Academic literature on the topic 'Bone, Paget, osteoclast, p62'

Abstract Marrow stromal cells in the bone microenvironment of multiple myeloma (MM) patients play a critical role in promoting both tumor growth and bone destruction. Binding of MM cells to marrow stromal cells, through VCAM-1 on stromal cells and α4β1 integrin on MM cells, results in increased production of TNF-α, receptor activator of NF-KB ligand (RANKL) and IL-6 by marrow stromal cells. These factors in turn increase osteoclast (OCL) formation and the growth of MM cells. Adhesive interaction between MM cells and marrow stromal cells also decrease the sensitivity of MM cells to chemotherapeutic agents. Many of the downstream effects of these adhesive interactions are mediated through the NF-KB, p38 MAPK and JNK signaling pathways. The adapter protein sequestosome-1 (p62) sits at the crossroads of these signaling pathways and mediates the effects of cytokines and other factors that activate NF-KB, p38 MAPK and JNK. However, the role that increased signaling through p62 plays in MM tumor growth and bone destruction is unknown. It is our hypothesis that increased signaling through p62 in marrow stromal cells is necessary for their capacity to increase the growth of MM cells and OCL formation in MM, and thus p62 may be an attractive therapeutic target for MM. To test these hypotheses, we established long-term Dexter-type marrow cultures to isolate marrow stromal cells from MM patients and normals and measured signaling through p62 and PKCζ activation in MM marrow. We found significantly elevated levels of phosho-PKCζ, total PKCζ and VCAM-1 in MM stromal cells. The enhanced IL-6 production resulted from increased p38 MAPK activity and the increased VCAM-1 expression was NF-KB dependent. We then examined the effects of blocking p62 activity in primary patients and normal stromal cells with p62 siRNA (10μg). We confirmed that p62 expression was decreased by at least 90% in both these stromal cells by Western blot analysis. Stromal cells expressing p62siRNA or control siRNA were cultured with or without MM1.S cells for 3 days in separate experiments. Knocking-down p62 in MM derived marrow stromal cells significantly decreased the levels of PKCζ, VCAM-1 and IL-6 in marrow stromal cells and markedly decreased stromal cell support of MM cell growth and OCL formation. Similarly, marrow stromal cells from p62−/− mice produced much lower levels of IL-6, TNF-α, VCAM-1 and RANKL and minimally supported MM cell growth and OCL formation compared to normal cells. Thus, increased signaling through p62 in marrow stromal cells in patients with MM plays an important role in the increased tumor growth and OCL formation in MM, and support p62 as an attractive therapeutic target for MM.

Functional analyses of PDB (Paget's disease of bone)-associated mutants of the p62 [also known as SQSTM1 (sequestosome 1)] signalling adaptor protein represent an interesting paradigm for understanding not only the disease mechanism in this skeletal disorder, but also the critical determinants of ubiquitin recognition by an ubiquitin-binding protein. The 11 separate PDB mutations identified to date all affect the C-terminal region of p62 containing the UBA domain (ubiquitin-associated domain), a ubiquitin-binding element. All of these mutations have deleterious effects on ubiquitin binding by p62 in vitro, and there is evidence of an inverse relationship between ubiquitin-binding function and disease severity. The effects on ubiquitin-binding function of most of the mutations can be attributed to either reduced UBA domain stability, and/or the mutations affecting the presumed ubiquitin-binding interface of the UBA domain. However, a subset of the mutations are more difficult to rationalize; several of these affect sequences of p62 outside of the minimal ubiquitin-binding region, providing insights into non-UBA domain sequences within the host protein which mediate ubiquitin-binding affinity. The p62 mutations are presumed to result in activation of (osteoclast) NF-κB (nuclear factor κB) signalling. Understanding how loss of ubiquitin-binding function of p62 impacts on signal transduction events in osteoclasts will undoubtedly further our understanding of the disease mechanism in PDB at the molecular level.

