Regulation Of Osteoblast Activity Research Articles

CHRONIC HIGH-FAT DIET CONSUMPTION ACCELERATES OSTEOBLAST DYSFUNCTION AND OSTEOPOROSIS BY GUT MICROBIAL METABOLITE TRIMETHYLAMINE-N-OXIDE

Obesity is correlated with the development of osteoporotic diseases. Gut microbiota-derived metabolite trimethylamine-n-oxide (TMAO) accelerates obesity-mediated tissue deterioration. This study was aimed to investigate what role TMAO may play in osteoporosis development during obesity.Mice were fed with high-fat diet (HFD; 60 kcal% fat) or chow diet (CD; 10 kcal% fat) or 0.2% TMAO in drinking water for 6 months. Body adiposis and bone microstructure were investigated using μCT imaging. Gut microbiome and serum metabolome were characterized using 16S rRNA sequencing and liquid chromatography-tandem mass spectrometry. Osteogenic differentiation of bone-marrow mesenchymal cells was quantified using RT-PCR and von Kossa staining. Cellular senescence was evaluated by key senescence markers p16, p21, p53, and senescence association β-galactosidase staining.HFD-fed mice developed hyperglycemia, body adiposis and osteoporosis signs, including low bone mineral density, sparse trabecular microarchitecture, and decreased biomechanical strength. HFD consumption induced gut microbiota dysbiosis, which revealed a high Firmicutes/Bacteroidetes ratio and decreased α-diversity and abundances of beneficial microorganisms Akkermansiaceae, Lactobacillaceae, and Bifidobacteriaceae. Serum metabolome uncovered increased serum L-carnitine and TMAO levels in HFD-fed mice. Of note, transplantation of fecal microbiota from CD-fed mice compromised HFD consumption-induced TMAO overproduction and attenuated loss in bone mass, trabecular microstructure, and bone formation rate. TMAO treatment inhibited trabecular and cortical bone mass and biomechanical characteristics; and repressed osteogenic differentiation capacity of bone-marrow mesenchymal cells. Mechanistically, TMAO accelerated mitochondrial dysfunction and senescence program, interrupted mineralized matrix production in osteoblasts.Gut microbial metabolite TMAO induced osteoblast dysfunction, accelerating the development of obesity-induced skeletal deterioration. This study, for the first time, conveys a productive insight into the catabolic role of gut microflora metabolite TMAO in regulating osteoblast activity and bone tissue integrity during obesity.

The miRNA-144-5p/IRS1/AKT axis regulates the migration, proliferation, and mineralization of osteoblasts: A mechanism of bone repair in diabetic osteoporosis.

Diabetic osteoporosis (DOP) is a disorder of bone metabolism induced by multiple mechanisms. Previous studies have revealed that microRNAs (miRNAs) play crucial roles in bone metabolism. MiRNA-144-5p has been proven to participate in the regulation of osteoblast activities; however, its specific mechanism in DOP has not been elucidated. This study investigated whether high glucose (HG) inhibited osteoblasts by regulating miRNA-144-5p. Our results showed that HG inhibited bone formation not only in vivo but also in vitro. We observed that HG severely hindered the migration, proliferation and mineralization of osteoblasts, while miRNA-144-5p was upregulated by way of the cell counting kit-8 assay, wound healing assay, alkaline phosphatase (ALP) activity assay and alizarin red staining. Double luciferase reporter experiments showed that miRNA-144-5p directly targeted insulin receptor substrate 1 (IRS1). The IRS1/AKT signaling pathway is closely related to osteoblasts' migration, proliferation, and mineralization. Silencing miRNA-144-5p promoted the mRNA, and protein expression of IRS1, thereby letting the expression of total AKT down, and then preventing phosphorylation of AKT into the nucleus to regulate migration, proliferation, and mineralization genes of osteoblasts. In conclusion, this study indicated that HG regulated the migration, proliferation, and mineralization of osteoblasts via the miRNA-144-5p/IRS1/AKT axis, which suggested a possible mechanism for DOP pathology.

Genetic analysis of osteoblast activity identifies Zbtb40 as a regulator of osteoblast activity and bone mass.

