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Project description:Ossification of the posterior longitudinal ligament (OPLL) is a progressive ectopic bone formation of ligaments and may lead to severe neurological symptoms. However, in addition to conservative therapy for neurological symptoms or surgical management, effective pharmacotherapy and preventive intervention for OPLL remains absent. Here, to reveal the potential therapeutic target we generate single-cell maps of T-OPLL patients. We found that type I IFN signaling pathway is significantly activated during the progression of ossification. Further, stimulation of IFN β obviously promoted the calcium deposition of murine MC3T3-E1 preosteoblasts. We profiled the transcriptome in MC3T3-E1 cells after osteogenic induction, and found that IFN β promoted the expression of Sox9 and SPP1. STAT1 positively regulated the expression of Sox9 and phosphorylated STAT1 occupied on the promoter of Sox9. We also observed the localization of Sox9 in the ossificated region of ligament from OPLL patients. Thus, Sox9 is a target gene of type I IFN pathway during osteogenic induction. Furthermore, blockage of IFNAR1 by anti-IFNAR1 neutralizing antibody significantly impeded osteoblast differentiation induced by IFN β and display efficacy against ossification. Together, these results demonstrated that type I IFN signaling pathway promotes osteoblast differentiation. Anti-IFNAR1 neutralizing antibody could be used as a potential therapy for OPLL by blocking type I IFN signaling pathway.
2023-11-30 | GSE241506 | GEO
Project description:Ossification of the posterior longitudinal ligament (OPLL) is a progressive ectopic bone formation of ligaments and may lead to severe neurological symptoms. However, in addition to conservative therapy for neurological symptoms or surgical management, effective pharmacotherapy and preventive intervention for OPLL remains absent. Here, to reveal the potential therapeutic target we generate single-cell maps of T-OPLL patients. We found that type I IFN signaling pathway is significantly activated during the progression of ossification. Further, stimulation of IFN β obviously promoted the calcium deposition of murine MC3T3-E1 preosteoblasts. We profiled the transcriptome in MC3T3-E1 cells after osteogenic induction, and found that IFN β promoted the expression of Sox9 and SPP1. STAT1 positively regulated the expression of Sox9 and phosphorylated STAT1 occupied on the promoter of Sox9. We also observed the localization of Sox9 in the ossificated region of ligament from OPLL patients. Thus, Sox9 is a target gene of type I IFN pathway during osteogenic induction. Furthermore, blockage of IFNAR1 by anti-IFNAR1 neutralizing antibody significantly impeded osteoblast differentiation induced by IFN β and display efficacy against ossification. Together, these results demonstrated that type I IFN signaling pathway promotes osteoblast differentiation. Anti-IFNAR1 neutralizing antibody could be used as a potential therapy for OPLL by blocking type I IFN signaling pathway.
2023-11-30 | GSE241505 | GEO
Project description:Dickkopf-1 (DKK1) is a powerful antagonist of canonical WNT signaling pathway, and is regarded as a biomarker for osteoporosis. Its expression is highly correlated with bone mass and osteoblasts maturation. In this study, mouse primary bone marrow cells and osteoblast cell lines were used. Luciferase reporter assay and western blotting methods were employed to validate if miRNA-433-3p epigenetically regulated DKK1 translation. Rat bone marrow derived osteoblasts were infected with lentivirus vector in which miR-433-3p was constructed. The authors constructed lentivirus mediated miRNA-433-3p stable expression and examined the alkaline phosphatase (ALP) activity and mineral deposition level in vitro. In situ hybridization method was used to observe miR-433-3p in primary osteoblasts. We built up an OVX rat model to mimic postmenopausal osteoporosis, and found aberrant circulating miR-433-3p and miR-106b, which were not reported previously. Results showed that miR-433-3p potentially regulated DKK1 mRNA, Furthermore, the correlation of serum DKK1 with circulating miR-433-3p level was significant (r = 0.7520, p = 0.046). In the luciferase reporter assay, we found that miR-433-3p siRNA decreased luminescence signal, indicating direct regulation of miR-433-3p on DKK1 mRNA. When the miR-433-3p binding site in DKK1 3'UTR was mutant, such reduction was prohibited. Western blotting result validated that miR-433-3p inhibited over 90% of DKK1 protein expression. Similarly, the change of protein expression was not observed in mutant group. The stable expression of lentivirus mediated miR-433-3p increased ALP activity and mineralization both in human and rat derived immortalized cells. We found that primary osteoblasts had higher miR-433-3p level compared with immortal cells through real-time PCR, as well as in situ hybridization experiment. Conclusively, our findings further emphasized the vital role of miR-433-3p in DKK1/WNT/?-catenin pathway through decreasing DKK1 expression and inducing osteoblasts differentiation.
