Expression of neuregulin 1, a member of the epidermal growth factor family, is expressed as multiple splice variants in the adult human cornea

Abstract

Purpose: 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.