Biochimica et Biophysica Acta

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1 Biochimic et Biophysic Act 1832 (2013) Contents lists ville t SciVerse ScienceDirect Biochimic et Biophysic Act journl homepge: Involvement of kinse PKC-zet in the p62/p62 P392L -driven ctivtion of NF-κB in humn osteoclsts Estelle Chmoux, Stephen McMnus, Gino Lerge, Mrtine Bisson, Sophie Roux Division of Rheumtology, Fculty of Medicine, University of Sherrooke, 3001, 12th Avenue North, Sherrooke, PQ, Cnd rticle info strct Article history: Received 1 July 2012 Received in revised form 22 Novemer 2012 Accepted 12 Decemer 2012 Aville online 22 Decemer 2012 Keywords: Pget's disese of one Humn osteoclst SQSTM1/p62 PKCζ p65 Muttions of the gene encoding sequestosome1 (SQSTM1/p62), clustering in or ner the UBA domin, hve een descried in Pget's disese of one (PDB); mong these the P392L sustitution is the most prevlent. Protein p62 medites severl cell functions, including the control of NF-κB signling, nd utophgy. This scffolding protein intercts with typicl PKCζ in the RANKL-induced signling complex. We hve previously shown tht osteoclsts (OCs) overexpressing the p62 P392L vrint were in constitutively ctivted stte, presenting ctivted kinse p-pkcζ/λ nd ctivted NF-κB prior to RANKL stimultion. In the present study, we investigted the reltionships etween PKCζ nd NF-κB ctivtion in humn OCs trnsfected with p62 vrints. We showed tht PKCζ nd p-pkcζ/λ co-loclize with p62, nd tht PKCζ is involved in the RANKL-induced NF-κB ctivtion nd in the RANKL-independent ctivtion of NF-κB oserved in p62 P392L -trnsfected cells. We lso oserved sl nd RANKL-induced increse in IκBα levels in the presence of the p62 P392L muttion tht contrsted with the NF-κB ctivtion. In this study we propose tht PKCζ plys role in the ctivtion of NF-κB yctingsp65(rela)kinsetser 536, independently of IκBα; this lterntive pthwy could e used preferentilly in the presence of the p62 P392L muttion, which my hinder the uiquitin protesome pthwy. Overll, our results highlight the importnce of p62-ssocited PKCζ in the overctive stte of pgetic OCs nd in the ctivtion of NF-κB, prticulrly in the presence of the p62 P392L muttion Elsevier B.V. All rights reserved. 1. Introduction Pget's disese of one (PDB) is skeletl disorder chrcterized y focl nd disorgnized increses in one turnover [1]. Osteoclsts (OCs) re the primry cells ffected in PDB s the initil phse of this one disese is chrcterized y excessive one resorption [2]. Pgetic OCs re oth lrger, nd more numerous thn norml OCs. They re lso hyperctive nd resistnt to poptosis [3]. This disese hs strong genetic component, nd muttions in the SQSTM1/p62 gene hve een identified in high proportion of PDB ptients, the most frequent eing the P392L muttion [4 6]. The SQSTM1/p62 gene encodes the uiquitin-inding protein sequestosome1, lso known s p62, which medites vrious cell functions, including the control of NF-κB signling, regultion of utophgy, nd gene trnscription [7 9]. The presence of multiple protein protein interction domins supports its vrious functions. In prticulr, p62 hs een descried s scffolding protein tht, long with TRAF6, intercts with the receptor ctivtor of NF-κB (RANK) signling complex, Arevitions: OC, osteoclst; PDB, Pget's disese of one; RANKL, receptor ctivtor of NF-κB lignd; UBA, uiquitin-ssocited domin; PKCζ, protein kinse C zet; PDK1, 3-phosphoinositide-dependent protein kinse 1; IκB, inhiitor of NF-κB; IKK, IκB kinse Corresponding uthor t: 3001, 12th Avenue N, Sherrooke, PQ, Cnd J1H5N4. Tel.: ; fx: E-mil ddress: Sophie.Roux@USherrooke.c (S. Roux). formed in response to RANK lignd (RANKL) stimultion, which is the key regultor of OC formtion, ctivity, nd survivl. All muttions tht hve een reported so fr in PDB ptients re clustered either within or ner the C-terminl region of the p62 protein tht emodies the uiquitin-ssocited (UBA) domin, nd ffect its uiquitin-inding cpcities [10 13]. The discovery of muttions in the SQSTM1/p62 gene in numerous ptients hs identified protein p62 s n importnt modultor of one turnover. We nd others hve recently shown tht p62 overexpression in PDB, nd the PDB-relted p62 P392L muttion led to impired control of OC survivl nd ctivities, thus highlighting the criticl role of p62 in cellulr dysfunction in this disese [3,14]. Trnsgenic niml models hve further demonstrted the impct of the p62 muttion in the development of PDB. Knocked-in mice expressing the p62 P394L gene (the murine equivlent of humn p62 P392L )develop focl osteolytic lesions, some of which resemle PDB lesions [15], lthough in nother study, co-expression of the mesles virus nucleocpsid gene (MVNP) in the osteoclst linege ws required [14]. In rodent OCs, erly events tht occur fter RANKL stimultion include the formtion of multiprotein complex contining p62, TRAF6 nd the typicl PKCζ [16]. This complex is required for RANKLinduced NF-κB ctivtion nd NFATc1 synthesis, oth resulting in OC differentition nd ctivtion [16]. We hve previously shown tht in response to RANKL stimultion, PKCζ nd its upstrem ctivtor PDK1 (3-phosphoinositide-dependent protein kinse 1) re ssocited with p62 in norml humn OCs. Moreover, in PDB OCs, this ssocition is /$ see front mtter 2012 Elsevier B.V. All rights reserved.

