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Excess glucocorticoid administration impairs osteogenic activities, which raises the risk of osteoporotic disorders. Epigenetic methylation of DNA and histone regulates the lineage commitment of progenitor cells. This study was undertaken to delineate the actions of histone lysine demethylase 6a (UTX) with regard to the glucocorticoid impediment of osteogenic differentiation. Osteogenic progenitor cells responded to supraphysiological glucocorticoid by elevating CpG dinucleotide methylation proximal to transcription start sites within Runx2 and osterix promoters and Wnt inhibitor Dickkopf-1 (Dkk1) expression concomitant with low UTX expression. 5′-Aza-deoxycystidine demethylation of Runx2 and osterix promoters abolished the glucocorticoid inhibition of mineralized matrix accumulation. Gain of UTX function attenuated the glucocorticoid-induced loss of osteogenic differentiation, whereas UTX silencing escalated adipogenic gene expression and adipocyte formation. UTX sustained osteogenic gene transcription through maintaining its occupancy to Runx2 and osterix promoters. It also mitigated the trimethylation of histone 3 at lysine 27 (H3K27me3), which reduced H3K27me3 enrichment to Dkk1 promoter and thereby lowered Dkk1 transcription. Modulation of β-catenin and Dkk1 actions restored UTX signaling in glucocorticoid-stressed cells. In vivo, UTX inhibition by exogenous methylprednisolone and GSK-J4 administration, an effect that disturbed H3K27me3, β-catenin, Dkk1, Runx2, and osterix levels, exacerbated trabecular microarchitecture loss and marrow adiposity. Taken together, glucocorticoid reduction of UTX function hindered osteogenic differentiation. Epigenetic hypomethylation of osteogenic transcription factor promoters and H3K27 contributed to the UXT alleviation of Dkk1 transcription and osteogenesis in glucocorticoid-stressed osteogenic progenitor cells. Control of UTX action has an epigenetic perspective of curtailing glucocorticoid impairment of osteogenic differentiation and bone mass. Key messages: [ABSTRACT FROM AUTHOR]

Abstract: Dickkopf-1 (DKK1) has been found to act as a potent Wnt signaling-inhibitory factor for regulating skeletal disorders. We investigated whether modulation of DKK1 expression by end-capped phosphorothioate DKK1 antisense oligonucleotide could alter estrogen loss-induced bone loss. Ovariectomized or sham-operated rats were given 20 μg/kg/day DKK1 sense or antisense oligonucleotide or vehicle for 28 days. Femurs and tibiae were dissected to assess bone mass, biomechanical strength, immunohistochemistry and ex vivo osteoclast formation. We found that DKK1 antisense oligonucleotide significantly abrogated the suppressing effect of ovariectomy on weight, mineral content, mineral density and peak load of femurs. DKK1 antisense oligonucleotide treatment reduced ovariectomy promotion of ex vivo osteoclast differentiation of primary M-CSF-dependent bone marrow macrophages. Histomorphometric observation demonstrated that DKK1 antisense oligonucleotide treatment increased osteoblast number and impaired ovariectomy-promoted trabecular bone loss and osteoclast number in bone tissue. Osteoblastic cells adjacent to endosteum of trabecular bone and chondrocytes at calcified cartilage expressed intensive DKK1 and RANKL and weak OPG immunostaining in ovariectomized rat bone microenvironments. Osteogenic cells and chondral cells displayed weak DKK1, RANKL and OPG expression of bone tissue after DKK1 antisense oligonucleotide treatment. Taken together, attenuation of DKK1 expression in ovariectomized rat bone tissue alleviated loss of bone mass and biomechanical property. The regulatory action of DKK1 antisense oligonucleotide treatment on bone tissue appeared to suppress the promoting effect of estrogen deficiency on osteoclastogenesis-stimulatory factor RANKL expression and osteoclast differentiation. Control of DKK1 signaling can be used in the future as an alternative strategy for protecting estrogen deficiency induction of bone loss. [Copyright &y& Elsevier]

