Gene expression of the neurotrophic pigment epithelium-derived factor in the human ciliary epithelium. Synthesis and secretion into the aqueous humor.
OBJECTIVE
To study the expression of the neurotrophic pigment epithelium-derived factor (PEDF), a protein with neurotrophic and neuronal-survival activities, by the human ocular ciliary epithelium.


METHODS
Total RNA extracted from human and bovine ocular tissues were screened by Northern blot analysis with cDNA probes for PEDF. Antibodies to PEDF were used to monitor its synthesis and secretion by metabolically labeling ciliary processes in vitro with 35S-methionine, followed by immunoprecipitation. Pigment epithelium-derived factor antibodies also were used to visualize the cellular distribution of PEDF along the human and bovine ciliary epithelium. Polymerase chain reaction (PCR) and reverse transcription (RT)-PCR was used to screen cDNA libraries of tissue and cell lines derived from the ciliary epithelium to demonstrate PEDF expression.


RESULTS
From a subtractive library of the human ocular ciliary body, the authors identified a cDNA clone exhibiting nucleotide homology with the PEDF. Northern blot analysis indicated that PEDF transcripts 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 transcriptionally active in cultured cell lines derived from the bilayer of the ciliary epithelium. Immunoprecipitation and Western blot (immunoblot) analyses with antisera to the PEDF protein demonstrated that a predominant PEDF form of 46 kDa is synthesized in the ciliary body and is secreted as a glycoprotein of 50 kDa. By indirect immunofluorescence 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 site of synthesis (ciliary epithelium) and accumulation (aqueous humor) of PEDF, and they emphasize its potential importance as a trophic factor in the neuro-differentiated functions of the human ciliary epithelium.
Ortego
J
J
Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Escribano
J
J
Becerra
S P
SP
Coca-Prados
M
M
D000818:Animals; D001082:Aqueous Humor / Q000378:metabolism*; D015152:Blotting, Northern; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002924:Ciliary Body / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D005136:Eye Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D006801:Humans; D007124:Immunoenzyme Techniques; D009414:Nerve Growth Factors / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011817:Rabbits; D015843:Serpins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014158:Transcription, Genetic; D000099174:Pigment Epithelium-Derived Factor
D017931:DNA Primers; D005136:Eye Proteins; D009414:Nerve Growth Factors; D011506:Proteins; D015843:Serpins; D000099174:Pigment Epithelium-Derived Factor