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1
Hiruma, Yuko, Noriyoshi Kurihara, Diane F. Jelinek, and David Roodman. "Increased Signaling through p62 in the Marrow Microenvironment Increases Myeloma Cell Growth and Osteoclast Formation." Blood 112, no. 11 (November 16, 2008): 642. http://dx.doi.org/10.1182/blood.v112.11.642.642.
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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.
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Layfield, R., J. R. Cavey, D. Najat, J. Long, P. W. Sheppard, S. H. Ralston, and M. S. Searle. "p62 mutations, ubiquitin recognition and Paget's disease of bone." Biochemical Society Transactions 34, no. 5 (October 1, 2006): 735–37. http://dx.doi.org/10.1042/bst0340735.
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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.
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Ishizuka, Fumito, Jolene Windle, David Roodman, and Noriyoshi Kurihara. "p62 as a Therapeutic Target for Myeloma Cell Growth and Osteoclast Formation." Blood 114, no. 22 (November 20, 2009): 2857. http://dx.doi.org/10.1182/blood.v114.22.2857.2857.
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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.
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Layfield, Robert. "The molecular pathogenesis of Paget disease of bone." Expert Reviews in Molecular Medicine 9, no. 27 (September 2007): 1–13. http://dx.doi.org/10.1017/s1462399407000464.
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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.
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Sundaram, Kumaran, Srinivasan Shanmugarajan, D. Sudhaker Rao, and Sakamuri V. Reddy. "Mutant p62P392L Stimulation of Osteoclast Differentiation in Paget's Disease of Bone." Endocrinology 152, no. 11 (August 30, 2011): 4180–89. http://dx.doi.org/10.1210/en.2011-1225.
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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.
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Layfield, Robert, and Mark S. Searle. "Disruption of ubiquitin-mediated processes in diseases of the brain and bone." Biochemical Society Transactions 36, no. 3 (May 21, 2008): 469–71. http://dx.doi.org/10.1042/bst0360469.
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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.
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Hiruma, Yuko, Tadashi Honjo, Diane F. Jelinek, Jolene J. Windle, Jaekyoon Shin, G. David Roodman, and Noriyoshi Kurihara. "Increased signaling through p62 in the marrow microenvironment increases myeloma cell growth and osteoclast formation." Blood 113, no. 20 (May 14, 2009): 4894–902. http://dx.doi.org/10.1182/blood-2008-08-173948.
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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.
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Sultana, Melanie A., Carmel Cluning, Wai-Sin Kwong, Nicole Polain, Nathan J. Pavlos, Thomas Ratajczak, John P. Walsh, Jiake Xu, and Sarah L. Rea. "The SQSTM1/p62 UBA domain regulates Ajuba localisation, degradation and NF-κB signalling function." PLOS ONE 16, no. 11 (November 4, 2021): e0259556. http://dx.doi.org/10.1371/journal.pone.0259556.
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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.
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Teramachi, Jumpei, Kyaw Ze Yar Myint, Rentian Feng, Xiangqun Xie, Jolene J. Windle, David Roodman, and Noriyoshi Kurihara. "Blocking the ZZ Domain of Sequestosome 1/p62 Suppress the Enhancement of Myeloma Cell Growth and Osteoclast Formation by Marrow Stromal Cells." Blood 118, no. 21 (November 18, 2011): 888. http://dx.doi.org/10.1182/blood.v118.21.888.888.
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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.
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Kurihara, Noriyoshi, Tadashi Honjo, Jolene J. Windle, J. Shin, and G. D. Roodman. "Targeting p62ZIP in Marrow Stromal Cells Is Highly Effective at Inhibiting Myeloma Cell Growth and Osteoclast Formation." Blood 106, no. 11 (November 16, 2005): 630. http://dx.doi.org/10.1182/blood.v106.11.630.630.
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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.
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Kurihara, Noriyoshi, Y. Hiruma, J. Windle, C. S. Hong, J. Shin, and David Roodman. "Targeting p62 in Marrow Stromal Cells Is Effective at Inhibiting Myeloma Cell Growth." Blood 108, no. 11 (November 16, 2006): 513. http://dx.doi.org/10.1182/blood.v108.11.513.513.