Vascular endothelial growth factor (VEGF) is an important regulator for angiogenesis and endochondral bone formation. Although low-intensity pulsed ultrasound (US) has been recently used for accelerating fracture healing, the effect of US stimulation on angiogenic factor production by osteoblasts remains undetermined. Here, we found that US elevation of VEGF-A expression in human osteoblasts to be mediated by nitric oxide (NO) and hypoxia-inducible factor-1α (HIF-1α). Human osteoblasts were treated with or without US stimulation (200 μs pulse, 1 kHz at 30 mW/cm2) for 20 min. Cells were subjected to assessment of VEGF-A expression, NO production, nitric oxide synthase (NOS) catalytic activities, and HIF-1α transactivation. Results showed that US significantly increased VEGF-A mRNA and protein levels in 6 h. US augmentation of VEGF level was transcriptionally mediated. Early inhibition of NO production, but not calcium or prostaglandin E2, significantly reduced US-enhanced VEGF-A levels. Osteoblasts responded to US treatment by increasing NO production, NOS catalytic activities, iNOS immunoexpression, nuclear HIF-1α activation, and binding to the VEGF-A promoter. Inhibition of NOS activity by N-nitro-l-arginine methyl ester (l-NAME) or blockade of guanylate cyclase activity by ODQ reduced US-augmented HIF-1α transactivation and VEGF-A levels. Conditioned medium harvested from US-treated osteoblasts promoted tube formation of human umbilical vein endothelial cells (HUVEC). Monoclonal VEGF-A antibody neutralization or l-NAME pretreatment reduced the promoting effect of conditioned medium on angiogenesis of HUVEC. Together, these findings show that NO plays an important role in mediating extracellular stimuli released by US and triggering intracellular response of osteoblasts to produce angiogenic factor after US treatment. [Copyright &y& Elsevier]

Glucocorticoid provokes bone mass loss and fatty marrow, accelerating osteoporosis development. Bromodomain protein BRD4, an acetyl–histone-binding chromatin reader, regulates stem cell and tissue homeostasis. We uncovered that glucocorticoid inhibited acetyl Lys-9 at the histone 3 (H3K9ac)-binding Runx2 promoter and decreased osteogenic differentiation, whereas bromodomain protein 4 (BRD4) and adipocyte formation were upregulated in bone-marrow mesenchymal progenitor cells. BRD4 knockdown improved H3K9ac occupation at the Runx2 promoter and osteogenesis, but attenuated glucocorticoid-mediated adipocyte formation together with the unaffected H3K9ac-binding PPARγ2 promoter. BRD4 regulated epigenome related to fatty acid metabolism and the forkhead box P1 (Foxp1) pathway, which occupied the PPARγ2 promoter to modulate glucocorticoid-induced adipocytic activity. In vivo, BRD4 inhibitor JQ-1 treatment mitigated methylprednisolone-induced suppression of bone mass, trabecular microstructure, mineral acquisition, and osteogenic differentiation. Foxp1 signaling, marrow fat, and adipocyte formation in glucocorticoid-treated skeleton were reversed upon JQ-1 treatment. Taken together, glucocorticoid-induced H3K9 hypoacetylation augmented BRD4 action to Foxp1, which steered mesenchymal progenitor cells toward adipocytes at the cost of osteogenic differentiation in osteoporotic skeletons. BRD4 inhibition slowed bone mass loss and marrow adiposity. Collective investigations convey a new epigenetic insight into acetyl histone reader BRD4 control of osteogenesis and adipogenesis in skeleton, and highlight the remedial effects of the BRD4 inhibitor on glucocorticoid-induced osteoporosis. [ABSTRACT FROM AUTHOR]

