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Case Orthop J. Author manuscript; available in PMC 2016 January 22. Published in final edited form as: Case Orthop J. 2011 ; 8(1): 45–50.
THE COP9 SIGNALOSOME, A NOVEL, ESSENTIAL REGULATOR OF SKELETAL DEVELOPMENT AND TUMORIGENESIS Lindsay Bashur, Ph.D., Dongxing Chen, M.S., and Guang Zhou, Ph.D. Department of Orthopaedics, Case Western Reserve University, Cleveland, Ohio
Abstract Author Manuscript
In recent years, the evolutionarily conserved complex named COP9 signalosome has emerged as an essential and versatile regulator of biological and pathological responses across species. Various components of COP9 signalosome, including CSN5 (also named JAB1), play critical roles in a wide array of developmental process. Using genetically engineered mouse models, our laboratory has recently identified CSN5/JAB1 as an essential regulator of skeletal development, in part through negatively regulating BMP signaling. BMP signaling is important for fracture healing and bone regeneration. BMPs have been used for treating acute fractures and for improving the success of spinal fusions. Therefore, harnessing the CSN5/JAB1-mediated inhibition of BMP signaling might be a novel strategy to improve the efficacy of BMP treatment and to reduce medical costs in the future. Furthermore, the up-regulation of COP9 signalosome has been associated with various cancers, including osteosarcoma. Thus, the COP9 signalosome can also be a valid target for treating osteosarcoma and other tumors.
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I. The structure and general function of COP9 signalosome
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The COP9 signalosome (CSN) complex is an evolutionarily conserved proteolysis regulator throughout eukaryotic organisms1. The CSN complex consists of eight subunits in higher eukaryotes. Based on their sizes, the CSN subunits were named from the largest, CSN1, to the smallest, CSN8. The CSN complex is critical to the development of Arabidopsis, S. cerevisiae, Drosophila, C. elegans, and Mus musculus1, 2. The mammalian CSN is involved in many biological processes, including embryonic development, cell cycle progression, organogenesis, and signal transduction3. The major functions of mammalian CSN can be attributed to: regulation of protein stability, regulation of gene transcription, and impact on intracellular distribution of proteins3. The most studied function of the CSN has been its regulation of protein degradation. The CSN controls the stability of downstream proteins by regulating the activity of a subgroup of E3 ubiquitin complexes, cullin-RING ligase (CRL) family (Figure 1)3, 4. Within the ubiquitinproteasome pathway, the CRL plays a critical role by controlling the degradation of cell-cycle regulators and transcriptional regulators1, 2. Through its deneddylation activity, the cleavage of Nedd8 (a small ubiquitin-like molecular) from the CRL, the CSN modulates the homeostasis of the CRL and in turn affects the stability and activity of downstream proteins (Figure 1)3, 4.
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II. Consequences of the dysregulation of various CSNs in the mouse and the human
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The essential function of the COP9 signalosome during mammalian development is best illustrated by the severe phenotypes in various Csn-deficient mouse models (Table 1). The constitutive deletion (“knockout”) of each individual subunit of the COP9 signalosome – including Csn2, Csn3, Csn5 (also named Jab1, see below), Csn6, and Csn8 – all resulted in early embryonic lethality in mice by embryonic (E) day 8.5. This underscores the nonredundant functions of different Csn subunits in early mouse embryogenesis5-9. The Csndeficient mouse embryos usually exhibited massive cell death, dys-regulated cell cycle, reduced proliferation, and altered gene expressions (Table 1). Within the CSN complex, Csn5/Jab1 plays critical roles both in general embryogenesis and in the differentiation of specific organs and tissues. When Csn5/Jab1 was deleted in the whole mouse embryos, mutant embryos were severely growth-retarded, disorganized, deficient in DNA damage repair, and started to be absorbed as early as E6.0. No viable Csn5/Jab1-deficient embryos were recovered at E8.57, 8. The T-cell specific deletion of Csn5/Jab1 in mice leads to defective T cell development at multiple stages with altered turnover of selected CSNcontrolled substrates, such as p53 (Table 1)10. The myeloidspecific deletion of Csn5/Jab1 showed that Csn5/Jab1 is involved in innate immune responses in macrophages in mice (Table 1)11. Thus, the loss-of-function study demonstrated that Csn5/Jab1 controls proliferation, survival, and function of different cell types in postnatal life. On the other hand, in a transgenic mouse model overexpressing Csn5/Jab1 under the control of a constitutive promoter, hematopoietic cells from the bone marrow of Csn5/Jab1 transgenic mice had a significantly larger stem cell population and exhibited higher and transplantable proliferative potential12. In general, the dosages of the CSN complex components need to be tightly controlled to ensure proper mammalian development and to prevent diseases such as cancers (Table 1, also see below) 3, 13.