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Abstract Release of growth factors from bone by osteoclastic bone destruction and by tumor-stromal cell interactions play critical roles in promoting myeloma cell growth. In particular, expression of the cytokines RANK ligand (RANKL), a potent inducer of osteoclast (OCL) formation, IL-6, tumor necrosis factor-α (TNF-α) and MCP-1 are upregulated in the bone microenvironment in response to myeloma (MM) cells as well as by adhesive interactions between myeloma cells and marrow stromal cells through VCAM-1 on stromal cells. Therefore, agents that can both inhibit OCL formation and block the effects of myeloma stromal cell interactions should have a major impact on both bone destruction and tumor growth. p62 plays a critical role in NF-κB activation induced by TNF-α, RANKL and IL-1 and is involved in multiple signaling pathways that result in enhanced tumor cell survival and bone destruction. It is our hypothesis that inhibiting p62 expression will profoundly diminish osteolytic bone destruction and myeloma growth in patients, by blocking production of RANKL, MCP-1, TNF-α and IL-6 in the tumor-bone microenvironment, and the upregulation of VCAM-1 on stromal cells. Therefore, we used p62 −/− mice to determine the effects of deleting p62 in stromal cells on the growth of myeloma cells. Marrow cells from p62 −/− or wild type mice were used to establish long-term Dexter-type marrow cultures to isolate marrow stromal cells. IL-6 and TNF-α production by p62 −/− stromal cells was decreased compared to WT stromal cells. To determine the effects of the lack of p62 on MM cell growth, GFP-labeled MM.1S cells were co-cultured with p62 −/− stromal cells. Growth of MM.1S cells was decreased by 70% in cocultures of p62 −/− mice, and IL-6 and TNF-α levels were not increased in cocultures of tumor cells with p62 −/− stromal cells. Next, we measured the relative expression levels of VCAM-1 on marrow stromal cells by Western blot in cocultures of human myeloma cells with marrow stromal cells. Stromal cells from the p62 −/− or wild type were cultured with and without MM.1S cells for 3 days in separate experiments. The levels of VCAM-1 in p62 −/− stromal cells were lower than p62 +/− stromal cells. In addition, VCAM-1 levels on p62 −/− bone marrow stromal cells were decreased compared to p62 +/− stromal cells when cocultured with MM cells. The addition of 25 ng/ml mouse TNF-α to p62 −/− stromal cells cocultured with MM.1S cells resulted in enhanced MM.1S growth and VCAM-1 production to similar levels as seen with p62 +/− stromal cells cocultured with MM.1S cells. We then determined the capacity of p62 −/− stromal cells to increase MCP-1 production, a chemoattract for myeloma cells, when they were cocultured with human myeloma cells for 48 hours. The conditioned media were collected after 48 hours of culture. Wild type stromal cells produced increased levels of MCP-1 when cocultured with MM.1S cells. MCP-1 levels in p62 −/− stromal cell conditioned media were decreased compared to wild type stromal cell cultures, regardless of whether MM.1S cells were present in the culture (MCP-1 in p62 +/− stromal cell culture, 980± 70pg/ml vs. p62 −/− 380± 10 pg/ml). These results show that p62 plays an important role in myeloma cell growth through regulation of production of cytokines that are upregulated in the marrow microenvironment in response to myeloma, and suggest that p62 is a novel target for treating myeloma.
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Teramachi, Jumpei, Jolene J. Windle, David Roodman, and Noriyoshi Kurihara. "The ZZ Domain of Sequestosome-1/p62 Plays An Important Role In Stromal Cell Support of Myeloma Cell Growth and Osteoclast Formation." Blood 116, no. 21 (November 19, 2010): 128. http://dx.doi.org/10.1182/blood.v116.21.128.128.
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Abstract Abstract 128 The marrow microenvironment provides a critical supportive role in myeloma (MM) and enhances both tumor growth and bone destruction through activation of multiple signaling pathways in stromal cells. We reported that sequestosome 1 (p62) plays a key role in the formation of signaling complexes that result in NF-kB, p38 MAPK, and PI3K activation in the marrow microenvironment of patients with MM. These results suggest that p62 is a potential therapeutic target for blocking the supportive effects of the marrow microenvironment in MM. 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, as a means to develop inhibitory peptides/molecules as potential therapeutic agents for MM. To pursue this objective, we generated deletion constructs of p62 that lacked specific p62 domains: ΔSH2, ΔPB1, ΔZZ, Δp38, ΔTBS and ΔUBA domains. We then transfected these constructs into a p62-knockout (KO) stromal cell-line we established from p62-KO mice and examined their RANKL, IL-6, and VCAM-1 expression induced by TNF-a. GFP-labeled MM1.S myeloma cells or normal CFU-GM, a source of OCL precursors, were then co-cultured with the p62-KO cells transduced with the different p62 deletion constructs and compared to wild type (WT) stromal cells. IL-6 production and VCAM-1 expression induced by TNF-a was 50% lower in non-transduced p62-KO stromal cells compared to WT stromal cells. Further, in contrast to WT cells, RANKL was not induced by TNF-a in the p62-KO stromal cell-line, and OCL formation in co-cultures of p62-KO stromal cells with CFU-GM was very low. Transduction of p62-KO stromal cells with the ΔSH2, ΔPB1, Δp38 and ΔUBA constructs restored stromal cell support of MM growth, VCAM-1 and IL-6 production. However, p62-KO stromal cells transduced with the ΔZZ construct did not increase MM cell growth, or increase IL-6 and VCAM-1 expression, or fully restore the capacity of the p62-KO stromal cells to support OCL formation. These results demonstrate that the ZZ domain of p62 is required for stromal cell support of MM cell growth, increased IL-6 and VCAM-1 expression, and OCL formation. These results suggest that dominant negative constructs or small molecules that target the ZZ domain of p62 should block p62 function and inhibit support of MM cells and OCL formation by the marrow microenvironment. Disclosures: Roodman: Amgen, Celgene, Acceleron & Millennium: Consultancy.
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Laurin, Nancy, Jacques P. Brown, Jean Morissette, and Vincent Raymond. "Recurrent Mutation of the Gene Encoding sequestosome 1 (SQSTM1/p62) in Paget Disease of Bone." American Journal of Human Genetics 70, no. 6 (June 2002): 1582–88. http://dx.doi.org/10.1086/340731.
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Hiruma, Yuko, Noriyoshi Kurihara, and David Roodman. "p62 Signaling Is Increased in Multiple Myeloma Microenvironment." Blood 110, no. 11 (November 16, 2007): 669. http://dx.doi.org/10.1182/blood.v110.11.669.669.
