scholarly journals DZNep Promotes Mouse Bone Defect Healing via Enhancing Both Osteogenesis and Osteoclastogenesis

2021 ◽  
Author(s):  
Xiankun Cao ◽  
Wenxin He ◽  
Kewei Rong ◽  
Shenggui Xu ◽  
Zhiqian Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Resorption ◽  
Bone Defect ◽  
Nuclear Translocation ◽  
Nuclear Factor Κb ◽  
Cell Counting ◽  
Nuclear Factor ◽  
Defect Healing ◽  
Bone Defect Healing

Abstract Background: EZH2 (Enhancer of zeste homolog 2) is a novel oncogene that can specifically trimethylate the histone H3 lysine 27 (H3K27me3) to transcriptionally inhibit the expression of downstream tumor-suppressing genes. As a small molecular inhibitor of EZH2, 3-Deazaneplanocin (DZNep) has been widely studied due to the role of tumor suppression. With the roles of epigenetic regulation of bone cells emerged in past decades, the property and molecular mechanism of DZNep on enhancing osteogenesis had been reported and attracted a great deal of attention recently. this study aims to elucidate the role of DZNep on EZH2-H3K27me3 axis and downstream factors during both osteoclasts and osteoblasts formation and the therapeutic possibility of DZNep on bone defect healing.Methods: Bone marrow drived macrophages (BMMs) cells were cultured and their responsiveness to DZNep was evaluated by Cell Counting Kit-8, TRAP staining assay, Bone Resorption Assay, Podosome Actin Belt. Bone marrow drived mesenchymal stem cells (BMSC) were cultured and their responsiveness to DZNep was evaluated by Cell Counting Kit-8, ALP and AR staining assay. The expression of nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), Wnt signaling pathway was determined by qPCR and western blotting. Mouse bone defect models were created, rescued by DZNep injection and the effectiveness was evaluated by X-ray and Micro-CT and Histological staining.Results: Consistent with the previous study that DZNep enhances osteogenesis via Wnt family member 1(Wnt1), Wnt6, and Wnt10a, our results showed that DZNep also promotes osteoblasts differentiation and mineralization through the EZH2-H3K27me3-Wnt4 axis. Furthermore, we identified that DZNep promoted the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast formation via facilitating the phosphorylation of IKKα/β, IκB, and subsequently NF-κB nuclear translocation, which credit to the EZH2-H3K27me3-Foxc1 axis. More importantly, the enhanced osteogenesis and osteoclastogenesis result in accelerated mice bone defect healing in vivo.Conclusion: DZNep targeting EZH2-H3K27me3 axis facilitated the healing of mice bone defect via simultaneously enhancing osteoclastic bone resorption and promoting osteoblastic bone formation.

scholarly journals DZNep promotes mouse bone defect healing via enhancing both osteogenesis and osteoclastogenesis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xiankun Cao ◽  
Wenxin He ◽  
Kewei Rong ◽  
Shenggui Xu ◽  
Zhiqian Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Resorption ◽  
Bone Defect ◽  
Nuclear Translocation ◽  
Nuclear Factor Κb ◽  
Cell Counting ◽  
Nuclear Factor ◽  
Defect Healing ◽  
Bone Defect Healing

Abstract Background Enhancer of zeste homolog 2 (EZH2) is a novel oncogene that can specifically trimethylate the histone H3 lysine 27 (H3K27me3) to transcriptionally inhibit the expression of downstream tumor-suppressing genes. As a small molecular inhibitor of EZH2, 3-Deazaneplanocin (DZNep) has been widely studied due to the role of tumor suppression. With the roles of epigenetic regulation of bone cells emerged in past decades, the property and molecular mechanism of DZNep on enhancing osteogenesis had been reported and attracted a great deal of attention recently. This study aims to elucidate the role of DZNep on EZH2-H3K27me3 axis and downstream factors during both osteoclasts and osteoblasts formation and the therapeutic possibility of DZNep on bone defect healing. Methods Bone marrow-derived macrophages (BMMs) cells were cultured, and their responsiveness to DZNep was evaluated by cell counting kit-8, TRAP staining assay, bone resorption assay, podosome actin belt. Bone marrow-derived mesenchymal stem cells (BMSC) were cultured and their responsiveness to DZNep was evaluated by cell counting kit-8, ALP and AR staining assay. The expression of nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), Wnt signaling pathway was determined by qPCR and western blotting. Mouse bone defect models were created, rescued by DZNep injection, and the effectiveness was evaluated by X-ray and micro-CT and histological staining. Results Consistent with the previous study that DZNep enhances osteogenesis via Wnt family member 1(Wnt1), Wnt6, and Wnt10a, our results showed that DZNep also promotes osteoblasts differentiation and mineralization through the EZH2-H3K27me3-Wnt4 axis. Furthermore, we identified that DZNep promoted the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast formation via facilitating the phosphorylation of IKKα/β, IκB, and subsequently NF-κB nuclear translocation, which credit to the EZH2-H3K27me3-Foxc1 axis. More importantly, the enhanced osteogenesis and osteoclastogenesis result in accelerated mice bone defect healing in vivo. Conclusion DZNep targeting EZH2-H3K27me3 axis facilitated the healing of mice bone defect via simultaneously enhancing osteoclastic bone resorption and promoting osteoblastic bone formation.

