 cells in 200 μl of whole media supplemented with 10 ng/ml m-CSF and RANKL in 96-well tissue culture plates. TAT-TI-JIP was added at the time of cell plating (day 1). TNF-α and LPS were added at day of the osteoclasts day 5 and day of at which the cells are and for tartrate-resistant acid phosphatase to osteoclasts acid phosphatase cells with or were as osteoclasts. Assay—Osteoclast precursors were plated onto tissue culture dishes in whole media supplemented with m-CSF. were then for and with the for a time the cells were in cell and was by of cell protein was used for Assay—Osteoclast precursors were plated onto tissue culture dishes in whole media supplemented with m-CSF. were for and with 10 ng/ml RANKL for the the cells were as protein was and were by of precursors were plated onto tissue culture dishes for in whole media supplemented with m-CSF. of two of cells were plated in this was with or at the time of one from was with or ng/ml cells were as protein was and were by Osteoclast Assay—Osteoclast precursors were plated in whole media supplemented with m-CSF. were or were with 10 ng/ml RANKL for 5 was from cells according to the of was to transcription under the conditions. of and of in 10 μl of in were to °C for 5 to and on The were then added at the or for a of of 5 5 of To were in a as °C for °C for 10 °C for 5 and °C to marrow osteoclast precursors were plated onto tissue culture in the of 10 ng/ml m-CSF and RANKL with or TAT-TI-JIP for the were as in the were at of μl of 10 μl of Inc. μl of 10 and and μl of were to time according to the in a time °C for °C for 10 °C for °C for for were time were as mouse and mouse mouse and mouse mouse receptor and mouse receptor mouse and mouse mouse and mouse mouse and mouse mouse and mouse mouse and mouse mouse and mouse mouse and mouse mouse and mouse and mouse and mouse cell were in the of an of 10% 10% and for 5 and to on The were to a and in in to The were with and to antibodies at and with the secondary horseradish peroxidase-conjugated antibodies at The were for and an was the protein expression was bones were from mice and in 10% for were then for days in in through and in were for to osteoclasts or for dUTP nick end labeling with the in apoptosis kit to with an Osteoclast IKKβ in and in in has been shown to be for osteoclast (10Ruocco M.G. Maeda S. Park J.M. Lawrence T. Hsu L.-C. Cao Y. Schett G. Wagner E.F. Karin M. J. Exp. Med. 2005; 201: 1677-1687Crossref PubMed Scopus (213) Google Scholar). To the mechanism through which IKKβ supports osteoclastogenesis, we generated mice with a of IKKβ in multiple hematopoietic lineages, including OCPs by crossing Cre transgenic mice M. Vacher J. Genesis. 2005; 41: 138-145Crossref PubMed Scopus (93) Google Scholar) with mice floxed IKKβ M. G. M. M. A. A. M. M. R. A. T. K. I. Nature. PubMed Scopus Google Scholar). this we on the osteoclast we will to the mice as or Cre+ mice possess a ability to osteoclasts in vivo as by a reduced of osteoclasts with by for in long bones This is further by lack of IKKβ protein in osteoclast precursors of mice We demonstrate that this defect is by bone OCPs in the of m-CSF and cells osteoclasts with IKKβ and IKKβ Furthermore, with TNF-α or LPS and is to rescue the osteoclast defect of with IKKβ OCPs show IKKβ protein expression but this result in impaired osteoclastogenesis to RANKL-induced and Osteoclast has previously been demonstrated to OCPs from apoptosis (10Ruocco M.G. Maeda S. Park J.M. Lawrence T. Hsu L.-C. Cao Y. Schett G. Wagner E.F. Karin M. J. Exp. Med. 2005; 201: 1677-1687Crossref PubMed Scopus (213) Google Scholar). We sought to determine OCPs are to apoptosis in response to RANKL and to the absence of IKKβ results in osteoclast To accomplish this, we and control OCPs in the of m-CSF and RANKL for or 5 days to induce osteoclast We by time the expression of several markers for osteoclast We a in the expression of mRNA for the osteoclast markers receptor matrix metalloproteinase and in cells with This to osteoclast markers in osteoclastogenic correlates with to an in expression of IKKβ mRNA in cells with in cells that of IKKβ with control osteoclast expression RANKL is to osteoclasts in in vitro This finding that IKKβ a function to osteoclastogenesis. to that IKKβ is essential at for differentiation and of OCPs. we cells were to apoptosis control we and control OCPs in the of whole media supplemented with fetal bovine and m-CSF to and by time the expression of mRNA for several anti-apoptotic S. J. Biol. PubMed Google Scholar). We in expression of mRNA for cellular inhibitor of apoptosis and X-linked inhibitor of apoptosis in with control cells To