Correction: Chaperonin 60 sustains osteoblast autophagy and counteracts glucocorticoid aggravation of osteoporosis by chaperoning RPTOR.

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Chaperonin 60 sustains osteoblast autophagy and counteracts glucocorticoid aggravation of osteoporosis by chaperoning RPTOR.

Glucocorticoid excess medication interrupts osteoblast homeostasis and exacerbates bone mass and microstructure loss ramping up the pathogenesis of osteoporotic disorders. Heat shock protein 60 (HSP60) is found to maintain protein function within cellular microenvironment upon encountering detrimental stress. In this study, we revealed that supraphysiological dexamethasone decreased HSP60 expression along with deregulated autophagy in osteoblasts cultures. This chaperonin is required to sustain autophagic markers Atg4, and Atg12 expression, LC3-II conversion, and autophagic puncta formation, and alleviated the glucocorticoid-induced loss of osteogenic gene expression and mineralized matrix accumulation. Regulator-associated protein of mTOR complex 1 (RPTOR) existed in HSP60 immunoprecipitate contributing to the HSP60-promoted autophagy and osteogenesis because knocking down RPTOR impaired autophagic influx and osteogenic activity. HSP60 shielded from RPTOR dysfunction by reducing the glucocorticoid-induced RPTOR de-phosphorylation, aggregation, and ubiquitination. In vivo, forced RPTOR expression attenuated the methylprednisolone-induced loss of osteoblast autophagy, bone mass, and trabecular microstructure in mice. HSP60 transgenic mice displayed increased cortical bone, mineral acquisition, and osteoblast proliferation along with higher osteogenesis of bone marrow mesenchymal cells than those of wild-type mice. HSP60 overexpression retained RPTOR signaling, sustained osteoblast autophagy, and compromised the severity of glucocorticoid-induced bone loss and sparse trabecular histopathology. Taken together, HSP60 is essential to maintain osteoblast autophagy, which facilitates mineralized matrix production. It fends off glucocorticoid-induced osteoblast apoptosis and bone loss by stabilizing RPTOR action to autophagy. This study offers a new insight into the mechanistic by which chaperonin protects against the glucocorticoid-induced osteoblast dysfunction and bone loss.

MicroRNA-128a represses chondrocyte autophagy and exacerbates knee osteoarthritis by disrupting Atg12.

Chondrocyte loss is a prominent feature of osteoarthritis (OA). Autophagy is indispensable in maintaining the metabolic activities of cells exposed to deleterious stress. The contribution of microRNA signaling to chondrocyte autophagy in OA development remains elusive. We uncovered an association between poor autophagy and increased miR-128a expressions in articular chondrocytes of patients with end-stage knee OA and in a rat anterior cruciate ligament transection (ACLT) model for OA development. Cartilage matrix degradation and severe OA histopathology was evident upon forced miR-128a expression within the articular compartment. Intra-articular injections with miR-128a antisense oligonucleotide stabilized chondrocyte autophagy and slowed ACLT-mediated articular tissue destruction, including cartilage erosion, synovitis, osteophyte formation, and subchondral plate damage. In vitro, miR-128 signaling hindered Atg12 expression, LC3-II conversion, and autophagic puncta formation through targeting the 3'-untranslated region of Atg12. It increased apoptotic programs, diminishing cartilage formation capacity of articular chondrocytes. Inactivating histone methyltransferase EZH2 reduced methyl histone H3K27 enrichment in the miR-128a promoter and upregulated miR-128a transcription in inflamed chondrocytes. Taken together, miR-128a-induced Atg12 loss repressed chondrocyte autophagy to aggravate OA progression. EZH2 inactivation caused H3K27 hypomethylation to accelerate miR-128a actions. Interruption of miR-128a signaling attenuated chondrocyte dysfunction and delayed OA development. Our data provide new insights into how miR-128a signaling affects chondrocyte survival and articular cartilage anabolism and highlight the potential of miR-128a targeting therapy to alleviate knee OA.

Complement component C3: Serologic signature for osteogenesis imperfecta. Analysis of a comparative proteomic study.

BACKGROUND/PURPOSE: Osteogenesis imperfecta (OI) is a disease characterized by low bone mass and bony fragility. This study investigated the serum proteomic profiles and their correlation with bone density for OI cases. METHODS: Twenty OI patients and 20 control participants were included. Comparative serum proteomic profiles were analyzed by two-dimensional electrophoresis and tandem mass spectrometry. Serum protein levels were measured by enzyme-linked immunosorbent assay. Cutoff values and areas under the curve were estimated by the receiver operating characteristic curve. Bone mineral density data was obtained from all OI patients. RESULTS: Candidate proteins identified by electrophoresis were complement component C3 (C3), vitamin D-binding protein (DBP), and haptoglobin (HP). Enzyme-linked immunosorbent assay validation showed that OI patients had decreased C3 and DBP and increased HP. The results were not affected by age or bisphosphonate use. Serum C3 levels significantly correlated with bone mineral density of the lumbar spine and hip. C3 had the greatest areas under the curve to distinguish OI from healthy controls. CONCLUSION: Serum C3, DBP, and HP are emerging serologic signatures for OI. Concentrations of serum C3 correlated with the T score of OI patients. C3 had the greatest areas under the curve of the three proteins to distinguish OI from healthy controls.

