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Procollagen I Synthesis in Human Skin Fibroblasts: Effect of Culture Conditions on Biosynthesis

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Procollagen I Synthesis in Human Skin Fibroblasts: Effect of Culture Conditions on Biosynthesis 0022-202X/ 80/7505-0425$02.00/ 0 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY, 75:425-430, 1980 Vol. 75, No. 5 Copyright © 1980 by The Williams & Wil!Ons Co. Printed in U.S.A. Procollagen I Synthesis in Human Skin Fibroblasts: Effect of Culture Conditions on Biosynthesis HARLEY FREIBERGER, M.D., DAVID GROVE, M.D., ARUNTHATHY SIVARAJAH, B.S., AND SHELDON R. PINNELL, M.D. Division of Dermatology, Department of M edicine, Dulle University Medical Cent e 1~ Durham, North Carolina, U. S.A . Human skin fibroblast culture promises to be a useful and diseased cell lines, we explored cultme conditions, including system for the investigation of the regulation of collagen the concentrations of serum, and ascorbic acid, in an attempt biosynthesis and the study of abnormalities in collagen to understand theii· effects on type-specific procollagen biosyn­ biosynthesis in connective tissue disorders. The effect of thesis. culture conditions on procollagen I biosynthesis has been determined. Optimal conditions for collagen bio­ synthesis were: 10% dialyzed heat-inactivated fetal calf MATERIALS AND METHODS serum, 0.15 mM ascorbate, and 0.078 mM ,8-am.inopro­ pionitrile. Newly synthesized procollagen I accumulated Fibroblast Culture in the medium at a linear rate for 18 hr. Preincubation of Normal human skin fibroblasts (CRL 1225-38-yr-old male, CRL cells in labelling media for 4 hr before adding radioactive 1229-33-yr-old female, CRL 1220- 15-yr-old female) were obtained proline enhanced synthesis. Collagenase digestion was from the American T ype Culture Collection (Rockville, MD) and grown used to study overall collagen biosynthesis. 94% of all in 100 mm plastic Petri dishes (Corning Glass Works, Corning, NY) in cco's modified Eagle medium (DME) containing 1 mg/ mJ glucose synthesized was found in the medium, and 6% Dulbe collagen (Grand Island Biological Co., Grand Island, NY). The medium was in the cell pellet. Under optimal conditions, collagen supplemented with 10% fetal calf serum (Irvine Scientific Sales Com­ comprised 24% of all protein in the medium, and 14% of pany, Fountain Valley, CA) which had been inactivated a t 56 °C for 30 protein produced by the whole culture. min, 50 units/ ml of potassium penicillin G (E.R. Squibb and Sons, Inc., New York, NY) and 50 J.Lg/ ml of streptomycin sulfate (Eli Lilly a nd Company, Indianapolis, IN). The c ultures were incubated at 37°C in Human skin fibroblast culture has proven to be a useful 5% C0 2-95% air atmosphere until confluent. Each experiment was means for studying collagen biosynthesis in inherited connec­ carried out with a single cell line at the same passage number- between tive tissue disorders. Genetic defects are expressed in the cells, 7 and 17. Two days following confluence the c ells were washed with even though they have been removed from their physiological phosphate buffered saline and changed to labeling medi a. Unl ess other­ surroundings. Thus, abnormalities in type-specific collagen syn­ wise noted these media were identical to the growth medium described l calf serum was dialyzed, t he g lucose co n­ thesis have been reported in osteogenesis imperfecta [1] and above e xcept that the feta as 4.5 mg/ ml, and sodium ascorbate 0.05-0.5 mM (Sigma (ecchymotic) [2,3]. Deficient centration w type IV Ehlers-Danlos syndrome Chemical Company, St. Louis, MO) and P-aminopropionitrile fumarate levels of enzymes essential for collagen biosynthesis have been (BAPN) 0.078 mM-0.39 mM (Aldrich Chemical Company, inc., Milwau­ reported in skin fibroblasts: of lysyl hydroxylase in type VI kee, WI) were added. In some experiments 20 mM tricine (Grand Island Ehlers-Danlos syndrome (hydroxylysine-deficient collagen dis­ Biological Co.) or 25 mM Hepes (Sigma) buffers were a dded and the ease) [ 4-7], of procollagen peptidase in type VII Ehlers-Danlos bicarbonate concentration was diminished to 24 mM . Except as other­ syndrome (procollagen peptidase deficiency) [8], and of lysyl wise indicated fibroblasts were preincubated f or 4-24 hr in labeling oxidase in type V Ehlers-Danlos synru·ome (X-linked) [9] and medium and labeled with 3 ml fresh labeling medium containing 25 J.L Ci 3 cutis laxa (X-linked) [10]. 