determine apoptosis in response to we and control OCPs to RANKL for a time of We the of PARP by as a of apoptosis. We the of PARP in cells whereas in control the of PARP is the time of RANKL which that RANKL has a pro-apoptotic on osteoclast precursors in IKKβ. We that IKKβ is for osteoclast differentiation and the of osteoclast precursors to To determine apoptosis of osteoclasts or OCPs from mice to the of osteoclasts in we of long bones of and control mice with the to apoptosis. We a of osteoclasts and OCPs in with control long bones and on our we that apoptosis to the osteoclast defect in mice. a in JNK were interested in potential pro-apoptotic signals induced by RANKL in OCPs. To this, we a by in and control OCPs with a time of RANKL or We that kinase signaling would possess a that would apoptosis. We noted several in the of protein phosphorylation in with control was an and of JNK phosphorylation in response to RANKL and and TNF-α in with control OCPs. and phosphorylation in the absence of IKKβ, that JNK is a function of IKKβ. We that kinase phosphatase protein and MKP5 protein are RANKL of cells with and MKP5 protein of RANKL of control OCPs This time correlates with of JNK phosphorylation RANKL and JNK phosphorylation is at of RANKL and which correlates with the absence of and MKP5 protein at this time in cells Y. M. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and MKP5 A. A. C. S. A. 1999; PubMed Scopus Google Scholar) as JNK induction may as an mechanism for JNK RANKL stimulation. We that the gain-of-function of JNK activation may result in apoptosis of of JNK RANKL-induced of Osteoclast and in Osteoclast activation has been to RANKL-induced apoptosis of osteoclasts A.C. Y. S. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). We hypothesized that because cells are to RANKL-induced inhibition of RANKL-mediated JNK activation in these cells would rescue osteoclastogenesis. We of a to JNK activation J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) RANKL stimulation. TAT-TI-JIP osteoclastogenesis at above This finding is the importance of c-Jun in osteoclast differentiation K. Wagner E.F. J. Cell Sci. PubMed Scopus Google Scholar, F. R. T. S. K. K. T. T. T. K. A. T. J. 2004; PubMed Scopus Google Scholar). TAT-TI-JIP osteoclastogenesis in cells at a of We hypothesized that this of TAT-TI-JIP is sufficient to the pro-apoptotic of JNK the activity of JNK c-Jun RANKL stimulation. of TAT-TI-JIP RANKL-induced PARP RANKL-induced phosphorylation of c-Jun a of TAT-TI-JIP inhibits RANKL-induced PARP in but it inhibits c-Jun phosphorylation which the inhibitory of this on osteoclastogenesis. results that JNK two distinct in osteoclast differentiation and TAT-TI-JIP inhibits apoptosis of OCPs induced by RANKL c-Jun we this for its potential to rescue osteoclastogenesis of OCPs in the absence of IKKβ. in the in vitro osteoclastogenesis TAT-TI-JIP osteoclastogenesis of cells For example, cells with RANKL 5% of the of osteoclasts by control OCPs TAT-TI-JIP of cells results in but of osteoclasts with control OCPs osteoclasts in in vitro culture OCPs that possess defects in osteoclast differentiation that are of apoptosis. we sought to determine rescue of osteoclastogenesis in through JNK inhibition results in a rescue of bone To accomplish this, we plated and osteoclast precursors on an bone in osteoclastogenic in the and absence of by cells were with by control osteoclasts that are in we cells with TAT-TI-JIP we that noted in cells of with our in vitro osteoclastogenesis data shown in of osteoclasts is that of that IKKβ through of JNK inhibition to osteoclastogenesis. previous we and others have shown that function is for osteoclastogenesis (4Iotsova V. Caamano J. Loy J. Young Y. Lewin A. Bravo R. Nat. Med. 1997; 3: 1285-1289Crossref PubMed Scopus (895) Google Scholar, M.G. Maeda S. Park J.M. Lawrence T. Hsu L.-C. Cao Y. Schett G. Wagner E.F. Karin M. J. Exp. Med. 2005; 201: 1677-1687Crossref PubMed Scopus (213) Google Scholar, 12Dai S. Hirayama T. Abbas S. Abu-Amer Y. J. Biol. Chem. 2004; 279: 37219-37222Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The diverse of the IKK and NF-κB that a in the of osteoclast It has been demonstrated that IKKβ OCPs from apoptosis in response to TNF-α (10Ruocco M.G. Maeda S. Park J.M. Lawrence T. Hsu L.-C. Cao Y. Schett G. Wagner E.F. Karin M. J. Exp. Med. 2005; 201: 1677-1687Crossref PubMed Scopus (213) Google Scholar). this finding is with previous the function of IKKβ in other W. Hu Y. Delhase M. T. M. Johnson R. Karin M. J. Exp. Med. 1999; PubMed Scopus Google Scholar), its downstream of RANK osteoclastogenesis has The most is that IKKβ in osteoclastogenesis that is the function of in the osteoclast to be It has been that IKKβ may be important for the and differentiation of OCPs (10Ruocco M.G. Maeda S. Park J.M. Lawrence T. Hsu L.