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.
Brown
Donald J
DJ
Department of Ophthalmology, College of Medicine, University of California at Irvine, Irvine, California 92868, USA. dbrown@uci.edu
Lin
Brian
B
Holguin
Bret
B
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