2 476 E. Chmoux et l. / Biochimic et Biophysic Act 1832 (2013) present prior to the ddition of RANKL. We hve lso shown tht the P392L muttion in p62 contriutes to the incresed ctivtion of kinses PKCζ/λ nd PDK1, long with sl ctivtion of NF-κB, independently of RANKL stimultion [3]. While the functions of RANKL-medited PKCζ ctivtion re not known, studies in vrious models hve shown tht ctivted PKCζ promotes cell survivl y ctivting the ERK nd NF-κB pthwys [17,18] or y intervening in poptosis cscdes [19,20]. Our present study ws intended to elucidte the reltionship etween the ctivtion of the RANKL signling kinse PKCζ nd tht of the NF-κBpthwy, nd the impct of the p62 P392L muttion in these interctions. 2. Mterils nd methods 2.1. Mterils Opti Egle's minimum essentil medi (Opti-MEM), penicillin, streptomycin, fungizone, glutmine, nd fetl clf serum (FCS) were purchsed from Wisent (Montrel, QC). Ficoll Pque ws purchsed from Amershm Biosciences (Montrel, QC), nd ntiody diluent from DAKO (Crpinteri, CA). Humn recominnt (hr) M-CSF, nd hrgm-csf were purchsed from R&D (R&D Systems, Minnepolis, MN); solule hrrankl ws produced in our lortory. Got polyclonl nti-p62 (P-15; directed ginst the C-terminus), rit polyclonl nti-nfκb (p50 suunit) nd nti-inhiitor of NF-κB (IκBα) ntiodies were purchsed from Snt Cruz Biotechnology (Snt Cruz, CA); mouse monoclonl nti-p62 (IF study), rit polyclonl ntiphospho (Ser 311 )-p65/rela nd mouse monoclonl nti-phospho-ikkβ ntiodies from Acm (Cmridge, MA); mouse monoclonl ntiphospho-iκbα nd rit monoclonl nti-phospho (Ser 536 )-p65/rela from Cell Signling Technology (Dnvers, MA). We used rit polyclonl ntiodies to detect PKCζ, phospho-pkcζ/λ (Cell Signling Technology), nd phospho-pkcλ/ι (Invitrogen, Burlington, ON). A myristoylted pseudosustrte peptide inhiitor of PKCζ (Myr-SIYRRGARRWRKL-OH) ws otined from Millipore (539624, Billeric, MA). The protesome inhiitor MG-132 ws purchsed from Cliochem (Mississug, ON), Di Aminido Phenyl lndol (DAPI) from Sigm (Okville, ON), nd Alex Fluor 633-conjugted phlloidin from Invitrogen Osteoclst cultures Blood ws hrvested from humn umilicl cord t delivery fter otining informed consent from prturient women, s pproved y our institution's review ord. Cord lood monocytes (CBMs) were isolted from heprinized lood y density-grdient centrifugtion, wshed, nd suspended in Opti-MEM with ntiiotics, glutmine, nd 2% FCS. They were plted t density of /ml on 8-well chmer/slides (L-Tek, Biosciences, Bedford, MA). After incuting overnight, the cells were gently wshed to remove ny non-dherent cells, nd cultured in Opti-MEM supplemented with GM-CSF (100 pg/ml) for the first 3 dys, nd then for further 3 weeks in the sme medium supplemented with M-CSF (25 ng/ml) nd RANKL (75 ng/ml). The medium ws chnged every 2 3 dys. These culture conditions generte multinucleted cells (MNC) tht express OC mrkers nd hve the ility to resor one [3,21]. In ll experiments, RANKL stimultions were performed 48 h fter the lst chnge of medium Constructs nd trnsfections The full coding sequence of SQSTM1 (p62 wt ) ws otined from IMAGE clone nd sucloned into pegfp-c2 vector. The P392L muttion (p62 P392L ) ws generted from the wild-type sequence y PCR-directed mutgenesis. These GFP-fused constructs hd previously een shown not to interfere with the norml sucellulr locliztion of p62 [22]. Differentited humn OCs were trnsfected in serum-free OPTI-MEM contining LipoLTX for 6 h with constructs contining n empty vector (EV), p62 wild type (p62 WT ) or mutted p62 P392L. Trnsfection efficiency ws ssessed y fluorescence microscopy 24 h fter the procedure, nd rnged etween 40% nd 70% in ll cultures. The effective expression of the plsmid ws ssessed in GFPimmunoprecipittes y p62 Western lotting (Supplementl Fig. 1) Immunofluorescence Cells were stimulted for vrious periods of time with 100 ng/ml of RANKL, wshed quickly with cold PBS, nd then fixed with 1% prformldehyde. After permeiliztion nd utofluorescence quenching, non-specific inding sites were locked with 2% Bovine Serum Alumin (BSA). Specific ntiodies directed ginst p62, PKCζ, p-pkcζ/λ, nd p-pkcλ/ι were incuted in ntiody diluent overnight t 4 C. Alex-546 (red) nti-rit ntiodies, Alex-488 (green) nti-mouse ntiodies or Alex Fluor 633-conjugted phlloidin were incuted for 2 h t room temperture. DAPI counter-stining ws performed, so tht OCs contining more thn three nuclei could e visulized. Sequences of pictures were tken with pproprite filters to show ll three colors, nd superimposed using Simple PCI softwre Cell frctiontion nd Western-lotting Cells were differentited s descried ove, nd stimulted with 100 ng/ml of RANKL for 45 min. Nucler nd cytoplsmic cell frctions were isolted using the Q-Proteome Cell Frctiontion kit (Qigen, Mississug, ON), ccording to the mnufcturer's instructions. Briefly, lysis ws performed in series of uffers, nd differentil centrifugtion mde it possile to recover cell frctions enriched with cytosolic or nucler proteins. Protein lystes were quntified y modified Brdford ssy (Bio Rd, Mississug, ONT) nd run through SDS-PAGE. Western-lots were performed y incuting primry ntiody (nti-p62, nti-p50, or nti-p-pkcζ/λ ntiodies where pproprite) overnight t 4 