Osteoporosis is a genetic disease characterized by progressive reductions in bone mineral density (BMD) leading to an increased risk of fracture. Over the last decade, genome-wide association studies (GWASs) have identified over 1000 associations for BMD. However, as a phenotype BMD is challenging as bone is a multicellular tissue affected by both local and systemic physiology. Here, we focused on a single component of BMD, osteoblast-mediated bone formation in mice, and identified associations influencing osteoblast activity on mouse Chromosomes (Chrs) 1, 4, and 17. The locus on Chr. 4 was in an intergenic region between Wnt4 and Zbtb40, homologous to a locus for BMD in humans. We tested both Wnt4 and Zbtb40 for a role in osteoblast activity and BMD. Knockdown of Zbtb40, but not Wnt4, in osteoblasts drastically reduced mineralization. Additionally, loss-of-function mouse models for both genes exhibited reduced BMD. Our results highlight that investigating the genetic basis of in vitro osteoblast mineralization can be used to identify genes impacting bone formation and BMD.

Down-regulated microRNA-141 facilitates osteoblast activity and inhibits osteoclast activity to ameliorate osteonecrosis of the femoral head via up-regulating TGF-β2

ABSTRACT Osteonecrosis of the femoral head (ONFH) is a pathological process that initially occurs in the weight-bearing field of the femoral head. Due to the unknown pathogenesis, this study was for the investigation of the effect of microRNA-141 (miR-141) targeting transforming growth factor-β2 (TGF-β2) on regulating osteoblast activity and osteoclast activity in steroid-induced ONFH. Tissues of ONFH and normal femoral head were collected for detecting the expression of miR-141 and TGF-β2. A rat model of ONFH was constructed by injection of hormones, and transfected with miR-141 inhibitors and overexpressed TGF-β2. The apoptosis of bone cells was detected by TUNEL staining. The expression of osteoprotegerin (OPG), osteoprotegerin ligand (OPGL), Bcl-2, Bax, Runx2, BMP2 and RANK were detected. Highly expressed miR-141 and lowly expressed TGF-β2 existed in femoral head tissues in ONFH. Inhibited miR-141 resulted in elevated TGF-β2 in femoral head tissues in ONFH of rats. Depressed miR-141 or overexpressed TGF-β2 inhibited the apoptosis of bone cells of rats with ONFH and induced elevated OPG, Bcl-2, BMP2, Runx2 and declined OPGL, Bax and RANK expression in the femoral head tissues of rats with ONFH. Altogether, we find that down-regulated miR-141 promotes osteoblast activity and inhibits osteoclast activity to ameliorate ONFH via up-regulated TGF-β2 expression.

Bone: a key aspect to understand phenomena in clinical hematology.

The bone marrow (BM) is located inside the bone. Now, it appears that bone tissue functionally communicates with the BM hematopoietic system. Osteoblast lineage cells serve as a part of the microenvironment for immature hematopoietic (stem/progenitor) cells. In contrast, mature hematopoietic cells such as neutrophils and macrophages play a critical role to regulate osteoblast activity. A progressive distortion of this precise inter-organ communication between hematopoietic and skeletal systems may lead to hematologic disorders. Recent studies have revealed that vitamin D receptor is a pivotal bridging molecule for this network and for the pathogenesis of myelofibrosis.

Marrowlogy: a key aspect for the deep understanding of clinical hematology

Mammals have developed bone marrow (BM) inside the bone tissue because of evolution. Now, it appears that bone tissue displays functional communication with the hematopoietic system. Osteoblast lineage cells serve as a part of the microenvironment for immature hematopoietic cells, whereas mature hematopoietic cells play important roles in regulating osteoblast activity. The nervous system maintains the balance between the hematopoietic and skeletal systems. An understanding of the multiple-organ network that exists between the BM and other systems is useful to elucidate phenomena in clinical hematology and even in other fields, an area which I propose to call "marrowlogy."

Ginkgolide B promotes osteoblast differentiation via activation of canonical Wnt signalling and alleviates osteoporosis through a bone anabolic way.

