April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
Myocilin Triggers Cell Migration Through Regulation of FAK Activity
Author Affiliations & Notes
  • H.-S. Kwon
    SMMG,LMDB, NEI/NIH, Bethesda, Maryland
  • S. Tomarev
    SMMG,LMDB, NEI/NIH, Bethesda, Maryland
  • Footnotes
    Commercial Relationships  H.-S. Kwon, None; S. Tomarev, None.
  • Footnotes
    Support  NEI Intramural grant
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 3634. doi:
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      H.-S. Kwon, S. Tomarev; Myocilin Triggers Cell Migration Through Regulation of FAK Activity. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3634.

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

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Purpose: : Our previous experiments demonstrated that addition of myocilin to cultured cells induced formation of stress fibers and increased levels of activated Rac1 and JNK. In this work we investigated the effects of myocilin on cell migration.

Methods: : HEK293 cells were transiently transfected with cDNA encoding FLAG-tagged full length human myocilin or its N-terminal or C-terminal proteolytic fragments under the control of a viral promoter. Full length FLAG-tagged myocilin was purified from conditioned medium (CM) of transfected cells using FLAG-agarose and HiTrap-S chromatography. Wound healing assays were performed with NIH3T3 and lens epithelial cell (FHL124) lines seeded on glass chamber slides. Trans-well migration assays were performed with 24-well cell culture plates and 6.5 mm-diameter inserts having 8 µm pore size. Active FAK was detected using an anti-phospho-tyrosine antibody that recognizes FAK phosphorylated at Tyr397.

Results: : Confluent monolayers of cultured cells were scratched with a pipette tip immediately before the addition of purified myocilin (3µg/ml) or vehicle. Addition of myocilin increased cell migration into the wound region by 3-fold as compared with control samples. Pre-incubation of myocilin with antibodies against myocilin reduced migration to control levels. Purified myocilin and CM from myocilin-expressing cells increased cell migration in the trans-well assay; the N-terminal fragment showed the highest stimulatory effect. Treatment of cells with CM from cells expressing full length myocilin or its fragments for 1 hr increased the levels of active phosphorylated FAK by 3-6 folds as compared with localized control samples. Active FAK was preferentially localized to the wound edge in wound healing assay.

Keywords: cell membrane/membrane specializations • wound healing • signal transduction 

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