III. CSN5/JAB1 and its interacting network
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Among the COP9 signalosome subunits, CSN5/JAB1 has been the most intensively studied one, largely due to its impressive capability of interacting with a wide array of proteins (Figure 2). JAB1, Jun activation domain-binding protein 1, was originally identified as a coactivator of c-Jun-containing AP1 complex14. JAB1 was later found to be identical to CSN5, as the 5th subunit and an essential part of the COP9 signalosome1. Like other CSN subunits, CSN5/JAB1 is highly conserved with over 60% identity between animal and plant counterparts13. Within the COP9 complex, CSN5/JAB1 is the only subunit that harbors a JAMM domain as the catalytic center to deconjugates Nedd8 modification from CRL (Figure 1)4. As described above, CSN5/JAB1 plays essential roles during mouse development. In both human cell lines and mouse embryos, the deletion of CSN5/JAB1 results in COP9 signalosome instability and the decay of various CSN components3. CSN5/JAB1 plays important roles in cell cycle, proliferation, apoptosis, DNA repair, angiogenesis, and signal transduction by modulating the stability and activity of numerous transcription factors (Figure 2)4, 15. Case Orthop J. Author manuscript; available in PMC 2016 January 22.
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CSN5/JAB1 can regulate the cytoplasm/ nuclear translocation, protein degradation, and DNA-binding activity of numerous transcription factors1, 2. As a potent transcriptional coregulator, CSN5/JAB1 can stabilize and promote the activity of some factors, including AP-1, HIF-1α, Rad51, and MDM2 (Figure 2). On the other hand, CSN5/JAB1 can stimulate the degradation and repress the function of various factors, such as p27, p53, Smad 4, Smad5, and Smad7 (Figure 2). However, it’s worthy noting that most of CSN5/JAB1 interaction studies so far have been limited to in vitro system or cell cultures. Therefore, for the majority of reported CSN5/JAB1 interactions, their physiological relevance during development and pathogenesis in vivo is still largely unknown.
IV. CSN5/JAB1 function in skeletogenesis and its effect on TGF-β/BMP signaling Author Manuscript Author Manuscript
During skeletogenesis, chondrocyte and osteoblast differentiation is governed by complex signal transduction pathways, including the transforming growth factor-β (TGF-β) superfamily16. Increasing evidence has demonstrated that CSN5/JAB1 might be a potent regulator of the TGF-β superfamily13. In humans, the TGF-β superfamily contains over 35 family members, including TGF-βs and bone morphogenic proteins (BMP)17, 18. They regulate proliferation, apoptosis, adhesion, migration, and a variety of developmental processes including bone and cartilage formation. Upon ligand binding, the receptors phosphorylate the receptor-regulated Smad mediators (R-Smads) Smads 2 and 3 for TGF-βs; and Smads 1, 5, and 8 for BMPsβ. The phosphorylated R-Smad proteins bind a common Smad, Smad4, to activate specific downstream target genes [20]. Another class of Smads, inhibitory Smads (I-Smads)--namely Smad6 and Smad7--negatively regulates TGF-β/BMP signaling by interfering with the activation of R-Smads. Mutations in TGF-β superfamily signaling components, such as the ligand GDF5, the type I BMP receptor BMPR1B, and the antagonist Noggin, all lead to severe skeletal developmental defects in humans, such as brachydactyly and chondrodysplasia, resulting in shortening of limbs and loss of joints and/or digits as identified in diseases21. A recurrent mutation in the BBMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva, a rare autosomal dominant disorder of skeletal malformations and progressive extraskeletal ossification22. Thus, the duration and intensity of TGF-β/BMP signaling has to be tightly controlled to ensure proper skeleton formation.
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Csn5/Jab1 can modulate TGF-β superfamily signaling that is mediated by TGF-β and BMP through direct interaction with various Smads. Csn5/Jab1 binds Smad4, induces degradation, and inhibits TGF-β-induced gene transcription23. On the other hand, Csn5/ Jab1 can also associate constitutively with inhibitory Smad7 to promote its degradation, leading to enhanced TGFβ-mediated transcriptional activity24. These conflicting results likely reflect the complex role of Csn5/Jab1 in TGF-β signaling. Additionally, Csn5/Jab1 can interact with Smad5 to negatively regulate BMP signaling25. Overexpression of Csn5/Jab1 resulted in an attenuation of BMP-dependent transcriptional response in vitro, suggesting that Csn5/ Jab1 acts as an inhibitor of BMP signaling25. However, the physiological relevance of various Csn5/Jab1-Smad interactions during development remains mostly unknown.