Abstract Abstract 2857 Poster Board II-833 We reported that sequestosome 1 (p62) plays a critical role in the formation of signaling complexes that result in NF-kB, p38 MAPK, and PI3K activation in the marrow microenvironment of patients with multiple myeloma (MM), and that p62 is a potential therapeutic target for MM. In contrast to treating patients with inhibitors of each of the multiple signaling pathways activated in marrow stromal cells by MM cells (e.g. NF-kB or p38 MAPK), blocking the function of p62 should inhibit the activation of the multiple pathways mediated by p62 and have a broader effect on the bone marrow microenvironment. The goal of this study was to identify the domains of p62 responsible for increased MM cell growth and osteoclast (OCL) formation mediated by NF-kB and p38 MAPK signaling in marrow stromal cells when they interact with myeloma cells, and develop inhibitory peptides as potential therapeutic agents that interfere with p62's role in these signaling complexes. To pursue this objective, we transfected p62−/− stromal cells with p62 deletion constructs and assessed their effects on NF-kB and p38 MAPK signaling induced by MM cells or TNF-a. p62−/− stromal cells support of MM growth or OCL formation was significantly decreased compared to WT stromal cells. We made a series of 5' deletion constructs of p62 that lacked specific p62 domains: ΔPB1 (Δ1) lacks homodimerization domain and binding to PKCz, ΔPB1, ZZ (Δ2) lacks PB1 and RIP1 binding domains, and ΔPB1, ZZ, TBS, (Δ3) the PB1, RIP1, p38 and TRAF6 binding domains have been deleted. These constructs were tested for their capacity to restore p62 function in p62−/−stromal cells and support MM cell growth and OCL formation. GFP-labeled MM1.S myeloma cells were cocultured with p62−/− and WT marrow stromal cells transduced with the different p62 deletion constructs. Transduction of p62−/− stromal cells with the full-length p62 construct restored the capacity of p62−/− stromal cells to enhance the growth of MM cells to levels induced by WT stromal cells. Transduction of p62−/− stromal cells with the Δ1 construct also restored stromal cell support of MM growth. Therefore, the PB1 domain is not important for this function. Transduction of p62−/− stromal cells with the Δ2 construct, resulted in an inability of the stromal cells to support MM cell growth. Additional loss of the p38 and TRAF6 binding domains did not further impair p62−/− stromal cells support of MM cell growth. These results suggest that the RIP1 binding domain plays a critical role in supporting the growth of MM cells by marrow stromal cells. We then examined the capacity of p62−/− stromal cells transduced with various p62 deletion constructs to support OCL formation. Normal CFU-GM, a source of OCL precursors, were cocultured with p62−/− stromal cells transfected with the different p62 cDNA deletion constructs. The Δ1 construct completely rescued p62−/− support of OCL formation. However, Δ2 construct transduced p62−/− stromal cells only partly restored stromal cell support of OCL formation. Transduction of the Δ3 construct did not restore the capacity of the p62−/− stromal cells to support OCL formation. Similarly, transduction of the Δ2 and Δ3 construction decreased WT stromal cell support of MM cell growth. We then tested the feasibility of using transduction domain (PTD) fusion peptides as a potential means of delivering dominant negative p62 constructs into stromal cells in vitro and in vivo to block MM cell growth and VCAM-1 expression induced by marrow stromal cells. PTD binding domain fusion peptides containing NEMO binding protein that blocks NF-kB activity was used as a model system to determine the feasibility of transducing marrow stromal cells with p62 constructs. Addition of PTD-NEMO fusion peptides to stromal cells significantly inhibited WT stromal cell to enhance MM cell growth and VCAM-1 expression on stromal cells, which is the capacity of dependent, in part, on NF-kB signaling. These results demonstrate that the ZZ, p38 MAPK and TRAF-6 domains of p62 together are required for stromal cell support of MM cell growth and OCL formation and suggest that PTD constructs containing dominant negatives for p62 may be a feasible method for blocking p62 function in the MM marrow microenvironment. Disclosures: Roodman: Novartis: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy; Celgene: Consultancy; Acceleron: Consultancy.

