Keratoconus is a corneal disorder characterized by excessive thinning of the stroma, severe irregular astigmatism and decreased visual acuity. It is a leading indication for corneal transplantation within the United States. Its pathogenesis is characterized by increased activities of degradative enzymes, altered processing of oxidative stress-related molecules, increased focal fibrosis and apoptosis. The underlying defect(s) that initiates these changes or ties them together is still not clear. During the past three years we have applied differential display technology, Smart cDNA synthesis and nucleic acid array analysis to keratoconus corneas and cell cultures. Approximately 3,000 genes have been screened for differential expression between normal and keratoconus. We found abnormalities in two distinct signal transduction pathways, (1) a receptor-like protein tyrosine phosphatase (leukocyte common antigen related protein- LAR) and (2) a receptor tyrosine kinase (ErbB3), its ligand (heregulin) and downstream factors, PYK2, EPB-l and TOB. Our data also show that KC corneas have increased inducible nitric oxide synthase (iNOS) and accumulation of peroxynitrite, a cytotoxic by-product of nitric oxide. We hypothesize that KC corneas have a defect in their ability to process free radicals and have up-regulation of these signal transduction pathways. This results in abnormal protein phosphorylation patterns, which contributes heavily to the pathogenesis of keratoconus. To test this hypothesis we propose the following specific aims:
Specific Aim #1 will identify the LAR isoforms within the normal and keratoconus corneas and charactize changes in tyrosine phosphorylation patterns as a result of LAR activity.
Specific Aim #2 will identify specific ErbB family members and heregulin isoforms present within keratoconus corneas and in vitro KC cell cultures. The nucleic acid array data demonstrating up-regulation of PYK2, JNK, EPB-l and TOB will be confirmed at the RNA (RT-PCR, Northern analyses, in situ hybridization) and protein levels (immunohistochemistry and Western blot analysis).
Specific Aim #3 will address whether nitric oxide donors or peroxynitrites can affect the tyrosine phosphatase (LAR) or the tyrosine kinase (ErbBIPYK2/JNK) pathways. Proteins that undergo changes in nitration as a result of peroxynitrite accumulation will be identified.
Specific Aim #4 will determine if the addition of heregulins, nitric oxide donors or peroxynitrites are capable of causing normal cells to change to the phenotype associated with KC. These studies will provide fundamental insights into KC pathogensis and may provide a basis for therapy.