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Although COP9 signalosome is critical for many cellular functions, its role in skeletogenesis is poorly understood due to the early embryonic lethality of various Csn subunits (Table 1). So far, only the CSN5/JAB1 function has been studied in bone and cartilage. In humans, CSN5/JAB1 is expressed in proliferating chondrocytes and osteoblasts25, 26. Using various genetically engineered mouse models, our laboratory has recently identified Csn5/Jab1 as a novel and essential regulator of successive steps of mouse skeletal development in vivo. Our results show that the limbspecific deletion of Csn5/Jab1 in mice led to severely shortened limbs at birth, strikingly mirroring the human birth defect phocomelia27. The ablation of Csn5/Jab1 specifically in differentiating chondrocytes leads to severe and generalized lethal chondrodysplasis (Figure 3). The Csn5/Jab1 mutants exhibited disorganized chondrocyte columns with increased apoptosis. Importantly, the Csn5/Jab1-deficient chondrocytes exhibited increased BMP signaling28. Thus, CSN5/JAB1 is required for normal skeletal development, likely in part by its negative effect on BMP signaling. BMP signaling has been shown to be critically important for all aspects of skeletal formation and regeneration, including fracture healing and articular cartilage repair29-33. BMP2- and BMP7-containing osteogenic implants have been used for bone regeneration worldwide for long-bone nonunions and acute fractures, and for improving the success of spinal fusions. Therefore, novel therapies based on controlling the CSN5/JAB1-mediated inhibition of BMP signaling might be able to improve the efficacy of BMP treatment in the future.
V. CSN5/JAB1 amplification in cancer cells
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So far the main research focus of CSN5/JAB1 has been its potential role in tumorigenesis. The detection of CSN5/JAB1 overexpression in a variety of human tumors suggests that it might be a significant contributor to cancer initiation and progression (Table 1)13. The role of CSN5/JAB1 in the pathogenesis of several cancer types might be linked to its effect on p27, a cell cycle inhibitor and tumor suppressor34. Low expression of p27 has been associated with excessive cell proliferation in many types of human tumors, including osteosarcoma13, 35. CSN5/JAB1 can bind p27 and facilitate p27 degradation, thus promoting cell cycle progression and proliferation (Figure 2)36. CSN5/JAB1 expression was low or absent in normal adult breast tissue, but was increased in 50% of primary breast tumors and 90% of metastatic lesions37. CSN5/JAB1 over-expression was also correlated with reduced levels of p27 and poor prognosis of breast cancer38. In addition to p27, the other targets of CSN5/JAB1 known to be involved in tumorigenesis include p53, MDM2, HIF-1α, Smad4, and Smad7 (Figure 2)13. Additionally, a genetic linkage study demonstrates that the coordinated amplification of CSN5/JAB1 and oncogene MYC induces a poor-prognosis expression pattern of 512 genes in breast cancer39. However, the causative role of CSN5/ JAB1 in tumorigenesis remains to be established in a whole animal system. Osteosarcoma, the primary malignancy of bone cells, most commonly affects rapidly growing bones in teenagers and also affects elderly individuals who have rapid bone turnover due to conditions such as Paget’s disease40. Osteosarcoma is the 5th most frequent malignancy in 15-to 19-year-olds, and a deeper understanding of the pathogenesis of osteosarcoma is critical for better diagnosis and treatment of these patients. CSN5/JAB is involved in DNA damage repair (Figure 2) and the down-regulation of CSN5/JAB1 in osteosarcoma cells rendered them more sensitive to γ-irradiation, suggesting that CSN5/ Case Orthop J. Author manuscript; available in PMC 2016 January 22.
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JAB1 can be a potential target in osteosarcoma treatment8. However, the role of CSN5/JAB1 in osteosarcoma pathogenesis is still largely unknown. The amplification of another COP9 signalosome subunit, CSN3, was significantly correlated with the large tumor size of osteosarcoma 41, and knockdown of CSN3 repressed the proliferation and metastasis of osteosarcoma cells in vitro42. However, the role of CSN3 in osteosarcoma pathogenesis in vivo remains to be determined.
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Recent mouse genetics studies demonstrated that p53 is the determining factor in the pathogenesis of osteosarcoma, while another tumor suppressor, Rb, serves an important supporting role43, 44. p53 is the most frequently mutated gene in human cancers, including osteosarcoma13, 45. Since the CSN is a potent inhibitor of p53 function13, a better understanding of the regulation of p53 by CSN5/JAB1 and other subunits of the CSN in bone might help us to improve osteosarcoma diagnosis and treatment. In various human cancer cell lines, the knockdown of CSN5/JAB1 inhibited the proliferation of tumor cells, suggesting that over-expression of CSN5/JAB1 not only serves as a marker of malignant transformation, but also actually contributes to tumorigenesis13. CSN5/JAB1 has been proposed as a candidate target for therapeutic intervention of various cancers46. It will be of great clinical relevance to determine whether CSN5/JAB1 itself can be a valid target of cancer treatment through future screening and characterization of effective CSN5/JAB1specific small molecular inhibitors.
