May 2003
Volume 44, Issue 13
ARVO Annual Meeting Abstract  |   May 2003
Characterization of Factors Released Upon Corneal Epithelial Injury and Their Role in Signaling
Author Affiliations & Notes
  • V. Trinkaus-Randall
    Ophthalmology and Biochemistry L903, Boston University Med School, Boston, MA, United States
  • L. Yang
    Biochemistry L903, Boston University Med School, Boston, MA, United States
  • V. Klepeis
    Pathology L903, Boston University Med School, Boston, MA, United States
  • Footnotes
    Commercial Relationships  V. Trinkaus-Randall, None; L. Yang, None; V. Klepeis, None.
  • Footnotes
    Support  NIH Grant EY06000, Massachusetts Lions Eye Research Fund and Research to Prevent Blindness
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 3270. doi:
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      V. Trinkaus-Randall, L. Yang, V. Klepeis; Characterization of Factors Released Upon Corneal Epithelial Injury and Their Role in Signaling . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3270.

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

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Abstract: : Purpose: Our goal was to identify the extracellular molecules released upon epithelial injury that induce activation of ERK1/2 and later signaling events. Previous studies using confocal microscopy demonstrated that injury caused a rapid increase in intracellular Ca2+ that propagated as a wave from the injury site. Methods: Corneal epithelial cells were cultured and injured using a scrape wound model. Wound media and or lysates were collected and analyzed for ERK1/2 activation and localization. To characterize the media, size fractionation spin columns were used and the filtrate and retentate were evaluated using live cell imaging, migration and biochemical assays. Cell cultures were also analyzed for expression of adhesion proteins in response to purines, pyrimidines or wound media. Results: We observed that injury to epithelial cells induced a rapid and transient activation of ERK1/2 (peak at 2 min) and activation was localized to the wound edge. Treatment of unwounded cells with wound media collected immediately after injury elicited a response that was inhibited by apyrase, and neither trypsin nor heat altered this response. EGF could not be detected in the wound media using an ELISA. Size fractionation of the wound media identified that a component less than 3 kDa was the predominant factor that induced the activation of ERK1/2. The same fraction also caused the release of Ca2+ while no activity was detected in higher molecular weight fractions. To further characterize the response, purines and pyrimidines were added to unwounded cells. Both ATP and UTP generated a very rapid and transient activation of ERK1/2, similar in time and amplitude to wound media. However, activation caused by UDP and ADP was weaker and later. The kinetics of ERK1/2 activation by these factors correlated with Ca2+ signaling events. ATPgammaS, a nonhydrolyzable form of ATP, caused a response that did not decrease as rapidly. In addition,the use of phospholipase C inhibitor, U73122, inhibited the signaling response generated by wound media, ATP and UTP. This corresponded with the presence of purinergic receptors (P2Y2, P2Y4 and P2Y6) which were identified in our cells using RT-PCR. At later time points ATP significantly enhanced cellular migration due to an alteration in the expression of adhesion proteins. Conclusions: These results indicate that injury induced activation of ERK1/2 is mediated by a purinergic P2Y signaling pathway that is activated immediately after injury and continues to mediate later events such as migration by altering the regulation of adhesion proteins.

Keywords: wound healing • cell-cell communication • signal transduction 

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