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Abstract: Excessive glucocorticoid administration accelerates osteoblast apoptosis and skeletal deterioration. Heat shock proteins (HSPs) regulate metabolic activities in osteoblastic cells. This study characterized the biological significance of HSP60 in glucocorticoid-induced bone loss. Rats were treated with glucocorticoid, HSP60 antisense oligonucleotides, or adenovirus-mediated HSP60 gene transfer. Bone mineral density, metaphyseal trabecular micro-architecture, and fragility were analyzed by dual X-ray absorptiometry, micro-computed tomography, and material testing, respectively. Differential proteomic profiles of bone tissue extracts were detected by bi-dimensional electrophoresis and mass spectrometry. Survival and proapoptotic signal transduction were quantified by immunoblotting. Glucocorticoid-treated rats had low bone mineral density and metaphyseal trabecular microstructure in association with downregulation of collagen 1α1 and HSP60 expressions in bone tissue. Gain of HSP60 function by adenovirus-mediated HSP60 gene transfer abrogated the deleterious effects of glucocorticoid treatment on bone mass, trabecular microstructure, and mechanical strength. Enhancement of HSP60 signaling attenuated the glucocorticoid-induced loss of trabecular bone volume, mineral acquisition reactions and osteoblast surface. HSP60 gene transfer activated ERK and Akt and reduced Bax and cytochrome c release, as well as caspase-3 cleavage, which attenuated the inhibitory effects of glucocorticoid treatment on osteoblast survival. Loss of HSP60 function by HSP60 antisense oligonucleotides accelerated mitochondrial apoptotic programs and osteoblast apoptosis. Knockdown of HSP60 induced loss of bone mass, micro-architecture integrity, and mechanical property. Taken together, loss of HSP60 signaling contributes to the glucocorticoid-induced enhancement of pro-apoptotic reactions, thereby accelerating osteoblast apoptosis and bone mass loss. Enhancement of HSP60 function is beneficial for protecting bone tissue against the glucocorticoid-induced inhibition of bone cell viability and bone formation. [Copyright &y& Elsevier]

Abstract: Aims: Long-term glucocorticoid administration is known to induce bone deterioration. Glycogen synthase kinase-3β (GSK-3β) signaling reportedly participates in bone remodeling. This study investigated whether GSK-3β inhibitor could regulate glucocorticoid-induced inhibition of osteoblast differentiation in vitro or bone mass in vivo. Main methods: MC3T3-E1 osteoblasts were treated with kinase-inactive GSK-3β mutant and 6-bromoindirubin-3′-oxim (BIO) and then exposed to 1µM dexamethasone. Survival and osteoblast differentiation of cell cultures were assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling, quantitative RT-PCR, and von Kossa staining. Mineral density, biomechanical properties and microenvironments of BIO- and glucocorticoid-treated rat bone tissues were analyzed using dual-energy X-ray absorptiometry, material testing, and histomorphometry, respectively. Key findings: Glucocorticoid decreased levels of phosphorylated Ser9-GSK-3β and β-catenin in osteoblast cultures. Kinase-inactive GSK-3β mutant and BIO treatments attenuated dexamethasone-induced inhibition of β-catenin, Runx2 abundance, and osteoblast differentiation but suppressed glucocorticoid-induced apoptosis of cell cultures. Exogenous BIO treatment alleviated methylprednisolone-induced impairment of mineral density, biomechanical strength, trabecular bone volume, osteoblast surface, and bone formation rate of rat bone tissue. BIO treatment also attenuated glucocorticoid-induced promotion of osteoclast surface and marrow adipocyte volume in bone tissue. Bone cells adjacent to glucocorticoid-stressed bone tissue displayed strong phosphorylated Ser9-GSK-3β and β-catenin immunostaining following BIO treatment. Significance: Inhibition of GSK-3β abrogated glucocorticoid-induced bone loss by increasing β-catenin- and Runx2-mediated osteoblast differentiation. Controlling GSK-3β signaling in bone cells may be a strategy for preventing glucocorticoid-induced osteopenia. [Copyright &y& Elsevier]

