GENE-EXPRESSION OF THE NEUROTROPHIC PIGMENT EPITHELIUM-DERIVED FACTORIN THE HUMAN CILIARY EPITHELIUM - SYNTHESIS AND SECRETION INTO THE AQUEOUS-HUMOR

Purpose. To study the expression of the neurotrophic pigment epitheliu m-derived factor (PEDF), a protein with neurotrophic and neuronal-surv ival activities, by the human ocular ciliary epithelium. Methods. Tota l RNA extracted from human and bovine ocular tissues were screened by Northern blot analysis with cDNA probes for PEDF. Antibodies to PEDF w ere used to monitor its synthesis and secretion by metabolically label ing ciliary processes in vitro with S-35-methionine, followed by immun oprecipitation. Pigment epithelium-derived factor antibodies also were used to visualize the cellular distribution of PEDF along the human a nd bovine ciliary epithelium. Polymerase chain reaction (PCR) and reve rse transcription (RT)-PCR was used to screen cDNA libraries of tissue and cell lines derived from the ciliary epithelium to demonstrate PED F expression. Results. From a subtractive library of the human ocular ciliary body, the authors identified a cDNA clone exhibiting nucleotid e homology with the PEDF. Northern blot analysis indicated that PEDF t ranscripts are present in all the ocular tissues in the human eye; in the bovine eye, it is expressed preferentially in the retinal pigment epithelium. RT-PCR and PCR demonstrated that the PEDF gene is still tr anscriptionally active in cultured cell lines derived from the bilayer of the ciliary epithelium. Immunoprecipitation and Western blot (immu noblot) analyses with antisera to the PEDF protein demonstrated that a predominant PEDF form of 46 kDa is synthesized in the ciliary body an d is secreted as a glycoprotein of 50 kDa. By indirect immunofluoresce nce and immunocytochemistry, PEDF antibodies decorated both cell types that comprise the ciliary epithelium (nonpigmented and pigmented) and , more distinctively, the plasma-membrane domain of nonpigmented cells in the pars plicata region. Conclusions. These results reveal a new s ite of synthesis (ciliary epithelium) and accumulation (aqueous humor) of PEDF, and they emphasize its potential importance as a trophic fac tor in the neuro-differentiated functions of the human ciliary epithel ium.