May 2004
Volume 45, Issue 13
ARVO Annual Meeting Abstract  |   May 2004
Connexin expression in the human corneal epithelium in vivo and in vitro
Author Affiliations & Notes
  • D.L. Shurman
    Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA
  • L. Glazewski
    Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA
  • J.D. Zieske
    Shepens Eye Research Institute, Boston, MA
  • G. Richard
    Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA
  • Footnotes
    Commercial Relationships  D.L. Shurman, None; L. Glazewski, None; J.D. Zieske, None; G. Richard, None.
  • Footnotes
    Support  National Foundation for Ectodermal Dysplasias, Research Award; NIH Grant AR02141
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 2225. doi:
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      D.L. Shurman, L. Glazewski, J.D. Zieske, G. Richard; Connexin expression in the human corneal epithelium in vivo and in vitro . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2225.

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

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Abstract: : Purpose: The genetic connexin disorder Keratitis–Ichthyosis–Deafness syndrome (KIDS) is caused by dominant missense mutations in the Cx26 gene GJB2, yet the pathomechanisms of corneal disease have not been elucidated. Therefore, we systematically investigated the expression of Cx26 and 16 other connexin genes in normal human cornea (in vivo) and cultured primary corneal epithelial cells at different stages of differentiation (in vitro). Methods: PCEC cultures were obtained from corneal rims after incubation with 0.1% dispase and cultured in keratinocyte serum–free medium with 0.09 mM Ca2+ or switched to KSFM with 0.3% fetal bovine serum and 1.8 mM Ca2+ to promote epithelial differentiation. We employed RT–PCR analysis from tRNA, immunohistochemisty (IHC), and fluorescent dye transfer assays to study the connexin expression and gap junction–mediated intercellular communication in human cornea and monolayers of PCEC. Results: Immunostaining of central cornea and limbus revealed overlapping yet distinct expression of Cx43 and Cx30 in the basal and suprabasal layers, sparing superficial cells. Cx26 staining was less pronounced and mostly limited to basal cells. There was no evidence for corneal expression of Cx31.1, Cx32, Cx37, Cx45, and Cx46, while immunostaining for Cx31 and Cx33 was inconclusive. In PCEC (low Ca2+) transcripts of ten connexin genes were detected by RT–PCR, albeit only Cx26 and Cx43 proteins showed a characteristic punctate plasma membrane staining. Despite the presence of Cx26 and Cx43, cells were poorly coupled by gap junctions, yielding an average dye transfer to 0.86 + 1.0 cells. A switch in Ca2+ concentration resulted in formation of epithelial sheets and strong upregulation of Cx26 and, to a lesser extent, Cx30 as evident by RT–PCR and IHC. Gap junction–mediated dye transfer was significantly increased with average dye spread to 5.72 + 3.1 neighboring cells (p<0.00001). Conclusions: The major gap junction proteins of the normal human corneal epithelium are Cx26, Cx30 and Cx43. Relatively undifferentiated PCEC abundantly express Cx26 and Cx43 yet remain uncoupled, while Ca2+ induced differentiation is associated with a significant increase in Cx26 and Cx30 expression and in cell–cell communication. Based on these observations, we speculate that KIDS mutations in Cx26 directly or indirectly interfere with the function of co–expressed Cx30, Cx43 or both. A resulting functional impairment might lead to perpetually induced expression of Cx26 as compensatory mechanism, thus producing the progressive corneal pathology in KIDS.

Keywords: gap junctions/coupling • cornea: epithelium • immunohistochemistry 

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