| S-EPMC5476290 | biostudies-literature
Project description:The contribution of deficient telomerase activity to age-related decline in osteoblast functions and bone formation is poorly studied. We have previously demonstrated that telomerase over-expression led to enhanced osteoblast differentiation of human bone marrow skeletal (stromal) stem cells (hMSC) in vitro and in vivo. Here, we investigated the signaling pathways underlying the regulatory functions of telomerase in osteoblastic cells. Comparative microarray analysis and Western blot analysis of telomerase-over expressing hMSC (hMSC-TERT) versus primary hMSC revealed significant up-regulation of several components of insulin-like growth factor (IGF) signaling. Specifically, a significant increase in IGF-induced AKT phosphorylation and alkaline phosphatase (ALP) activity were observed in hMSC-TERT. Enhanced ALP activity was reduced in presence of IGF1 receptor inhibitor: picropodophyllin. In addition, telomerase deficiency caused significant reduction in IGF signaling proteins in osteoblastic cells cultured from telomerase deficient mice (Terc(-/-)). The low bone mass exhibited by Terc(-/-) mice was associated with significant reduction in serum levels of IGF1 and IGFBP3 as well as reduced skeletal mRNA expression of Igf1, Igf2, Igf2r, Igfbp5 and Igfbp6. IGF1-induced osteoblast differentiation was also impaired in Terc(-/-) MSC. In conclusion, our data demonstrate that impaired IGF/AKT signaling contributes to the observed decreased bone mass and bone formation exhibited by telomerase deficient osteoblastic cells.
| S-EPMC4602053 | biostudies-literature
Project description:The circadian clock regulates various physiological processes, including bone metabolism. The nuclear receptors Reverbs, comprising Rev-erb? and Rev-erb?, play a key role as transcriptional regulators of the circadian clock. In this study, we demonstrate that Rev-erbs negatively regulate differentiation of osteoclasts and osteoblasts. The knockdown of Rev-erb? in osteoclast precursor cells enhanced receptor activator of nuclear factor-?B ligand (RANKL)-induced osteoclast formation, as well as expression of nuclear factor of activated T cells 1 (NFATc1), osteoclast-associated receptor (OSCAR), and tartrate-resistant acid phosphatase (TRAP). The overexpression of Rev-erb? leads to attenuation of the NFATc1 expression via inhibition of recruitment of c-Fos to the NFATc1 promoter. The overexpression of Rev-erb? in osteoblast precursors attenuated the expression of osteoblast marker genes including Runx2, alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC). Rev-erb? interfered with the recruitment of Runx2 to the promoter region of the target genes. Conversely, knockdown of Reverb? in the osteoblast precursors enhanced the osteoblast differentiation and function. In addition, Rev-erb? negatively regulated osteoclast and osteoblast differentiation by suppressing the p38 MAPK pathway. Furthermore, intraperitoneal administration of GSK4112, a Rev-erb agonist, protects RANKL-induced bone loss via inhibition of osteoclast differentiation in vivo . Taken together, our results demonstrate a molecular mechanism of Rev-erbs in the bone remodeling, and provide a molecular basis for a potential therapeutic target for treatment of bone disease characterized by excessive bone resorption.
| S-EPMC6999712 | biostudies-literature
Project description:Osteoporosis is a severe bone disease characterized by a decrease in the density and structure of bones, with high risks of fractures. Pilose antler peptide (PAP), extracted and purified from deer antlers, can promote regeneration and fracture healing, and strengthen sinews and bone. To determine whether PAP can promote osteoblast development and to elucidate the molecular mechanisms underlying its functions, the present study investigated the effects of PAP on osteoblast proliferation, differentiation and mineralization, and the role of the insulin signaling pathway using MTT assay, alkaline phosphatase activity assay, western blot analysis and reverse transcription-quantitative PCR. The present results suggested that PAP promoted osteoblast proliferation, differentiation and mineralization in vitro via the insulin signaling pathway. The effect of PAP on insulin signaling in osteoblasts may be mediated via the ERK pathway and partially by the PI3K/Akt pathway. The present results indicated that PAP could potentially be developed as an alternative treatment strategy for bone diseases related to diabetes characterized by insulin signaling impairment.
