Inhibition of Microtubule Assembly in Osteoblasts Stimulates Bone Morphogenetic Protein 2 Expression and Bone Formation through Transcription Factor Gli2

Zhao, Ming; Ko, Sun-Young; Liu, Jin Hua; Chen, Di; Zhang, Jianghong; Wang, Baolin; Harris, S. E.; Oyajobi, Babatunde O.; Mundy, Gregory R.

doi:10.1128/mcb.01566-08

Cited by 46 publications

(66 citation statements)

References 58 publications

(102 reference statements)

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“…It has previously been demonstrated that microtubule inhibitors stimulate osteoblast differentiation and increase bone mass in mice through elevated levels of BMP-2 (14,15). We show that parbendazole significantly increased BMP2 expression (Fig.…”

Section: Parbendazole Inhibition Of Microtubule Polymerization Is Reqsupporting

confidence: 60%

Connectivity Map-based discovery of parbendazole reveals targetable human osteogenic pathway

Brum

Peppel

Leije

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Osteoporosis is a common skeletal disorder characterized by low bone mass leading to increased bone fragility and fracture susceptibility. In this study, we have identified pathways that stimulate differentiation of bone forming osteoblasts from human mesenchymal stromal cells (hMSCs). Gene expression profiling was performed in hMSCs differentiated toward osteoblasts (at 6 h). Significantly regulated genes were analyzed in silico, and the Connectivity Map (CMap) was used to identify candidate bone stimulatory compounds. The signature of parbendazole matches the expression changes observed for osteogenic hMSCs. Parbendazole stimulates osteoblast differentiation as indicated by increased alkaline phosphatase activity, mineralization, and up-regulation of bone marker genes (alkaline phosphatase/ALPL, osteopontin/SPP1, and bone sialoprotein II/IBSP) in a subset of the hMSC population resistant to the apoptotic effects of parbendazole. These osteogenic effects are independent of glucocorticoids because parbendazole does not up-regulate glucocorticoid receptor (GR) target genes and is not inhibited by the GR antagonist mifepristone. Parbendazole causes profound cytoskeletal changes including degradation of microtubules and increased focal adhesions. Stabilization of microtubules by pretreatment with Taxol inhibits osteoblast differentiation. Parbendazole up-regulates bone morphogenetic protein 2 (BMP-2) gene expression and activity. Cotreatment with the BMP-2 antagonist DMH1 limits, but does not block, parbendazole-induced mineralization. Using the CMap we have identified a previously unidentified lineage-specific, bone anabolic compound, parbendazole, which induces osteogenic differentiation through a combination of cytoskeletal changes and increased BMP-2 activity.Connectivity Map | osteoblast | mesenchymal stem cell | cytoskeleton | osteoporosis

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Section: Parbendazole Inhibition Of Microtubule Polymerization Is Reqsupporting

confidence: 60%

Connectivity Map-based discovery of parbendazole reveals targetable human osteogenic pathway

Brum

Peppel

Leije

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Furthermore, in the presence of CS1, a tubulin component (TUBA4A) was also found to be up-regulated compared with sHA1. A disturbed assembly of microtubule was shown to enhance BMP2 and bone formation (68). No exclusive regulations after HA treatment could be observed.…”

Section: Influence Of Various Gags On the Matrix Vesicle Proteomementioning

confidence: 99%

Osteoblast-released Matrix Vesicles, Regulation of Activity and Composition by Sulfated and Non-sulfated Glycosaminoglycans

Schmidt

Kliemt

Preissler

et al. 2016

Molecular & Cellular Proteomics

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Our aging population has to deal with the increasing threat of age-related diseases that impair bone healing. One promising therapeutic approach involves the coating of implants with modified glycosaminoglycans (GAGs) that mimic the native bone environment and actively facilitate skeletogenesis. In previous studies, we reported that coatings containing GAGs, such as hyaluronic acid (HA) and its synthetically sulfated derivative (sHA1) as well as the naturally low-sulfated GAG chondroitin sulfate (CS1), reduce the activity of bone-resorbing osteoclasts, but they also induce functions of the bone-forming cells, the osteoblasts. However, it remained open whether GAGs influence the osteoblasts alone or whether they also directly affect the formation, composition, activity, and distribution of osteoblast-released matrix vesicles (MV), which are supposed to be the active machinery for bone formation. Here, we studied the molecular effects of sHA1, HA, and CS1 on MV activity and on the distribution of marker proteins. Furthermore, we used comparative proteomic methods to study the relative protein compositions of isolated MVs and MV-releasing osteoblasts. The MV proteome is much more strongly regulated by GAGs than the cellular proteome. GAGs, especially sHA1, were found to severely impact vesicle-extracellular matrix interaction and matrix vesicle activity, leading to stronger extracellular matrix formation and mineralization. This