MicroRNA-29a promotion of nephrin acetylation ameliorates hyperglycemia-induced podocyte dysfunction.

Podocyte dysfunction is a detrimental feature in diabetic nephropathy, with loss of nephrin integrity contributing to diabetic podocytopathy. MicroRNAs (miRs) reportedly modulate the hyperglycemia-induced perturbation of renal tissue homeostasis. This study investigated whether regulation of histone deacetylase (HDAC) actions and nephrin acetylation by miR-29 contributes to podocyte homeostasis and renal function in diabetic kidneys. Hyperglycemia accelerated podocyte injury and reduced nephrin, acetylated nephrin, and miR-29a levels in primary renal glomeruli from streptozotocin-induced diabetic mice. Diabetic miR-29a transgenic mice had better nephrin levels, podocyte viability, and renal function and less glomerular fibrosis and inflammation reaction compared with diabetic wild-type mice. Overexpression of miR-29a attenuated the promotion of HDAC4 signaling, nephrin ubiquitination, and urinary nephrin excretion associated with diabetes and restored nephrin acetylation. Knockdown of miR-29a by antisense oligonucleotides promoted HDAC4 action, nephrin loss, podocyte apoptosis, and proteinuria in nondiabetic mice. In vitro, interruption of HDAC4 signaling alleviated the high glucose-induced apoptosis and inhibition of nephrin acetylation in podocyte cultures. Furthermore, HDAC4 interference increased the acetylation status of histone H3 at lysine 9 (H3K9Ac), the enrichment of H3K9Ac in miR-29a proximal promoter, and miR-29a transcription in high glucose-stressed podocytes. In conclusion, hyperglycemia impairs miR-29a signaling to intensify HDAC4 actions that contribute to podocyte protein deacetylation and degradation as well as renal dysfunction. HDAC4, via epigenetic H3K9 hypoacetylation, reduces miR-29a transcription. The renoprotective effects of miR-29a in diabetes-induced loss of podocyte integrity and renal homeostasis highlights the importance of post-translational acetylation reactions in podocyte microenvironments. Increasing miR-29a action may protect against diabetic podocytopathy.

MicroRNA-29a ameliorates glucocorticoid-induced suppression of osteoblast differentiation by regulating ß-catenin acetylation.

Excess glucocorticoid treatment induces loss of osteoblast differentiation. Post-translational modification of ß-catenin reportedly regulates osteogenic activities in bone cells. This study was undertaken to test whether miR-29a signaling regulates the acetylation status of ß-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. ß-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 ß-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 ß-catenin deacetylation and ubiquitination and restored nuclear ß-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 ß-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 ß-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.

MicroRNA-29a protects against glucocorticoid-induced bone loss and fragility in rats by orchestrating bone acquisition and resorption.

OBJECTIVE: Excessive glucocorticoid treatment increases the incidence of osteopenia and osteonecrosis. MicroRNAs (miRNAs) reportedly target messenger RNA expression and regulate osteoblastogenesis and skeletal development. We undertook this study to investigate whether miR-29a regulates glucocorticoid-mediated bone loss. METHODS: Rats were given methylprednisolone, lentivirus-mediated miR-29a precursor, or lentivirus-mediated miR-29a inhibitor. Dual x-ray absorptiometry, micro-computed tomography, material testing, and enzyme-linked immunosorbent assay were performed to quantify bone mass, microarchitecture, peak load, and serum Dkk-1 levels. Differential miRNA expression profiles were detected using polymerase chain reaction arrays. The abundance of signaling molecules was assessed using immunoblotting. RESULTS: Glucocorticoid treatment induced loss of bone mineral density and trabecular microstructure in association with reduced miR-29a expression. Treatment with miR-29a precursor attenuated the adverse effects of glucocorticoid on bone mass, trabecular bone volume fraction, and biomechanical load-bearing capacity of bone tissue. Gain of miR-29a function alleviated the detrimental effects of glucocorticoid treatment on mineral acquisition and ex vivo osteoblast differentiation, and also reduced osteoclast surface, ex vivo osteoclast differentiation, and RANKL expression in bone microenvironments. Knockdown of miR-29a accelerated osteoclast resorption, cortical bone porosity, bone fragility, and loss of ex vivo osteogenic differentiation capacity. MicroRNA-29a regulated the abundance of Wnt signaling components (Wnt-3a, glycogen synthase kinase 3ß, and ß-catenin), the Wnt inhibitor Dkk-1, Akt, and phosphorylated ERK, and the expression of the osteogenic factors RUNX-2 and insulin-like growth factor 1 in bone tissue. CONCLUSION: MicroRNA-29a signaling protected against glucocorticoid-induced disturbance of Wnt and Dkk-1 actions and improved osteoblast differentiation and mineral acquisition. Promotion of miR-29a signaling is an alternative strategy for alleviating glucocorticoid-induced bone deterioration.

