Expression of neuregulin 1, a member of the epidermal growth factor family, is expressed as multiple splice variants in the adult human cornea.
OBJECTIVE
To determine whether neuregulin 1 (Nrg-1) is expressed in the normal adult human cornea.


METHODS
cDNA for Nrg-1 was obtained by direct amplification of RNA isolated from human corneal cell cultures. After sequencing, the likely exon/intron structure was determined by comparison to genomic sequence. RNA was purified from isolated corneal epithelium, corneal stroma, and primary cultures of both epithelial cells and stromal fibroblasts. Quantitative real-time polymerase chain reactions (qPCR) were performed to determine the overall levels of Nrg-1. A combination of fluorescent primers, restriction endonucleases, and image analysis was used to determine the proportion of each splice variant. Finally, the receptor family known to interact with Nrg-1 was examined to confirm its expression in corneal tissue.


RESULTS
RT-PCR and Western blot analyses demonstrated that Nrg-1 and its receptor are expressed in adult corneal tissue and cultured cells derived from this tissue. qPCR suggested that epithelial cells and stromal cells produce equivalent levels of Nrg-1, but distinct variants were present that differ in proportion with each source of RNA.


CONCLUSIONS
Eight distinct forms of Nrg-1 were expressed in the adult human cornea that differ by the alternate use of four exons. This altered the predicted coding sequence in three domains of Nrg-1. These domains are known to direct ligand/receptor interaction and the trafficking, processing, and release of Nrg-1 from the cell. Finally, there was a preference of exon usage that varied by location in the cornea and this pattern changed when cells were placed into culture.
Department of Ophthalmology, College of Medicine, University of California at Irvine, Irvine, California 92868, USA. dbrown@uci.edu
Donald J
DJ
Brown
Brian
B
Lin
Bret
B
Holguin
D000293:Adolescent; D000328:Adult; D017398:Alternative Splicing; D015153:Blotting, Western; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology / Q000378:metabolism*; D003319:Corneal Stroma / Q000378:metabolism; D019573:Epithelium, Corneal / Q000378:metabolism; D006801:Humans; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism*; D020890:Neuregulin-1; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014019:Tissue Donors
C094131:NRG1 protein, human; D009419:Nerve Tissue Proteins; D020890:Neuregulin-1