V. Future directions of the COP9 studies
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In the past decade, our understanding of the role of COP9 signalosome has greatly expanded. The COP9 signalosome is essential for embryogenesis and the functions of a diverse group of cells, including skeletal cells. Overall, in progenitor cell populations, the CSN appears to maintain those cells in an undifferentiated state and to keep them proliferating. In differentiating cells, the CSN is required for proper cell cycle progression and cell survival. The CSN, especially CSN5/JAB1, likely also contributes to tumorigenesis by modulating the stability and activity of numerous targets. The dosages of various CSN subunits appear to be critical for the COP9 signalosome functions and dys-regulated CSN expression is associated with developmental defects and cancers in mice and humans. The CSN function under normal development and in pathological conditions is likely to be celltype specific and contextdependent. Despite recent progress, several important questions of the CSN remain unanswered: what is the underlying mechanism of CSN5/JAB1 function during the successive steps of skeletogenesis? What is the precise mechanism of CSNmediated cell death? How is the CSN itself regulated? Is CSN abnormality associated with any human birth defect? What might be the role of CSN5/JAB1 in the pathogenesis of osteosarcoma? Answers to these critical questions will provide further insights into the COP9 signalosome function and facilitate the development of novel therapeutic options to treat human diseases, including osteosarcoma.
Acknowledgements Work in Dr. Guang Zhou’s laboratory was supported by a NIH grant (R03DE19190). We thank Valerie Schmedlen for editorial assistance.
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Figure 1. Structural organization of COP9 signalosome
Lightning symbol represents Csn5/Jab1 enzymatic activity. Abbreviations: CRL, cullinRING ligase; N8, Nedd8 (neural precursor cell-expressed developmentally downregulated-8).
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Figure 2. Functions and targets of JAB1/CSN5
Plus (+) signs indicate JAB1/CSN5-mediated positive effect; Minus (−) signs indicate JAB1/ CSN5-mediated negative effect. Abbreviations: AP-1, activator protein 1; BMP, bone morphogenetic protein; HIF-1a, hypoxia inducible factor-1a; MDM2, murine double minute 2; TGF-β, transforming growth factor-β.
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Severe and generalized chondrodysplasis of the mutant mice in which Csn5/Jab1, an integral part of COP signalosome, was specifically ablated in chondrocytes.
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Table 1
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Consequences of dys-regulated CSN subunits in the mouse and the human.
Author Manuscript Author Manuscript
Gene
Alteration
Phenotypes
References
Csn2 mice
KO
Embryonic lethality by E7.5; Deficient cell proliferation; ↑p53, p21, and cyclin E
[5]
Csn3 mice
KO
Embryonic lethality by E8.5; ↑Apoptosis; ↓Csn8 expression
[6]
CSN3 human
Overexpression
CSN3 amplification significantly correlated with large tumor size of osteosarcoma
[41]
Csn5/Jab1 mice
KO
Embryonic lethality by E8.5; Impaired proliferation; ↑DNA damage and apoptosis; ↓p53, p27, and cyclin E
[7], [8]
Csn5/Jab1 mice
Overexpression driven by a constitutive CAG promoter
↑Proliferation and maintenance of hematopoietic progenitors
[12]
Csn5/Jab1 mice
cKO in chondrocytes
Neonatal lethal chondrodysplasia with disorganized chondrocytes; ↑Apoptosis
[28]
Csn5/Jab1 mice
cKO in T cells
Impaired T cell development; ↑Apoptosis; ↓p53, IkB-a, and b-catenin
[10]
Csn5/Jab1 mice
cKO in myeloid cells
↓Mortality in polymicrobial sepsis; ↓TLR-and TNF-a-mediated MAPK signaling in macrophage; ↑Antioxidation genes
[11]
Csn5/Jab1 human
Overexpression
CSN5 amplification associated with poor outcomes in breast cancer, lung cancer, and pancreatic adenocarcinoma
[13]
Csn6 mice
KO
Embryonic lethality by E7.5; ↑p53 activity
[9]
CSN6 human
Overexpression
Upregulated CSN6 expression significantly correlated with enhanced expression of MDM2, a p53 inhibitor, in breast and thyroid tumors
[9]
Csn8 mice
KO
Embryonic lethality by E7.5
[47]
KO: constitutive knockout; cKO: conditional knockout.