-C. Cao Y. Schett G. Wagner E.F. Karin M. J. Exp. Med. 2005; 201: 1677-1687Crossref PubMed Scopus (213) Google Scholar). We show that OCPs in IKKβ a defect in osteoclast which is with impaired induction of mRNA for the osteoclast and RANKL stimulation. We show that OCPs are to apoptosis in response to RANKL stimulation. on our time this is to result from impaired NF-κB-mediated transcription of anti-apoptotic the pro-apoptotic function of RANKL in osteoclast precursors has been in which of IKKβ induction of osteoclast differentiation markers was impaired in vitro osteoclastogenesis was We the expression of mRNA for protein M. T. Y. K. K. K. T. K. Y. M. Y. T. J. Exp. Med. 2005; PubMed Scopus Google Scholar) and the of the J. D. T. T. T. B. J. Y. Nat. Med. PubMed Scopus Google Scholar), two to be important in osteoclast in these cells to determine a was for the of these markers was to in this of cells induction of other osteoclast may be impaired in our data that RANKL-induced apoptosis is a major to the osteoclast defect in these We hypothesized that the kinase signaling downstream of RANK in cells would reveal pro-apoptotic signaling several signaling we noted in that JNK a and phosphorylation RANKL in OCPs that lack IKKβ. JNK activation has been with RANKL-induced apoptosis previously A.C. Y. S. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar), we that cells apoptosis as a result of RANKL-mediated JNK activation. by inhibiting we osteoclastogenesis in We that cells RANKL-induced apoptosis RANKL at a to the mature for two the of osteoclasts of cells that of Abu-Amer and J. TAT-TI-JIP only the osteoclast defect of added to the culture at the time as RANKL stimulation. with TAT-TI-JIP RANKL JNK inhibition rescue osteoclastogenesis of the absence of IKKβ protein is essential for the gain-of-function of JNK in response to RANKL is For example, it is that a downstream of IKKβ signaling and IKKβ is for inhibition of JNK RANKL stimulation. we demonstrate that fine-tuned inhibition of JNK in cells osteoclastogenesis. Our data a RANKL of OCPs to activation and of JNK through kinase phosphatase the absence of IKKβ, RANKL-mediated and MKP5 are to and of JNK phosphorylation and activation in apoptosis. may that blockade of JNK through inhibition of the JNK will result in and RANKL-induced JNK activity that is The mechanism of apoptosis in response to RANKL is well It has been demonstrated previously that in response to TNF-α in JNK activation to of the in of and apoptosis Y. L. Y. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). RANKL of OCPs result in it is that JNK activity in the absence of IKKβ to apoptosis of OCPs RANKL stimulation. expression of IKKβ in cells osteoclast it is that inhibition of apoptosis will rescue osteoclastogenesis in the absence of IKKβ. the of a JNK inhibitor on osteoclastogenesis in a of IKKβ expression that differentiation to but apoptosis. We our deletion of IKKβ was a in this to kinase several potential the absence of IKKβ and JNK activation For example, of NF-κB to JNK activation as which inhibits activation of JNK S. F. C. S. K. S. T. E. W.J. L. G. Nat. Cell Biol. 2004; PubMed Scopus Google Scholar). is a of NF-κB that JNK activation in response to TNF-α G. Y. B. Li Z. Karin M. A. Nature. 2001; PubMed Scopus Google Scholar). Furthermore, has been to a in NF-κB-mediated inhibition of JNK activation by Nat. Cell Biol. 2007; PubMed Scopus Google Scholar), this has been reduced and it will be to these of IKKβ and JNK in RANKL signaling osteoclastogenesis. It is important to that c-Jun activation is for osteoclastogenesis K. Wagner E.F. J. Cell Sci. PubMed Scopus Google Scholar). c-Jun activation to a and which induces expression of and differentiation of osteoclasts F. R. T. S. K. K. T. T. T. K. A. T. J. 2004; PubMed Scopus Google Scholar). inhibition of the pathway is a for of J. 2004; PubMed Scopus (79) Google Scholar). of the effects of the two of JNK activation in the it will be critical to the and means of RANKL-mediated JNK activation. Our results highlight the necessity of IKKβ in osteoclastogenesis. We demonstrate that IKKβ is important for both differentiation and of osteoclasts. Given that we are to rescue osteoclastogenesis in cells through inhibition of we that IKKβ at through of JNK activity to cell osteoclastogenesis. Our results that of JNK and inhibition of IKKβ in OCPs are potential means to

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