Osteoporosis has become a worldwide problem as the population ages. Although many advances have been made in the treatment of osteoporosis in the past few years, the outcome are sometimes disturbing because of the adverse effects of these treatments. Further studies are still needed to identify novel alternate agents to improve the therapeutic effect. Ginkgolide B (GB), a derivative of Ginkgo biloba leaves, has numerous pharmacological effects, including anticancer and anti‐inflammation activities. However, the effect of GB on the regulation of osteoblast activity and bone formation effect has not yet been investigated. In this study, we showed the in vitro and in vivo effects of GB on osteoblast differentiation and bone formation. We found that GB promotes osteoblast differentiation of Bone Mesenchymal Stem Cells (BMSCs) and MC3T3‐E1 cells in vitro in a Wnt/β‐catenin‐dependent manner. In an in vivo study, we constructed a cranial defect model in rats and treated with GB. Histomorphometric and histological analyses confirmed that the usage of GB significantly promotes bone formation. Further study on ovariectomy (OVX) rats demonstrated that GB is capable of alleviating ovariectomy‐induced bone loss by enhancing osteoblast activity. Our findings indicate that GB is a potential therapeutic agent of osteoporosis through an anabolic way in bone.

Mouse genome-wide association and systems genetics identifies Lhfp as a regulator of bone mass

Bone mineral density (BMD) is a strong predictor of osteoporotic fracture. It is also one of the most heritable disease-associated quantitative traits. As a result, there has been considerable effort focused on dissecting its genetic basis. Here, we performed a genome-wide association study (GWAS) in a panel of inbred strains to identify associations influencing BMD. This analysis identified a significant (P = 3.1 x 10−12) BMD locus on Chromosome 3@52.5 Mbp that replicated in two separate inbred strain panels and overlapped a BMD quantitative trait locus (QTL) previously identified in a F2 intercross. The association mapped to a 300 Kbp region containing four genes; Gm2447, Gm20750, Cog6, and Lhfp. Further analysis found that Lipoma HMGIC Fusion Partner (Lhfp) was highly expressed in bone and osteoblasts. Furthermore, its expression was regulated by a local expression QTL (eQTL), which overlapped the BMD association. A co-expression network analysis revealed that Lhfp was strongly connected to genes involved in osteoblast differentiation. To directly evaluate its role in bone, Lhfp deficient mice (Lhfp-/-) were created using CRISPR/Cas9. Consistent with genetic and network predictions, bone marrow stromal cells (BMSCs) from Lhfp-/- mice displayed increased osteogenic differentiation. Lhfp-/- mice also had elevated BMD due to increased cortical bone mass. Lastly, we identified SNPs in human LHFP that were associated (P = 1.2 x 10−5) with heel BMD. In conclusion, we used GWAS and systems genetics to identify Lhfp as a regulator of osteoblast activity and bone mass.

The long noncoding RNA lnc-ob1 facilitates bone formation by upregulating Osterix in osteoblasts.

Long noncoding RNAs (lncRNAs) have emerged as integral regulators of physiology and disease, but specific roles of lncRNAs in bone disease remain largely unknown. Here, we show that lnc-ob1 regulates osteoblast activity and bone formation in mice by upregulating the osteogenic transcription factor Osterix. Expression of lnc-ob1 is enriched in osteoblasts and upregulated during osteoblastogenesis. We demonstrate that osteoblast-specific knock-in of lnc-ob1 enhances bone formation and increases bone mass. Pharmacological overexpression of lnc-ob1 specifically in osteoblasts confers resistance to ovariectomy-induced osteoporosis in mice. In humans, expression of the homologue, lnc-OB1, decreases with age in osteoblasts of patients with osteoporosis. Mechanistically, lnc-ob1 upregulates the expression of Osterix in mouse and human osteoblasts, probably via inhibition of H3K27me3 methylation. Our data indicate that lnc-OB1 regulates bone formation and might be a drug target for the treatment of osteoporosis.

Mesenchymal Cell-Derived Juxtacrine Wnt1 Signaling Regulates Osteoblast Activity and Osteoclast Differentiation.

ABSTRACTHuman genetic evidence demonstrates that WNT1 mutations cause osteogenesis imperfecta (OI) and early‐onset osteoporosis, implicating WNT1 as a major regulator of bone metabolism. However, its main cellular source and mechanisms of action in bone remain elusive. We generated global and limb bud mesenchymal cell–targeted deletion of Wnt1 in mice. Heterozygous deletion of Wnt1 resulted in mild trabecular osteopenia due to decreased osteoblast function. Targeted deletion of Wnt1 in mesenchymal progenitors led to spontaneous fractures due to impaired osteoblast function and increased bone resorption, mimicking the severe OI phenotype in humans with homozygous WNT1 mutations. Importantly, we showed for the first time that Wnt1 signals strictly in a juxtacrine manner to induce osteoblast differentiation and to suppress osteoclastogenesis, in part via canonical Wnt signaling. In conclusion, mesenchymal cell‐derived Wnt1, acting in short range, is an essential regulator of bone homeostasis and an intriguing target for therapeutic interventions for bone diseases. © 2019 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.

