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Improved Methodology for Studying Postnatal Osteogenesis via Intramembranous Ossification in a Murine Bone Marrow Injury Model

Long bone injuries heal through either endochondral or intramembranous bone formation pathways. Unlike the endochondral pathway that requires a cartilage ...

UConn School of Medicine

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.

Loss of Cbl-PI3K interaction in mice prevents significant bone loss following ovariectomy.

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 Osterix 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.

Loss of Cbl-PI3K interaction modulates the periosteal response to fracture by enhancing osteogenic commitment and differentiation.

The neurogenic locus notch homolog protein (Notch)-2 receptor is a determinant of B-cell allocation, and gain-of-NOTCH2-function mutations are associated with Hajdu-Cheney syndrome (HCS), a disease presenting with osteoporosis and acro-osteolysis. We generated a mouse model reproducing the HCS mutation (Notch2HCS), and heterozygous global mutant mice displayed gain-of-Notch2 function. In the mutant spleen, the characteristic perifollicular rim marking the marginal zone (MZ), which is the interface between the nonlymphoid red pulp and the lymphoid white pulp, merged with components of the white pulp. As a consequence, the MZ of Notch2HCS mice occupied most of the splenic structure. To explore the mechanisms involved, lymphocyte populations from the bone marrow and spleen were harvested from heterozygous Notch2HCS mice and sex-matched control littermates and analyzed by flow cytometry. Notch2HCS mice had an increase in CD21/35CD23 splenic MZ B cells of approximately fivefold and a proportional decrease in splenic follicular B cells (CD21/35CD23) at 1, 2, and 12 months of age. Western blot analysis revealed that Notch2HCS mutant splenocytes had increased phospho-Akt and phospho-Jun N-terminal kinase, and gene expression analysis of splenic CD19 B cells demonstrated induction of Hes1 and Hes5 in Notch2HCS mutants. Anti-Notch2 antibodies decreased MZ B cells in control and Notch2HCS mice. In conclusion, Notch2HCS mutant mice have increased mature B cells in the MZ of the spleen.

The Hajdu Cheney Mutation Is a Determinant of B-Cell Allocation of the Splenic Marginal Zone.

Mice harboring Notch2 mutations replicating Hajdu-Cheney syndrome (Notch2) have osteopenia and exhibit an increase in splenic marginal zone B cells with a decrease in follicular B cells. Whether the altered B-cell allocation is responsible for the osteopenia of Notch2 mutants is unknown. To determine the effect of NOTCH2 activation in B cells on splenic B-cell allocation and skeletal phenotype, a conditional-by-inversion (COIN) Hajdu-Cheney syndrome allele of Notch2 (Notch2) was used. Cre recombination generates a permanent Notch2 allele expressing a transcript for which sequences coding for the proline, glutamic acid, serine, and threonine-rich (PEST) domain are replaced by a stop codon. CD19-Cre drivers were backcrossed into Notch2 to generate CD19-specific Notch2 mutants and control Notch2 littermates. There was an increase in marginal zone B cells and a decrease in follicular B cells in the spleen of CD19;Notch2 mice, recapitulating the splenic phenotype of Notch2 mice. The effect was reproduced when the NOTCH1 intracellular domain was induced in CD19-expressing cells (CD19;Rosa mice). However, neither CD19;Notch2 nor CD19;Rosa mice had a skeletal phenotype. Moreover, splenectomies in Notch2 mice did not reverse their osteopenic phenotype. In conclusion, Notch2 activation in CD19-expressing cells determines B-cell allocation in the spleen but has no skeletal consequences.

Induction of the Hajdu-Cheney Syndrome Mutation in CD19 B Cells in Mice Alters B-Cell Allocation but Not Skeletal Homeostasis.

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 (Cbl, YF) have increased bone volume partly due to decreased osteoclast-mediated bone resorption. The Cbl 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.

Cbl-PI3K interaction regulates Cathepsin K secretion in osteoclasts.

Diabetics are at increased risk for fracture, and experience severely impaired skeletal healing characterized by delayed union or nonunion of the bone. The periosteum harbors osteochondral progenitors that can differentiate into chondrocytes and osteoblasts, and this connective tissue layer is required for efficient fracture healing. While bone marrow-derived stromal cells have been studied extensively in the context of diabetic skeletal repair and osteogenesis, the effect of diabetes on the periosteum and its ability to contribute to bone regeneration has not yet been explicitly evaluated. Within this study, we utilized an established murine model of type I diabetes to evaluate periosteal cell differentiation capacity, proliferation, and availability under the effect of a diabetic environment. Periosteal cells from diabetic mice were deficient in osteogenic differentiation ability in vitro, and diabetic mice had reduced periosteal populations of mesenchymal progenitors with a corresponding reduction in proliferation capacity following injury. Additionally, fracture callus mineralization and mature osteoblast activity during periosteum-mediated healing was impaired in diabetic mice compared to controls. We propose that the effect of diabetes on periosteal progenitors and their ability to aid in skeletal repair directly impairs fracture healing.

Diabetes impairs periosteal progenitor regenerative potential.