Cannabinoid receptor 1 mediates glucocorticoid-induced bone loss in rats by perturbing bone mineral acquisition and marrow adipogenesis.

OBJECTIVE: Prolonged glucocorticoid treatment increases the risk of osteopenic disorders. Bone loss and marrow fat accumulation are prominent features of glucocorticoid-induced skeletal destruction. Cannabinoid receptor 1 (CB(1) ) has been found to regulate energy expenditure and adipose tissue lipogenesis. We undertook this study to investigate whether CB(1) signaling regulates glucocorticoid-induced bone loss. METHODS: Rats were administered glucocorticoid, CB(1) antisense oligonucleotide, CB(1) sense oligonucleotide, or the CB(1) antagonist AM251. Bone mineral density, microstructure, biomechanical strength, and signaling transduction were assessed by dual x-ray absorptiometry, micro-computed tomography, material testing, and immunoblotting, respectively. Primary bone marrow stromal cells were isolated for assessment of ex vivo osteoblast and adipocyte differentiation. RESULTS: Glucocorticoid administration accelerated bone deterioration and fatty marrow formation in association with up-regulation of CB(1) expression. Genetic and pharmacologic blockade of CB(1) by CB(1) antisense oligonucleotide and AM251 attenuated the deleterious effects of glucocorticoid treatment on bone mineral density, trabecular microarchitecture, and mechanical properties. CB(1) antagonism improved osteoblast survival, osteoblast surface, and bone mineral acquisition, but abrogated marrow adiposity. Knockdown of CB(1) restored osteogenic differentiation capacity and attenuated the promoting effects of glucocorticoid on adipogenic differentiation in primary bone marrow mesenchymal cells. CB(1) signaling modulated ERK, JNK, and Akt activation as well as runt-related transcription factor 2 and peroxisome proliferator-activated receptor γ2 signaling. Adiponectin signaling was activated by CB(1) regulation of bone formation and fatty marrow. CONCLUSION: CB(1) mediates glucocorticoid-induced suppression of bone formation and marrow fat homeostasis. CB(1) antagonism reduces adipogenic and apoptotic reactions in bone microenvironments, thereby abrogating the deleterious effects of glucocorticoid treatment on bone integrity. Modulation of CB(1) signaling has therapeutic potential for preventing glucocorticoid-induced osteopenic disorders.

Cannabinoid receptor 1 regulates ERK and GSK-3ß-dependent glucocorticoid inhibition of osteoblast differentiation in murine MC3T3-E1 cells.

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 1 µM 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-3ß, and Akt activation as well as Runx2 and IGF-I expression. Inhibition of GSK-3ß by the kinase-inactive GSK-3ß 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-3ß-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.

Heat shock protein 60 protects skeletal tissue against glucocorticoid-induced bone mass loss by regulating osteoblast survival.

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.

Inhibition of glycogen synthase kinase-3beta attenuates glucocorticoid-induced bone loss.

AIMS: Long-term glucocorticoid administration is known to induce bone deterioration. Glycogen synthase kinase-3beta (GSK-3beta) signaling reportedly participates in bone remodeling. This study investigated whether GSK-3beta 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-3beta mutant and 6-bromoindirubin-3'-oxim (BIO) and then exposed to 1microM 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-3beta and beta-catenin in osteoblast cultures. Kinase-inactive GSK-3beta mutant and BIO treatments attenuated dexamethasone-induced inhibition of beta-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-3beta and beta-catenin immunostaining following BIO treatment. SIGNIFICANCE: Inhibition of GSK-3beta abrogated glucocorticoid-induced bone loss by increasing beta-catenin- and Runx2-mediated osteoblast differentiation. Controlling GSK-3beta signaling in bone cells may be a strategy for preventing glucocorticoid-induced osteopenia.

Comparative serum proteome expression of osteonecrosis of the femoral head in adults.

Osteonecrosis of the femoral head (ONFH) is a skeletal disorder characterized by ischemic deterioration, bone marrow edema and eventually femoral head collapse. The systemic regulation of ONFH in adult patients has not been examined. Serum proteomic is an innovative tool that potentially detects simultaneous expressions of serum proteins in pathological contexts. We compared the serum proteome profiles of 11 adult patients with ONFH (3 females and 8 males) and 11 healthy volunteers (3 females and 8 males). The proteins in the aliquots of sera were subjected to isoelectric focusing, two-dimensional gel electrophoresis and silver staining. The protein spots were matched and quantified using an imaging analysis system. The differentially expressed protein spots were subjected to in-gel trypsin digestion. The peptide mass fingerprints were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF) and a bioinformation search. We found that ONFH patients showed significantly higher abundances of kininogen 1 variant, complement factor C3 precursor, and complement factor H and lower levels of antithrombin III chain B, apolipoprotein A--IV precursor, and gelsolin isoform alpha precursor. These proteins of interest were reported to modulate thrombotic/fibrinolytic reactions, oxidative stress, vessel injury, tissue necrosis or cell apoptosis in several tissue types under pathological contexts. Taken together, the occurrence of ONFH was associated with various serum protein expressions. Our high--throughput serum proteomic findings indicated that multiple pathological reactions presumably occurred in ONFH.