AbstractPaget disease of bone (PDB) is a condition characterised by increased bone remodelling at discrete lesions throughout the skeleton. The primary cellular abnormality in PDB involves a net increase in the activity of bone-resorbing osteoclasts, with a secondary increase in bone-forming osteoblast activity. Genetic factors are known to play an important role, with mutations affecting different components of the RANK–NF-κB signalling pathway having been identified in patients with PDB and related disorders. Whilst the disease mechanism in these cases is likely to involve aberrant RANK-mediated osteoclast NF-κB signalling, the precise relationship between other potential contributors, such as viruses and environmental factors, and the molecular pathogenesis of PDB is less clear. This review considers the roles of these different factors in PDB, and concludes that a fuller understanding of their contributions to disease aetiology is likely to be central to future advances in the clinical management of this debilitating skeletal disorder.

Paget's disease of the bone (PDB) is an autosomal dominant trait with genetic heterogeneity, characterized by abnormal osteoclastogenesis. Sequestosome 1 (p62) is a scaffold protein that plays an important role in receptor activator of nuclear factor κB (RANK) signaling essential for osteoclast (OCL) differentiation. p62P392L mutation in the ubiquitin-associated (UBA) domain is widely associated with PDB; however, the mechanisms by which p62P392L stimulate OCL differentiation in PDB are not completely understood. Deubiquitinating enzyme cylindromatosis (CYLD) has been shown to negatively regulate RANK ligand-RANK signaling essential for OCL differentiation. Here, we report that CYLD binds with the p62 wild-type (p62WT), non-UBA mutant (p62A381V) but not with the UBA mutant (p62P392L) in OCL progenitor cells. Also, p62P392L induces expression of c-Fos (2.8-fold) and nuclear factor of activated T cells c1 (6.0-fold) transcription factors critical for OCL differentiation. Furthermore, p62P392L expression results in accumulation of polyubiquitinated TNF receptor-associated factor (TRAF)6 and elevated levels of phospho-IκB during OCL differentiation. Retroviral transduction of p62P392L/CYLD short hairpin RNA significantly increased TRAP positive multinucleated OCL formation/bone resorption activity in mouse bone marrow cultures. Thus, the p62P392L mutation abolished CYLD interaction and enhanced OCL development/bone resorption activity in PDB.

A role for ubiquitin in the pathogenesis of human diseases was first suggested some two decades ago, from studies that localized the protein to intracellular protein aggregates, which are a feature of the major human neurodegenerative disorders. Although several different mechanisms have been proposed to connect impairment of the UPS (ubiquitin–proteasome system) to the presence of these ‘ubiquitin inclusions’ within diseased neurones, their significance in the disease process remains to be fully clarified. Ubiquitin inclusions also contain ubiquitin-binding proteins, such as the p62 protein [also known as SQSTM1 (sequestosome 1)], which non-covalently interacts with the ubiquitinated protein aggregates and may serve to mediate their autophagic clearance. p62 is a multifunctional protein and, in the context of bone-resorbing osteoclasts, is an important scaffold in the RANK [receptor activator of NF-κB (nuclear factor κB)]–NF-κB signalling pathway. Further, mutations affecting the UBA domain (ubiquitin-associated domain) of p62 are commonly found in patients with the skeletal disorder PDB (Paget's disease of bone). These mutations impair the ability of p62 to bind to ubiquitin and result in disordered osteoclast NF-κB signalling that may underlie the disease aetiology. Recent structural insights into the unusual mechanism of ubiquitin recognition by the p62 UBA domain have helped rationalize the mechanisms by which different PDB mutations exert their negative effects on ubiquitin binding by p62, as well as providing an indication of the ubiquitin-binding selectivity of p62 and, by extension, its normal biological functions.

