April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Inhibition Of Hypoxia Inducible Factor 2 Alpha In Human Corneal Epithelial Cells Delays Cell Cycle At G2 Phase Even Under Normoxia
Author Affiliations & Notes
  • Hideyuki Miyashita
    Ophthalmology, Keio University Sch of Med, Shinjyuku-ku, Japan
  • Miyuki Kubota
    Ophthalmology, Keio University Sch of Med, Shinjyuku-ku, Japan
  • Kazuo Tsubota
    Ophthalmology, Keio University Sch of Med, Shinjyuku-ku, Japan
  • Shigeto Shimmura
    Ophthalmology, Keio University Sch of Med, Shinjyuku-ku, Japan
  • Footnotes
    Commercial Relationships  Hideyuki Miyashita, None; Miyuki Kubota, None; Kazuo Tsubota, None; Shigeto Shimmura, None
  • Footnotes
    Support  JSPS Grant-in-Aid for Scientific Research (KAKENHI 20791272)
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 282. doi:
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      Hideyuki Miyashita, Miyuki Kubota, Kazuo Tsubota, Shigeto Shimmura; Inhibition Of Hypoxia Inducible Factor 2 Alpha In Human Corneal Epithelial Cells Delays Cell Cycle At G2 Phase Even Under Normoxia. Invest. Ophthalmol. Vis. Sci. 2011;52(14):282.

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

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Abstract

Purpose: : Hypoxia inducible factor 2 alpha (HIF2A), also called EPAS, is an oxygen sensitive transcriptional factor and partly shares functions with HIF1A. Previously, we reported that inhibition of HIF2A, but not HIF1A, in human corneal limbal epithelial cells decreases the cell proliferation. In this study, the relationship between HIF2A inhibition and cell cycle was investigated.

Methods: : Normal human corneal limbal epithelial cells (NLE) and cell line HCET were used. HIF1A and HIF2A were knocked down by using siRNA. On the following day, paired plates were cultured under normoxia (21% O2) or hypoxia (2% O2) by using multi-gas incubator, respectively. Cell proliferation was evaluated by counting cell number at day1-4 (HCET) and about day 9 (NLE). Cell cycle was analyzed by staining cells with DNA-binding fluorescent dye DAPI followed with flow cytometry, or direct counting of mitotic cells. Expression of cyclin dependent kinase CDK1/cdc2 was analyzed with Western blot analysis.

Results: : Knock down of HIF1A did not affect proliferation of NLE and HCET, and HIF2A knock-down significantly decreased proliferation. Unexpectedly, HIF2A knock down inhibited cell growth even under normoxia. Cell cycle analysis showed that HIF2A knock-down decreased cell population in the S phase and M phase. G1 phase, which is known to be controlled by HIF1A and HIF2A via c-Myc activity, was not affected. Instead, cell population in G2 phase increased. Western blot analysis showed that inhibitory phosphorylated CDK1 (phosho-Y15-CDK1) was upregulated in HIF2A knock down cells.

Conclusions: : HIF2A may control cell cycle progression of human corneal epithelial cells at G2 phase even under normoxia.

Keywords: cornea: epithelium • hypoxia • proliferation 
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