Role of Cbl-PI3K Interaction during Skeletal Remodeling in a Murine Model of Bone Repair.
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ABSTRACT:
SUBMITTER: Scanlon V
PROVIDER: S-EPMC4578922 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cbl is an adaptor protein and E3 ligase that plays both positive and negative roles in several signaling pathways that affect various cellular functions. Tyrosine 737 is unique to Cbl and phosphorylated by Src family kinases. Phosphorylated CblY737 creates a binding site for the p85 regulatory subunit of phosphatidylinositol 3 kinase (PI3K) that also plays an important role in the regulation of bone homeostasis. To investigate the role of Cbl-PI3K interaction in bone homeostasis, we examined knock-in mice in which the PI3K binding site on Cbl was ablated due to the substitution of tyrosine 737 to phenylalanine (Cbl(YF/YF), YF mice). We previously reported that bone volume in these mice is increased due to decreased osteoclast function (Adapala et al., J Biol Chem 285:36745-36758, 19). Here, we report that YF mice also have increased bone formation and osteoblast numbers. In ex vivo cultures bone marrow-derived YF osteoblasts showed increased Col1A expression and their proliferation was also significantly augmented. Moreover, proliferation of MC3T3-E1 cells was increased after treatment with conditioned medium generated by culturing YF bone marrow stromal cells. Expression of stromal derived factor-1 (SDF-1) was increased in YF bone marrow stromal cells compared to wild type. Increased immunostaining of SDF-1 and CXCR4 was observed in YF bone marrow stromal cells compared to wild type. Treatment of YF condition medium with neutralizing anti-SDF-1 and anti-CXCR4 antibodies attenuated MC3T3-E1 cell proliferation. Cumulatively, these results show that abrogation of Cbl-PI3K interaction perturbs bone homeostasis, affecting both osteoclast function and osteoblast proliferation.
| S-EPMC3191294 | biostudies-literature
Project description:Cbl and Cbl-b are E3 ubiquitin ligases and adaptor proteins, which perform regulatory roles in bone remodeling. Cbl-/- mice have delayed bone development due to decreased osteoclast migration. Cbl-b-/- mice are osteopenic due to increased bone resorbing activity of osteoclasts. Unique to Cbl, but not present in Cbl-b, is tyrosine 737 in the YEAM motif, which upon phosphorylation provides a binding site for the regulatory p85 subunit of PI3K. Substitution of tyrosine 737 with phenylalanine (Y737F, CblYF/YF mice) prevents Y737 phosphorylation and abrogates the Cbl-PI3K interaction. We have previously reported that CblYF/YF mice had increased bone volume due to defective bone resorption and increased bone formation. Here we show that the lumbar vertebra from CblYF/YF mice did not have significant bone loss following ovariectomy. Our data also suggests that abrogation of Cbl-PI3K interaction in mice results in the loss of coupling between bone resorption and formation, since ovariectomized CblYF/YF mice did not show significant changes in serum levels of c-terminal telopeptide (CTX), whereas the serum levels of pro-collagen type-1 amino-terminal pro-peptide (P1NP) were decreased. In contrast, following ovariectomy, Cbl-/- and Cbl-b-/- mice showed significant bone loss in the tibiae and L2 vertebrae, concomitant with increased serum CTX and P1NP levels. These data indicate that while lack of Cbl or Cbl-b distinctly affects bone remodeling, only the loss of Cbl-PI3K interaction protects mice from significant bone loss following ovariectomy.
| S-EPMC4149851 | biostudies-literature
Project description:Effective bone resorption by osteoclasts is critical for balanced bone remodeling. We have previously reported that mice harboring a substitution mutation of tyrosine 737 to phenylalanine in the adapter protein Cbl (CblY737F, YF) have increased bone volume partly due to decreased osteoclast-mediated bone resorption. The CblY737F mutation abrogates interaction between Cbl and the p85 subunit of PI3K. Here, we studied the mechanism for defective resorptive function of YF mutant osteoclasts. The YF osteoclasts had intact actin cytoskeletons and sealing zones. Expression and localization of proteins needed for acidification of the resorptive lacunae were also comparable between the WT and YF osteoclasts. In contrast, secretion of Cathepsin K, a major protease needed to degrade collagen, was diminished in the conditioned media derived from YF osteoclasts. The targeting of Cathepsin K into LAMP2-positive vesicles was also compromised due to decreased number of LAMP2-positive vesicles in YF osteoclasts. Further, we found that in contrast to WT, conditioned media derived from YF osteoclasts promoted increased numbers of alkaline phosphatase positive colonies, and increased expression of osteogenic markers in WT calvarial cultures. Cumulatively, our results suggest that the Cbl-PI3K interaction regulates Cathepsin K secretion required for proper bone resorption, and secretion of factors which promote osteogenesis.
