Core-binding factor beta is required for osteoblast differentiation during fibula fracture healing

Background Growing evidence has implicated core-binding factor beta (Cbfb) as a contributor to osteoblast differentiation, which plays a key role in fracture healing. Herein, we aimed to assess whether Cbfb affects osteoblast differentiation after fibula fracture. Methods Initially, we established a Cbfb conditional knockout mouse model for subsequent studies. Immunohistochemical staining was conducted to detect the expression of proliferating cell nuclear antigen (PCNA) and collagen II in the fracture end. Next, we isolated and cultured osteoblasts from specific Cbfb conditional knockout mice for BrdU analysis, alkaline phosphatase (ALP) staining, and von Kossa staining to detect osteoblast viability, differentiation, and mineralization, respectively. Western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to detect the expression of osteoblast differentiation-related genes. Results The Cbfb conditional knockout mice exhibited downregulated expression of PCNA and collagen II, reduced ALP activity, and mineralization, as well as diminished expression of osteoblast differentiation-related genes. Further, Cbfb knockout exerted no obvious effects on osteoblast proliferation. Conclusions Overall, these results substantiated that Cbfb could promote fibula fracture healing and osteoblast differentiation and thus provided a promising therapeutic target for clinical treatment of fibula fracture.

The p53/miR-145a Axis Promotes Cellular Senescence and Inhibits Osteogenic Differentiation by Targeting Cbfb in Mesenchymal Stem Cells

The osteogenic differentiation capacity of senescent bone marrow mesenchymal stem cells (MSCs) is reduced. p53 not only regulates cellular senescence but also functions as a negative regulator in bone formation. However, the role of p53 in MSCs senescence and differentiation has not been extensively explored. In the present study, we investigated the molecular mechanism of p53 in MSCs senescence and osteogenic differentiation. We found that p53 was upregulated during cellular senescence and osteogenic differentiation of MSCs respectively induced by H2O2 and BMP9. Similarly, the expression of p53-induced miR-145a was increased significantly. Furthermore, Overexpression of miR-145a in MSCs promoted cellular senescence and inhibited osteogenic differentiation. Then, we identified that p53-induced miR-145a inhibited osteogenic differentiation by targeting core binding factor beta (Cbfb), and the restoration of Cbfb expression rescued the inhibitory effects of miRNA-145a. In summary, our results indicate that p53/miR-145a axis exert its functions both in promoting senescence and inhibiting osteogenesis of MSCs, and the novel p53/miR-145a/Cbfb axis in osteogenic differentiation of MSCs may represent new targets in the treatment of osteoporosis.

The Role of p53-induced miR-145a in Senescence and Osteogenesis of Mesenchymal Stem Cells

Background: The osteogenic differentiation capacity of senescent bone marrow mesenchymal stem cells (MSCs) is diminished. However, little is known about the mechanisms. p53 not only regulates cellular senescence but also, as a negative regulator, is involved in bone formation. This study investigated the molecular mechanism of p53 in cellular senescence and osteogenesis.Methods: The expression of p53 and its downstream gene p21 were measured during cellular senescence and osteogenesis differentiation of primary bone marrow MSCs. Then miR-145a was picked out from those p53-induced miRNAs as its expression change in bone marrow MSCs senescence and osteogenesis induced by H2O2 and BMP-9, respectively. The function of p53 induced miR-145a was analyzed by transfected with miRNA mimic in osteogenic differentiation of MSCs. Western blot and luciferase reporter assay were used for validating the target of miR-145a. Results: p53, its downstream effector p21, and p53-induced miR-145a were significantly upregulated during primary MSCs senescence and osteogenesis. Overexpression of miR-145a promoted cellular senescence and inhibited osteogenic differentiation of MSCs. p53-induced miR-145a inhibited osteogenic differentiation by targeting core binding factor beta (Cbfb), and the restoration of Cbfb expression rescued the inhibitory effects of miRNA-145a on osteogenesis.Conclusions: p53-induced miR-145a functions both in promoting senescence and inhibiting osteogenesis of MSCs, and the novel pathway p53/miR-145a/Cbfb in osteogenic differentiation of MSCs may represent new targets in the treatment of osteoporosis.