Inhibition of JAK2/STAT3 signaling suppresses bone marrow stromal cells proliferation and osteogenic differentiation, and impairs bone defect healing

2018 ◽  
Vol 399 (11) ◽  
pp. 1313-1323 ◽  
Author(s):  
Xin Yu ◽  
Zhi Li ◽  
Qilong Wan ◽  
Xin Cheng ◽  
Jing Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Bone Defect ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Defect Healing ◽  
Stat3 Signaling ◽  
Bone Defect Healing

Abstract Mesenchymal stem cells (MSCs) undergo osteogenic differentiation during bone defect healing. However, the role of JAK2/STAT3 in the osteogenic differentiation of MSCs and bone defect healing is still not fully understood. In this study, we aimed to analyze the effect of AG490, a JAK2-specific inhibitor, on MSCs proliferation and osteogenic differentiation as well as in bone defect healing. We used AG490 to inhibit the JAK2/STAT3 signaling in a mice bone marrow stromal cells (BMSCs) culture. AG490 inhibited BMSCs proliferation and osteogenic differentiation markers, i.e. Col1α, Alp and Ocn expression in mRNA and protein levels. Inhibition of JAK2 reduced ALP activity and matrix mineralization in BMSCs culture. Inhibition of JAK2 reduced phosphorylation of STAT3, AKT, P38, and JNK phosphorylation. Immunohistochemistry showed high numbers of pJAK2, pSTAT3 and ALP positive cells and AG490 reduced this effect in vivo. Histology and μ-computed tomography (CT) data showed that AG490 treatment inhibits bone regeneration and bone defect healing. Our results clearly showed the inhibitory effect of AG490 on proliferation and osteogenic differentiation of BMSCs, bone regeneration and bone defect healing. Moreover, AG490 inhibited phosphorylation of STAT3, P38, JNK and AKT. This suggests the possible role of JAK2/STAT3 signaling in hypoxia-induced osteogenic differentiation of MSCs and bone defect healing.

scholarly journals IKKα plays a major role in canonical NF-kB signalling in colorectal cells

2022 ◽  
Author(s):  
Jack A Prescott ◽  
Kathryn Balmanno ◽  
Jennifer P Mitchell ◽  
Hanneke Okkenhaug ◽  
Simon J Cook
Keyword(s):  
Cell Lines ◽  
Nuclear Translocation ◽  
Transcriptional Response ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Knock Out ◽  
Iκb Kinase ◽  
Sw620 Cells ◽  
Hct116 Cells