Purpose: MicroRNAs (miRNAs) are associated with various pathologic conditions and can serve as diagnostic or therapeutic biomarkers. This study tried to identify the differentially expressed miRNAs to predict the possible pathomechanisms involved in osteonecrosis of the femoral head (ONFH).Methods: We compared the peripheral blood miRNAs in 46 patients with ONFH and 85 healthy controls by microarray and droplet digital polymerase chain reaction (ddPCR). Putative interacted networks between the differentially responded miRNAs were analyzed by web-based bioinformatics prediction tools.Results: Microarray identified 51 differentially expressed miRNAs with at least twofold change (upregulation in 34 and downregulation in 17), and the results were validated by ddPCR using six selected miRNAs. Bioinformatics genetic network analysis focusing on the six miRNAs found the upregulated miR-18a and miR-19a are associated with angiogenesis after induction of ischemia; the upregulated miR-138-1 can inhibit osteogenic differentiation of mesenchymal stem cells; the most targeted genes, p53 and SERBP1, are associated with hypoxia and hypofibrinolysis.Conclusions: This study combined the miRNA analysis with the bioinformatics and predicts that hypoxia, inhibited osteogenesis of stem cells, and dysregulated angiogenesis might be orchestrated through the miRNA interacting circuits in the pathogenesis of ONFH. [ABSTRACT FROM AUTHOR]

Subchondral bone deterioration and osteophyte formation attributable to excessive mineralization are prominent features of end-stage knee osteoarthritis (OA). The cellular events underlying subchondral integrity diminishment remained elusive. This study was undertaken to characterize subchondral mesenchymal stem cells (SMSCs) isolated from patients with end-stage knee OA who required total knee arthroplasty. The SMSCs expressed surface antigens CD29, CD44, CD73, CD90, CD105, and CD166 and lacked CD31, CD45, and MHCII expression. The cell cultures exhibited higher proliferation and greater osteogenesis and chondrogenesis potencies, whereas their population-doubling time and adipogenic lineage commitment were lower than those of bone marrow MSCs (BMMSCs). They also displayed higher expressions of embryonic stem cell marker OCT3/4 and osteogenic factors Wnt3a, β-catenin, and microRNA-29a (miR-29a), concomitant with lower expressions of joint-deleterious factors HDAC4, TGF-β1, IL-1β, TNF-α, and MMP3, in comparison with those of BMMSCs. Knockdown of miR-29a lowered Wnt3a expression and osteogenic differentiation of the SMSCs through elevating HDAC4 translation, which directly regulated the 3′-untranslated region of HDAC4. Likewise, transgenic mice that overexpressed miR-29a in osteoblasts exhibited a high bone mass in the subchondral region. SMSCs in the transgenic mice showed a higher osteogenic differentiation and lower HDAC4 signaling than those in wild-type mice. Taken together, high osteogenesis potency existed in the SMSCs in the osteoarthritic knee. The miR-29a modulation of HDAC4 and Wnt3a signaling was attributable to the increase in osteogenesis. This study shed an emerging light on the characteristics of SMSCs and highlighted the contribution of SMSCs in the exacerbation of subchondral integrity in end-stage knee OA. Key messages: [ABSTRACT FROM AUTHOR]

Objective: Strong vascular endothelial growth factor (VEGF) expression of osteoprogenitors was found in callus site during fracture healing. The aim of this study was to investigate whether VEGF modulates the angiogenesis and osteogenesis in shockwave-promoted fracture healing in rabbits. Materials and Methods: Twenty-seven Japanese rabbits were used in the study. A fracture of left tibia with 5 mm gap was created, and the fracture was stabilized with an external fixator. The rabbits were randomly divided into three groups. Group I was the control group and received no shockwave therapy. Group II received shockwave therapy, and group III was pretreated with bevacizumab, a monoclonal antibody against VEGF, before receiving shockwave. Radiographs of the tibia were obtained at 1, 4, and 8 wk. Bone mineral density was performed at 8 wk. The rabbits were euthanized at 8 wk, and the bone specimens were subjected to histomorphological examination and immunohistochemical analysis. Results: At 8 wk, radiographs showed considerably better bone healing and remodeling of the fracture in group II compared with groups I and III, whereas no discernable difference was noted between group I and group III. The BMD values were significantly higher in group II than groups I and III, but no difference noted between group I and group III. In histomorphological examination, significant increases in bone tissue was were noted in group II compared with groups I and III, but no difference was noted between group I and group III. In immunohistochemical analysis, significant increases in VEGF, vWF, PCNA, BMP-2 and osteocalcin, and a decrease in TUNEL expression were observed in group II compared with groups I and III, but no statistical difference was noted between group I and group III. Conclusion: Significant increases in VEGF and angiogenic and osteogenic growth factors were noted in shockwave-promoted bone healing. Pre-treatment with bevacizumab inhibited VEGF and in turn, attenuated the effect of shockwave. It appears that VEGF modulates angiogenesis and osteogenesis in shockwave-promoted bone healing in rabbits. [Copyright &y& Elsevier]

