Effects of hypotonic swelling on the cellular distribution and expression of pICln in human nonpigmented ciliary epithelial cells

Abstract

Purpose. It has been proposed that pI Cln, a highly acidic protein, is a candidate gene product related to the swelling-activated chloride (Cl -) channel I cl.swell in mammalian cells. However, no consensus has been reached as to whether this relationship is direct or indirect. Recently the cDNA for pI Cln was isolated from human ciliary epithelial cells. To learn more about the structure-function of pI Cln we attempted to: i) overexpress pI Cln as a fusion protein in bacteria; ii) carry out its purification; iii) generate polyclonal antibodies to study its expression and cellular localization in the ciliary epithelial cells; and iv) determine whether cell-swelling affects pI Cln expression in ciliary epithelial cells. Methods. The open reading frame (ORF) of human pI Cln was subcloned in the pET-20b(+) plasmid and established as a recombinant vector in E. coli BL21(DE3)pLysS cells. Upon induction with iso-propyl-ß-thio-galactopyranoside (IPTG), pI Cln was isolated as a His-Tag fusion protein and purified to homogeneity. Polyclonal antibodies were raised in rabbits after immunization with pI Cln purified protein, and its expression and cellular distribution in ciliary epithelial cells determined by Western blot, immunoprecipitation and indirect immunofluorescence respectively. Cell-swelling effect on ciliary epithelial cells was carried out upon treatment of cultured cells with hypotonic solution up to 60 min and pI Cln expression measured by Northern and Western blot analysis. Results. By Western blot analysis or immunoprecipitation, pI Cln antisera recognized a main band of 37-kDa in total cell extracts from ciliary body or metabolically labeled ciliary epithelial cells. By indirect immunofluorescence, pI Cln antibodies stained the cytoplasm of NPE in the intact tissue, and the perinuclear region of cultured ciliary epithelial cells. When subjected to hypotonic treatment, NPE cells did not induce translocation of pI Cln protein from the cytoplasm into the plasma membrane, nor changes in pI Cln expression at the protein level, but did down regulate up to 30% the level of pI Cln mRNA in continued hypotonic treatment. Conclusions. These observations indicate that, contrary to previous suggestions, the pI Cln protein is not likely to be in contact with the plasma membrane of ciliary epithelial cells, and its influence on Cl - -channel activity is more likely to be expressed indirectly, (i.e. through cytoskeletal restructuring).