VCP/p97 increases BMP signaling by accelerating ubiquitin ligase Smurf1 degradation.

The bone morphogenetic protein (BMP)-Smad signaling pathway plays a crucial role in the control of bone homeostasis by regulating osteoblast activity. It is known that the ubiquitin ligase Smad ubiquitination regulatory factor (Smurf)1 is a master negative regulator of BMP signaling, but how its stability and activity are regulated remains poorly understood. Our study showed that valosin-containing protein/p97, the mutations of which lead to rare forms of Paget's disease of bone (PDB)-like syndrome-such as inclusion body myopathy (IBM) associated with Paget's disease of bone and frontotemporal dementia (IBM-PFD)-together with its adaptor nuclear protein localization (NPL)4, specifically interact with Smurf1 and deliver the ubiquitinated Smurf1 for degradation. Depletion of either p97 or NPL4 resulted in the elevation of Smurf1 protein level and decreased BMP signaling accordingly. Mechanically, a typical proline, glutamic acid, serine, and threonine motif specifically existing in Smurf1 is necessary for its recognition and degradation by p97, and this process is dependent on p97 ATPase activity. More importantly, compared with p97 WT, PDB-associated mutation of p97 (mainly A232E) harboring the higher ATPase activity of p97 further promoted Smurf1 degradation, thus increasing BMP signaling activity. Our findings first establish a link between p97 and Smurf1, providing an in-depth understanding of how Smurf1 is regulated, as well as the mechanism of p97-related bone diseases.-Li, H., Cui, Y., Wei, J., Liu, C., Chen, Y., Cui, C.-P., Li, L., Zhang, X., Zhang, L. VCP/p97 increases BMP signaling by accelerating ubiquitin ligase Smurf1 degradation.

SMAD1/5 signaling in osteoclasts regulates bone formation via coupling factors.

Bone remodeling occurs via coupling between bone resorption by osteoclasts and bone formation by osteoblasts. The mechanisms that regulate osteoclast signals to osteoblasts are not well understood. Published studies have reported that BMP signaling in osteoclasts regulate osteoclast coupling targets. To investigate the necessity of canonical BMP signaling on osteoclast differentiation and coupling, we mated Smad1fl/fl; Smad5fl/fl mice to c-Fms-Cre mice. We analyzed male mice at 3 months of age to determine the skeletal phenotype of the Smad1fl/fl; Smad5fl/fl;c-Fms-Cre (SMAD1/5 cKO) mice. There was a 1.2-fold decrease in trabecular BV/TV in SMAD1/5 cKO. Analyses of osteoclast serum markers in SMAD1/5 cKO mice, showed a significant increase in CTX-1 (1.5 fold) and TRAP ELISA (3 fold) compared to control mice. In these same mice, there was a 1.3-fold increase in cortical thickness. Consistent with the increase in cortical thickness, we found a 3-fold increase in osteoblast activity as measured by P1NIP ELISA assay from SMAD1/5 cKO mice. To explain the changes in cortical thickness and P1NP activity, we determined conditioned media from SMAD1/5 cKO osteoclast cultures enhanced mineralization of an osteoblast cell line and coupling factors expressed by osteoclasts that regulate osteoblast activity Wnt1 (4.5-fold increase), Gja1 (3-fold increase) and Sphk1 (1.5-fold increase) were all upregulated in osteoclasts from SMAD1/5 cKO compared to control osteoclasts. Lastly osteoclasts treated with dorsomorphin, a chemical inhibitor of SMAD1/5 signaling, demonstrates an increase in Wnt1 and Gja1 expression similar to the SMAD1/5 cKO mice. Previous studies demonstrated that TGF-β signaling in osteoclasts leads to increases in WNT1 expression by osteoclasts. Therefore, our data suggest that TGF-β and BMP signaling pathways in osteoclasts could act in an antagonistic fashion to regulate osteoblast activity through WNT1 and other coupling factors.