Author Manuscript Case Orthop J. Author manuscript; available in PMC 2016 January 22.
Case Orthop J. Author manuscript; available in PMC 2016 January 22. Published in final edited form as: Case Orthop J. 2011 ; 8(1): 45–50.
THE COP9 SIGNALOSOME, A NOVEL, ESSENTIAL REGULATOR OF SKELETAL DEVELOPMENT AND TUMORIGENESIS Lindsay Bashur, Ph.D., Dongxing Chen, M.S., and Guang Zhou, Ph.D. Department of Orthopaedics, Case Western Reserve University, Cleveland, Ohio
Abstract Author Manuscript
In recent years, the evolutionarily conserved complex named COP9 signalosome has emerged as an essential and versatile regulator of biological and pathological responses across species. Various components of COP9 signalosome, including CSN5 (also named JAB1), play critical roles in a wide array of developmental process. Using genetically engineered mouse models, our laboratory has recently identified CSN5/JAB1 as an essential regulator of skeletal development, in part through negatively regulating BMP signaling. BMP signaling is important for fracture healing and bone regeneration. BMPs have been used for treating acute fractures and for improving the success of spinal fusions. Therefore, harnessing the CSN5/JAB1-mediated inhibition of BMP signaling might be a novel strategy to improve the efficacy of BMP treatment and to reduce medical costs in the future. Furthermore, the up-regulation of COP9 signalosome has been associated with various cancers, including osteosarcoma. Thus, the COP9 signalosome can also be a valid target for treating osteosarcoma and other tumors.
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I. The structure and general function of COP9 signalosome
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The COP9 signalosome (CSN) complex is an evolutionarily conserved proteolysis regulator throughout eukaryotic organisms1. The CSN complex consists of eight subunits in higher eukaryotes. Based on their sizes, the CSN subunits were named from the largest, CSN1, to the smallest, CSN8. The CSN complex is critical to the development of Arabidopsis, S. cerevisiae, Drosophila, C. elegans, and Mus musculus1, 2. The mammalian CSN is involved in many biological processes, including embryonic development, cell cycle progression, organogenesis, and signal transduction3. The major functions of mammalian CSN can be attributed to: regulation of protein stability, regulation of gene transcription, and impact on intracellular distribution of proteins3. The most studied function of the CSN has been its regulation of protein degradation. The CSN controls the stability of downstream proteins by regulating the activity of a subgroup of E3 ubiquitin complexes, cullin-RING ligase (CRL) family (Figure 1)3, 4. Within the ubiquitinproteasome pathway, the CRL plays a critical role by controlling the degradation of cell-cycle regulators and transcriptional regulators1, 2. Through its deneddylation activity, the cleavage of Nedd8 (a small ubiquitin-like molecular) from the CRL, the CSN modulates the homeostasis of the CRL and in turn affects the stability and activity of downstream proteins (Figure 1)3, 4.
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II. Consequences of the dysregulation of various CSNs in the mouse and the human
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The essential function of the COP9 signalosome during mammalian development is best illustrated by the severe phenotypes in various Csn-deficient mouse models (Table 1). The constitutive deletion (“knockout”) of each individual subunit of the COP9 signalosome – including Csn2, Csn3, Csn5 (also named Jab1, see below), Csn6, and Csn8 – all resulted in early embryonic lethality in mice by embryonic (E) day 8.5. This underscores the nonredundant functions of different Csn subunits in early mouse embryogenesis5-9. The Csndeficient mouse embryos usually exhibited massive cell death, dys-regulated cell cycle, reduced proliferation, and altered gene expressions (Table 1). Within the CSN complex, Csn5/Jab1 plays critical roles both in general embryogenesis and in the differentiation of specific organs and tissues. When Csn5/Jab1 was deleted in the whole mouse embryos, mutant embryos were severely growth-retarded, disorganized, deficient in DNA damage repair, and started to be absorbed as early as E6.0. No viable Csn5/Jab1-deficient embryos were recovered at E8.57, 8. The T-cell specific deletion of Csn5/Jab1 in mice leads to defective T cell development at multiple stages with altered turnover of selected CSNcontrolled substrates, such as p53 (Table 1)10. The myeloidspecific deletion of Csn5/Jab1 showed that Csn5/Jab1 is involved in innate immune responses in macrophages in mice (Table 1)11. Thus, the loss-of-function study demonstrated that Csn5/Jab1 controls proliferation, survival, and function of different cell types in postnatal life. On the other hand, in a transgenic mouse model overexpressing Csn5/Jab1 under the control of a constitutive promoter, hematopoietic cells from the bone marrow of Csn5/Jab1 transgenic mice had a significantly larger stem cell population and exhibited higher and transplantable proliferative potential12. In general, the dosages of the CSN complex components need to be tightly controlled to ensure proper mammalian development and to prevent diseases such as cancers (Table 1, also see below) 3, 13.