C. HRP-conjugted secondry ntiodies were used to chieve detection with chemiluminescent system. The memrnes were stripped nd proed gin when necessry, nd nti-nucler lmin or nti-ctin ntiodies were used s controls for the nucler nd cytoplsmic frctions respectively Immunoprecipittion nd Western-lotting Cells were cultured s descried ove. After stimultion, the cells were lysed for 20 min in uffer contining NP40, nd cocktil of protese/phosphtse inhiitors. Lystes were pre-clered with got or rit serum followed y A/G grose ed precipittion. Lystes were then sujected to overnight immunoprecipittion (IP) t 4 C with ntiodies directed ginst p62, GFP or IκBα. Immune complexes were recovered with protein A/G grose eds, nd loded onto SDS-PAGE. Western-lots were performed y incuting specific primry ntiodies overnight t 4 C. HRP-conjugted secondry ntiodies were used to chieve detection with chemiluminescent system. Resulting films were scnned nd densitometric nlyses were performed with ImgeJ softwre Kinse ssy The PKC kinse ssy ws dpted from pulished method [23]. OCs cultured in medium supplemented with M-CSF nd RANKL were incuted in the presence of myristoylted pseudosustrte (Myr- SIYRRGARRWRKL-OH) t 10 μm or diluent control for 30 min. Cells were disrupted y NP-40 lysis s previously descried, nd lystes were immunoprecipitted with nti-pkcζ or nti-pkcλ/ι ntiodies. Immunoprecipittes were wshed in lysis uffer (with NP-40 nd NCl) nd resuspended in 20 μl kinse uffer (20 mm HEPES, ph 7.2, 10 mm MgCl 2, 5 mm DTT, 20 nm ATP, 5% NP-40) contining dephosphorylted myelin sic protein (MBP), PKC sustrte, nd μl [γ-32p]atp (10 mci/ml). After 15 min, rections were stopped

3 E. Chmoux et l. / Biochimic et Biophysic Act 1832 (2013) y dding oiling Lemmli smple uffer 2 μl, nd smples were migrted on SDS-PAGE gel prior to utordiogrphy Sttisticl nlysis Results were expressed s the men±stndrd devition (SD), nd the significnce ws determined y pired Student's t-test or nlysis of vrince (ANOVA) with Tukey's post-test where pproprite. Sttisticl significnce ws defined s p5. 3. Results 3.1. Phospho-PKCζ/λ inds to nd co-loclizes with p62 To investigte the reltionships etween PKCζ nd the p62-driven ctivtion of NF-κB in the ctivtion of OCs, we first nlyzed the interctions etween the ctivted form of PKCζ (p-pkcζ) nd p62 in norml OCs. In OC cultures derived from CBMs, short-term kinetic study indicted tht RANKL stimultion induced the inding of p-pkcζ/λ to p62 s shown in time-dependent ssocition of p62 immunoprecipittes (Fig. 1). Using immunofluorescence, p62 nd p-pkcζ/λ were oth shown to e co-loclized in or round the nuclei 45 min fter RANKLstimultion, s were p62 nd PKCζ;p-PKCλ/ι, lthough expressed in osteoclsts, did not co-loclize with p62 in these conditions, ut rther with the ctin ring t the cell periphery (Fig. 1) PKCζ is involved in the RANKL-induced ctivtion of NF-κB in osteoclsts nd their precursors p62 plys criticl role in RANKL-induced NF-κB ctivtion in OCs. So we next investigted whether the RANKL-induced p62/pkcζ interctions were ctully relted to the ctivtion of NF-κB, using myristoylted pseudosustrte for PKCζ in NF-κB ctivtion ssys. The specificity of this inhiitor for PKCζ ws determined in OCs y performing kinse ssy (Fig. 2). OCs from CBMs were then cultured in the presence of the myristoylted pseudosustrte efore undergoing RANKL stimultion, nd then lysed nd frctionted into nuclei- or cytoplsmic-enriched frctions. As shown in Fig. 2, dding RANKL to non-trnsfected OC cultures incresed the nucler trnsloction of the NF-κB p50 suunit (rtio of nucler/cytoplsmic p50: 6±4 vs. 0.24±9 in untreted cells, p5). This effect ws prevented y preincuting the cells with PKCζ inhiitor efore dding RANKL, suggesting tht PKCζ is involved in the RANKLinduced NF-κB ctivtion in humn OCs. We hd previously reported tht the p62 P392L muttion enhnced the ssocition etween p62 nd ctivted p-pkcζ/λ, nd tht NF-κB ctivtion ws constitutively incresed in OCs expressing the mutted form of p62 (p62 P392L ) [3]. In this study, we therefore investigted the potentil role of the p62 P392L muttion in the PKCζ-relted NF-κB ctivtion using OC cultures trnsfected with p62 vrints or n empty vector (EV). In EV-trnsfected cells, we oserved results similr to Fig. 1. PKCζ nd p62 interctions in humn osteoclsts. At the end of CBM cultures, the cells were either stimulted with RANKL (100 ng/ml) for the time indicted or left untreted. ) Immunoprecipittions (IPs) were conducted with n nti-p62 ntiody in cell lystes. Western lots (WBs) with nti-p-pkcζ/λ nd nti-p62 ntiodies re shown. Opticl densities (ODs) for nds corresponding to p-pkcζ/λ were corrected with the OD otined for nds corresponding to p62, nd computed in grphicl representtions (3 independent experiments). Anlyses re reported s men rtio±sd. *p5; p1; *p01 vs. nonstimulted cells. ) Immunofluorescence studies were performed using ntiodies directed ginst p62, PKCζ, p-pkcζ/λ, p-pkcλ/ι. filments were stined with Alex Fluor 633-conjugted phlloidin. Imges tken with pproprite filters revel p62 (Alex-488, green), PKCζ nd p-pkcζ/λ, p-pkcλ/ι (Alex-594, red) nd nuclei (DAPI, lue). Typicl cells re shown nd represent out 75% of MNCs oserved in 2 experiments.