R-spondin-2 is a Wnt agonist that regulates osteoblast activity and bone mass

The R-spondin family of proteins are Wnt agonists, and the complete embryonic disruption of Rspo2 results in skeletal developmental defects that recapitulate the phenotype observed with Lrp5/6 deficiency. Previous work has shown that R-spondin-2 (Rspo2, RSPO2) is both highly expressed in Wnt-stimulated pre-osteoblasts and its overexpression induces osteoblast differentiation in the same cells, supporting its putative role as a positive autocrine regulator of osteoblastogenesis. However, the role of Rspo2 in regulating osteoblastogenesis and bone formation in postnatal bone has not been explored. Here we show that limb-bud progenitor cells from Rspo2 knockout mice undergo reduced mineralization during osteoblastogenesis in vitro and have a corresponding alteration in their osteogenic gene expression profile. We also generated the first Rspo2 conditional knockout (Rspo2floxed) mouse and disrupted Rspo2 expression in osteoblast-lineage cells by crossing to the Osteocalcin-Cre mouse line (Ocn-Cre + Rspo2f/f). Ocn-Cre + Rspo2f/f male and female mice at 1, 3, and 6 months were examined. Ocn-Cre + Rspo2f/f mice are decreased in overall body size compared to their control littermates and have decreased bone mass. Histomorphometric analysis of 1-month-old mice revealed a similar number of osteoblasts and mineralizing surface per bone surface with a simultaneous decrease in mineral apposition and bone formation rates. Consistent with this observation, serum osteocalcin in 3-month-old Ocn-Cre + Rspo2f/f was reduced, and bone marrow-mesenchymal stem cells from Ocn-Cre + Rspo2f/f mice undergo less mineralization in vitro. Finally, gene expression analysis and immunohistochemistry of mature bone shows reduced beta-catenin signaling in Ocn-Cre + Rspo2f/f. Overall, RSPO2 reduces osteoblastogenesis and mineralization, leading to reduced bone mass.

IL-17A induces osteoblast differentiation by activating JAK2/STAT3 in ankylosing spondylitis

BackgroundIL-17A has recently emerged as a potential target that regulates the extensive inflammation and abnormal bone formation observed in ankylosing spondylitis (AS). Blocking IL-17A is expected to inhibit bony ankylosis. Here, we investigated the effects of anti IL-17A agents in AS.MethodsTNFα, IL-17A, and IL-12/23 p40 levels in serum and synovial fluid from patients with ankylosing spondylitis (AS), rheumatoid arthritis (RA), osteoarthritis (OA), or healthy controls (HC) were measured by ELISA. Bone tissue samples were obtained at surgery from the facet joints of ten patients with AS and ten control (Ct) patients with noninflammatory spinal disease. The functional relevance of IL-17A, biological blockades, Janus kinase 2 (JAK2), and non-receptor tyrosine kinase was assessed in vitro with primary bone-derived cells (BdCs) and serum from patients with AS.ResultsBasal levels of IL-17A and IL-12/23 p40 in body fluids were elevated in patients with AS. JAK2 was also highly expressed in bone tissue and primary BdCs from patients with AS. Furthermore, addition of exogenous IL-17A to primary Ct-BdCs promoted the osteogenic stimulus-induced increase in ALP activity and mineralization. Intriguingly, blocking IL-17A with serum from patients with AS attenuated ALP activity and mineralization in both Ct and AS-BdCs by inhibiting JAK2 phosphorylation and downregulating osteoblast-involved genes. Moreover, JAK2 inhibitors effectively reduced JAK2-driven ALP activity and JAK2-mediated events.ConclusionsOur findings indicate that IL-17A regulates osteoblast activity and differentiation via JAK2/STAT3 signaling. They shed light on AS pathogenesis and suggest new rational therapies for clinical AS ankylosis.

Histone demethylase LSD1 regulates bone mass by controlling WNT7B and BMP2 signaling in osteoblasts

Multiple regulatory mechanisms control osteoblast differentiation and function to ensure unperturbed skeletal formation and remodeling. In this study we identify histone lysine-specific demethylase 1(LSD1/KDM1A) as a key epigenetic regulator of osteoblast differentiation. Knockdown of LSD1 promoted osteoblast differentiation of human mesenchymal stem cells (hMSCs) in vitro and mice lacking LSD1 in mesenchymal cells displayed increased bone mass secondary to accelerated osteoblast differentiation. Mechanistic in vitro studies revealed that LSD1 epigenetically regulates the expression of WNT7B and BMP2. LSD1 deficiency resulted in increased BMP2 and WNT7B expression in osteoblasts and enhanced bone formation, while downregulation of WNT7B- and BMP2-related signaling using genetic mouse model or small-molecule inhibitors attenuated bone phenotype in vivo. Furthermore, the LSD1 inhibitor tranylcypromine (TCP) could increase bone mass in mice. These data identify LSD1 as a novel regulator of osteoblast activity and suggest LSD1 inhibition as a potential therapeutic target for treatment of osteoporosis.