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“…To examine whether conditional deletion of Kif3a (disruption of ciliogenesis) has an impact on Hh signaling in osteoblasts, 1.5610 6 cells were transfected with 3.0 mg Gli-responsive luciferase reporter construct (8xGli-Luc) (Zhao et al, 2009), 3.0 mg pcDNA3.1 empty vector and 0.6 mg Renilla luciferase-null (RL-null) as an internal control plasmid by electroporation using a Cell Line Nucleofector Kit R according to the manufacturer's protocol (Amaxa, Inc., Gaithersburg, MD). The cells were cultured in a-MEM supplemented with 1% FBS and the relative luciferase activity of cell lysates was measured using a luciferase assay kit (Promega, Madison, WI) 72 hours after transfection in the presence or absence of 1 mg/ml of recombinant mouse Shh N-terminus (Shh-N) for the last 8 hours Xiao, Z. et al, 2010).…”

Section: Transient Transfection and Western Blot Analysismentioning

confidence: 99%

Disruption of Kif3a in osteoblasts results in defective bone formation and osteopenia

Qiu

Xiao

Cao

et al. 2012

Journal of Cell Science

102

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SummaryWe investigated whether Kif3a in osteoblasts has a direct role in regulating postnatal bone formation. We conditionally deleted Kif3a in osteoblasts by crossing osteocalcin (Oc; also known as Bglap)-Cre with Kif3a flox/null mice. Conditional Kif3a-null mice (Kif3a Oc-cKO ) had a 75% reduction in Kif3a transcripts in bone and osteoblasts. Conditional deletion of Kif3a resulted in the reduction of primary cilia number by 51% and length by 27% in osteoblasts. Kif3aOc-cKO mice developed osteopenia by 6 weeks of age unlike Kif3a flox/+ control mice, as evidenced by reductions in femoral bone mineral density (22%), trabecular bone volume (42%) and cortical thickness (17%). By contrast, Oc-Cre;Kif3a flox/+ and Kif3a flox/null heterozygous mice exhibited no skeletal abnormalities. Loss of bone mass in Kif3aOc-cKO mice was associated with impaired osteoblast function in vivo, as reflected by a 54% reduction in mineral apposition rate and decreased expression of Runx2, osterix (also known as Sp7 transcription factor 7; Sp7), osteocalcin and Dmp1 compared with controls. Immortalized osteoblasts from Kif3aOc-cKO mice exhibited increased cell proliferation, impaired osteoblastic differentiation, and enhanced adipogenesis in vitro. Osteoblasts derived from Kif3aOc-cKO mice also had lower basal cytosolic calcium levels and impaired intracellular calcium responses to fluid flow shear stress. Sonic hedgehog-mediated Gli2 expression and Wnt3a-mediated b-catenin and Axin2 expression were also attenuated in Kif3aOc-cKO bone and osteoblast cultures. These data indicate that selective deletion of Kif3a in osteoblasts disrupts primary cilia formation and/or function and impairs osteoblast-mediated bone formation through multiple pathways including intracellular calcium, hedgehog and Wnt signaling.

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Inhibition of Microtubule Assembly in Osteoblasts Stimulates Bone Morphogenetic Protein 2 Expression and Bone Formation through Transcription Factor Gli2

Cited by 46 publications

References 58 publications

Connectivity Map-based discovery of parbendazole reveals targetable human osteogenic pathway

Connectivity Map-based discovery of parbendazole reveals targetable human osteogenic pathway

Osteoblast-released Matrix Vesicles, Regulation of Activity and Composition by Sulfated and Non-sulfated Glycosaminoglycans

Disruption of Kif3a in osteoblasts results in defective bone formation and osteopenia

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