| S-EPMC6966112 | biostudies-literature
Project description:The regulation of bone formation and detailed mechanisms are still largely elusive, and the roles of microRNAs in this process have attracted much attention. Recently, a specific subtype of CD31hiendomucinhi (CD31hiEMCNhi) endothelium has been identified to promote bone formation, together with osteoblast development. However, the role of microRNA143 in the generation of CD31hi EMCNhi endothelium and bone formation remains unknown. In this study, we found that miR-143 was expressed both in osteoblast cells and CD31hiEMCNhi endothelial cells. Serum miR-143 level was negatively correlated with age in humans. Overexpression of miR-143 promoted osteoblast formation and angiogenic effects. Furthermore, CD31hiEmcnhi vessels and osteoblast formation were significantly inhibited in miR-143 knockout mice. Mechanistically, inhibitor HDAC7 was directly targeted by miR-143 and knockdown of HDAC7 was found to rescue the function of miR-143 deficiency. Thus, miR-143 promotes angiogenesis coupling with osteoblast differentiation by targeting HDAC7, which may serve as a potential target in angiogenic and osteogenic diseases.
| S-EPMC7062786 | biostudies-literature
Project description:Gliomas are brain and spinal cord malignancies characterized by high malignancy, high recurrence and poor prognosis, the underlying mechanisms of which remain largely elusive. Here, we found that the Sry-related high mobility group box (Sox) family transcription factor, Sox9, was upregulated and correlated with poor prognosis of clinical gliomas. Sox9 promotes migration and invasion of glioma cells and in vivo development of xenograft tumors from inoculated glioma cells. Sox9 functions downstream of the transforming growth factor-β (TGF-β) pathway, in which TGF-β signaling prevent proteasomal degradation of the Sox9 protein in glioma cells. These findings provide novel insight into the wide interplay between TGF-β signaling and oncogenic transcription factors, and have implications for targeted therapy and prognostic assessment of gliomas.
| S-EPMC7886799 | biostudies-literature
Project description:BackgroundRespiratory syncytial virus (RSV) infects infants and children, predisposing them to development of asthma during adulthood. Epithelial neuroendocrine phenotypes may be associated with development of asthma. This study hopes to ascertain if RSV infection promotes epithelial neuroendocrine phenotypes through the NODAL signaling pathway.MethodsThe GSE6802 data set was obtained from the GEO database, and the differential genes were analyzed using the R language. An in vitro model was constructed with RSV infected human respiratory epithelial cells, and then real-time qPCR and immunofluorescence were used to detect the expression of different epithelial biomarkers and airway neuropeptides. The acute and chronic infection model of RSV infection was established by intranasal injection of RSV into guinea pigs. Immunohistochemistry and Western blot were used to detect the expression of pulmonary neuroendocrine cells markers ENO2 and neuropeptides.ResultsThe expression levels of ENO2, SP, CGRP, and NODAL/ACTRII were significantly higher in the RSV infection group than those of the control group, which were abrogated by siRNA-NODAL. In vivo, we found that the expression levels of ENO2, SP, and CGRP were significantly higher than that of the control group.ConclusionRSV promotes epithelial neuroendocrine phenotypes through the NODAL signaling pathway.
| S-EPMC8445725 | biostudies-literature
Project description:The BMP/Smad signaling pathway plays an important role in the viability and differentiation of osteoblast; however, it is not clear whether this pathway is involved in the fluoride-induced osteoblast differentiation. In this study, we investigated the role of BMP/Smad signaling pathway in fluoride-induced osteoblast-like Saos-2 cells differentiation. Cells were exposed to fluoride of different concentrations (0, 0.1, 0.2, 0.4, 0.8, and 1.6 mM), and cell proliferation was determined using WST assays. The expression of osteoblast marker genes such as osteocalcin (BGP) and bone alkaline phosphatase (BALP) were detected by qRT-PCR. We found that fluoride enhanced the proliferation of Saos-2 cells in a dose-dependent manner and 0.2 mM of fluoride resulted in a higher expression of osteoblast marker genes. In addition, immunofluorescence analysis showed that the promotion effects of 0.2 mM of fluoride on Saos-2 cells differentiation were associated with the activation of the BMP/Smad pathway. Expression of phosphorylated Smad1/5(p-Smad1/5) was higher in cells exposed to 0.2 mM of fluoride. Plasmid expression vectors encoding the short hairpin RNA (shRNA) targeting Smad4 gene were used to block the BMP/Smad pathway, which resulted in a significantly reduced expression of BGP and BALP as well as their corresponding mRNA. The mRNA levels after transfection remained low even in the presence of fluoride. The present results reveal that BMP/Smad signaling pathway was altered during the period of osteogenesis, and that the activities of p-Smad1/5 were required for Saos-2 cells viability and differentiation induced by fluoride.
| S-EPMC3763164 | biostudies-literature