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Project description:Cbl family proteins, Cbl and Cbl-b, are E3 ubiquitin ligases and adaptor proteins, which play important roles in bone-resorbing osteoclasts. Loss of Cbl in mice decreases osteoclast migration, resulting in delayed bone development where as absence of Cbl-b decreases bone volume due to hyper-resorptive osteoclasts. A major structural difference between Cbl and Cbl-b is tyrosine 737 (in YEAM motif) only on Cbl, which upon phosphorylation interacts with the p85 subunit of phosphatidylinositol-3 Kinase (PI3K). In contrast to Cbl(-/-) and Cbl-b(-/-) , mice lacking Cbl-PI3K interaction due to a Y737F (tyrosine to phenylalanine, YF) mutation showed enhanced osteoclast survival, but defective bone resorption. To investigate whether Cbl-PI3K interaction contributes to distinct roles of Cbl and Cbl-b in osteoclasts, mice bearing CblY737F mutation in the Cbl-b(-/-) background (YF/YF;Cbl-b(-/-) ) were generated. The differentiation and survival were augmented similarly in YF/YF and YF/YF;Cbl-b(-/-) osteoclasts, associated with enhanced PI3K signaling suggesting an exclusive role of Cbl-PI3K interaction, independent of Cbl-b. In addition to PI3K, the small GTPase Ras also regulates osteoclast survival. In the absence of Cbl-PI3K interaction, increased Ras GTPase activity and Ras-PI3K binding were observed and inhibition of Ras activation attenuated PI3K mediated osteoclast survival. In contrast to differentiation and survival, increased osteoclast activity observed in Cbl-b(-/-) mice persisted even after introduction of the resorption-defective YF mutation in YF/YF;Cbl-b(-/-) mice. Hence, Cbl and Cbl-b play mutually exclusive roles in osteoclasts. Whereas Cbl-PI3K interaction regulates differentiation and survival, bone resorption is predominantly regulated by Cbl-b in osteoclasts.
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Project description:The Wnt genes encode a highly conserved class of signaling factors required for the development of several types of tissues including musculoskeletal and neural structures. There is increasing evidence that Wnt signaling is critical for bone mass accrual, bone remodeling, and fracture repair. Wnt proteins bind to cell-surface receptors and activate signaling pathways which control nuclear gene expression; this Wnt-regulated gene expression controls cell growth and differentiation. Many of the components of the Wnt pathway have recently been characterized, and specific loss-of-function or gain-of-function mutations in this pathway in mice and in humans have resulted in disorders of deficient or excess bone formation, respectively. Pharmacologically targeting components of the Wnt signaling pathway will allow for the manipulation of bone formation and remodeling and will have several orthopedic applications including enhancing bone formation in nonunion and osteoporosis and restricting pathologic bone formation in osteogenic tumors and heterotopic ossification.
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Project description:Casitas B-lineage lymphoma (CBL) encodes an E3 ubiquitin ligase and signaling adaptor that regulates receptor and nonreceptor tyrosine kinases. Recurrent CBL mutations occur in myeloid neoplasms, including 10% to 20% of chronic myelomonocytic leukemia (CMML) cases, and selectively disrupt the protein's E3 ubiquitin ligase activity. CBL mutations have been associated with poor prognosis, but the oncogenic mechanisms and therapeutic implications of CBL mutations remain incompletely understood. We combined functional assays and global mass spectrometry to define the phosphoproteome, CBL interactome, and mechanism of signaling activation in a panel of cell lines expressing an allelic series of CBL mutations. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced CBL phosphorylation, phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) recruitment, and downstream phosphatidylinositol 3-kinase (PI3K)/AKT signaling in CBL-mutant cells. Signaling adaptor domains of CBL, including the tyrosine kinase-binding domain, proline-rich region, and C-terminal phosphotyrosine sites, were all required for the oncogenic function of CBL mutants. Genetic ablation or dasatinib-mediated inhibition of LYN reduced CBL phosphorylation, CBL-PIK3R1 interaction, and PI3K/AKT signaling. Furthermore, we demonstrated in vitro and in vivo antiproliferative efficacy of dasatinib in CBL-mutant cell lines and primary CMML. Overall, these mechanistic insights into the molecular function of CBL mutations provide rationale to explore the therapeutic potential of LYN inhibition in CBL-mutant myeloid malignancies.