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Abstract The bone microenvironment plays a critical role in promoting both tumor growth and bone destruction in myeloma (MM). Marrow stromal cells produce factors, which stimulate both the growth of MM cells and osteoclastic bone destruction and are key regulators of these processes. Marrow stromal cells produce these factors in increased amounts when they bind MM cells through adhesive interactions mediated via VCAM-1 on stromal cells and β1 integrins on the surface of MM cells. We have examined the role of sequestosome-1 (p62), a recently described member of the NF-κB signaling pathway, in this process, since it sits at the crossroads of multiple signaling pathways potentially involved in both osteoclastogenesis and MM cell growth. In previous studies, we found that stromal cells lacking p62 minimally supported the growth of MM cells or osteoclast (OCL) formation compared to wild-type p62 containing stromal cells. We have further shown that stromal cells lacking p62 produce much lower levels of TNF-α, interleukin (IL)-6, MCP-1 and RANK ligand (RANKL), factors which increase OCL formation, and express lower levels of VCAM-1. However, the mechanisms responsible for this decreased cytokine production and VCAM-1 expression in multiple myeloma patient stromal cells lacking p62 are unknown. Further, it is unknown that targeting PKCζ, a downstream enzyme activity activated by p62, will have similar effects on myeloma patient stromal cells. Our hypotheses are that inhibiting PKCζ expression will block the effects of cytokines produced in the MM-bone microenvironment in response to TNF-α, decrease VCAM-1 expression in stromal cells, and markedly diminish osteolytic bone destruction and MM growth in MM patients. To test these hypotheses, we established long-term Dexter-type marrow cultures to isolate marrow stromal cells from MM patient and normals and screened for p62 and PKCζ/λ activation in MM marrow. We measured levels of PKCζ and p62 by Western blot analysis in marrow stromal cells from 13 patients and 11 healthy controls. We found significantly elevated levels of phosho-PKCζ/λ and total PKCζ in MM patients although the levels varied greatly among the patients. We next measured the relative expression levels of VCAM-1 on marrow stromal cells by Western blot in these marrow stromal cells. Since inhibiting p62 expression should profoundly diminish osteolytic bone destruction and myeloma growth in patients, by blocking production of RANKL, MCP-1, TNF-α and IL-6 in the tumor-bone microenvironment, and the upregulation of VCAM-1 on stromal cells, we assessed the effects of blocking p62 activity in human stromal cells. p62 siRNA (40 nM) was transduced into human stromal cells using a commercial transfection substrate. We confirmed that p62 expression was decreased by at least 50% in these stromal cells by Western blot analysis using an anti-p62 antibody. Stromal cells from the p62siRNA and control siRNA treatment and non-treatment stromal cells were cultured with or without MM1s cells for 3 days in separate experiments. The levels of VCAM-1 in p62siRNA transducer stromal cells were lower than control siRNA and untreated stromal cells. In addition, IL-6 production by MM1.s was increased when cocultured with control stromal cells but not with p62siRNA treated bone marrow stromal cells. These results show that p62 plays an important role in myeloma cell growth induced by cytokines that are upregulated in the marrow microenvironment and myeloma cells in patients with myeloma. Therefore, p62 is an attractive therapeutic target for MM growth and bone destruction.
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Liu, Chang, Yuan He, Xiaobing Xu, and Baorong He. "Phospholipase Cγ Signaling in Bone Marrow Stem Cell and Relevant Natural Compounds Therapy." Current Stem Cell Research & Therapy 15, no. 7 (October 14, 2020): 579–87. http://dx.doi.org/10.2174/1574888x14666191107103755.
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Excessive bone resorption has been recognized play a major role in the development of bone-related diseases such as osteoporosis, rheumatoid arthritis, Paget's disease of bone, and cancer. Phospholipase Cγ (PLCγ) family members PLCγ1 and PLCγ2 are critical regulators of signaling pathways downstream of growth factor receptors, integrins, and immune complexes and play a crucial role in osteoclast. Ca2+ signaling has been recognized as an essential pathway to the differentiation of osteoclasts. With growing attention and research about natural occurring compounds, the therapeutic use of natural active plant-derived products has been widely recognized in recent years. In this review, we summarized the recent research on PLCγ signaling in bone marrow stem cells and the use of several natural compounds that were proven to inhibit RANKL-mediated osteoclastogenesis via modulating PLCγ signaling pathways.
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Höppner, Jakob, Katja Steff, Felix Lobert, Christoph M. Heyer, Berthold P. Hauffa, and Corinna Grasemann. "Rhizomelia and Impaired Linear Growth in a Girl with Juvenile Paget Disease: The Natural History of the Condition." Hormone Research in Paediatrics 94, no. 3-4 (2021): 151–58. http://dx.doi.org/10.1159/000517164.
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In ultra-rare bone diseases, information on growth during childhood is sparse. Juvenile Paget disease (JPD) is an ultra-rare disease, characterized by loss of function of osteoprotegerin (OPG). OPG inhibits osteoclast activation via the receptor activator of nuclear factor-κB (RANK) pathway. In JPD, overactive osteoclasts result in inflammatory-like bone disease due to grossly elevated bone resorption. Knowledge on the natural history of JPD, including final height and growth, is limited. Most affected children receive long-term antiresorptive treatment, mostly with bisphosphonates, to contain bone resorption, which may affect growth. In this study, we report the follow-up of height, growth velocity, and skeletal maturation in a 16-year-old female patient with JPD. The patient was treated with cyclic doses of pamidronate starting at 2.5 years of age and with 2 doses of denosumab at the age of 8 years, when pamidronate was paused. In the following years, a sustainable decline in a height <i>z</i>-score and a stunted pubertal growth spurt; despite appropriate maturation of the epiphyseal plates of the left hand, the proximal right humerus and both femora were observed. Whether this reflects the growth pattern in JPD or might be associated to the antiresorptive treatments is unclear, since there is very limited information available on the effect of bisphosphonates and denosumab on growth and the growth plate in pediatric patients. Studies are needed to understand the natural history of an ultra-rare bone disease and to assess the effects of antiresorptive treatment on the growing skeleton.
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Mannino, Federica, and Post Doc. "ODP599 Modulation of Wnt/b-catenin and Autophagy in an in vitro Model of Glucocorticoid-induced Osteoporosis." Journal of the Endocrine Society 6, Supplement_1 (November 1, 2022): A186. http://dx.doi.org/10.1210/jendso/bvac150.384.