Adhesive interactions between multiple myeloma (MM) cells and marrow stromal cells activate multiple signaling pathways including nuclear factor κB (NF-κB), p38 mitogen-activated protein kinase (MAPK), and Jun N-terminal kinase (JNK) in stromal cells, which promote tumor growth and bone destruction. Sequestosome-1 (p62), an adapter protein that has no intrinsic enzymatic activity, serves as a platform to facilitate formation of signaling complexes for these pathways. Therefore, we determined if targeting only p62 would inhibit multiple signaling pathways activated in the MM microenvironment and thereby decrease MM cell growth and osteoclast formation. Signaling through NF-κB and p38 MAPK was increased in primary stromal cells from MM patients. Increased interleukin-6 (IL-6) production by MM stromal cells was p38 MAPK-dependent while increased vascular cell adhesion molecule-1 (VCAM-1) expression was NF-κB–dependent. Knocking-down p62 in patient-derived stromal cells significantly decreased protein kinase Cζ (PKCζ), VCAM-1, and IL-6 levels as well as decreased stromal cell support of MM cell growth. Similarly, marrow stromal cells from p62−/− mice produced much lower levels of IL-6, tumor necrosis factor-α (TNF-α), and receptor activator of NF-κB ligand (RANKL) and supported MM cell growth and osteoclast formation to a much lower extent than normal cells. Thus, p62 is an attractive therapeutic target for MM.

The LIM-domain containing protein Ajuba and the scaffold protein SQSTM1/p62 regulate signalling of NF-κB, a transcription factor involved in osteoclast differentiation and survival. The ubiquitin-associated domain of SQSTM1/p62 is frequently mutated in patients with Paget’s disease of bone. Here, we report that Ajuba activates NF-κB activity in HEK293 cells, and that co-expression with SQSTM1/p62 inhibits this activation in an UBA domain-dependent manner. SQSTM1/p62 regulates proteins by targeting them to the ubiquitin-proteasome system or the autophagy-lysosome pathway. We show that Ajuba is degraded by autophagy, however co-expression with SQSTM1/p62 (wild type or UBA-deficient) protects Ajuba levels both in cells undergoing autophagy and those exposed to proteasomal stress. Additionally, in unstressed cells co-expression of SQSTM1/p62 reduces the amount of Ajuba present in the nucleus. SQSTM1/p62 with an intact ubiquitin-associated domain forms holding complexes with Ajuba that are not destined for degradation yet inhibit signalling. Thus, in situations with altered levels and localization of SQSTM1/p62 expression, such as osteoclasts in Paget’s disease of bone and various cancers, SQSTM1/p62 may compartmentalize Ajuba and thereby impact its cellular functions and disease pathogenesis. In Paget’s, ubiquitin-associated domain mutations may lead to increased or prolonged Ajuba-induced NF-κB signalling leading to increased osteoclastogenesis. In cancer, Ajuba expression promotes cell survival. The increased levels of SQSTM1/p62 observed in cancer may enhance Ajuba-mediated cancer cell survival.

