June 2015
Volume 56, Issue 7
ARVO Annual Meeting Abstract  |   June 2015
TGF-β-activated kinase 1 is essential for tumor necrosis factor-α-induced epithelial mesenchymal transition in ARPE-19 cells
Author Affiliations & Notes
    Kumamoto University, Kumamoto, Japan
  • Eri Takahashi
    Kumamoto University, Kumamoto, Japan
  • Akira Haga
    Kumamoto University, Kumamoto, Japan
  • Hidenobu Tanihara
    Kumamoto University, Kumamoto, Japan
  • Footnotes
    Commercial Relationships KOKI MATSUBARA, None; Eri Takahashi, None; Akira Haga, None; Hidenobu Tanihara, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 4240. doi:
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      KOKI MATSUBARA, Eri Takahashi, Akira Haga, Hidenobu Tanihara; TGF-β-activated kinase 1 is essential for tumor necrosis factor-α-induced epithelial mesenchymal transition in ARPE-19 cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4240.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: To examine the role of TGF-β-activated kinase 1 (TAK1) in tumor necrosis factor-α (TNF-α)-induced EMT in human retinal pigment epithelial (ARPE-19) cells.

Methods: ARPE-19 cells were incubated with/ without TNF-α and TAK1 inhibitor, (5Z)-7-oxozeaenol in serum-free medium. After 24 hours trasnsfection with TAK1 siRNA (siTAK1) or control siRNA (siCTL), cells were stimulated with TNF-α for 48 hours in serum-free medium. Whole cell lysates were subjected to immunoblot analysis with antibodies to TAK1, phospho-p38 mitogen-activated protein kinase (p-p38MAPK), cytokeratin18 (CK18), fibronectin, or α-smooth muscle actin (α-SMA). p38MAPK and β-actin were used as loading controls. Cells were subjected to immunostaining analysis with N-cadherin after 24 hours TNF-α and (5z)-7-oxozeaenol treatment. For wound healing assay in vitro, a confluent cell monolayer was scratched by a plastic tip and cells migrating into wounded area were photographed.

Results: Immunofluorescence analysis showed that N-cadherin was located at cell-cell junction in control cells and the linear staining was disrupted by TNF-α stimulation. TNF-α-treated cells maintained the cell-cell contact in the presence of (5z)-7-oxozeaenol. In addition, (5z)-7-oxozeaenol inhibited TNF-α-induced high motility in wound healing assay. Immunoblot analysis showed that transfection of siTAK1 depleted endogenous TAK1 expression and TNF-α-induced high levels of mesenchymal markers (fibronectin and α-SMA) were decreased in siTAK1-transfetecd cells. Epithelial marker, CK18 was downregulated by TNF-α and siTAK1 inhibited the reduction of CK18 in the presence of TNF-α. Furthermore, TNF-α promoted phosphorylation of p38MAPK through TAK1.

Conclusions: Our findings indicate that TAK1 is essential for TNF-α-induced EMT in ARPE-19 cells and is a potential therapeutic target of proliferative fibrosis in vitreoretinal disorders.


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