CLONING AND IN-SITU HYBRIDIZATION OF RABBIT DECORIN IN CORNEAL TISSUES

Purpose. To develop molecular probes and to identify the cell types re sponsible for decorin synthesis in healing cornea. Methods. Adult rabb it cornea and rabbit corneal stromal cell (keratocyte) culture cDNA li braries were constructed. The libraries were screened with commerciall y available human cDNA and oligonucleotide probes. Positive clones wer e sequenced to obtain a fun length rabbit decorin cDNA. Synthetic olig onucleotides for rabbit decorin were chosen as probes for Northern blo t analysis and in situ hybridization of healing rabbit corneas. Result s. The cDNA sequences of the positive clones from the two libraries we re identical in areas of overlap. The combined cDNA sequence indicated a 1.5-kb length with a complete open reading frame for decorin. The c DNA and deduced amino acid sequences are 90% and 88% identical, respec tively, to previously reported human fibroblast and bovine bone decori n sequences. A hypervariable region near the N-terminal has little hom ology to decorins of other species or other rabbit protein. Northern b lot analysis detected a 2.0-kb and a 2.3-kb band in mRNA from rabbit k eratocyte cultures. Decorin mRNA was detected in keratocytes of normal and healing rabbit corneas by in situ hybridization. Label in the hea ling tissue was markedly increased above normal. Normal endothelium an d epithelium in normal and healing cornea failed to show label. Conclu sions. Decorin mRNA from normal adult rabbit cornea is identical to de corin mRNA from keratocytes in culture and is highly homologous to dec orin from other animal species. In situ hybridization indicated an upr egulation of decorin message in cells adjacent to and within the heali ng tissue. Both stroma-derived and endothelium-derived cells in the wo und synthesize message for decorin.