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Abstract Background During bone aging and osteoporosis, formation and resorption are not tightly coupled; the main cells involved in bone remodeling are osteoblasts and osteoclast but also osteocytes play a pivotal role in this process. Osteocytes are the most abundant cell type in bone and are mainly responsible for sensing mechanical signals on the bones, controlling osteoblast and osteoclast activities through cell-to-cell communication and via secreted factors. In particular, osteocytes regulate bone resorption, thanks to the production of RANKL, reducing osteoclast activity. Ellagic acid, a natural polyphenolic compound derived from pomegranate, could modulate cell function via specific estrogen receptor b; activation of ERb plays a critical role in bone remodeling suppressing osteoclast differentiation and function, promoting osteoblast proliferation through the Wnt/b-catenin pathway, increasing osteoprotegerin levels. In addition, stimulation of ERb plays an anti-inflammatory effect, increasing the expression of IL-10 and reducing the expression of proinflammatory cytokines, such as IL-1β and TNF-α, and can regulate the expression of autophagy inhibiting the PI3K/Akt/mTOR pathway. The hypothesis here tested is that ellagic acid, through ERb modulation, could induce Wnt/b-catenin and autophagy pathways in osteocytes challenged with dexamethasone to mimic glucocorticoid-induced osteoporosis. Materials and methods The osteocyte cells MLO-A5 were differentiated into osteocytes under appropriate culturing conditions. Cells were treated with Ellagic acid (1µM) following dexamethasone (1µM) challenge for 24h to induce an in vitro model of osteoporosis. At the end of the treatment period, cell viability was evaluated by MTT assay; qPCR was performed to evaluate the expression of the genes involved in osteocyte function (SOST, RANKL, Destrin and Dmp1) and Wnt/B-catenin signaling pathway (Wnt5a, Wnt10b, B-Catenin and DKK1); Western Blot was performed to evaluate the expression of proteins involved in apoptosis (cleaved-Caspase3) and autophagy (Beclin-1, LC3 and p62). In addition, the expression of Sclerostin and nuclear translocation of B-Catenin was evaluated by immunofluorescence. Results Ellagic acid reduced the gene expression of SOST, RANKL, Dmp1 and increased the expression of Destrin compared to untreated cells stimulated with dexamethasone; caused a significant increase of Wnt5a, Wnt10b and B-Catenin expression and reduced significantly the expression of DKK1. CGS21680 inhibited dexamethasone-induced apoptosis, reducing the expression of Caspase3, and increased the expression of Beclin-1 and LC3 compared to cell treated with dexamethasone. Finally, treatment with Ellagic acid stimulated the nuclear translocation of B-Catenin, promoting the transcription of genes involved in osteogenesis. Conclusion These preliminary data suggest that stimulation of ERb through Ellagic acid could modulate bone remodelling through activation of Wnt/b-catenin pathway and autophagy, providing evidence on the possible use of this natural compound as a new therapeutic approach for osteoporosis. Presentation: No date and time listed
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Silbermann, Rebecca, Wei Zhao, Julie L. Eiseman, Jan H. Beumer, Xiang-Qun Xie, Peng Yang, Kyaw-Zeyar Myint, et al. "A Novel Sequestosome-1/p62 ZZ Domain Inhibitor Induces New Bone Formation In The Presence Of Myeloma In Vivo." Blood 122, no. 21 (November 15, 2013): 684. http://dx.doi.org/10.1182/blood.v122.21.684.684.
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Abstract Multiple myeloma (MM) cells activate multiple signaling pathways in bone marrow stromal cells (BMSC), which results in enhanced tumor growth, increased osteoclast (OCL) formation and activity, and profound inhibition of bone formation by osteoblasts (OBL). We recently showed that sequestosome-1 (p62) acts as a signaling hub for these pathways, including MM-cell induced NFκβ, p38MAPK, and PI3K activation in MM patient BMSC, and that the p62 ZZ domain is required for BMSC enhancement of MM cell growth, IL-6 production, VCAM-1 expression and OCL formation. To target the ZZ domain we developed a small molecule inhibitor (CMPD3) that specifically blocked p62-ZZ domain mediated protein interactions required for BMSC support of tumor / OCL formation, but had no effect on CFU-blasts. We now report characterization of CMPD3 in an in vivomodel of MM. Murine MM cells (5TGM1-tk-gfp) were implanted intra-tibially in female SCID mice and male C57BL/KaLwRij mice and allowed to engraft for 2 weeks. Pilot PK studies using a multiple dose MTD of 60mg/kg IV in female SCID mice bearing MM cells demonstrated a terminal t1/2 of CMPD3 in plasma of greater than 5 hours (h), and extratibial tumor CMPD3 concentrations ranging between 5 and 10-fold higher than plasma. A pilot efficacy study in male C57BL/KaLwRij mice based on the multiple dose MTD demonstrated that mice treated with CMPD3 lost weight compared with vehicle treated mice. CMPD3 treatment did not alter activity scores of the mice