Inhibitor of kappa B (IκB) kinase β (IKKβ) has long been viewed as the dominant IKK in the canonical nuclear factor-κB (NF-κB) signalling pathway, with IKKα being more important in non-canonical NF-κB activation. Here we have investigated the role of IKKα and IKKβ in canonical NF-κB activation in colorectal cells using CRISPR-Cas9 knock-out cell lines, siRNA and selective IKKβ inhibitors. IKKα and IKKβ were redundant for IκBα phosphorylation and turnover since loss of IKKα or IKKβ alone had little (SW620 cells) or no (HCT116 cells) effect. However, in HCT116 cells IKKα was the dominant IKK required for basal phosphorylation of p65 at S536, stimulated phosphorylation of p65 at S468, nuclear translocation of p65 and the NF-κB-dependent transcriptional response to both TNFα and IL-1α. In these cells IKKβ was far less efficient at compensating for the loss of IKKα than IKKα was able to compensate for the loss of IKKβ. This was confirmed when siRNA was used to knock-down the non-targeted kinase in single KO cells. Critically, the selective IKKβ inhibitor BIX02514 confirmed these observations in WT cells and similar results were seen in SW620 cells. Notably, whilst IKKα loss strongly inhibited TNFα-dependent p65 nuclear translocation, IKKα and IKKβ contributed equally to c-Rel nuclear translocation indicating that different NF-κB subunits exhibit different dependencies on these IKKs. These results demonstrate a major role for IKKα in canonical NF-κB signalling in colorectal cells and may be relevant to efforts to design IKK inhibitors, which have focused largely on IKKβ to date.

scholarly journals Activation of nuclear factor-κB during doxorubicin-induced apoptosis in endothelial cells and myocytes is pro-apoptotic: the role of hydrogen peroxide

2002 ◽  
Vol 367 (3) ◽  
pp. 729-740 ◽  
Author(s):  
Suwei WANG ◽  
Srigiridhar KOTAMRAJU ◽  
Eugene KONOREV ◽  
Shasi KALIVENDI ◽  
Joy JOSEPH ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Nuclear Translocation ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Apoptotic Pathway ◽  
Toxic Side Effect ◽  
Rat Cardiomyocytes ◽  
A Cell ◽  
Induced Apoptosis

Doxorubicin (DOX) is a widely used anti-tumour drug. Cardiotoxicity is a major toxic side effect of DOX therapy. Although recent studies implicated an apoptotic pathway in DOX-induced cardiotoxicity, the mechanism of DOX-induced apoptosis remains unclear. In the present study, we investigated the role of reactive oxygen species and the nuclear transcription factor nuclear factor κB (NF-κB) during apoptosis induced by DOX in bovine aortic endothelial cells (BAECs) and adult rat cardiomyocytes. DOX-induced NF-κB activation is both dose- and time-dependent, as demonstrated using electrophoretic mobility-shift assay and luciferase and p65 (Rel A) nuclear-translocation assays. Addition of a cell-permeant iron metalloporphyrin significantly suppressed NF-κB activation and apoptosis induced by DOX. Overexpression of glutathione peroxidase, which detoxifies cellular H2O2, significantly decreased DOX-induced NF-κB activation and apoptosis. Inhibition of DOX-induced NF-κB activation by a cell-permeant peptide SN50 that blocks translocation of the NF-κB complex into the nucleus greatly diminished DOX-induced apoptosis. Apoptosis was inhibited when IκB mutant vector, another NF-κB inhibitor, was added to DOX-treated BAECs. These results suggest that NF-κB activation in DOX-treated endothelial cells and myocytes is pro-apoptotic, in contrast with DOX-treated cancer cells, where NF-κB activation is anti-apoptotic. Removal of intracellular H2O2 protects endothelial cells and myocytes from DOX-induced apoptosis, possibly by inhibiting NF-κB activation. These findings suggest a novel mechanism for enhancing the therapeutic efficacy of DOX.

scholarly journals Autologous bone marrow clot as an alternative to autograft for bone defect healing

2019 ◽  
Vol 8 (3) ◽  
pp. 107-117 ◽  
Author(s):  
Z. X. H. Lim ◽  
B. Rai ◽  
T. C. Tan ◽  
A. K. Ramruttun ◽  
J. H. Hui ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Defect ◽  
Autologous Bone Marrow ◽  
Defect Healing ◽  
Bone Defect Healing ◽  
Autologous Bone

scholarly journals Role of Prostaglandin E in Receptor Activator of Nuclear Factor-κB Ligand (RANKL) Expression in Osteoblasts Induced by Cell Adhesion to Bone Marrow B-lymphocytes