| S-EPMC8063090 | biostudies-literature
Project description:After cartilage injury and repair, the subchondral bone plate remodels. Skeletal maturity likely impacts both bone remodeling and inherent cartilage repair capacity. The objective of this study was to evaluate subchondral bone remodeling as a function of injury type, repair scenario, and skeletal maturity in a Yucatan minipig model. Cartilage defects (4?mm) were created bilaterally in the trochlear groove. Treatment conditions included a full thickness chondral defect (full chondral defect, n?=?3 adult/3 juvenile), a partial thickness (?50%) chondral defect (PCD, n?=?3/3), and FCD treated with microfracture (MFX, n?=?3/3). At 6 weeks postoperatively, osteochondral samples containing the lesion site were imaged by micro-computed tomography (CT) and analyzed by histology and immunohistochemistry. Via micro-CT, FCD and MFX groups showed increased bone loss in juveniles compared with adults. Quantification of histology using the ICRS II scoring system showed equal overall assessment for the FCD groups and better overall assessment in juvenile animals treated with MFX compared with adults. All FCD and MFX groups were inferior to control samples. For the PCD injury, both age groups had values close to the control values. For the FCD groups, there were greater alterations in the subchondral bone in juveniles compared with adults. Staining for collagen II showed more intense signals in juvenile FCD and MFX groups compared with adults. This large animal study of cartilage repair shows the significant impact of skeletal maturity on the propensity of subchondral bone to remodel as a result of chondral injury. This will improve selection criteria for animal models for studying cartilage injury, repair, and treatment.
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Project description:OBJECTIVE:To investigate whether ?-catenin signaling in chondrocytes regulates osteoclastogenesis, thereby contributing to postnatal bone growth and bone remodeling. METHODS:Mice with conditional knockout (cKO) or conditional activation (cAct) of chondrocyte-specific ?-catenin were generated. Changes in bone mass, osteoclast numbers, and osteoblast activity were examined. The mechanisms by which ?-catenin signaling in chondrocytes regulates osteoclast formation were determined. RESULTS:The ?-catenin cKO mice developed localized bone loss, whereas cAct mice developed a high bone mass phenotype. Histologic findings suggested that these phenotypes were caused primarily by impaired osteoclast formation, rather than impaired bone formation. Further molecular signaling analyses revealed that ?-catenin signaling controlled this process by regulating the expression of the RANKL and osteoprotegerin (OPG) genes in chondrocytes. Activation of ?-catenin signaling in chondrocytes suppressed Rankl gene transcription through a glucocorticoid receptor-dependent mechanism. The severe bone loss phenotype observed in ?-catenin cKO mice was largely restored by treatment with human recombinant OPG or transgenic overexpression of Opg in chondrocytes. CONCLUSION:?-catenin signaling in chondrocytes plays a key role in postnatal bone growth and bone remodeling through its regulation of osteoclast formation.
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Project description:The periosteum contains multipotent skeletal progenitors that contribute to bone repair. The signaling pathways regulating the response of periosteal cells to fracture are largely unknown. Phosphatidylinositol-3 Kinase (PI3K), a prominent lipid kinase, is a major signaling protein downstream of several factors that regulate osteoblast differentiation. Cbl is an E3 ubiquitin ligase and a major adaptor protein that binds to the p85 regulatory subunit and modulates PI3K activity. Substitution of tyrosine 737 to phenylalanine (Y737F) in Cbl abolishes the interaction between Cbl and p85 subunit without affecting the Cbl's ubiquitin ligase function. Here, we investigated the role of PI3K signaling during the very early stages of fracture healing using OsterixRFP reporter mice. We found that the absence of PI3K regulation by Cbl resulted in robust periosteal thickening, with increased proliferation of periosteal cells. While the multipotent properties of periosteal progenitors to differentiate into chondrocytes and adipocytes did not change, osteogenic differentiation in the absence of Cbl-PI3K interaction was highly augmented. The increased stability and nuclear localization of Osterix observed in periosteal cells lacking Cbl-PI3K interaction may explain this enhanced osteogenic differentiation since the expression of Osterix transcriptional target genes including osteocalcin and BSP are increased in YF cells. Overall, our findings highlight a hitherto unexplored and novel role for Cbl and PI3K in modulating the osteogenic response of periosteal cells during the early stages of fracture repair.
| S-EPMC5819877 | biostudies-literature
Project description:Bone defects can originate from a variety of causes, including trauma, cancer, congenital deformity, and surgical reconstruction. Success of the current "gold standard" treatment (i.e., autologous bone grafts) is greatly influenced by insufficient or inappropriate bone stock. There is thus a critical need for the development of new, engineered materials for bone repair. This review describes the use of natural and synthetic hydrogels as scaffolds for bone tissue engineering. We discuss many of the advantages that hydrogels offer as bone repair materials, including their potential for osteoconductivity, biodegradability, controlled growth factor release, and cell encapsulation. We also discuss the use of hydrogels in composite devices with metals, ceramics, or polymers. These composites are useful because of the low mechanical moduli of hydrogels. Finally, the potential for thermosetting and photo-cross-linked hydrogels as three-dimensionally (3D) printed, patient-specific devices is highlighted. Three-dimensional printing enables controlled spatial distribution of scaffold materials, cells, and growth factors. Hydrogels, especially natural hydrogels present in bone matrix, have great potential to augment existing bone tissue engineering devices for the treatment of critical size bone defects.
| S-EPMC4675359 | biostudies-literature