Core-binding factor beta is required for osteoblast differentiation during fibula fracture healing

Background: Growing evidence has implicated core-binding factor beta (Cbfb) as a contributor to the osteoblast differentiation, which plays a key role in fracture healing. Here, we conducted the present study with the main objective to assess whether Cbfb affects osteoblast differentiation after fibula fracture. Methods: Initially, Cbfb conditional knockout mouse model was established. Immunohistochemical staining was carried out to detect the expression of proliferating cell nuclear antigen (PCNA) and Collagen II in the fracture end. Then osteoblasts were isolated from specific Cbfb conditional knockout mice and cultured. BrdU method, Alkaline phosphatase (ALP) staining and Von Kossa staining were followed to detect osteoblast proliferation, differentiation and mineralization, respectively. Western blot analysis and RT-qPCR were used to detect the expression of osteoblast differentiation-related genes. Cbfb conditional knockout mouse model was successfully constructed. Results: The mice treated with Cbfb knockout were shown to exhibit significantly decreased expression of PCNA and Collagen II, ALP activity and mineralization, as well as inhibited expression of Runx2, ALP, BglaPl, SPPl, Osteocalcin, Atf4 and Osterix. Further, Cbfb knockout showed no effects on osteoblast differentiation. Conclusion: Overall, these results demonstrated that the Cbfb could potentially promote fibula fracture healing and osteoblast differentiation and thus could comprise a potential means of impeding the progression of fibula fracture.

Inhibition of Vif-Mediated Degradation of APOBEC3G through Competitive Binding of Core-Binding Factor Beta

ABSTRACT Vif counteracts the host restriction factor APOBEC3G (A3G) and other APOBEC3s by preventing the incorporation of A3G into progeny virions. We previously identified Vif mutants with a dominant-negative (D/N) phenotype that interfered with the function of wild-type Vif, inhibited the degradation of A3G, and reduced the infectivity of viral particles by increased packaging of A3G. However, the mechanism of interference remained unclear, in particular since all D/N Vif mutants were unable to bind Cul5 and some mutants additionally failed to bind A3G, ruling out competitive binding to A3G or the E3 ubiquitin ligase complex as the sole mechanism. The goal of the current study was to revisit the mechanism of D/N interference by Vif mutants and analyze the possible involvement of core binding factor beta (CBFβ) in this process. We found a clear correlation of D/N properties of Vif mutants with their ability to engage CBFβ. Only mutants that retained the ability to bind CBFβ exhibited the D/N phenotype. Competition studies revealed that D/N Vif mutants directly interfered with the association of CBFβ and wild-type Vif. Furthermore, overexpression of CBFβ counteracted the interference of D/N Vif mutants with A3G degradation by wild-type Vif. Finally, overexpression of Runx1 mimicked the effect of D/N Vif mutants and inhibited the degradation of A3G by wild-type Vif. Taken together, we identified CBFβ as the key player involved in D/N interference by Vif. IMPORTANCE Of all the accessory proteins encoded by HIV-1 and other primate lentiviruses, Vif has arguably the strongest potential as a target for antiviral therapy. This conclusion is based on the observation that replication of HIV-1 in vivo is critically dependent on Vif. Thus, inhibiting the function of Vif via small-molecule inhibitors or other approaches has significant therapeutic potential. We previously identified dominant-negative (D/N) Vif variants whose expression interferes with the function of virus-encoded wild-type Vif. We now show that D/N interference involves competitive binding of D/N Vif variants to the transcriptional cofactor core binding factor beta (CBFβ), which is expressed in cells in limiting quantities. Overexpression of CBFβ neutralized the D/N phenotype of Vif. In contrast, overexpression of Runx1, a cellular binding partner of CBFβ, phenocopied the D/N Vif phenotype by sequestering endogenous CBFβ. Thus, our results provide proof of principle that D/N Vif variants could have therapeutic potential.