Biosynthesis of Sulphated Macromolecules by Rabbit Epithelium. II. Relationship to Basement Membrane Formation Lens

Rabbit lens epithelial cells display a similar "cobblestone" morphology and produce the same complement of sulphated macromolecules (also see Heathcote, J. G., and R. W. Orkin, 1984, J. Cell Biol., 99:852-860) whether grown on plastic or glass, dried films of gelatin or type IV collagen with laminin, or on gels of type I collagen. There was no evidence of basement membrane formation by these cells when they were grown on plastic, glass, or dried films. In contrast, cultures that had been grown on gels deposited a discrete basement membrane that followed the contours of the basal surfaces of the cells and in addition, they secreted amorphous basement membrane-like material that diffused into the interstices of the gel and associated with the collagen fibrils of the gel. A significant proportion (~70%) of the heparan sulphate proteoglycan fraction that was secreted into the culture medium (fraction MI) when the cells were grown on plastic became associated with the cell-gel layer in the gel cultures. Further, when basement membrane was isolated by detergent extraction, >90% of the 35S-labeled material present was in this heparan sulphate proteoglycan. In the preceding paper (1), we have described the sulphated macromolecules synthesized and secreted by confluent rabbit lens epithelial cells grown on a plastic (tissue culture dish) substratum. Among the secreted macromolecules is a heparan sulphate proteoglycan (designated MI) that can be precipitated from the medium by the addition of (NH4)2SO4 to 30% saturation. Although confined under these culture conditions to the medium compartment of the cultures, this component resembles the proteoglycans described in the basement membrane-like stroma of the EHS "sarcoma" (2), in the basement membrane of murine mammary epithelial cells (3), and in the basement membrane-like matrix deposited in cell culture by the murine teratocarcinoma-derived cell line, PYS-2 (4). In this paper we show that rabbit lens epithelial cells cultured on a plastic substratum or on dried films of gelatin or laminin plus type IV collagen, do not deposit a basement membrane. In contrast, when grown on hydrated gels of type I collagen, the cells deposit basement membrane-like material that follows the contour of the basal cell surfaces. Production of this basement membrane is associated with the deposition of a heparan sulphate proteoglycan, similar to MI (see referTHE JOURNAL OF CELL BIOLOGY • VOLUME 99 SEPTEMBER 1984 861-869 © The Rockefeller University Press 0021-952518410910861109 $1.00 ence 1), in the cell layer. We have also compared the sulphated macromolecules produced by the epithelial cells in vitro with those synthesized by intact rabbit lenses in organ culture. MATERIALS AND METHODS The materials used were generally as described in the previous paper (1). L-[53H]proline was purchased from New England Nuclear (Boston, MA). Gelatin (USP reagent, granular) was obtained from Eastman Kodak Co. (Rochester, NY) and laminin and type IV collagen from the EHS "sarcoma" (see references 5 and 6) were generous gifts from Dr. Hynda Kleinman, National Institute of Dental Research (Bethesda, MD). Pepsin-digested bovine dermal type I collagen (Vitrogen) was purchased from Collagen Corp. (Palo Alto, CA). Sodium deoxycholate was obtained from Sigma Chemical Co. (St. Louis, MO). NCS tissue solubilizer was purchased from Amersham Corp., Arlington Heights, IL. Preparation of Artificial Substrata: To prepare films of laminin and type IV collagen, 100 ul of a solution of laminin (1 mg/ml in 0.4 M NaCI, 0.05 M Tris-HCl, pH 7.4) an d 25 #1 of a solution of collagen (1 mg/ml in 0.5 M acetic acid) were brought to a final volume of 500 #1 with distilled water. The surface of 35-mm plastic tissue culture dishes was covered with the mixture (500 #l/dish) and allowed to dry in a laminar flow hood overnight under ultraviolet light. Dried gelatin films were prepared by applying 1 ml of a sterile solution of 0.1% gelatin in distilled water per 35-ram tissue culture dish for 1 h at room temperature, The solution was then aspirated off and the plates were allowed to dry overnight in a laminar flow hood under ultraviolet light. 861 on F ebuary 1, 2013 jcb.rress.org D ow nladed fom Published September 1, 1984