Bone remodeling occurs through the interactions of three major cell lineages, osteoblasts, which mediate bone formation, osteocytes, which derive from osteoblasts, sense mechanical force and direct bone turnover, and osteoclasts, which mediate bone resorption. However, multiple additional cell types within the bone marrow, including macrophages, T lymphocytes and B lymphocytes influence the process. The bone marrow microenvironment, which is supported, in part, by bone cells, forms a nurturing network for B lymphopoiesis. In turn, developing B lymphocytes influence bone cells. Bone health during homeostasis depends on the normal interactions of bone cells with other lineages in the bone marrow. In disease state these interactions become pathologic and can cause abnormal function of bone cells and inadequate repair of bone after a fracture. This review summarizes what is known about the development of B lymphocytes and the interactions of B lymphocytes with bone cells in both health and disease.

Interactions of B-lymphocytes and bone cells in health and disease.

Despite the remarkable regenerative capacity of skeletal tissues, nonunion of bone and failure of fractures to heal properly presents a significant clinical concern. Stem and progenitor cells are present in bone and become activated following injury; thus, elucidating mechanisms that promote adult stem cell-mediated healing is important. Wnt-associated adult stem marker Lgr6 is implicated in the regeneration of tissues with well-defined stem cell niches in stem cell-reliant organs. Here, we demonstrate that Lgr6 is dynamically expressed in osteoprogenitors in response to fracture injury. We used an Lgr6-null mouse model and found that Lgr6 expression is necessary for maintaining bone volume and efficient postnatal bone regeneration in adult mice. Skeletal progenitors isolated from Lgr6-null mice have reduced colony-forming potential and reduced osteogenic differentiation capacity due to attenuated cWnt signaling. Lgr6-null mice consist of a lower proportion of self-renewing stem cells. In response to fracture injury, Lgr6-null mice have a deficiency in the proliferation of periosteal progenitors and reduced ALP activity. Further, analysis of the bone regeneration phase and remodeling phase of fracture healing in Lgr6-null mice showed impaired endochondral ossification and decreased mineralization. We propose that in contrast to not being required for successful skeletal development, Lgr6-positive cells have a direct role in endochondral bone repair.

Wnt-associated adult stem cell marker Lgr6 is required for osteogenesis and fracture healing.

Fracture management largely relies on the bone's inherent healing capabilities and, when necessary, surgical intervention. Currently, there are limited osteoinductive therapies to promote healing, making targeting skeletal stem/progenitor cells (SSPCs) a promising avenue for therapeutic development. A limiting factor for this approach is our incomplete understanding of the molecular mechanisms governing SSPCs' behavior. We have recently identified that the Leucine-rich repeat-containing G-protein coupled receptor 6 (Lgr6) is expressed in sub-populations of SSPCs, and is required for maintaining bone volume during adulthood and for proper fracture healing. Lgr family members (Lgr4-6) are markers of stem cell niches and play a role in tissue regeneration primarily by binding R-Spondin (Rspo1-4). This interaction promotes canonical Wnt (cWnt) signaling by stabilizing Frizzled receptors. Interestingly, our findings here indicate that Lgr6 may also influence cWnt-independent pathways. Remarkably, Lgr6 expression was enhanced during Bmp-mediated osteogenesis of both human and murine cells. Using biochemical approaches, RNA sequencing, and bioinformatic analysis of published single-cell data, we found that elements of BMP signaling, including its target gene, pSMAD, and gene ontology pathways, are downregulated in the absence of Lgr6. Our findings uncover a molecular interdependency between the Bmp pathway and Lgr6, offering new insights into osteogenesis and potential targets for enhancing fracture healing.

Signaling pathways associated with Lgr6 to regulate osteogenesis.

Long bone injuries heal through either endochondral or intramembranous bone formation pathways. Unlike the endochondral pathway that requires a cartilage template, the process of intramembranous ossification involves the direct conversion of skeletal stem and progenitor cells (SSPCs) into bone-forming osteoblasts. There are limited surgical methods to model this process in experimental mice. Here, we have improved upon a bone marrow injury model in mice to facilitate the study of bone repair via intramembranous ossification and to assess postnatal regulators of osteogenesis. This method is highly reproducible and user-friendly, and it allows temporal assessment of new bone formation in a short period (3-7 days post-injury) using μCT and frozen section histology. Furthermore, the contributions of SSPCs and mature osteoblasts can be readily assessed using a combination of fluorescent reporter mice and this intramembranous bone marrow injury model. In clinical contexts, intramembranous bone formation is relevant for healing critical size defects, stress fractures, cortical defects, trauma from tumor resections, and joint replacements.

Archana Sanjay

Department of Orthopedic Surgery

UConn School of Medicine

Improved Methodology for Studying Postnatal Osteogenesis via Intramembranous Ossification in a Murine Bone Marrow Injury Model

Archana Sanjay

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Orthopedic Surgery

UConn School of Medicine

Improved Methodology for Studying Postnatal Osteogenesis via Intramembranous Ossification in a Murine Bone Marrow Injury Model

Department of Orthopedic Surgery