Abstract Abstract 888 The marrow microenvironment enhances both tumor growth and bone destruction in multiple myeloma (MM) through MM cell-induced activation of multiple signaling pathways in bone marrow stromal cells (BMSC) by TNFα. We reported that sequestosome-1 (p62) acts as a signaling hub for NF-kB, MAPK and PI3K activation in BMSC of MM patients and enhances MM growth and osteoclast (OCL) formation. p62 is composed of 5 domains that are involved in protein–protein interactions required for formation of these signaling complexes, but which domain of p62 mediates increase MM growth and OCL formation is unclear. Therefore, deletion constructs of p62 that lacked each of the 5 domains (PB1, ZZ, p38, TBS or UBA) were transfected into a p62−/− stromal cell line. We found that the ZZ domains mediated BMSC enhancement of MM cell growth, IL-6 production, VCAM-1 expression and OCL formation. Using virtual modeling of the ZZ domain, we identified 6 candidate p62-ZZ inhibitory molecules and tested them for their capacity to block enhanced MM cell growth, OCL formation, IL-6 production, and VCAM-1 expression on BMSC induced by TNFα. When MM1.S, RPMI8266, ANBL6 MM cell-lines or CD138+ primary MM cells were cultured in the absence of BMSC with p62-ZZ inhibitor #3 (10mM), this inhibitor directly induced cell death. The p62-ZZ inhibitor-induced cell death was characterized by an increase in reactive oxygen species (ROS) production by inhibiting NF-kB activation, and apoptotic cell death by triggering the activation of caspases 3, 7, and 9. This inhibitor had an IC50 of 4.6mM for MM1.S survival. In contrast, CFU-Blast formation by human CD34+ cells was not inhibited by p62-ZZ inhibitor #3 (10mM). p62-ZZ inhibitor #3 (10mM) treatment of human OCL precursors derived from CFU-GM, inhibited OCL formation by blocking precursor proliferation. To examine the specificity of the p62-ZZ inhibitor #3, we tested its effects on OCL formation by CD11b+ mononuclear cells from wild type (WT) and p62−/− mice cultured with TNFα for 7 days. p62-ZZ inhibitor #3 blocked WT OCL formation but did not block p62−/− OCL formation. The p62-ZZ inhibitor also blocked VCAM-1 expression and IL-6 production by normal and MM patient stromal cells induced by TNFα compared to vehicle. Importantly, the inhibitor (10mM) did not block stromal cell proliferation. Further, the inhibitor blocked TNFα induced PKCζ phosphorylation in stromal cells and MM1.S when the cells were pretreated with the p62-ZZ inhibitor for 3 hours. These results demonstrate that p62-ZZ inhibitor #3 specifically blocks both stromal cells independent and dependent MM cell growth and OCL formation but does not affect hematopoietic or stromal cell growth. These results support p62 as a potential novel therapeutic target for MM. Disclosures: Roodman: Amgen: Consultancy; Millennium: Consultancy.

Abstract Marrow stromal cells play a critical role in osteolytic bone destruction in multiple myeloma, and promote tumor growth. In particular, adhesive interactions between myeloma cells and marrow stromal cells increase RANK ligand (RANKL), a potent inducer of osteoclast formation, IL-6 and TNF-α production by marrow stromal cells. IL-6 enhances the growth and prevents apoptosis of myeloma cells, and TNF increases production of RANKL and IL-6. Recently, a new member of NF-κB signaling pathway, p62ZIP, has been identified. p62ZIP plays a critical role in NF-κB activation induced by TNF-α and RANKL and is involved in multiple signaling pathways that result in enhanced IL-6 production, tumor cell survival and bone destruction. It is our hypothesis that inhibiting p62ZIP expression will profoundly diminish myeloma growth by blocking the effects of IL-6 produced in the tumor-bone microenvironment in response to TNF-α. Therefore, we used p62ZIP−/− mice to determine the effects of deleting p62ZIP in stromal cells on the growth of myeloma cells. Marrow cells from p62ZIP −/− or wild type mice were used to establish long-term Dexter-type marrow cultures to isolate marrow stromal cells. Marrow stromal cells from p62ZIP −/− or wild type mice were cocultured for 48 h with a GFP-labeled human MM.1S myeloma cell line (MM.1S) and murine and human RANKL, IL-6 and TNF-α levels were determined in conditioned media from these cocultures using commercial ELISA assays. Cocultures of MM.1S with wild type marrow stromal cells resulted in much greater upregulation of murine IL-6 than p62−/− stromal cell coculture (IL-6 in p62−/− stromal cell cultures; 114+70 vs. WT 1900+9 pg/ml). In addition, deleting p62ZIP in stromal cells markedly decreased the growth of tumor cells. Coculture with wild type stromal cells induce 1.4-fold greater increase in MM.1S cell growth at 72 h compared to p62−/− stromal cells. Further, addition of neutralizing antibodies to TNF-α and IL-6 to the cocultures of MM.1S cells with WT stromal cells similarly affected the growth of the MM.1S. Since TNF-alpha can increase the expression of adhesion molecules on stromal cells and tumor cells, we measured expression levels of ICAM-1 and VCAM-1 by Western blot. VCAM-1 and ICAM-1 levels on p62ZIP−/− bone marrow stromal cells were not changed compared to WT stromal cells. We then determined the capacity of p62 −/− cells to support OCL formation by normal spleen CFU-blast. OCL formation was decreased about 50 % in cocultures containing p62−/− stromal cells treated with PTH-rp, IL-6 and TNF-α compare with WT stromal cell culture. These results show that p62 plays an important role in myeloma cell growth and OCL formation induced by cytokines that are upregulated in the marrow microenvironment in patients with myeloma.