or plasma IgG2b levels, a marker of tumor burden. Both compartmental and non-compartmental pharmacokinetic analysis of plasma concentrations of CMPD3 in the male C57BL/KaLwRij mice on the multiple dosing regime after doses of 27 mg/kg/day or 40 mg/kg/day ip resulted in a plasma terminal t1/2 of 10 -12 h and a clearance of 1188 ml/h/kg. An expanded efficacy study using CMPD3 doses of 27mg/kg/day or 40mg/kg/day, or vehicle ip for 5 days/week for 2 weeks demonstrated no change in body weights, tumor scores, or plasma IgG2b levels in any animals. Radiographic analysis of tibiae confirmed the presence of lytic bone lesions in all animals, and of 27 evaluable animals at the end of treatment, 6 CMPD3 treated and 2 vehicle treated mice developed grade 4 tumors (defined as tumor expanding outside the tibia and impairing the use of the limb). 4 of the 6 CMPD3 treated mice with grade 4 tumors and 1 of the 2 vehicle treated animals had radiographic evidence of new bone formation on the anterior tibial surface. µCT analysis of the tibiae from all CMPD3 vs. vehicle treated mice showed that the ratio of new bone volume to total bone volume (NBV/TBV) was increased in CMPD3 treated mice regardless of tumor score (1 - 4) (p=0.015). Stratification of NBV/TBV based on tumor score showed that CMPD3 treated animals with a tumor score of 4 had significantly more new bone formation than animals with tumor scores of 1 - 3 (p<0.01). NBV/TBV was not increased in the non-injected tibiae, indicating a lack of systemic effect on non tumor-bearing bone. Thus, CMPD3 induces new bone formation in bones bearing MM and suggest that p62 ZZ inhibition modulates tumor induced OBL suppression. Non-compartmental analysis of the maximal plasma concentration of CMPD3 at the 40mg/kg dose was 5.7µM, while the in vitro IC50 of CMPD3 for 5TGM1 cells was 4.35µM. This difference may explain why IgG2b levels did not change with CMPD3 treatment. In contrast, the IC50 in OCL formation assays was 0.1µM, suggesting that CMPD3’s inhibition of OCL formation in vivo may also contribute to increased bone. These findings demonstrate that CMPD3 can modulate tumor cell – bone cell interactions in MM in vivo, and support development of CMPD3 as a potential anabolic bone agent for MM. Disclosures: Roodman: Amgen: Consultancy; Eli Lilly and Co.: Research Funding.
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Borsani, Elisa, Veronica Bonazza, Barbara Buffoli, Pier Francesco Nocini, Massimo Albanese, Francesca Zotti, Francesco Inchingolo, Rita Rezzani, and Luigi F. Rodella. "Beneficial Effects of Concentrated Growth Factors and Resveratrol on Human Osteoblasts In Vitro Treated with Bisphosphonates." BioMed Research International 2018 (2018): 1–13. http://dx.doi.org/10.1155/2018/4597321.
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Bisphosphonates are primary pharmacological agents against osteoclast-mediated bone loss and widely used in the clinical practice for prevention and treatment of a variety of skeletal conditions, such as low bone density and osteogenesis imperfecta, and pathologies, such as osteoporosis, malignancies metastatic to bone, Paget disease of bone, multiple myeloma, and hypercalcemia of malignancy. However, long-term bisphosphonate treatment is associated with pathologic conditions including osteonecrosis of the jaw, named BRONJ, which impaired bone regeneration process. Clinical management of BRONJ is controversy and one recent approach is the use of platelet concentrates, such as Concentrated Growth Factors, alone or together with biomaterials or antioxidants molecules, such as resveratrol. The aim of the present study was to investigate the in vitro effects of Concentrated Growth Factors and/or resveratrol on the proliferation and differentiation of human osteoblasts, treated or not with bisphosphonates. Human osteoblasts were stimulated for 3 days in complete medium and for 21 days in mineralization medium. At the end of the experimental period, the in vitro effect on osteoblast proliferation and differentiation was evaluated using different techniques such as MTT, ELISA for the quantification/detection of osteoprotegerin and bone morphogenetic protein-2, immunohistochemistry for sirtuin 1 and collagen type I, and the Alizarin Red S staining for the rate of mineralization. Results obtained showed that Concentrated Growth Factors and/or resveratrol significantly increased osteoblast proliferation and differentiation and that the cotreatment with Concentrated Growth Factors and resveratrol had a protective role on osteoblasts treated with bisphosphonates. In conclusion, these data suggest that this approach could be promised in the clinical management of BRONJ.
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Silbermann, Rebecca, Dan Zhou, Jumpei Teramachi, Xiang-Qun Xie, G. David Roodman, and Noriyoshi Kurihara. "The p62-ZZ Domain Inhibitor XRK3F2 Alters Myeloma-Induced Suppression of Osteoblast Differentiation and Is Highly Cytotoxic to Myeloma Cells in Combination with Bortezomib." Blood 124, no. 21 (December 6, 2014): 2083. http://dx.doi.org/10.1182/blood.v124.21.2083.2083.