Abstract: Excess glucocorticoid treatment induces loss of osteoblast differentiation. Post-translational modification of β-catenin reportedly regulates osteogenic activities in bone cells. This study was undertaken to test whether miR-29a signaling regulates the acetylation status of β-catenin in the glucocorticoid-mediated osteoblast dysfunction. Murine osteoblast cultures were incubated under osteogenic conditions with or without supraphysiological glucocorticoid, miR-29a precursor, antisense oligonucleotides or histone deacetylase 4 (HDAC4) RNA interferences. Osteoblast differentiation was determined by alkaline phosphatase activity, calcium deposition, and von Kossa stain. β-Catenin acetylation and miR-29a transcription were detected by immunoblotting, chromatin immunoprecipitation and quantitative PCR. Protein interaction was detected by fluorescence protein ligation assay. Supraphysiological glucocorticoid treatment repressed osteoblast differentiation and induced loss of miR-29a expression and acetylated β-catenin levels in osteoblast cultures. Gain of miR-29a function attenuated the deleterious effects of glucocorticoid on osteogenic gene expression and mineralized nodule formation, whereas knockdown of miR-29a signaling accelerated loss of osteoblast differentiation capacity. miR-29a reduced HDAC4 signaling and attenuated the glucocorticoid-mediated β-catenin deacetylation and ubiquitination and restored nuclear β-catenin levels. Glucocorticoid-induced loss of miR-29a signaling occurred through transcriptional and translational regulation. Interruption of HDAC4 signaling attenuated the glucocorticoid-induced hypoacetylation of histone H3 at lysine 9 (H3K9Ac) and restored the enrichment of H3K9Ac in miR-29a proximal promoter region and miR-29a transcription in cell cultures. Taken together, excess glucocorticoid-induced loss of miR-29a signaling accelerates β-catenin deacetylation and ubiquitination that impairs osteogenic activities of osteoblast cultures. miR-29a and HDAC4 reciprocal regulation of H3K9 acetylation contributes to the acetylation status of β-catenin and miR-29a expression. Enhancement of miR-29a signaling is an alternative strategy for protecting against the adverse actions of excess glucocorticoid on differentiation capacity of osteogenic cells. [Copyright &y& Elsevier]

Abstract: Supraphysiological glucocorticoid administration accelerates loss of survival and differentiation in osteoblastic cells, thereby increasing the risks of osteopenic or osteonecrotic disorders. Neuroendocrine component type 1 cannabinoid receptor (CB1) is found to regulate bone mass. This study characterized the biological role of CB1 in glucocorticoid-induced suppression of osteoblast differentiation. Murine MC3T3-E1 osteoblasts were incubated under osteogenic conditions in the presence or absence of 1μM glucocorticoid, RNA interference, CB1 antagonist AM251, and agonist WIN55212-2. Cell survival was detected by formazan synthesis and TUNEL staining. Osteoblast differentiation was quantified by mineralized matrix accumulation and expression of the osteogenic factors Runx2 and osteocalcin. Expression of signaling molecules was assessed by immunoblotting. Glucocorticoid increased CB1 expression in association with decreased osteocalcin expression and mineralized nodule deposition. CB1 RNA interference and AM251 attenuated the deleterious actions of glucocorticoid treatment on survival and osteogenic activities, whereas activating CB1 by WIN55212-2 impaired osteoblast differentiation. CB1 signaling regulated JNK, ERK, GSK-3β, and Akt activation as well as Runx2 and IGF-I expression. Inhibition of GSK-3β by the kinase-inactive GSK-3β mutant or activation of ERK by the active MEK-1 mutant abrogated glucocorticoid-induced inhibition of osteoblast differentiation. Glucocorticoid-induced CB1 expression occurred via glucocorticoid receptor-dependent transcriptional and translational regulation. Gain of Runx2 function and loss of MKP-1 action attenuated glucocorticoid-induced enhancement of CB1 expression. Taken together, CB1 regulation of ERK and GSK-3β-dependent pathways participates in glucocorticoid inhibition of Runx2 signaling and osteoblast differentiation. Runx2 reciprocally regulates glucocorticoid-induced promotion of CB1 signaling. Our findings provide new insights into the role of the neuroendocrine component CB1 in glucocorticoid-induced osteoblast dysfunction. [Copyright &y& Elsevier]