III. CSN5/JAB1 and its interacting network
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Among the COP9 signalosome subunits, CSN5/JAB1 has been the most intensively studied one, largely due to its impressive capability of interacting with a wide array of proteins (Figure 2). JAB1, Jun activation domain-binding protein 1, was originally identified as a coactivator of c-Jun-containing AP1 complex14. JAB1 was later found to be identical to CSN5, as the 5th subunit and an essential part of the COP9 signalosome1. Like other CSN subunits, CSN5/JAB1 is highly conserved with over 60% identity between animal and plant counterparts13. Within the COP9 complex, CSN5/JAB1 is the only subunit that harbors a JAMM domain as the catalytic center to deconjugates Nedd8 modification from CRL (Figure 1)4. As described above, CSN5/JAB1 plays essential roles during mouse development. In both human cell lines and mouse embryos, the deletion of CSN5/JAB1 results in COP9 signalosome instability and the decay of various CSN components3. CSN5/JAB1 plays important roles in cell cycle, proliferation, apoptosis, DNA repair, angiogenesis, and signal transduction by modulating the stability and activity of numerous transcription factors (Figure 2)4, 15. Case Orthop J. Author manuscript; available in PMC 2016 January 22.
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CSN5/JAB1 can regulate the cytoplasm/ nuclear translocation, protein degradation, and DNA-binding activity of numerous transcription factors1, 2. As a potent transcriptional coregulator, CSN5/JAB1 can stabilize and promote the activity of some factors, including AP-1, HIF-1α, Rad51, and MDM2 (Figure 2). On the other hand, CSN5/JAB1 can stimulate the degradation and repress the function of various factors, such as p27, p53, Smad 4, Smad5, and Smad7 (Figure 2). However, it’s worthy noting that most of CSN5/JAB1 interaction studies so far have been limited to in vitro system or cell cultures. Therefore, for the majority of reported CSN5/JAB1 interactions, their physiological relevance during development and pathogenesis in vivo is still largely unknown.
IV. CSN5/JAB1 function in skeletogenesis and its effect on TGF-β/BMP signaling Author Manuscript Author Manuscript
During skeletogenesis, chondrocyte and osteoblast differentiation is governed by complex signal transduction pathways, including the transforming growth factor-β (TGF-β) superfamily16. Increasing evidence has demonstrated that CSN5/JAB1 might be a potent regulator of the TGF-β superfamily13. In humans, the TGF-β superfamily contains over 35 family members, including TGF-βs and bone morphogenic proteins (BMP)17, 18. They regulate proliferation, apoptosis, adhesion, migration, and a variety of developmental processes including bone and cartilage formation. Upon ligand binding, the receptors phosphorylate the receptor-regulated Smad mediators (R-Smads) Smads 2 and 3 for TGF-βs; and Smads 1, 5, and 8 for BMPsβ. The phosphorylated R-Smad proteins bind a common Smad, Smad4, to activate specific downstream target genes [20]. Another class of Smads, inhibitory Smads (I-Smads)--namely Smad6 and Smad7--negatively regulates TGF-β/BMP signaling by interfering with the activation of R-Smads. Mutations in TGF-β superfamily signaling components, such as the ligand GDF5, the type I BMP receptor BMPR1B, and the antagonist Noggin, all lead to severe skeletal developmental defects in humans, such as brachydactyly and chondrodysplasia, resulting in shortening of limbs and loss of joints and/or digits as identified in diseases21. A recurrent mutation in the BBMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva, a rare autosomal dominant disorder of skeletal malformations and progressive extraskeletal ossification22. Thus, the duration and intensity of TGF-β/BMP signaling has to be tightly controlled to ensure proper skeleton formation.
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Csn5/Jab1 can modulate TGF-β superfamily signaling that is mediated by TGF-β and BMP through direct interaction with various Smads. Csn5/Jab1 binds Smad4, induces degradation, and inhibits TGF-β-induced gene transcription23. On the other hand, Csn5/ Jab1 can also associate constitutively with inhibitory Smad7 to promote its degradation, leading to enhanced TGFβ-mediated transcriptional activity24. These conflicting results likely reflect the complex role of Csn5/Jab1 in TGF-β signaling. Additionally, Csn5/Jab1 can interact with Smad5 to negatively regulate BMP signaling25. Overexpression of Csn5/Jab1 resulted in an attenuation of BMP-dependent transcriptional response in vitro, suggesting that Csn5/ Jab1 acts as an inhibitor of BMP signaling25. However, the physiological relevance of various Csn5/Jab1-Smad interactions during development remains mostly unknown.