4 478 E. Chmoux et l. / Biochimic et Biophysic Act 1832 (2013) Myr-peptide PKC inhiitor IP: PKC / - + IP: PKC - + p-mbp WB: p Nucler frction Cytoplsmic frction Lmin p50 No RL RL No RL RL Rtio nucler vs cytoplsmic p * No RL RL No RL RL c WB: + PKC inhiitor EV p62 WT p62 P392L RANKL (100ng/ml) PKC inhiitor Nucler frction p50 Lmin + PKC inhiitor Cytoplsmic frction p50 No RANKL RANKL * Rtio nucler vs cytoplsmic p50 PKC inhiitor EV p62 WT p62 P392L Fig. 2. Effect of PKCζ inhiitor on NF-κB nucler trnsloction. OC cultures supplemented with M-CSF nd RANKL were incuted in the presence of myristoylted pseudosustrte (Myr-SIYRRGARRWRKL-OH) t 10 μm or diluent control for 30 min. PKCζ or PKCλ/ι immunoprecipittes were nlyzed in kinse ssy using myelin sic protein (MBP) s sustrte. The reltive rtios re indicted (vs sence of PKCζ inhiitor). The dt shown re representtive of 2 independent experiments (). At the end of CBM cultures, the cells were trnsfected with pegfp-c2 plsmid contining p62 wt, p62 P392L, or n empty pegfp vector (EV), or were not trnsfected. Non-trnsfected cells (), or cells trnsfected with vectors contining p62 vrints (c) were preincuted with myristoylted PKCζ pseudo-sustrte t 10 μm for 30 min, nd then stimulted with RANKL (RL) 100 ng/ml for 30 min or left untreted (no RL). Lystes were seprted into frctions enriched with nucler or cytoplsmic proteins. Western lots were performed with n nti-p50 (NF-κB) suunit ntiody, nd fter memrne stripping, with nti-ctin or nti-lmin ntiodies for the cytoplsmic or nucler frctions respectively. Western lots (WB) of the cytoplsmic nd nucler frctions re shown. ODs were mesured with ImgeJ softwre, nd the rtios of p50 to lmin (nucler frction) over p50 to ctin (cytoplsmic frction) were computed. Anlyses re reported s the men rtio±sd. *p5, p1 vs untreted, p5, p1 sence vs presence of PKCζ inhiitor, p5 vs EV. The dt re representtive of 3 independent experiments.

5 E. Chmoux et l. / Biochimic et Biophysic Act 1832 (2013) those in non-trnsfected cells (Fig. 2c). When p62 wt ws overexpressed, we oserved non-significnt increse in the nucler expression of p50. No further increse ws detected fter dding RANKL, nor ws this modified y the presence of the PKCζ inhiitor. In contrst, cells trnsfected with the mutnt form of p62 (p62 P392L ) exhiited significntly higher nucler expression of p50 compred to EV-trnsfected cells, even prior to RANKL stimultion (8±0.1 p62 P392L vs. 0.27±3 EV, p5). Moreover, fter dding RANKL, the rtio of nucler/cytoplsmic p50 ws further incresed to 9± 9 in p62 P392L cells, nd ws significntly greter thn tht in the untreted cells (8±0.1, p5). Preincuting p62 P392L -expressing cells with the PKCζ inhiitor mrkedly reduced the nucler expression of p50 oth in the presence of RANKL (0.19±4) nd in its sence (0.36±7), to vlues not significntly different from those oserved in untreted, EV-trnsfected cells. This indictes overll tht PKCζ plys criticl role in oth the sl nd RANKL-induced overctivtion of NF-κB in OCs expressing the p62 P392L muttion P62 P392L muttion modifies the cnonicl NF-κB signling pthwy We next considered how PKCζ could intervene in NF-κB ctivtion, prticulrly in the presence of the p62 P392L muttion. The ctivtion of NF-κB normlly involves the inctivtion of IκB (Inhiitor of NF-κB), its relese from NF-κB, nd the susequent nucler trnsloction of IκB-free NF-κB suunits (p50/p65), required for RANKLstimulted OCs to differentite nd function properly. The p62- driven NF-κB ctivtion my involve either its scffold properties fcilitting kinse cscde ctivtion to free IκBα, or its polyuiquitin chin inding properties fcilitting IκBα protesome degrdtion. To further investigte the role of p62 nd of the p62 P392L muttion in the ctivtion of NF-κB, we first evluted the expression of IκBα in OCs with nd without RANKL stimultion. As p62 my contriute to the protesome-medited degrdtion of IκB, we nlyzed IκBα expression in cells which hd een preincuted with MG132, potent protesome inhiitor. As shown in Fig. 3, IκBα ws timedependently degrded within the first 30 min fter dding RANKL. However, when MG132 ws dded to the culture medium 30 min efore dding RANKL, no ovious degrdtion of IκBα ws seen fter the ddition of RANKL (Fig. 3). The sme results were otined in cells trnsfected with n empty vector, thus suggesting tht the trnsfection process did not modify IκBα degrdtion. However, when p62 wt ws overexpressed in humn OCs, lower levels of IκBα were detected in contrst to control cells trnsfected with n empty vector; these remined stle in the presence of RANKL, ut incresed considerly in the presence of protesome inhiitor (Fig. 3). These results suggest tht p62 overexpression induced ccelerted degrdtion of IκBα, ut tht this ws locked in the presence of MG-132. In cells crrying the p62 P392L muttion, incresed expression of IκBα ws detected in non-stimulted cells t level significntly higher thn tht in p62 wt trnsfected