title: Calyculin A induces apoptosis and stimulates phosphorylation of p65NF-kappaB in human osteoblastic osteosarcoma MG63 cells.
abstract: Previously, we reported that okadaic acid, a specific inhibitor of serine/threonine protein phosphatases, induced apoptosis in human osteoblastic cells. However, it is not clear whether calyculin A, another inhibitor of protein phosphatases, would induce apoptosis in human osteoblastic cells and if so, which mechanisms are involved and whether the phosphorylation status of NF-kappaB could be affected by the treatment with calyculin A. In this report, we demonstrate that calyculin A induced apoptosis in MG63 cells, as judged by WST-8 assay, nuclear fragmentation, and DNA ladder formation. Expression of PTEN, FasL, and FasR mRNA was stimulated by calyculin A treatment in MG63 cells. Calyculin A also enhanced the phosphorylation level of NF-kappaB, as judged from the results of Western blot analysis and an in vitro dephosphorylation assay. Western blot analysis with anti-phospho-p65NF-kappaB antibody disclosed that the NF-kappaB was phosphorylated on serine 536 in cytosol and translocated into nucleus with calyculin A-treatment. The phosphorylation status of p65NF-kappaB was further confirmed by using the phosphorylation site-mutated p65NF-kappaB gene transfected into HEK293 cells. Unlike TNF-alpha, calyculin A treatment did not degraded IkappaBalpha within 10 min, while it degraded IkappaBalpha at 2-h treatment. Our findings indicate that calyculin A elicit phosphorylation of NF-kappaB on serine 536 in MG63 cells, resulting in the translocation of phospho-NF-kappaB to the nucleus, thereby promoting transcriptional activity of NF-kappaB-related genes.
authors.affiliation: Department of Histology, Institute of Health Biosciences, The University of Tokushima Graduate School, Kuramoto, Tokushima 770-8504, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tanaka
authors.forename: Kaya
authors.initials: K
authors.lastname: Yoshida
authors.forename: Hirohiko
authors.initials: H
authors.lastname: Okamura
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Morimoto
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Nagata
authors.forename: Tatsuji
authors.initials: T
authors.lastname: Haneji
mesh_terms: D017209:Apoptosis; D001665:Binding Sites; D001859:Bone Neoplasms / Q000188:drug therapy*; D045744:Cell Line, Tumor; D002470:Cell Survival; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008387:Marine Toxins; D009154:Mutation; D010006:Osteoblasts / Q000378:metabolism*; D012516:Osteosarcoma / Q000188:drug therapy*; D010080:Oxazoles / Q000494:pharmacology*; D010766:Phosphorylation; D012694:Serine / Q000737:chemistry; D051996:Transcription Factor RelA / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D051996:Transcription Factor RelA; D012694:Serine; C059041:calyculin A