Skeletal cell YAP and TAZ combinatorially promote bone development.

The functions of the paralogous transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in bone are controversial. Each has been observed to promote or inhibit osteogenesis in vitro, with reports of both equivalent and divergent functions. Their combinatorial roles in bone physiology are unknown. We report that combinatorial YAP/TAZ deletion from skeletal lineage cells, using Osterix-Cre, caused an osteogenesis imperfecta-like phenotype with severity dependent on allele dose and greater phenotypic expressivity with homozygous TAZ vs. YAP ablation. YAP/TAZ deletion decreased bone accrual and reduced intrinsic bone material properties through impaired collagen content and organization. These structural and material defects produced spontaneous fractures, particularly in mice with homozygous TAZ deletion and caused neonatal lethality in dual homozygous knockouts. At the cellular level in vivo, YAP/TAZ ablation reduced osteoblast activity and increased osteoclast activity, in an allele dose-dependent manner, impairing bone accrual and remodeling. Transcriptionally, YAP/TAZ deletion and small-molecule inhibition of YAP/TAZ interaction with the transcriptional coeffector TEAD reduced osteogenic and collagen-related gene expression, both in vivo and in vitro. These data demonstrate that YAP and TAZ combinatorially promote bone development through regulation of osteoblast activity, matrix quality, and osteoclastic remodeling.-Kegelman, C. D., Mason, D. E., Dawahare, J. H., Horan, D. J., Vigil, G. D., Howard, S. S., Robling, A. G., Bellido, T. M., Boerckel, J. D. Skeletal cell YAP and TAZ combinatorially promote bone development.

Sclerostin level in rheumatoid arthritis patients and its relationship to disease severity and bone mineral density

Background Bone loss in rheumatoid arthritis is caused by increased bone resorption without increasing bone formation. The Wnt pathway is important in the control of bone formation through the regulation of osteoblast activity. Sclerostin is an important regulator of the Wnt pathway by blocking Wnt binding to its receptor and thereby , inhibiting bone formation. Aim Of The Work was to correlate the relation between level of serum sclerostin and bone mineral density with disease severity in rheumatoid arthritis patients. Subjects The study was conducted on 50 subjects divided into two groups: Group I : Thirty patients of rheumatoid arthritis subjects diagnosed according to 2010 ACR / EULAR diagnostic criteria. Group II : Twenty persons as a control group. Methods All patients were subjected to ; thorough medical history taking, DAS -28, disability Index, complete blood count (CBC), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), RF, antinuclear antibody (ANA), Anti-CCP Antibodies, Human Sclerostin levels using ELISA technique, Plain X- ray on both hands and feets, U/S on both hands, and (DEXA) scan. Results There was a statistical significant difference between the two studied groups regarding the age, gender, sclerostin level, and DAS-28 (P>0.05). Conclusion Most of patients were under treatment with disease modifying anti-rheumatic drugs (DMARDs) as methotrexate, fracture risk was not assessed, and measurements for renal function were not measured. However, it is possible that circulating sclerostin levels may not reflect changes of sclerostin at a local level. Despite the many questions that remain, pre-clinical studies and clinical trial results would imply that sclerostin antibodies will emerge as a dominant first- line treatment in the management of osteoporosis.

Exosomal miR-141-3p regulates osteoblast activity to promote the osteoblastic metastasis of prostate cancer.