cells, nd which remined reltively high with or without RANKL stimultion in the presence of the protesome inhiitor. This increse in IκBα ws surprising, contrsting with the incresed ctivtion of NF-κB oserved under sl conditions in these cells P62 P392L muttion ffects IκBα degrdtion The high level of IκB oserved in cells expressing p62 P392L might hve een result of defect in the IκBα degrdtion process. Indeed, p62 my contriute to the degrdtion of IκB s result of its ssocition with the protesome, nd the presence of UBA domin t its C-terminus, which inds non-covlently to polyuiquitin chins [24]. Thus, the p62 P392L muttion could interfere with the degrdtion of IκBα vi ltered interctions etween mutted UBA domin tht my not ind properly to uiquitinted IκBα. We therefore investigted whether p62 could e ssocited with IκBα, nd whether the p62 P392L muttion could modify this interction. Cells were stimulted for vrious lengths of time with 100 ng/ml of RANKL, lysed nd sujected to immunoprecipittion using ntip62 ntiodies in non-trnsfected cells. Western lots were performed with nti-iκbα ntiodies. As shown in Fig. 4, p62 nd IκBα were ssocited physiclly in time-dependent mnner in non-trnsfected cells stimulted with RANKL. Accordingly, the reverse immunoprecipittion of IκBα lso reveled the presence of p62. Similr experiments were conducted in cells trnsfected with n EV, wild-type form of p62 or p62 vector crrying the p62 P392L muttion (Fig. 4). As these p62 mutnts were fused to GFP encoding sequence, we precipitted the ectopic p62 using n nti-gfp ntiody. After RANKL stimultion of cells overexpressing p62 wt, high level of ssocition of p62 with IκBα ws detected (3 times higher thn in non-treted cells, p5). This ws not oserved in cells trnsfected with n EV, thus indicting tht the GFP flg did not modify p62/iκbα interction. In contrst, in cells expressing the p62 P392L vrint, the interction of p62 with IκBα in the presence of RANKL ws not significntly different from tht in the untreted cells, thus indicting tht the p62 muttion lters the function of p62 in NF-κB signling y decresing its cpcity to ind IκBα, nd thus to shuttle this fctor towrds the protesome PKCζ my ct s n IKK kinse in osteoclsts Atypicl PKCs nd p62 re prt of the RANKL-signling complex, nd PKCζ could potentilly ct s n IKK (IκB kinse) in the p62-driven ctivtion of NF-κB in OCs, s hs een shown in non-oc cells [25,26]. Hence,PKCζ could contriute to the upstrem ctivtion/phosphoryltion of IKK, which results in the ctivtion of NF-κB, nd if so, the p62 P392L muttion my hve n impct on this function. To explore this possiility, we studied the phosphoryltion of IKKβ, theregultorysuunitof theikkcomplex, inthepresence of n inhiitor of PKCζ, nd then stimulted for 3 min or 30 min with RANKL (Fig. 5, ). While no significnt chnges in the levels of p-ikkβ nd p-iκbα were oserved fter short-term RANKL stimultion (3 min) (Supplementl Fig. 2), the expression of p-ikkβ ws significntly incresed fter 30-min RANKL stimultion in EV-trnsfected OCs, nd this increse ws prevented in the presence of the inhiitor of PKCζ in these cells. No significnt modultions of p-ikkβ were oserved in p62 wt -trnsfected OCs. In p62 P392L -trnsfected OCs, the sl expression of p-ikkβ ws significntly lower in the presence of the inhiitor of PKCζ, nd incresed fter RANKL-stimultion. However, p-ikkβ expression ws not significntly different in p62 P392L -trnsfected OCs compred to EVtrnsfected cells, whether in the presence of the inhiitor of PKCζ or not. While these results demonstrte possile role of PKCζ s n IKK kinse in the RANKL-induced ctivtion of NF-κB in OCs, they did not suggest tht this kinse ctivity mkes ny mjor contriution to the ctivtion of NF-κB oserved in the presence of the p62 P392L muttion PKCζ cts s p65 kinse independently of IκBα We found tht in the presence of p62 P392L, the expression of IκBα incresed even in the sence of RANKL, nd tht n ltertion of IκBα degrdtion my e t lest prtilly responsile for this. However, this increse in IκBα contrsts with the simultneous incresed ctivtion of NF-κB oserved under sl or RANKL-stimulted conditions in the p62 P392L -expressing cells. Becuse of the involvement of PKCζ in the ctivtion of NF-κB oserved in these cells, we hypothesized tht PKCζ my directly phosphorylte p65 (RelA) to ctivte the NF-κB pthwy, independently of IκB [27]. The expression of p-ser 536 or p-ser 311 p65 ws evluted y Western lot in nontrnsfected cells, nd in cells trnsfected with p62 vrints or EV. In non-trnsfected OCs, the expression of p-ser 536 p65, ut not tht of p-ser 311 p65, ws significntly incresed in the presence of RANKL,