Biophysical stimulation alters bone-forming cell activity, bone formation and remodeling. The effect of piezoelectric microvibration stimulation (PMVS) intervention on osteoporosis development remains uncertain. We investigated whether 60 Hz, 120 Hz, and 180 Hz PMVS (0.05 g, 20 min/stimulation, 3 stimulations/week for 4 consecutive weeks) intervention affected bone integrity in ovariectomized (OVX) mice or osteoblastic activity. PMVS (120 Hz)-treated OVX mice developed fewer osteoporosis conditions, including bone mineral density loss and trabecular microstructure deterioration together with decreased serum resorption marker CTX-1 levels, as compared to control OVX animals. The biomechanical strength of skeletal tissue was improved upon 120 Hz PMVS intervention. This intervention compromised OVX-induced sparse trabecular bone morphology, osteoblast loss, osteoclast overburden, and osteoclast-promoting cytokine RANKL immunostaining and reversed osteoclast inhibitor OPG immunoreactivity. Osteoblasts in OVX mice upon PMVS intervention showed strong Wnt3a immunoreaction and weak Wnt inhibitor Dkk1 immunostaining. In vitro, PMVS reversed OVX-induced loss in von Kossa-stained mineralized nodule formation, Runx2, and osteocalcin expression in primary bone-marrow stromal cells. PMVS also promoted mechanoreceptor Piezo1 expression together with increased microRNA-29a and Wnt3a expression, whereas Dkk1 rather than SOST expression was repressed in MC3T3-E1 osteoblasts. Taken together, PMVS intervention promoted Piezo1, miR-29a, and Wnt signaling to upregulate osteogenic activity and repressed osteoclastic bone resorption, delaying estrogen deficiency-induced loss in bone mass and microstructure. This study highlights a new biophysical remedy for osteoporosis. [ABSTRACT FROM AUTHOR]

Bone turnover is sophisticatedly balanced by a dynamic coupling of bone formation and resorption at various rates. The orchestration of this continuous remodeling of the skeleton further affects other skeletal tissues through organ crosstalk. Chronic excessive bone resorption compromises bone mass and its porous microstructure as well as proper biomechanics. This accelerates the development of osteoporotic disorders, a leading cause of skeletal degeneration-associated disability and premature death. Bone-forming cells play important roles in maintaining bone deposit and osteoclastic resorption. A poor organelle machinery, such as mitochondrial dysfunction, endoplasmic reticulum stress, and defective autophagy, etc., dysregulates growth factor secretion, mineralization matrix production, or osteoclast-regulatory capacity in osteoblastic cells. A plethora of epigenetic pathways regulate bone formation, skeletal integrity, and the development of osteoporosis. MicroRNAs inhibit protein translation by binding the 3′-untranslated region of mRNAs or promote translation through post-transcriptional pathways. DNA methylation and post-translational modification of histones alter the chromatin structure, hindering histone enrichment in promoter regions. MicroRNA-processing enzymes and DNA as well as histone modification enzymes catalyze these modifying reactions. Gain and loss of these epigenetic modifiers in bone-forming cells affect their epigenetic landscapes, influencing bone homeostasis, microarchitectural integrity, and osteoporotic changes. This article conveys productive insights into biological roles of DNA methylation, microRNA, and histone modification and highlights their interactions during skeletal development and bone loss under physiological and pathological conditions. [ABSTRACT FROM AUTHOR]