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Abstract Multiple myeloma (MM) bone disease results in increased osteoclastic bone destruction and irreversible osteoblast (OB) suppression. Cell-cell interactions between MM cells and bone marrow stromal cells (BMSC), which are OB precursors, activate signaling pathways in BMSC that result in enhanced MM cell growth, osteoclast formation, and inhibition of OB differentiation. OB inhibition results from suppression of key OB transcription factors, such as RUNX2, and induction of Gfi1, a transcriptional repressor of RUNX2. We reported that the ZZ domain of p62 (sequestosome-1) is required for BMSC support of MM cell growth and acts as a signaling hub for the formation of BMSC signaling complexes activated by MM cells and TNFα, resulting in NFκB, p38MAPK, and PI3K activation. We developed a small molecule inhibitor targeting the ZZ domain, XRK3F2 (previously CMPD3) that blocks p62-ZZ domain-mediated protein interactions, including TNFα-mediated signaling in BMSC. Further, we found that XRK3F2 was directly cytoxic to MM cells at high concentrations (5TGM1 murine MM cell IC50 4.35μM). Recently, we reported that administration of XRK3F2 two weeks after intratibial injection of 5TGM1 MM cells induces dramatic new cortical bone formation adjacent to MM cells in an immunocompetent in vivo model of MM bone disease. Additionally, bones from XRK3F2 treated animals not inoculated with tumor did not demonstrate new bone formation. This suggests that XRK3F2 changes the local effect of MM on bone at concentrations that were not sufficient for MM cell death. We now report that XRK3F2 alters MM-induced suppression of OB differentiation, and that the combination of XRK3F2 and bortezomib increases in vitroMM cell cytotoxicity. To determine if the dramatic bone formation induced by XRK3F2 resulted from reduced production of OB inhibitors by MM cells, enhanced BMSC differentiation to OB, or alteration of MM cell-BMSC interactions, we treated 5TGM1 cells, MC4 (murine stromal) cells, and 5TGM1-MC4 co-cultures with XRK3F2 and evaluated expression of RUNX2, Gfi1, and other key OB differentiation genes by qPCR. XRK3F2 treatment of 5TGM1 cells did not change 5TGM1 production of the OB inhibitors IL-7 or TNFα, and did not alter osteogenic differentiation of MC4 cells. However, XRK3F2 treatment of 5TGM1-MC4 co-cultures performed with a transwell insert increased MC4-derived RUNX2 expression 3-fold, as compared with vehicle treated co-cultures, and fully blocked the induction of Gfi1 in MC4 cells by MM cells (MC4-5TGM1 co-culture resulted in 12.4-fold increase in MC4 cell Gfi1 expression as compared with MC4 expression of Gfi1 without MM cell co-culture.) XRK3F2 treatment of MC4 cells also blocked TNFα-induced upregulation of Gfi1 (TNFα treatment increased MC4 cell Gfi1 expression 16.4-fold compared with control.) These results suggest that XRK3F2 blocks the TNFα-induced suppression of OB differentiation observed in MM cell-BMSC co-cultures through it’s effects on TNFα-mediated signaling facilitated by the p62-ZZ domain. To determine if we could further enhance the effects of XRK3F2 on bone formation and tumor burden, we evaluated if bortezomib, an anti-MM drug with potential bone anabolic effects, enhanced XRK3F2-induced MM cell cytotoxicity. Human MM cell lines were treated with combinations of XRK3F2 and bortezomib at concentrations below the IC50 of each drug for each cell line. H929, MM1.S, U266, ANBL6, and RPMI8226 cells were treated for 48 hours with XRK3F2, bortezomib (1nM), or the combination, and analyzed by MTT assay for viability. All cell lines demonstrated a highly significant decrease in viability in response to treatment with the XRK3F2-bortezomib combination as compared with either alone (p < 0.01). These results suggest that XRK3F2 or its derivatives, in combination with bortezomib or other proteasome inhibitors, should have a profound therapeutic effect on MM tumor burden and bone disease. Disclosures Silbermann: Amgen: Consultancy; Celgene: Research Funding. Roodman:Eli Lilly and Co.: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees.
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Teramachi, J., R. Silbermann, P. Yang, W. Zhao, K. S. Mohammad, J. Guo, J. L. Anderson, et al. "Blocking the ZZ domain of sequestosome1/p62 suppresses myeloma growth and osteoclast formation in vitro and induces dramatic bone formation in myeloma-bearing bones in vivo." Leukemia 30, no. 2 (August 19, 2015): 390–98. http://dx.doi.org/10.1038/leu.2015.229.
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Wang, Shumin, Eri O. Maruyama, John Martinez, Justin Lopes, Trunee Hsu, Wencheng Wu, Wei Hsu, and Takamitsu Maruyama. "MicroRNA-27a is essential for bone remodeling by modulating p62-mediated osteoclast signaling." eLife 12 (February 8, 2023). http://dx.doi.org/10.7554/elife.79768.
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The ability to simultaneously modulate a set of genes for lineage-specific development has made microRNA an ideal master regulator for organogenesis. However, most microRNA deletions do not exhibit obvious phenotypic defects possibly due to functional redundancy. MicroRNAs are known to regulate skeletal lineages as the loss of their maturation enzyme Dicer impairs bone remodeling processes. Therefore, it is important to identify specific microRNA essential for bone homeostasis. We report the loss of MIR27a causing severe osteoporosis in mice. MIR27a affects osteoclast-mediated bone resorption but not osteoblast-mediated bone formation during skeletal remodeling. Gene profiling and bioinformatics further identify the specific targets of MIR27a in osteoclast cells. MIR27a exerts its effects on osteoclast differentiation through modulation of Squstm1/p62 whose mutations have been linked to Paget's disease of bone. Our findings reveal a new MIR27a-p62 axis necessary and sufficient to mediate osteoclast differentiation and highlight a therapeutic implication for osteoporosis.
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Oh, Sue Young, Namju Kang, Jung Yun Kang, Ki Woo Kim, Jong-Hoon Choi, Yu-Mi Yang, and Dong Min Shin. "Sestrin2 Regulates Osteoclastogenesis via the p62-TRAF6 Interaction." Frontiers in Cell and Developmental Biology 9 (March 26, 2021). http://dx.doi.org/10.3389/fcell.2021.646803.