Paget’s disease of bone (PDB) is an age-related metabolic bone disease characterized by focal lesions of increased bone resorption and formation, eventually leading to bone deformities. The cause of PDB and the mechanisms that give rise to focal lesions are yet to be understood, but findings suggest that the disease is driven by aberrant, highly nucleated, osteoclasts (OCs). In recent years evidences of a genetic involvement were found: mutations in UBA domain of SQSTM1, which encodes for p62, have been reported in both familial and sporadic cases of PDB (P392L most commonly). Although, their actual pathogenicity has been controversial in experimental studies. Moreover, mutations only involve a part of PDB cases and, although some novel genes have been more recently associated to PDB (e.g.ZNF687), the genetic background of PDB remains in part unknown. In an attempt to establish an experimental model of PDB and better understand p62 role in the disease, we compared two genetically modified murine models, systemic p62 knock-out (p62KO) and mutated p62-P394L (P394L) mice. To further characterize the genetic background of PDB we investigated PDB-associated genes and novel genes in SQSTM1-negative patients. In vitro bone marrow-derived macrophages (BMMs) showed a reduced RANKL-induced osteoclastogenesis (OCgenesis) in p62KO-mice, also seen by TRAP staining on bone sections. BMMs of P394L mice showed a higher sensitivity to RANKL and an increase in OC size and number of nuclei, resembling PDB. Such alterations did not result in a bone phenotype at 6 months of age in either model. However, we found that, with ageing, 47% of P394L mice do develop focal osteolytic lesions. Surprisingly, 78% of p62KO mice developed severe lesions. Although further histological characterization is needed, both animals showed focal, PDB-like, osteolytic/sclerotic features. In vitro analysis of aged p62KO BMMs no longer showed a reduction in OCgenesis potential. Taken together, our findings suggest that p62 mutations in UBA cause a loss of function mechanism in PDB, further exacerbated by total loss of the protein. In support of our hypothesis, proteomics showed that aged p62KO and P394L BMMs are primed for OCgenesis and both present similar expression profiles. Investigating possible molecular mechanisms, we found that UBA-dependent p62 functions of autophagy and NF-κB signalling are not altered in either p62KO and P394L cells. Genetic analysis of 34 patients was performed on genes SQSTM1, TNFRSF11A, VCP, ZNF687 and two variants on TM7SF4 and RIN3. The majority of our cohort was negative for rare mutations on such genes, apart from three cases, carrying TNFRSF11A_M566L, SQSTM1_S275N and ZNF687_P937R. Finally, taking advantage of a large pedigree of a severely affected PDB family we performed Exome NGS to identify novel causal gene. Analysis of impact, familial segregation and allelic frequency identified a novel mutation: PFN1_D107Rfs*3, that causes loss of C-terminal domain of PFN1. This gene encodes for profilin1, a regulator of actin polymerization and cell motility. Given the essential role of cytoskeleton reorganization in OCs biology and the previous findings of bone focal deformities in PFN1 OC-conditional knock-out mice, we started investigating its potential pathogenicity. Silencing of PFN1 in murine BMMs resulted in larger OCs with a higher number of nuclei and increased resorption activity. Screening of PFN1 mutations on other PDB cases is ongoing. Overall, our data demonstrated that both p62 depletion and P394L mutation and are sufficient to cause PDB-like disease in mice. Based on the available molecular data the likely role of p62 in PDB is UBA-dependent but autophagy and NF-κB independent. The genetic background of PDB remains largely unknown, as demonstrated by our screening. Finally, the gene discovery part of the project allowed to identify a likely novel gene for PDB, associated with an early onset and aggressive phenotype.

