Gene expression of proteases and protease inhibitors in the human ciliary epithelium and ODM-2 cells.
Complementary DNAs (cDNAs), corresponding to the human proteinases cathepsins D and O and proteinase inhibitors alpha2-macroglobulin and PP5/TFPI-2, have recently been isolated and identified from a subtractive human ciliary body library. In the present study we determined: (i) their pattern of expression in the human eye; (ii) the ability of the ciliary body and/or ciliary epithelial cells to synthesize and secrete cathepsin D and alpha1-antitrypsin in vitro; and (iii) whether alpha1-antitrypsin expression in cultured ciliary epithelial cells is modulated by protein kinase C activation. Northern analysis demonstrated that the ciliary body expresses high levels of cathepsins D and O, alpha2-macroglobulin, alpha1-antitrypsin and PP5/TFPI-2 transcripts. Western blot analysis and immunoprecipitation experiments with cathepsin D and alpha1-antitrypsin antibodies indicated that metabolically labeled ciliary body explants and/or ciliary epithelial cells in vitro with 35S-methionine, synthesize and secrete these proteins. Cultured nonpigmented ciliary epithelial ODM-2 cells, in response to phorbol-12-myristate 13-acetate (PMA), but not to the non-protein kinase C binding phorbol ester 4 alpha-phorbol didecanoate (PDBu), elicited up-regulation (up to 5-fold) of transcription, synthesis and secretion of alpha1-antitrypsin. These results provide in vitro evidence that the ciliary epithelium synthesizes and secretes a selective group of proteinases and proteinase inhibitors detected also in aqueous humor. The expression of at least of one of the proteinase inhibitors, alpha1-antitrypsin, can be modulated in response to phorbol ester.
Ortego
J
J
Department of Ophthalmology and Visual Science, Yale University School of Medicine, 330 Cedar St., New Haven, CT 06510, USA.
Escribano
J
J
Coca-Prados
M
M
D000368:Aged; D015152:Blotting, Northern; D015153:Blotting, Western; D002273:Carcinogens / Q000494:pharmacology; D002402:Cathepsin D / Q000235:genetics; D056657:Cathepsin K; D002403:Cathepsins / Q000235:genetics; D002460:Cell Line; D002924:Ciliary Body / Q000201:enzymology*; D003546:Cysteine Endopeptidases / Q000235:genetics; D018076:DNA, Complementary / Q000032:analysis; D010450:Endopeptidases / Q000235:genetics*; D004847:Epithelial Cells / Q000201:enzymology; D015870:Gene Expression / Q000187:drug effects; D006023:Glycoproteins / Q000235:genetics; D006801:Humans; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011257:Pregnancy Proteins / Q000235:genetics; D011480:Protease Inhibitors; D015842:Serine Proteinase Inhibitors / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000515:alpha 1-Antitrypsin / Q000235:genetics; D000511:alpha-Macroglobulins / Q000235:genetics
D002273:Carcinogens; D018076:DNA, Complementary; D006023:Glycoproteins; D011257:Pregnancy Proteins; D011480:Protease Inhibitors; D015842:Serine Proteinase Inhibitors; D000515:alpha 1-Antitrypsin; D000511:alpha-Macroglobulins; C021240:tissue-factor-pathway inhibitor 2; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C534130:CTSK protein, human; D056657:Cathepsin K; D002402:Cathepsin D; D013755:Tetradecanoylphorbol Acetate