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The receptor activator of nuclear factor-kappa B ligand (RANKL) mediates osteoclast differentiation and functions by inducing Ca2+ oscillations, activating mitogen-activated protein kinases (MAPKs), and activating nuclear factor of activated T-cells type c1 (NFATc1) via the RANK and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) interaction. Reactive oxygen species (ROS) also plays an important role during osteoclastogenesis and Sestrin2, an antioxidant, maintains cellular homeostasis upon stress injury via regulation of ROS, autophagy, and inflammation. However, the role of Sestrin2 in osteoclastogenesis remains unknown. In this study, we investigated the role of Sestrin2 in the RANKL-RANK-TRAF6 signaling pathway during osteoclast differentiation. Deletion of Sestrin2 (Sesn2) increased bone mass and reduced the number of multinucleated osteoclasts on bone surfaces. RANKL-induced osteoclast differentiation and function decreased in Sesn2 knockout (KO) bone marrow-derived monocytes/macrophages (BMMs) due to inhibition of NFATc1 expression, but osteoblastogenesis was not affected. mRNA expression of RANKL-induced specific osteoclastogenic genes and MAPK protein expression were lower in Sesn2 KO BMMs than wild-type (WT) BMMs after RANKL treatment. However, the Sesn2 deletion did not affect ROS generation or intracellular Ca2+ oscillations during osteoclastogenesis. In contrast, the interaction between TRAF6 and p62 was reduced during osteoclasts differentiation in Sesn2 KO BMMs. The reduction in the TRAF6/p62 interaction and TRAP activity in osteoclastogenesis in Sesn2 KO BMMs was recovered to the WT level upon expression of Flag-Sesn2 in Sesn2 KO BMMs. These results suggest that Sestrin2 has a novel role in bone homeostasis and osteoclasts differentiation through regulation of NFATc1 and the TRAF6/p62 interaction.
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Qin, Huan, Wenwen Zhao, Yang Jiao, Haoyi Zheng, Hao Zhang, Jingyu Jin, Qiu Li, Xiuping Chen, Xia Gao, and Yantao Han. "Aqueous Extract of Salvia miltiorrhiza Bunge-Radix Puerariae Herb Pair Attenuates Osteoporosis in Ovariectomized Rats Through Suppressing Osteoclast Differentiation." Frontiers in Pharmacology 11 (January 21, 2021). http://dx.doi.org/10.3389/fphar.2020.581049.
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Traditional herb pair Salvia miltiorrhiza Bunge-Radix Puerariae (DG) owns various biological activities including anti-inflammatory and anti-oxidative stress. Oxidative stress is one high-risk factor for osteoporosis, then effect of DG on osteoporosis and underlying mechanisms was explored both in vivo and in vitro. Firstly, the predication from network pharmacology hinted that DG has the potential for ameliorating osteoporosis. Consistent with predication, DG significantly restored bone loss and deficiency of type II collagen, decreased TRAP and Cathepsin K positive areas in femur. Meanwhile it improved important characteristics of microarchitectural deterioration of tissue, reduced the numbers of NFATc1-positive osteoclast in the vertebra as well as decreased the serum osteoclast-specific cytokine RANKL and OPG release in OVX rats exhibiting its protective effect against osteoporosis. In vitro, DG noticeably decreased osteoclastic-special marker protein expressions of RANK, c-Fos and NFATc1. Furthermore, autophagy pathway p62/LC3B, ROS production and NF-κB were all activated by RANKL stimulation and blocked by DG pretreatment. Moreover, autophagy inhibitors, ROS scavenger, Ca2+ chelator and NF-κB inhibitor remarkably suppressed c-Fos and NFATc1 expressions. Taken together, DG may ameliorate osteoporosis by regulating osteoclast differentiation mediated by autophagy and oxidative stress. This study provided a mechanistic basis for DG treating osteoporosis and offered a safe dose for DG in preventing and improving bone diseases.
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Wen, Cailing, Yuheng Zhou, Yanting Xu, Huijing Tan, Caixia Pang, Haiqian Liu, Kaifei Liu, et al. "The Regulatory Role of GBF1 on Osteoclast Activation Through EIF2a Mediated ER Stress and Novel Marker FAM129A Induction." Frontiers in Cell and Developmental Biology 9 (August 25, 2021). http://dx.doi.org/10.3389/fcell.2021.706768.
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Bone-resorbing activities of osteoclasts (OCs) are highly dependent on actin cytoskeleton remodeling, plasma membrane reorganization, and vesicle trafficking pathways, which are partially regulated by ARF-GTPases. In the present study, the functional roles of Golgi brefeldin A resistance factor 1 (GBF1) are proposed. GBF1 is responsible for the activation of the ARFs family and vesicular transport at the endoplasmic reticulum–Golgi interface in different stages of OCs differentiation. In the early stage, GBF1 deficiency impaired OCs differentiation and was accompanied with OCs swelling and reduced formation of mature OCs, indicating that GBF1 participates in osteoclastogenesis. Using siRNA and the specific inhibitor GCA for GBF1 knockdown upregulated endoplasmic reticulum stress-associated signaling molecules, including BiP, p-PERK, p-EIF2α, and FAM129A, and promoted autophagic Beclin1, Atg7, p62, and LC3 axis, leading to apoptosis of OCs. The present data suggest that, by blocking COPI-mediated vesicular trafficking, GBF1 inhibition caused intense stress to the endoplasmic reticulum and excessive autophagy, eventually resulting in the apoptosis of mature OCs and impaired bone resorption function.
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Li, Jie, Xinle Li, Daquan Liu, Kazunori Hamamura, Qiaoqiao Wan, Sungsoo Na, Hiroki Yokota, and Ping Zhang. "eIF2α signaling regulates autophagy of osteoblasts and the development of osteoclasts in OVX mice." Cell Death & Disease 10, no. 12 (December 2019). http://dx.doi.org/10.1038/s41419-019-2159-z.
