Enhanced expression of a transmembrane phosphotyrosine phosphatase (LAR) in keratoconus cultures and corneas

The purpose of the study was to identify genes that are differentially expr essed in normal versus keratoconus corneas. Total RNA isolated from corneal stromal cell cultures was reverse-transcribed and then amplified by the po lymerase chain reaction (PCR) using defined, arbitrary primers. The product s were displayed on polyacrylamide gels and bands that were differentially expressed were excised, reamplified and subcloned. The resulting clones wer e sequenced and utilized as probes for Northern blots with cultured cell RN A or Southern blots of corneal cDNA. One of the products that appeared to b e more highly expressed in keratoconus cultures and corneas displayed 100% homology with leukocyte common antigen related protein (LAR), a transmembra ne phosphotyrosine phosphatase. Western analyses and immunohistochemistry w ith monoclonal and/or polyclonal antibodies to LAR were used to examine ker atocyte cultures and fresh frozen normal, keratoconus and pseudophakic bull ous corneas. We identified a gene product with 100% homology to LAR that is expressed at the RNA level in keratoconus corneas and cell cultures but is found only a t low or undetectable levels in normal cultures and normal and pseudophakic bullous keratopathy (PBK) corneas. By Western blotting and immunofluoresce nce with specific LAR antibodies, the protein was identified in keratoconus stromal cell cultures but not in normal cultures. When fresh frozen tissue was examined, LAR protein was localized to numerous stromal cells througho ut central keratoconus corneas, while no central staining was seen in norma l or bullous keratopathy corneas. LAR, a transmembrane phosphotyrosine phosphatase, is more highly expressed in keratoconus corneas and stromal cell cultures as demonstrated by differe ntial display, Northern analyses, immunohistochemistry and Western blotting . (C) 1999 Academic Press.