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Although COP9 signalosome is critical for many cellular functions, its role in skeletogenesis is poorly understood due to the early embryonic lethality of various Csn subunits (Table 1). So far, only the CSN5/JAB1 function has been studied in bone and cartilage. In humans, CSN5/JAB1 is expressed in proliferating chondrocytes and osteoblasts25, 26. Using various genetically engineered mouse models, our laboratory has recently identified Csn5/Jab1 as a novel and essential regulator of successive steps of mouse skeletal development in vivo. Our results show that the limbspecific deletion of Csn5/Jab1 in mice led to severely shortened limbs at birth, strikingly mirroring the human birth defect phocomelia27. The ablation of Csn5/Jab1 specifically in differentiating chondrocytes leads to severe and generalized lethal chondrodysplasis (Figure 3). The Csn5/Jab1 mutants exhibited disorganized chondrocyte columns with increased apoptosis. Importantly, the Csn5/Jab1-deficient chondrocytes exhibited increased BMP signaling28. Thus, CSN5/JAB1 is required for normal skeletal development, likely in part by its negative effect on BMP signaling. BMP signaling has been shown to be critically important for all aspects of skeletal formation and regeneration, including fracture healing and articular cartilage repair29-33. BMP2- and BMP7-containing osteogenic implants have been used for bone regeneration worldwide for long-bone nonunions and acute fractures, and for improving the success of spinal fusions. Therefore, novel therapies based on controlling the CSN5/JAB1-mediated inhibition of BMP signaling might be able to improve the efficacy of BMP treatment in the future.
V. CSN5/JAB1 amplification in cancer cells
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So far the main research focus of CSN5/JAB1 has been its potential role in tumorigenesis. The detection of CSN5/JAB1 overexpression in a variety of human tumors suggests that it might be a significant contributor to cancer initiation and progression (Table 1)13. The role of CSN5/JAB1 in the pathogenesis of several cancer types might be linked to its effect on p27, a cell cycle inhibitor and tumor suppressor34. Low expression of p27 has been associated with excessive cell proliferation in many types of human tumors, including osteosarcoma13, 35. CSN5/JAB1 can bind p27 and facilitate p27 degradation, thus promoting cell cycle progression and proliferation (Figure 2)36. CSN5/JAB1 expression was low or absent in normal adult breast tissue, but was increased in 50% of primary breast tumors and 90% of metastatic lesions37. CSN5/JAB1 over-expression was also correlated with reduced levels of p27 and poor prognosis of breast cancer38. In addition to p27, the other targets of CSN5/JAB1 known to be involved in tumorigenesis include p53, MDM2, HIF-1α, Smad4, and Smad7 (Figure 2)13. Additionally, a genetic linkage study demonstrates that the coordinated amplification of CSN5/JAB1 and oncogene MYC induces a poor-prognosis expression pattern of 512 genes in breast cancer39. However, the causative role of CSN5/ JAB1 in tumorigenesis remains to be established in a whole animal system. Osteosarcoma, the primary malignancy of bone cells, most commonly affects rapidly growing bones in teenagers and also affects elderly individuals who have rapid bone turnover due to conditions such as Paget’s disease40. Osteosarcoma is the 5th most frequent malignancy in 15-to 19-year-olds, and a deeper understanding of the pathogenesis of osteosarcoma is critical for better diagnosis and treatment of these patients. CSN5/JAB is involved in DNA damage repair (Figure 2) and the down-regulation of CSN5/JAB1 in osteosarcoma cells rendered them more sensitive to γ-irradiation, suggesting that CSN5/ Case Orthop J. Author manuscript; available in PMC 2016 January 22.
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JAB1 can be a potential target in osteosarcoma treatment8. However, the role of CSN5/JAB1 in osteosarcoma pathogenesis is still largely unknown. The amplification of another COP9 signalosome subunit, CSN3, was significantly correlated with the large tumor size of osteosarcoma 41, and knockdown of CSN3 repressed the proliferation and metastasis of osteosarcoma cells in vitro42. However, the role of CSN3 in osteosarcoma pathogenesis in vivo remains to be determined.
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Recent mouse genetics studies demonstrated that p53 is the determining factor in the pathogenesis of osteosarcoma, while another tumor suppressor, Rb, serves an important supporting role43, 44. p53 is the most frequently mutated gene in human cancers, including osteosarcoma13, 45. Since the CSN is a potent inhibitor of p53 function13, a better understanding of the regulation of p53 by CSN5/JAB1 and other subunits of the CSN in bone might help us to improve osteosarcoma diagnosis and treatment. In various human cancer cell lines, the knockdown of CSN5/JAB1 inhibited the proliferation of tumor cells, suggesting that over-expression of CSN5/JAB1 not only serves as a marker of malignant transformation, but also actually contributes to tumorigenesis13. CSN5/JAB1 has been proposed as a candidate target for therapeutic intervention of various cancers46. It will be of great clinical relevance to determine whether CSN5/JAB1 itself can be a valid target of cancer treatment through future screening and characterization of effective CSN5/JAB1specific small molecular inhibitors.
