Translatable mRNA for GP 140 ( a Subunit of Type Vl Collagen ) Is Absent in SV 40 Transformed Fibroblasts

Production of GP140, a major component of the extracellular matrix of cultured fibroblasts, is markedly decreased in SV40 transformed cells as compared with normal cells (Carter, W. G., 1982, J. Biol. Chem., 257:13805-13815). To determine at what step the biosynthesis is inhibited, we compared the levels of functional mRNA for GP140 in normal and transformed fibroblasts. Translation of total RNA from Wl38 cells in a reticulocyte lysate, followed by immunoprecipitation with affinity-purified antibodies to GP140, yielded a single polypeptide with an Mr of 125,000. This polypeptide was identified as GP140 based on its immunoreactivity, collagenase sensitivity, and comigration on polyacrylamide gels with GP140 synthesized by cells in the presence of tunicamycin and 2,2'-bipyridyl. No cell-free synthesis of GP140 was observed with total RNA from SV40 transformed Wl38 cells, indicating that these cells contain very low levels of GP140-specific mRNA. The biosynthesis of GP140 might therefore be blocked at the transcriptional level. GP140 is a major glycoprotein of the extracellular matrix of cultured fibroblasts (1-4). The protein has been shown to form covalent and/or noncovalent interactions with several other matrix components including GP250, fibronectin, and collagen type I. When isolated under nonreducing conditions, GPI40 can promote stable attachment and spread fibroblasts in vitro (2). This activity, however, is lost upon reduction, suggesting that the correct conformation of the protein provided by intact disulfide bonds is important for the interaction of GPI40 with the cell surface. A soluble form of GP140, which differs from the matrix form in its lower degree of intermolecular disulfide bonding, is found in the conditioned media of cultured fibroblasts (3, 4). GP140 contains equal amounts of hydroxyproline and hydroxylysine, indicating that it is a collagen-like protein (2). Recently, three different laboratories have demonstrated by immunological, biochemical, and electron microscopical techniques that GPI40 is related to type VI collagen (5-7), a dumbbell-shaped molecule with a short, collagenous triple helix separating two large globular domains (8). Previous work from this laboratory has established that GP 140 is absent from the extracellular matrix of SV40 transformed fibroblasts and various human tumor cells, and that very little is found in the conditioned media of such cells (3, 4). The loss of GPI40 appeared to be due to a decreased synthesis rather than an increased degradation, since matrix derived from normal cells was not degraded at an unusual rate in the presence of transformed cells, and since virtually no cytoplasmic precursor form of GP140 was detected in transformed cells. The goal of the present study was therefore to determine at what step the production of GP140 may be inhibited. We show by in vitro translation that the level of functional mRNA for GP140 is decreased more than 20-fold in SV40 transformed WI38 cells as compared with normal cells. The biosynthesis of GPI40 might thus be blocked at the transcriptional level. MATERIALS AND METHODS Cell Culture: Normal fibroblasts from embryonic human lung (W138 cells) and SV40 transformants of these cells (W138 VA 13 cells) were obtained from the American Type Culture Collection (Rockville, MD). A204 cells from a human rhabdomyosarcoma and 251 MG cells from a human astrocytoma were provided by Dr. K. Alitalo (University of Helsinki). All cells were propagated at 37*C under an atmosphere of 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 ug/ml streptomycin. For metabolic labeling, the cells were rinsed twice with methionine-free medium containing 0.5 mg/ml bovine serum albumin instead of fetal calf serum, and incubated for 5-14 h in this medium with 100 uCi/ml, 1,090 Ci/mmol [3~S]methionine (New England Nuclear, Boston, MA). For metabolic labeling in the presence of 2 #g/ml tunicamycin (CalbiochemBehring Corp., La Jolla, CA) and 0.2 mM 2,2'-bipyridyl (Sigma Chemical Co., St. Louis, MO), cells were preincubated with these inhibitors for 2-5 h before addition of [~SS]methionine. Cell-free Translation: Total cellular RNA was extracted by the guanidine HC1 method (9) or by the SDS/proteinase K method ( l 0). A reticulocyte lysate was prepared from anemic rabbits and treated with micrococeal nuclease THE JOURNAL OF CELL BIOLOGY VOLUME 100 FEBRUARY 1985 638-641 638 © The Rockefeller University Press . 0021-9525/85/02/0638/04 $1.00 on July 8, 2017 jcb.rress. org D ow nladed fom essentially as described by Pelham and Jackson (11). For in vitro translation, 19.6 ~l reticulocyte lysate was incubated for 2 h at 30"C in a final volume of 50 #l containing 100 #Ci [~SS]methionine (1,090 Ci/mmol) (New England Nuclear), 50-800 #g/ml of total cellular RNA, 7.5 mM creatine phosphate, 1.25 mM spermidine, 1.87 mM dithiothreitol, 88 mM potassium acetate, 0.3 mM magnesium acetate, 23.4 #M of all amino acids except methionine, and 18.7 mM HEPES (pH 7.6). Before addition to the translation reaction, the RNA was heated to 80"C for 6 s. In some experiments, 100 U/ml of the ribonuclease inhibitor RNasin (Promega Biotec, Madison, WI) was added. Immunoprecipitation: Antisera to a preparation of GPl40 from the matrix of WI38 cells were raised in rabbits (3) and affinity-purified on Sepharose-immobilized GPI40 that had been isolated from human placenta (5). A rabbit antiserum to vinculin was a generous gift from Dr. L. R. Rohrschneider (Fred Hutchinson Cancer Research Center, Seattle, WA). Proteins were precipitated from the translation mixtures (40-/~1 aliquots) or from the conditioned media of WI38 cells (250-~1 aliquots) as described (12). The amount of antibody required for quantitative precipitation was determined in a previous experiment. When immunoprecipitation was successively repeated with the same aliquot of translation mixture, only negligible quantities of GPI40 were recovered in the second precipitate. For control experiments, nonspecific rabbit immunoglobulins (Sigma Chemical Co.) were used. Digestion of the immunoprecipitates with bacterial collagenase (315 U/mg, chromatographically purified) (Worthington, Freehold, NJ) was done in the presence of 2 mg/ml bovine serum albumin as described 02). Gel Electrophoresis: Protein was analyzed on 7% SDS polyacrylamide gels essentially as described by Laemmli (13). Gels were fixed in 50% methanol, 10% acetic acid, allowed to swell to their original size in 10% methanol, 10% acetic acid, and exposed to Kodak X-Omat AR film (Eastman Kodak Co., Rochester, NY) after we dried them on filter paper. For quantitative comparison, areas corresponding to radioactive bands were excised from the dried gels, dissolved in 0.2 ml of 70% perchloric acid, 0.4 ml of 30% hydrogen peroxide (2-3 h at 75"C), and counted in 5 ml of Formula 963 scintillation fluid (New England Nuclear).