6 480 E. Chmoux et l. / Biochimic et Biophysic Act 1832 (2013) RANKL (min) RANKL (min) WB: I B kd 40 kd Control cells MG132-treted cells Rtio I B / ctin 2.5 No RANKL RANKL RANKL (min) EV p62 wt p62 P392L EV p62 wt p62 P392L RANKL WB: I B 35 kd 40 kd No RANKL RANKL Control cells MG132-treted cells Rtio I B / ctin $$$ $$ $$ $$$ $$ EV p62 wt p62 P392L EV p62 wt p62 P392L Fig. 3. Study of IκB expression. At the end of CBM cultures, the cells were trnsfected with pegfp-c2 plsmid contining p62 wt, p62 P392L, or n empty pegfp vector (EV), or were not trnsfected. Non-trnsfected cells () or cells trnsfected with vectors contining p62 vrints () were preincuted with MG-132 (protesome inhiitor) t 10 nm for 1 h efore the experimenttion where pproprite. The cells were then treted with RANKL (100 ng/ml) for the length of time indicted (), or for 45 min (), or left untreted (no RANKL). Whole cell lystes were sujected to Western lotting using n nti-iκbα ntiody. After stripping, the memrnes were relotted using nti-ctin ntiodies. After quntifiction, the ODs otined for ech nd were computed, nd sujected to two-wy nlysis of vrince. The rtios of IκBα to ctin were reported (n=3 independent experiments). p1, *p01 RANKL vs no stimultion; p5, p1, p01 MG132 vs no pre-tretment; p1, p01, p62 WT or p62 P392L vs EV; p01 p62 P392L vs p62 wt. nd this increse ws prevented in the presence of PKCζ inhiitor (Fig. 6). In trnsfected cells, the expression of p-ser 536 p65 ws incresed in the presence of RANKL, with stronger ctivtion occurring in the presence of the p62 P392L vrint (Fig. 6). In ddition, preincuting p62 P392L -expressing cells with the PKCζ inhiitor mrkedly reduced the expression of p-ser 536 p65 oth in the presence of RANKL nd in its sence, to vlues not significntly different from those oserved in untreted, EV-trnsfected cells. These results suggest tht PKCζ ws involved not only in the p62-driven ctivtion of NF-κB induced y RANKL, ut lso in the sl ctivtion of NF-κB

7 E. Chmoux et l. / Biochimic et Biophysic Act 1832 (2013) IP p62 WB: I B IP I B WB: p62 p62 I B RANKL kd 35 kd IP rtio I B / p * * RANKL (min) IP GFP EV p62 wt p62 P392L WB: RANKL I B GFP 35 kd no RANKL * IP rtio I B / p62 EV p62 WT p62 P392L Fig. 4. Interctions etween p62 nd IκBα. At the end of the CBM cultures, the cells were trnsfected with pegfp-c2 plsmid contining p62 wt, p62 P392L, or n empty pegfp vector (EV), or were not trnsfected. Non-trnsfected cells () or cells trnsfected with vectors contining p62 vrints () were stimulted with RANKL (100 ng/ml) for 0, 10, 15, 30 nd 60 min. s descried ove, nd then lysed. Immunoprecipittions were conducted on the lystes with n nti-p62 ntiody. Agrose-ed precipittes were then loded onto SDS-PAGE, nd Western lots were performed with n nti-iκbα ntiody. After visuliztion, the memrnes were stripped nd proed gin with n nti-p62 ntiody. The reverse experiment, consisting of precipitting IκBα nd reveling p62 ws performed to confirm the specificity. Opticl densities were mesured with ImgeJ softwre, nd the rtios of IκBα to p62 were reported. Anlyses re reported s the men rtio±sd. *p5, p1, *p01 vs non-treted. The dt shown re representtive of 3 independent experiments. oserved in the presence of the p62 P392L muttion, through the p65/ RelA pthwy. Activtion of this pthwy could explin the NF-κB ctivtion in OCs even in the presence of high levels of IκBα, nd could e preferentilly used in the presence of the p62 P392L muttion. 4. Discussion The formtion of ternry complex etween TRAF6, p62 nd typicl PKCs in response to RANKL stimultion hs een reported in rodent OCs [16]. In humn OCs RANKL stimultion hs een shown to induce the formtion of multiprotein complex tht not only contins PKCζ nd p62, ut lso PDK1, the sustrtes of which include PKCζ [3]. In PDB OCs, nd in OCs overexpressing wild-type nd p62 P392L, the formtion of ctivted kinse/p62 complexes is constitutive, lthough only the mutted gene led to n incresed sl level of NF-κB ctivtion [3]. These dt identify p62 s n importnt meditor in osteoclstogenesis, nd suggest tht p62 P392L contriutes to the formtion of over-ctive OCs through signling pthwys involving PDK1, PKCζ/λ nd NF-κB. To explore the reltionship etween the constitutive ctivtion of PKCζ, nd tht of NF-κB previously reported in the presence of the p62 P392L muttion [3], NF-κB ctivtion ws investigted fter inhiiting PKCζ. In norml OCs, RANKL incresed nucler trnsloction of the p50 suunit of NF-κB, nd this ws prevented y PKCζ inhiitor, indicting tht prt of the RANKL-induced ctivtion of NF-κB is relted to PKCζ. In OCs overexpressing p62 P392L, ut not in p62 wt OCs, n increse in the ctivtion of NF-κB ws oserved even prior to RANKL stimultion, nd this ws considerly reduced in the presence of PKCζ inhiitor. Thus PKCζ ppers to e involved not only in the RANKL-induced ctivtion of NF-κB, ut lso in the sl, non-stimulted, ctivtion of NF-κB oserved in p62 P392L -trnsfected cells. Among PKCs, PKCλ might lso hve n importnt role in OC signling. Like PKCζ, PKCλ hs een shown to interct with p62. In ddition, the loss of PKCζ does not impir RANKL-induced osteoclstogenesis [16]. Thus, it hs een speculted tht, t lest in mice, p62/pkcλ my e necessry for NF-κB ctivtion [28].

