Calyculin A induces apoptosis and stimulates phosphorylation of p65NF-κB in human osteoblastic osteosarcoma MG63 cells

Hiroaki Tanaka, Kaya Yoshida, Hirohiko Okamura, Hiroyuki Morimoto, Toshihiko Nagata, Tatsuji Haneji

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10 Citations (Scopus)

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-κB 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-κB, as judged from the results of Western blot analysis and an in vitro dephosphorylation assay. Western blot analysis with antiphospho-p65NF-κB antibody disclosed that the NF-κB was phosphorylated on serine 536 in cytosol and translocated into nucleus with calyculin A-treatment. The phosphorylation status of p65NF-κB was further confirmed by using the phosphorylation site-mutated p65NF-κB gene transfected into HEK293 cells. Unlike TNF-α, calyculin A treatment did not degraded IeκBγ within 10 min, while it degraded IκBα at 2-h treatment. Our findings indicate that calyculin A elicit phosphorylation of NF-κB on serine 536 in MG63 cells, resulting in the translocation of phospho-NF-κB to the nucleus, thereby promoting transcriptional activity of NF-κB-related genes.

Original languageEnglish
Pages (from-to)389-396
Number of pages8
JournalInternational journal of oncology
Volume31
Issue number2
Publication statusPublished - Aug 1 2007

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Keywords

  • Apoptosis
  • Calyculin A
  • NF-κB
  • Phosphatase
  • Phosphorylation

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

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