Résumé : La maladie osseuse de Paget (MP) est un désordre squelettique caractérisé par une augmentation focale et désorganisée du remodelage osseux. Les ostéoclastes (OCs) de MP sont plus larges, actifs et nombreux, en plus d’être résistants à l’apoptose. Même si la cause précise de la MP demeure inconnue, des mutations du gène SQSTM1, codant pour la protéine p62, ont été décrites dans une proportion importante de patients avec MP. Parmi ces mutations, la substitution P392L est la plus fréquente, et la surexpression de p62P392L dans les OCs génère un phénotype pagétique partiel. La protéine p62 est impliquée dans de multiples processus, allant du contrôle de la signalisation NF-κB à l’autophagie. Dans les OCs humains, un complexe multiprotéique composé de p62 et des kinases PKCζ et PDK1 est formé en réponse à une stimulation par Receptor Activator of Nuclear factor Kappa-B Ligand (RANKL), principale cytokine impliquée dans la formation et l'activation des OCs. Nous avons démontré que PKCζ est impliquée dans l’activation de NF-κB induite par RANKL dans les OCs, et dans son activation constitutive en présence de p62P392L. Nous avons également observé une augmentation de phosphorylation de Ser536 de p65 par PKCζ, qui est indépendante d’IκB et qui pourrait représenter une voie alternative d'activation de NF-κB en présence de la mutation de p62. Nous avons démontré que les niveaux de phosphorylation des régulateurs de survie ERK et Akt sont augmentés dans les OCs MP, et réduits suite à l'inhibition de PDK1. La phosphorylation des substrats de mTOR, 4EBP1 et la protéine régulatrice Raptor, a été évaluée, et une augmentation des deux a été observée dans les OCs pagétiques, et est régulée par l'inhibition de PDK1. Également, l'augmentation des niveaux de base de LC3II (associée aux structures autophagiques) observée dans les OCs pagétiques a été associée à un défaut de dégradation des autophagosomes, indépendante de la mutation p62P392L. Il existe aussi une réduction de sensibilité à l’induction de l'autophagie dépendante de PDK1. De plus, l’inhibition de PDK1 induit l’apoptose autant dans les OCs contrôles que pagétiques, et mène à une réduction significative de la résorption osseuse. La signalisation PDK1/Akt pourrait donc représenter un point de contrôle important dans l’activation des OCs pagétiques. Ces résultats démontrent l’importance de plusieurs kinases associées à p62 dans la sur-activation des OCs pagétiques, dont la signalisation converge vers une augmentation de leur survie et de leur fonction de résorption, et affecte également le processus autophagique.