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AbstractBone loss in postmenopausal osteoporosis is induced chiefly by an imbalance of bone-forming osteoblasts and bone-resorbing osteoclasts. Salubrinal is a synthetic compound that inhibits de-phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). Phosphorylation of eIF2α alleviates endoplasmic reticulum (ER) stress, which may activate autophagy. We hypothesized that eIF2α signaling regulates bone homeostasis by promoting autophagy in osteoblasts and inhibiting osteoclast development. To test the hypothesis, we employed salubrinal to elevate the phosphorylation of eIF2α in an ovariectomized (OVX) mouse model and cell cultures. In the OVX model, salubrinal prevented abnormal expansion of rough ER and decreased the number of acidic vesiculars. It regulated ER stress-associated signaling molecules such as Bip, p-eIF2α, ATF4 and CHOP, and promoted autophagy of osteoblasts via regulation of eIF2α, Atg7, LC3, and p62. Salubrinal markedly alleviated OVX-induced symptoms such as reduction of bone mineral density and bone volume fraction. In primary bone-marrow-derived cells, salubrinal increased the differentiation of osteoblasts, and decreased the formation of osteoclasts by inhibiting nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). Live cell imaging and RNA interference demonstrated that suppression of osteoclastogenesis is in part mediated by Rac1 GTPase. Collectively, this study demonstrates that ER stress-autophagy axis plays an important role in OVX mice. Bone-forming osteoblasts are restored by maintaining phosphorylation of eIF2α, and bone-resorbing osteoclasts are regulated by inhibiting NFATc1 and Rac1 GTPase.
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Li, Mengye, Shengxiang Fu, Zhongyuan Cai, Danyang Li, Li Liu, Di Deng, Rongrong Jin, and Hua Ai. "Dual regulation of osteoclastogenesis and osteogenesis for osteoporosis therapy by iron oxide hydroxyapatite core/shell nanocomposites." Regenerative Biomaterials 8, no. 5 (August 9, 2021). http://dx.doi.org/10.1093/rb/rbab027.
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Abstract Osteoporosis is a skeletal disorder resulted in significant structural and functional changes, arousing a wide concern for the high prevalence and cost. Imbalance between osteoclastogenesis and osteogenesis have been verified as a main pathology etiology and considered an efficient therapy target in both clinical and pre-clinical studies. In recent years, inorganic nanomaterials have shown provable activities on osteoclastogenesis inhibition and osteogenesis promotion, respectively. Hence, in this study, a class of hydroxyapatite coated superparamagnetic iron oxide nanoparticles (SPIO@HA) were developed with a core−shell structure for targeting both osteoclastogenesis and osteogenesis. The optimal ratio of SPIO@15HA (Fe/Ca = 1:15, mol/mol) was screened to obtain dual function for inducing both bone formation and preventing bone resorption. The obtained nanocomposites significantly prevented the bone loss of ovariectomized (OVX) mice and increased bone mineral density (BMD) by 9.4%, exhibiting high bone accumulation in magnetic resonance imaging evaluation and reasonable biosafety profile. The mechanism study revealed that SPIO@15HA can suppress bone marrow monocyte derived osteoclast differentiation through TRAF6−p62−CYLD signaling complex regulation. Meanwhile, it could activate MSC osteogenic differentiation by TGF-β, PI3K-AKT and calcium signaling pathway regulation. Moreover, incubation of SPIO@15HA with MSC resulted in several cytokines overexpression such as osteoprotegerin (OPG), CSF2, CCL2 etc., which are responsible for maintaining the bone remodeling balance. The dual function of as-prepared SPIO@15HA may find a new way for designing of inorganic components containing core/shell nanomaterials for osteoporosis treatment.
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Cheng, Cheng, Lan Weiss, Henri Leinonen, Alyaa Shmara, Hong Z. Yin, Timothy Ton, Annie Do, et al. "VCP/p97 inhibitor CB-5083 modulates muscle pathology in a mouse model of VCP inclusion body myopathy." Journal of Translational Medicine 20, no. 1 (January 8, 2022). http://dx.doi.org/10.1186/s12967-021-03186-6.
Full textAbstract:
Abstract Background Pathogenic gain of function variants in Valosin-containing protein (VCP) cause a unique disease characterized by inclusion body myopathy with early-onset Paget disease of bone and frontotemporal dementia (also known as Multisystem proteinopathy (MSP)). Previous studies in drosophila models of VCP disease indicate treatment with VCP inhibitors mitigates disease pathology. Earlier-generation VCP inhibitors display off-target effects and relatively low therapeutic potency. New generation of VCP inhibitors needs to be evaluated in a mouse model of VCP disease. In this study, we tested the safety and efficacy of a novel and potent VCP inhibitor, CB-5083 using VCP patient-derived myoblast cells and an animal model of VCP disease. Methods First, we analyzed the effect of CB-5083 in patient-derived myoblasts on the typical disease autophagy and TDP-43 profile by Western blot. Next, we determined the maximum tolerated dosage of CB-5083 in mice and treated the 2-month-old VCPR155H/R155H mice for 5 months with 15 mg/kg CB-5083. We analyzed motor function monthly by Rotarod; and we assessed the end-point blood toxicology, and the muscle and brain pathology, including autophagy and TDP-43 profile, using Western blot and immunohistochemistry. We also treated 12-month-old VCPR155H/+ mice for 6 months and performed similar analysis. Finally, we assessed the potential side effects of CB-5083 on retinal function, using electroretinography in chronically treated VCPR155H/155H mice. Results In vitro analyses using patient-derived myoblasts confirmed that CB-5083 can modulate expression of the proteins in the autophagy pathways. We found that chronic CB-5083 treatment is well tolerated in the homozygous mice harboring patient-specific VCP variant, R155H, and can ameliorate the muscle pathology characteristic of the disease. VCP-associated pathology biomarkers, such as elevated TDP-43 and p62 levels, were significantly reduced. Finally, to address the potential adverse effect of CB-5083 on visual function observed in a previous oncology clinical trial, we analyzed retinal function in mice treated with moderate doses of CB-5083 for 5 months and documented the absence of permanent ocular toxicity. Conclusions Altogether, these findings suggest that long-term use of CB-5083 by moderate doses is safe and can improve VCP disease-associated muscle pathology. Our results provide translationally relevant evidence that VCP inhibitors could be beneficial in the treatment of VCP disease.