2009 ◽  
Vol 55 (5) ◽  
pp. 832-837 ◽  
Author(s):  
Michiko Hirata ◽  
Suguru Harada ◽  
Chiho Matsumoto ◽  
Morichika Takita ◽  
Chisato Miyaura ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Adhesion ◽  
B Lymphocytes ◽  
Nuclear Factor Κb ◽  
Prostaglandin E ◽  
Nuclear Factor ◽  
Rankl Expression ◽  
Receptor Activator

scholarly journals Role of osteogenic Dickkopf-1 in bone remodeling and bone healing in mice with type I diabetes mellitus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nick Hildebrandt ◽  
Juliane Colditz ◽  
Caio Dutra ◽  
Paula Goes ◽  
Juliane Salbach-Hirsch ◽  
...  
Keyword(s):  
Bone Loss ◽  
Bone Formation ◽  
Bone Defect ◽  
Serum Levels ◽  
Diabetic Control ◽  
Male Mice ◽  
Defect Healing ◽  
Bone Defect Healing ◽  
Dickkopf 1

AbstractType 1 diabetes mellitus (T1DM) is associated with low bone mass and a higher risk for fractures. Dickkopf-1 (Dkk1), which inhibits Wnt signaling, osteoblast function, and bone formation, has been found to be increased in the serum of patients with T1DM. Here, we investigated the functional role of Dkk1 in T1DM-induced bone loss in mice. T1DM was induced in 10-week-old male mice with Dkk1-deficiency in late osteoblasts/osteocytes (Dkk1f/f;Dmp1-Cre, cKO) and littermate control mice by 5 subsequent injections of streptozotocin (40 mg/kg). Age-matched, non-diabetic control groups received citrate buffer instead. At week 12, calvarial defects were created in subgroups of each cohort. After a total of 16 weeks, weight, fat, the femoral bone phenotype and the area of the bone defect were analyzed using µCT and dynamic histomorphometry. During the experiment, diabetic WT and cKO mice did not gain body weight compared to control mice. Further they lost their perigonadal and subcutaneous fat pads. Diabetic mice had highly elevated serum glucose levels and impaired glucose tolerance, regardless of their Dkk1 levels. T1DM led to a 36% decrease in trabecular bone volume in Cre− negative control animals, whereas Dkk1 cKO mice only lost 16%. Of note, Dkk1 cKO mice were completely protected from T1DM-induced cortical bone loss. T1DM suppressed the bone formation rate, the number of osteoblasts at trabecular bone, serum levels of P1NP and bone defect healing in both, Dkk1-deficient and sufficient, mice. This may be explained by increased serum sclerostin levels in both genotypes and the strict dependence on bone formation for bone defect healing. In contrast, the number of osteoclasts and TRACP 5b serum levels only increased in diabetic control mice, but not in Dkk1 cKO mice. In summary, Dkk1 derived from osteogenic cells does not influence the development of T1DM but plays a crucial role in T1DM-induced bone loss in male mice by regulating osteoclast numbers.

scholarly journals Overexpression of γ-Glutamyltransferase in Transgenic Mice Accelerates Bone Resorption and Causes Osteoporosis

Endocrinology ◽  
2007 ◽  
Vol 148 (6) ◽  
pp. 2708-2715 ◽  
Author(s):  
Kiyoshi Hiramatsu ◽  
Yutaro Asaba ◽  
Sunao Takeshita ◽  
Yuji Nimura ◽  
Sawako Tatsumi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Resorption ◽  
Transgenic Mice ◽  
Bone Destruction ◽  
Nuclear Factor Κb ◽  
Osteoclast Formation ◽  
Expression Cloning ◽  
Nuclear Factor ◽  
Receptor Activator

We previously identified γ-glutamyltransferase (GGT) by expression cloning as a factor inducing osteoclast formation in vitro. To examine its pathogenic role in vivo, we generated transgenic mice that overexpressed GGT in a tissue-specific manner utilizing the Cre-loxP recombination system. Systemic as well as local production of GGT accelerated osteoclast development and bone resorption in vivo by increasing the sensitivity of bone marrow macrophages to receptor activator of nuclear factor-κB ligand, an essential cytokine for osteoclastogenesis. Mutated GGT devoid of enzyme activity was as potent as the wild-type molecule in inducing osteoclast formation, suggesting that GGT acts not as an enzyme but as a cytokine. Recombinant GGT protein increased receptor activator of nuclear factor-κB ligand expression in marrow stromal cells and also stimulated osteoclastogenesis from bone marrow macrophages at lower concentrations. Thus, GGT is implicated as being involved in diseases characterized by accelerated osteoclast development and bone destruction and provides a new target for therapeutic intervention.

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