V. Future directions of the COP9 studies
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In the past decade, our understanding of the role of COP9 signalosome has greatly expanded. The COP9 signalosome is essential for embryogenesis and the functions of a diverse group of cells, including skeletal cells. Overall, in progenitor cell populations, the CSN appears to maintain those cells in an undifferentiated state and to keep them proliferating. In differentiating cells, the CSN is required for proper cell cycle progression and cell survival. The CSN, especially CSN5/JAB1, likely also contributes to tumorigenesis by modulating the stability and activity of numerous targets. The dosages of various CSN subunits appear to be critical for the COP9 signalosome functions and dys-regulated CSN expression is associated with developmental defects and cancers in mice and humans. The CSN function under normal development and in pathological conditions is likely to be celltype specific and contextdependent. Despite recent progress, several important questions of the CSN remain unanswered: what is the underlying mechanism of CSN5/JAB1 function during the successive steps of skeletogenesis? What is the precise mechanism of CSNmediated cell death? How is the CSN itself regulated? Is CSN abnormality associated with any human birth defect? What might be the role of CSN5/JAB1 in the pathogenesis of osteosarcoma? Answers to these critical questions will provide further insights into the COP9 signalosome function and facilitate the development of novel therapeutic options to treat human diseases, including osteosarcoma.
Acknowledgements Work in Dr. Guang Zhou’s laboratory was supported by a NIH grant (R03DE19190). We thank Valerie Schmedlen for editorial assistance.
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Figure 1. Structural organization of COP9 signalosome
Lightning symbol represents Csn5/Jab1 enzymatic activity. Abbreviations: CRL, cullinRING ligase; N8, Nedd8 (neural precursor cell-expressed developmentally downregulated-8).
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Figure 2. Functions and targets of JAB1/CSN5
Plus (+) signs indicate JAB1/CSN5-mediated positive effect; Minus (−) signs indicate JAB1/ CSN5-mediated negative effect. Abbreviations: AP-1, activator protein 1; BMP, bone morphogenetic protein; HIF-1a, hypoxia inducible factor-1a; MDM2, murine double minute 2; TGF-β, transforming growth factor-β.
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Severe and generalized chondrodysplasis of the mutant mice in which Csn5/Jab1, an integral part of COP signalosome, was specifically ablated in chondrocytes.
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Table 1
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Consequences of dys-regulated CSN subunits in the mouse and the human.
Author Manuscript Author Manuscript
Gene
Alteration
Phenotypes
References
Csn2 mice
KO
Embryonic lethality by E7.5; Deficient cell proliferation; ↑p53, p21, and cyclin E
[5]
Csn3 mice
KO
Embryonic lethality by E8.5; ↑Apoptosis; ↓Csn8 expression
[6]
CSN3 human
Overexpression
CSN3 amplification significantly correlated with large tumor size of osteosarcoma
[41]
Csn5/Jab1 mice
KO
Embryonic lethality by E8.5; Impaired proliferation; ↑DNA damage and apoptosis; ↓p53, p27, and cyclin E
[7], [8]
Csn5/Jab1 mice
Overexpression driven by a constitutive CAG promoter
↑Proliferation and maintenance of hematopoietic progenitors
[12]
Csn5/Jab1 mice
cKO in chondrocytes
Neonatal lethal chondrodysplasia with disorganized chondrocytes; ↑Apoptosis
[28]
Csn5/Jab1 mice
cKO in T cells
Impaired T cell development; ↑Apoptosis; ↓p53, IkB-a, and b-catenin
[10]
Csn5/Jab1 mice
cKO in myeloid cells
↓Mortality in polymicrobial sepsis; ↓TLR-and TNF-a-mediated MAPK signaling in macrophage; ↑Antioxidation genes
[11]
Csn5/Jab1 human
Overexpression
CSN5 amplification associated with poor outcomes in breast cancer, lung cancer, and pancreatic adenocarcinoma
[13]
Csn6 mice
KO
Embryonic lethality by E7.5; ↑p53 activity
[9]
CSN6 human
Overexpression
Upregulated CSN6 expression significantly correlated with enhanced expression of MDM2, a p53 inhibitor, in breast and thyroid tumors
[9]
Csn8 mice
KO
Embryonic lethality by E7.5
[47]
KO: constitutive knockout; cKO: conditional knockout.
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