8 482 E. Chmoux et l. / Biochimic et Biophysic Act 1832 (2013) Fig. 5. Expression of p-ikk in the presence of PKCζ inhiitor. At the end of CBM cultures, the cells were trnsfected with pegfp-c2 plsmid contining p62 wt,p62 P392L,ornempty pegfp vector (EV). Cells were preincuted with myristoylted PKCζ pseudo-sustrte t 10 μm for 30 min, nd then stimulted with RANKL 100 ng/ml for 30 min or left untreted (No RANKL). ) Whole cell lystes were sujected to Western lotting using n nti-p- IKKβ, nd fter memrne stripping, with nti-ctin ntiodies. Western lots (WB) re shown. ) ODs were mesured with ImgeJ softwre. Protein expressions were normlized reltive to the respective ctin intensity, nd the results re expressed s the expression reltive to tht of the untreted EV-trnsfected cells (men±sd). * p5, p1 vs untreted, p5, sence vs presence of PKCζ inhiitor. The dt shown re representtive of 5 independent experiments. The ctivtion of NF-κB involves the IKK-medited phosphoryltion of IκB, its relese from NF-κB, nd the susequent nucler trnsloction of IκB-free NF-κB suunits (p50/p65). Phosphoryltion trgets IκB for uiquitintion nd proteosoml degrdtion. In the present pper, we oserved in OC cultures tht the expression of IκBα ws greter in the presence of the p62 P392L muttion thn in p62 wt -trnsfected cells. p62 is known to shuttle proteins towrds the protesome for degrdtion [24], nd muttions in the UBA domin my interfere with the proteosoml degrdtion of polyuiquitinylted proteins directly, or indirectly through UBA structurl chnges [29 31]. Our results indicte tht p62 contriutes to IκBα degrdtion y direct ssocition. The high levels of IκBα detected in presence of the p62 P392L muttion re thus likely relted to less effective inding of IκBα to p62, resulting in defective IκBα clernce in mutted Ocs, in ddition to the NF-κB-induced IκB gene expression [27]. Consistent with the findings of studies using other models, it seems tht functionl UBA domin is required to chieve p62-dependent protesoml functions [32]. However, impired IκBα degrdtion should led to decrese in the ctivtion of the NF-κB remining in the cytoplsm [33]. This hs een reported in rodent OCs, where proteosoml inhiitors suppressed RANKL-induced osteoclstogenesis y ltering IκBα degrdtion [34]. As the effect of the p62 P392L muttion is in contrst n over-ctivtion of NF-κB, our findings suggest tht lthough IκBα clernce is indeed impired, this could e outweighed in p62 P392L cells y other mechnisms tht stimulte NF-κB ctivtion. Depending on the system, PKCζ my ctivte NF-κB y cting s n IKK kinse, or my ct downstrem of IKK y controlling the trnscriptionl ctivity of the NF-κB complex [9]. In non-oc cells, PKCζ hs een shown to e involved in the p62-driven NF κb ctivtion, responsile for the phosphoryltion of the IκB kinse IKKβ [25,35],nd IKKβ could e recruited to the multiprotein complex including p62, TRAF6 nd PKCζ [32]. In ddition, IKKβ overctivtion is le to drive osteoclstogenesis even in the sence of RANKL [36]. Our results suggest tht PKCζ my ct s n IKK kinse in the RANKL-induced ctivtion of NF-κB in OCs, ut did not suggest tht this kinse ctivity mkes ny mjor contriution to the ctivtion of NF-κB oserved in the presence of the p62 P392L muttion. Mechnisms other thn the cytosolic degrdtion of IκB my regulte NF-κB ctivtion, such s the phosphoryltion of p65 (Rel A), one suunit of the NF-κB dimer [37]. The phosphoryltion of p65 my occur t specific serine residues in nucleus or cytoplsm, depending on the kinse, stimulus nd cell type [38]. The direct phosphoryltion of p65 enhnces its trnsctivtion potentil, ut my lso reduce its ffinity for IκB, nd induce its trnsloction to the nucleus independently of IκB degrdtion [27]. Vrious kinses hve een reported to phosphorylte p65, such s MAP3K NIK (NF-κB-inducing kinse) [39] or IKKs t residue Ser536 [40], csein kinse II t Ser529 [41] nd PKCζ t Ser311 [42]. In humn Sos cells, p53- stimulted RSK1 (riosoml S6 kinse-1) hs een shown to phosphorylte nucler p65 t Ser536, reducing its ffinity for IκBα, nd leding to decresed IκBα-medited nucler export of the p-p65/ p50 complex [43]. In murine OC precursors, RANKL hs een reported to induce p65 phosphoryltion t Ser536 vi TAK1 (TGF β-ctivted kinse 1)-MKK6-p38 pthwy, independently of the RANKL-induced phosphoryltion nd degrdtion of IκB [44]. In our study, we evluted the expression of p65 phosphorylted t Ser 536 nd Ser 311 in CBMderived OCs, nd we found tht the RANKL-induced p-ser 536 p65 levels were ltered y inhiiting PKCζ. This ws further highlighted in OCs expressing p62 P392L muttion, suggesting tht the PKCζ/p65 pthwy