Exosomes from cancer cells, which contain microRNA and reach metastasis loci prior to cancer cells, stimulate the formation of a metastatic microenvironment. Previous studies have shown that exosomal miR-141-3p is associated with metastatic prostate cancer (PCa). However, the role and regulatory mechanism of miR-141-3p in the microenvironment of bone metastases require further study. In this study, we performed a series of experiments in vivo and in vitro to determine whether exosomal miR-141-3p from MDA PCa 2b cells regulates osteoblast activity to promote osteoblastic metastasis. We demonstrate that extracts obtained from cell culture supernatants contained exosomes and that miR-141-3p levels were significantly higher in MDA PCa 2b cell exosomes. Via confocal imaging, numerous MDA PCa 2b exosomes were observed to enter osteoblasts, and miR-141-3p was transferred to osteoblasts through MDA PCa 2b exosomes in vitro. Exosomal miR-141-3p from MDA PCa 2b promoted osteoblast activity and increased osteoprotegerin OPG expression. miR-141-3p suppressed the protein levels of the target gene DLC1, indicating its functional significance in activating the p38MAPK pathway. In animal experiments, exosomal miR-141-3p had bone-target specificity and promoted osteoblast activity. Mice injected with miR-141-3p-mimics exosomes developed apparent osteoblastic bone metastasis. Exosomal miR-141-3p from MDA PCa 2b cells promoted osteoblast activity and regulated the microenvironment of bone metastases, which plays an important role in the formation of bone metastases and osteogenesis damage in PCa. Clarifying the specific mechanism of bone metastasis will help generate new possibilities for the treatment of PCa.

Estradiol activates chloride channels via estrogen receptor-α in the cell membranes of osteoblasts.

Estrogen plays important roles in regulation of bone formation. Cl- channels in the ClC family are expressed in osteoblasts and are associated with bone physiology and pathology, but the relationship between Cl- channels and estrogen is not clear. In this study the action of estrogen on Cl- channels was investigated in the MC3T3-E1 osteoblast cell line. Our results show that 17β-estradiol could activate a current that reversed at a potential close to the Cl- equilibrium potential, with a sequence of anion selectivity of I- > Br- > Cl- > gluconate, and was inhibited by the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoate and 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid. Knockdown of ClC-3 Cl- channel expression by a specific small interfering RNA to ClC-3 attenuated activation of the 17β-estradiol-induced Cl- current. Extracellular application of membrane-impermeable 17β-estradiol-albumin conjugates activated a similar current. The estrogen-activated Cl- current could be inhibited by the estrogen receptor (ER) antagonist fulvestrant (ICI 182780). The selective ERα agonist, but not ERβ agonist, activated a Cl- current similar to that induced by 17β-estradiol. Silencing ERα expression prevented activation of estrogen-induced currents. Immunofluorescence and coimmunoprecipitation experiments demonstrated that ClC-3 Cl- channels and ERα were colocalized and closely related in cells. Estrogen promoted translocation of ClC-3 and ERα to the cell membrane from the nucleus. In conclusion, our findings show that Cl- channels can be activated by estrogen via ERα on the cell membrane and suggest that the ClC-3 Cl- channel may be one of the targets of estrogen in the regulation of osteoblast activity.

The association between sclerostin and incident type 2 diabetes risk: a cohort study.

To determine whether sclerostin is associated with fasting glucose, insulin levels, insulin resistance or increased risk of incident type 2 diabetes. Type 2 diabetic patients have a higher risk of fractures. Recent studies suggest sclerostin, a regulator of osteoblast activity, is associated with diabetes. Sclerostin levels were obtained from 1778 individuals with no history of type 2 diabetes participating in the population-based Canadian Multicentre Osteoporosis Study (CaMos) cohort. Participants were followed until diagnosis of type 2 diabetes, death or end of the study period (31 December 2013). The relationship of sclerostin with fasting glucose, insulin levels and homoeostatic model assessment-insulin resistance (HOMA-IR) was studied in linear regression models. Cox proportional hazards models were used to determine the association of sclerostin levels and the risk of incident type 2 diabetes during a mean 7·5 years of follow-up. Fasting glucose, fasting insulin levels and HOMA-IR were weakly correlated with sclerostin levels (Spearman's correlation coefficient: 0·11, P < 0·05; -0·09, P < 0·05; and -0·07, P = 0·02, respectively). Multiple linear regression analyses confirmed a significant association between sclerostin and fasting insulin and HOMA-IR but no significant association with fasting glucose levels. Sclerostin levels were not found to be significantly associated with the risk of incident type 2 diabetes (HR: 1·30; 95% CI: 0·37-4·57). We observed an association between sclerostin levels with fasting insulin levels and HOMA-IR, but there was no clear association with type 2 diabetes risk. Further studies are needed to understand the role of sclerostin in type 2 diabetes.