Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.108 (TAT)
 2,389 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this study we investigated the effect of NF-kB signaling blockade on polymethylmethacrylate (PMMA) particle-induced osteoclastogenesis in vitro. We first established effective blockade of NF-kB activity as tested by electrophoretic mobility shift assays (EMSA). Particle-induced NF-kB activation in murine osteoclast precursor cells (CSF-1-dependent bone marrow macrophages) was markedly reduced by co-treatment of the cells with the NF-kB inhibitors N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and Calpain Inhibitor I (CPI). This inhibition of NF-kB activity was associated with blockade of p50 NF-kB subunit nuclear translocation. We then established a direct NF-kB inhibition approach by utilizing a TAT-bound, mutant IkB (TAT:IkB(46-317)), and demonstrated an inhibitory effect evidenced by decreased NF-kB DNA binding activity. Having established that these strategies (TPCK, CPI, TAT: IkB(46-317)) effectively block NF-kB activation, we next investigated the effect of these agents on particle-stimulated osteoclast formation. PMMA particle stimulation of mature osteoclast formation from RANKL-primed osteoclast precursor cells was blocked by all three inhibitors. To further test the efficacy of NF-kB blockade, experiments were performed with the TAT:IkB(46-317) mutant peptide in whole bone marrow cultures that contain supporting stromal cells. Again, this inhibitor efficiently blocked particle-induced osteoclastogenesis. Thus, we have shown that pharmaceutical and molecular blockade of NF-kB activation inhibits PMMA particle-directed osteoclastogenesis in vitro.
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PMID:NF-kB signaling blockade abolishes implant particle-induced osteoclastogenesis. 1465 54

Osteoclastogenesis (OCG) results from the fusion of monocytes after stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). Migration of monocytes into close proximity precedes critical fusion events that are required for osteoclast formation. Cellular migration requires leading-edge actin cytoskeleton assembly that drives cellular locomotion. Filamin A (FLNa) cross-links F-actin filaments in the leading edge of migrating cells and also has been shown to regulate signal transduction during cell migration. However, little is known about the possible role of FLNa in osteoclastogenesis. Our objective in this study was to investigate the role of FLNa in osteoclastogenesis. Bone marrow monocytes isolated from the tibiae and femora of wild type (WT) and Flna-null mice were cultured for 6 days with M-CSF and RANKL, and osteoclasts were identified by tartrate-resistant acid phosphatase (TRACP) staining. The Flna-null mouse skeletal phenotype was characterized using dual-energy X-ray absorptiometry (DXA) to analyze the skeleton, as well as tests on blood chemistry. Osteoclast levels in vivo were quantified by counting of TRACP-stained histologic sections of distal femora. To elucidate the mechanisms by which Flna regulates osteoclastogenesis, migration, actin polymerization, and activation of Rho GTPases, Rac1, Cdc42, and RhoA were assessed in monocytes during in vitro OCG. Deficiencies in migration were rescued using constitutively active Rac1 and Cdc42 TAT fusion proteins. The RANKL signaling pathway was evaluated for activation by monitoring nuclear translocation of NF kappaB and c-jun and expression of key osteoclast genes using quantitative real-time polymerase chain reaction (qRT-PCR). Our results show that Flna-null monocytes formed fewer osteoclasts in vitro, and those that were formed were smaller with fewer nuclei. Decreased OCG was reflected in vivo in TRACP-stained histologic bone sections. Flna-null monocytes experienced impaired migratory ability. When OCG was performed at increasing starting cellular plating densities in order to decrease intercellular distances, there was progressive rescue of Flna-null osteoclast formation comparable with WT levels, confirming that Flna regulates monocyte migration prefusion. Activation of the actin cytoskeleton regulators Rac1, Cdc42, and RhoA and actin free-barbed end generation were partially or completely abrogated in Flna-null monocytes; however, monocyte migration was restored on rescuing with constitutively active Rac1 and Cdc42 TAT fusion proteins. We conclude that filamin A is required for osteoclastogenesis by regulating actin dynamics via Rho GTPases that control monocyte migration.
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PMID:Filamin A regulates monocyte migration through Rho small GTPases during osteoclastogenesis. 1992 39