In vivo and in vitro expression of connexins in the human corneal epithelium.
OBJECTIVE
This study is designed to provide a comprehensive expression profile of connexins in the human corneal epithelium (in vivo) and in cultured primary corneal epithelial cells (PCECs) (in vitro). It also evaluates the pathologic effects of a pathogenic missense mutation in Cx26, which causes keratitis-ichthyosis-deafness syndrome (KIDS), a rare genetic disorder with corneal involvement.


METHODS
RT-PCR analysis, immunohistochemistry, and fluorescent dye transfer assays were used to determine the expression pattern and gap junction intercellular communication in PCECs and human cornea. Differentiation-dependent differences in connexin expression of PCECs after a calcium switch were verified by real-time RT-PCR. The common KIDS mutation Cx26(D50N) was studied by determining transient expression in PCECs.


RESULTS
In vivo immunostaining revealed widespread and overlapping expression of Cx43 and -30 in the basal and suprabasal layers. Cx26 staining was limited to the lower suprabasal cells, whereas Cx31.1 localized to the apical surface of basal cells in the central cornea and to the lower and middle suprabasal cells in the limbal region. Immunostaining for nine other connexins, including Cx50, was negative. In PCEC, nine connexin genes were detectable by RT-PCR, however, only Cx26, -30, and -43 formed visible gap junction plaques. High-Ca(2+) culture conditions were accompanied by a 1.6- to 2.2-fold upregulation of expression of Cx26, -30, and -43 and a significant increase in gap-junction-mediated dye transfer. Transient expression of mutant Cx26(D50N) in PCECs resulted in cytoplasmic accumulation and lack of gap junction plaque formation and was not altered by coexpression of wild-type (wt)Cx26 or -30.


CONCLUSIONS
Gap junction communication in the human corneal epithelium is mediated by Cx26, -30, -31.1, and -43. Poorly differentiated PCECs are uncoupled, and Ca(2+) induced differentiation is associated with an upregulation of connexin expression and intercellular communication. The transfection experiments suggest that KIDS Cx26(D50N) impairs intracellular formation and transport of connexons formed by Cx26 and -30, consistent with a dominant negative effect.
Shurman
Daniel L
DL
Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA. daniel.shurman@jefferson.edu
Glazewski
Lisa
L
Gumpert
Anna
A
Zieske
James D
JD
Richard
Gabriele
G
D000328:Adult; D000368:Aged; D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D000072259:Connexin 26; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D019573:Epithelium, Corneal / Q000187:drug effects / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D006801:Humans; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D020125:Mutation, Missense; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D015854:Up-Regulation
D017630:Connexins; C000607042:GJB2 protein, human; D012333:RNA, Messenger; D000072259:Connexin 26; D002118:Calcium