June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Pathway analysis provides insights into keratin 6A-mediated inflammatory network in human corneal epithelial cells
Author Affiliations & Notes
  • William Martin Carrera
    School of Medicine, Case Western Reserve University, Cleveland, Ohio, United States
  • Jonathan K Chan
    Department of Ophthalmic Research, Cleveland Clinic Cole Eye Institute and Lerner Research Institute, Cleveland, Ohio, United States
  • K P Connie Tam
    Department of Ophthalmic Research, Cleveland Clinic Cole Eye Institute and Lerner Research Institute, Cleveland, Ohio, United States
    Department of Ophthalmology, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio, United States
  • Footnotes
    Commercial Relationships   William Carrera, None; Jonathan Chan, None; K P Connie Tam, US Patent No. 9,187,541 B2 - “Anti-Microbial Peptides and Methods of Use Thereof” (P)
  • Footnotes
    Support  NIH-NEI R01EY023000 and Cleveland Clinic Seed Fund (K. P. C. Tam); NIH-NEI P30 Core Grant (IP30EY025585-01A1) and Unrestricted Grant from The Research to Prevent Blindness, Inc. (Cole Eye Institute)
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 5770. doi:
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    • Get Citation

      William Martin Carrera, Jonathan K Chan, K P Connie Tam; Pathway analysis provides insights into keratin 6A-mediated inflammatory network in human corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5770.

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

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Abstract

Purpose : In recent years, keratin proteins have been shown to regulate various cellular and physiological processes. Little is known about their potential involvement in immune system regulation. Here, we performed a proteomic and bioinformatic analysis of protein binding partners of keratin 6A (K6A) to explore potential mechanisms of its regulation of the corneal immune response.

Methods : Multilayered telomerase immortalized human corneal epithelial cells were treated with culture media or purified flagellin, lipoteichoic acid, or lipopolysaccharide for 16 hours. Immunoprecipitation of K6A was then performed with cell lysates. Mass spectrometry identified proteins that co-precipitated with K6A, which were subsequently analyzed with the PANTHER classification system and with Ingenuity Pathway Analysis. A right-tailed Fisher Exact Test with a p-value cutoff of 0.05 was used for statistical analysis.

Results : Mass spectrometry of K6A immunoprecipitates identified 506 proteins as potential binding partners of K6A, including 11 defense/immunity proteins, 95 nucleic acid binding proteins, and 35 receptor and signaling proteins. Antigen treatment led to increased co-precipitation of an average of 211 proteins, and decreased co-precipitation of an average of 147 proteins. Co-precipitation increased by greater than 5-fold with 13 proteins, most of which were involved in cellular defense, survival, adhesion, metabolism, and protein translation. Co-precipitation decreased by greater than 5-fold with 18 proteins, most of which were cytoskeletal proteins and histones, or were proteins involved in cellular metabolism, DNA repair, and protein repair, trafficking, or ubiquitination. The most significant canonical pathways were involved in EIF2 signaling, epithelial adherens junction signaling and remodeling, Germ cell-Sertoli cell junction signaling, protein ubiquitination, EIF4 regulation and p70S6k regulation. Our analysis identified ELKS, HNRNPA1, COPS5, ESR1 and EGFR as potential mediators of K6A’s immunoregulatory effects in the human corneal epithelium.

Conclusions : Our functional network and pathway analysis identified several proteins and pathways of interest that provide mechanistic insights by which keratin 6A may regulate corneal inflammation. Further studies are needed to confirm the interactions between keratin and these regulatory pathways.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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