contriutes to the sl ctivtion of NF-κB in these cells. Our oservtions of PKCζ nd IκBα expression/degrdtion highlight criticl divergence etween OCs expressing mutted nd non-mutted p62: when the p62 UBA domin is ffected, PKCζ my e preferentil pthwy to NF-κB ctivtion through the phosphoryltion of p65. While the mechnisms involved in the over-ctivtion of PKCζ in the presence of p62 P392L muttion remin to e investigted, one possiility is tht the conformtionl chnges resulting from muttions in the UBA domin could modify the protein-protein interctions involved in the multiprotein complex formed round p62, nd thus promote the interction etween the p62 N-terminl PB1 domin nd PKCζ, nd its ctivtion [30,45]. Previous studies hd indicted tht overexpression of the p62 P392L mutnt in HEK293 or Cos-1 cells incresed sl nd RANKL-induced NF-κB ctivtion, more thn overexpression of the p62 wild type mutnt [46]. However, little is known out the effects of the p62 P392L muttion on osteoclst signling in humns, lthough somes studies hve demonstrted the impct of the p62 mutnt on OC precursor signling. An increse in the RANKL-induced NF-κB, p38 MAPK nd NFATc1 ctivtion ws reported in murine OC precursors in the presence of the p62 P392L or p62 P394L muttion [14,47]. We nlyzed here the impct of the p62 P392L muttion on humn mture OCs, ut its effects on signling pthwys during OC differentition is n importnt spect tht requires further studies. Although the presence of the p62 P392L sustitution cnnot ccount for ll spects of the pgetic osteoclst phenotype [14], it clerly ffects osteoclst ehvior nd signling pthwys. p62 overexpression in PBD OCs contriutes to the overctive phenotype of these cells, nd the p62 P392L muttion induces some specific effects [3]. Overll, our findings provide new insights into the p62-linked pthwys leding to NF-κB ctivtion in humn OCs, nd highlight the crucil role of PKCζ in the uncontrolled ctivtion of PDB OCs, which my contriute to the incresed resistnce to OC poptosis nd to the excessive one resorption oserved in pgetic lesions.

9 E. Chmoux et l. / Biochimic et Biophysic Act 1832 (2013) WB (1) p-ser 536 p65 WB (2) p-ser 311 p65 No RL RL No RL RL + PKC inhiitor rtio p-ser 536 p65 / ctin Fold chnge rtio p-ser 311 p65 / ctin Fold chnge * No RL RL No RL RL EV p62 WT + PKC inhiitor p62 P392L PKC inhiitor RANKL (100ng/ml) WB (1) p-ser 536 p No RANKL RANKL IP rtio p-ser 536 p65 / ctin Fold chnge PKC inhiitor * EV p62 WT p62 P392L Fig. 6. Effect of PKCζ inhiitor on p65 phosphoryltion. At the end of CBM cultures, the cells were trnsfected with pegfp-c2 plsmid contining p62 wt, p62 P392L, or n empty pegfp vector (EV), or were not trnsfected. Non-trnsfected cells (), or cells trnsfected with vectors contining p62 vrints () were preincuted with myristoylted PKCζ pseudo-sustrtes t 10 μm for 30 min, nd then stimulted with RANKL (RL) 100 ng/ml for 30 min or left untreted (No RL). Western lots were performed with n nti- p-p65 (RelA) suunit ntiody detecting NF-κB p65 when phosphorylted either t Ser 536 or Ser 311, nd fter memrne stripping, with nti-ctin ntiodies s loding control. Western lots (WB) re shown. ODs were mesured with ImgeJ softwre. Protein expressions were normlized reltive to the respective ctin intensity, nd the results re expressed reltive to those for the untreted EV-trnsfected cells (men±sd). *p5, p1 vs untreted, p5, sence vs presence of PKCζ inhiitor, p5 vs EV. The dt re representtive of 3 to 4 independent experiments. Supplementry dt to this rticle cn e found online t dx.doi.org/ /j.dis Acknowledgements This study ws funded y grnts from the Cndin Institutes of Helth Reserch (CIHR) (S.R.), nd S.R. ws supported y the FRSQ (Fonds de l Recherche en Snté du Quéec). We re grteful to Dr P. McDonld (University of Sherrooke) for the ssistnce with kinse ssys. References [1] S. Seitz, M. Priemel, J. Zustin, F.T. Beil, J.Semler, H. Minne, T. Schinke, M. Amling, Pget's disese of one: histologic nlysis of 754 ptients, J. Bone Miner. Res. 24 (2009) [2] G.D. Roodmn, J.J. Windle, Pget disese of one, J. Clin. Invest. 115 (2005) [3] E. Chmoux, J. Couture, M. Bisson, J. Morissette, J.P. Brown, S. Roux, The p62 P392L muttion linked to Pget's disese induces ctivtion of humn osteoclsts, Mol. Endocrinol. 23 (2009) [4] P.Y. Chung, W. Vn Hul, Pget's disese of one: evidence for complex pthogenetic interctions, Semin. Arthritis Rheum. 41 (2012) [5]L.J.Hocking,G.J.Lucs,A.Droszewsk,J.Mngion,M.Olvesen,T.Cundy,G.C. Nicholson, L. Wrd, S.T. Bennett, W. Wuyts, W. Vn Hul, S.H. Rlston, Domin-specific

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