Abstract : Paget’s disease of bone (PDB) is a skeletal disorder characterized by focal and disorganized increases in bone turnover. In PDB, osteoclasts are larger, more active, more numerous, and resistant to apoptotic stimuli. While no single root cause has been identified, mutations to the gene encoding the p62 protein, SQSTM1, have been described in a significant population of patients with PDB. Among these mutations, the P392L substitution is the most prevalent, and overexpression of p62P392L in osteoclasts generates at least a partial pagetic phenotype in vitro. Normally this protein mediates a number of cell functions, from control of NF-κB signaling to autophagy. In human osteoclasts, a multiprotein complex containing p62 and protein kinases PKCζ and PDK1 (the principal kinase of Akt), form in response to stimulation by receptor activator of nuclear factor kappa-B ligand (RANKL), the principal osteoclastogenic-signaling cytokine. We found that PKCζ is involved in RANKL-induced activation of NF-κB, and that it contributed to a basal activation of NF-κB observed in p62P392L mutants. This may be regulated in part by a PKCζ dependent increase in p65 phosphorylation at Ser536 which we characterized, independent of IκB. This could represent one alternative pathway by which mutant p62 leads to increased NF-κB activation. We observed increased basal phosphorylation of survival regulators ERK and Akt in PDB that was reduced upon PDK1 inhibition. The activity of 4EBP1 and Raptor, associated with mTOR activity, were also altered in pagetic osteoclasts and regulated by PDK1 inhibition. We then identified autophagic defects common to pagetic osteoclasts; with higher basal levels of LC3II (associated with autophagic structures), regardless of p62 mutation, and reduced sensitivity to autophagy induction in PDB. These results suggest an accumulation of non-degradative autophagosomes. Inhibition of PDK1 not only induced apoptosis in PDB and controls, but significantly reduced resorption in PDB, and with regards to autophagy, PDK1 inhibition was more potent in PDB than in controls. Therefore PDK1/Akt signaling represents an important checkpoint to PDB osteoclast activation. In sum, these results demonstrate the importance of several p62-associated kinases in the over-activation of pagetic osteoclasts, through increased survival and altered signaling. As p62 mutations alone do not account for most cases of PDB, the characterization of these pathways may identify a common factor linking pagetic osteoclasts. Therefore these studies represent a novel approach to osteoclast apoptosis, activation, and autophagy associated with PDB.

Paget’s disease (PDB) is the second most common metabolic bone disease after osteoporosis, affecting up to 3% of adults over age 55. It is characterized by focal lesions of bone resorbed by hyperactive osteoclasts coupled with rapid formation of highly disorganized, low quality bone formed by osteoblasts. Such lesions cause skeletal deformity, fractures, and other symptoms that significantly decrease quality of life. In 2001, mutations in the SQSTM1/p62 gene were found in a subset of Paget’s patients. The work summarized in this dissertation sought to answer two broad questions: what is the function of p62 in normal bone homeostasis and how do PDB-associated mutations alter it? These studies took advantage of two mouse models: p62 knock-out (KO) mice, and p62P394L “knock-in” (KI) mice carrying the most common PDB-associated mutation. KO, KI, and wildtype (WT) controls were aged to one year for skeletal-histological characterization. No differences were observed in a variety of bone parameters between WT and KO bones, while bones from age-matched KI mice exhibited a 33% decrease in bone volume and a 25% increase in osteoclast formation. In vivo, TNF-α caused a potent induction of osteoclastogenesis in calvariae of WT and KI, but not KO, mice. In vitro, RANKL induced osteoclast formation in a dose-dependent manner in WT and KI, but not KO, cultures. Gene expression profiling of RANKL-treated osteoclast progenitors from WT, KO, and KI mice was then performed to identify the changes in signaling pathways responsible for these effects. Surprisingly, gene expression patterns from all three groups were consistent with robust activation of NFκB signaling in RANKL-treated samples, indicating that p62 is dispensable for RANKL activation of NFκB. Interestingly, gene expression patterns in KO cells suggested impaired proliferation and response to reactive oxygen species (ROS), a finding which was confirmed in cell culture experiments. In contrast, KI cells displayed enrichment for genes associated with the unfolded protein response, consistent with p62’s role in ubiquitin-mediated protein degradation via proteolysis and autophagy. These studies have therefore generated several novel hypotheses concerning the role of p62 in both normal bone homeostasis and Paget’s disease of bone.