2- 3, H-proline (New England Nuclear, Boston, MA) for an additional h, and Human skin fibroblasts devote a significant portion of theii· 6-72 hr. The medium was harvested, combined wi th cell was ddition of protease inhibitors: 1 mM N-ethyl­ to coll agen [11-14]. Types I and stored at -20°C after a protein synthesizing capacity maleimide (NEM) (Sigma), 1 mM phenylmethylsulfonyl fluoride III procollagen are released into the medium dming culture. (PMSF) (Sigma), and 1 mM disodium ethylenedinitrilotetraacetic acid Although a small amount of procollagen is converted to collagen (EDTA) (Matheson, Coleman, and Bell , Norwood, OH). The ce ll pell et and deposited in fibrillar form in the cell layer, most remains as was harvested with 0.05% trypsin-0.02% EDTA and an aliquot of the procollagen in the medium [15-17]. Each fibroblast seems ca­ cells was counted in a hemocytometer. All results were c orrected f or pable of simultaneously synthesizing both types I and III col­ ceU number. lagen [3]. Since human skin fibroblast cultme promises to be a useful Diethylam.inoethyl (DEAE) Cellulose Column Chromatography tool for studying regulation of collagen biosynthesis in normal Procollagen was isolated by DEAE chromatography modified from Uitto, Lichtenstein, a nd Bauer [18]. Samples were e quilibrated b y dialysis for 18 hr at 4 oc with 70 vol of DEAE buffer ( 2 M m ea, 0.4 mM 1 mM NEM, I mM PMSF, 25 mM Tris HCl, pH 7. 5). (PMSF Manuscript received March 20, 1980; accepted for publication June EDTA, was added in isopropanol at the start of dialysis, and to t he gradient 10, 1980. solution just before chromatography [19]) . Samples were a pplied to a This work was supported by grants from the National Institutes of 1.6 x 5.0 em column of DEAE cellulose (DE52-Whatman; Maids tone Health, grant 2R01 AM 17128 and Cardiology Training grant 5T32 , England). The sample was eluted at 4 °C wi th a 400 mllinear salt HL07101 and by the Howard Hughes Medical Institute Laboratory, the Kent gradient (0-0.2 M NaCI, or in some e xperiments, 0-0.18 M LiCI) in Department of Medicine, at Duke University Medical Center. DEAE buffer, pH 7.5, with the same protease inhibitors, at a fl ow rate This is publication number 52 of the Dermatological Research Lab­ of 100 ml per hr, followed b y a wash of 1 M NaCI in DEAE buffer. 0.5 oratories at Duke University Medical Center. ml aliquots of 5 ml fractions were combined with 3.5 ml of Aquasol-2 Reprint requests to: Sheldon R. Pinnell, M.D., Box 3135 Hospital, (New England Nuclear , Boston, MA) and counted in a P ackard Tri­ ity Medical Center, Durham, NC 27710. Duke Univers Carb liquid scintillation counter at a n effi ciency of 20%. Abbreviations: BAPN: P-aminopropionitrile Pep sinization CMC: carboxymethyl cellulose NEM: N-ethylmaleimide In some cases elution fractions from DEAE column clu-omatography PMSF: phenylmethylsulfonyl fluoride or cell pellets from fibroblast culture were pepsinized in 0.5 M acetic 425 426 FREIBERGER ET AL Vol. 75, No. 5 acid. Pepsin (2720 !L/mg; Worthington Biochemical Corporation, Free­ indistinguishable results. The fraction of co llagenase soluble counts hold, NJ) was employed in a concentration of 50 !Lg/ ml for 6 hr at 4 °C. (cpm) was calculated from the formula: (Collagenase-digested cprn­ Samples were adjusted to pH 7.5 with NaOH and allowed to stand 30 blank cpm)/(Aliquot cpm-blank cpm). Percent collagen in a fraction min at 4°C to insure pepsin inactivation. could be calculated using a rearrangement of the formula of Diegelmann and Peterkosky [22]: % collagen= !:J. cpm/[5.4- (4.4 X !:J. cpm)]. Amino acid analysis was carried out on lyophilized samples of me­ SDS-Polyacrylamide Slab Gel Electrophoresis dium, on procollagen I recovered from DEAE column separations, and on 0.5 N acetic acid-soluble and -insoluble cell pellet fractions. Samples The procedure was modified from Laemmli [20]. DEAE-coiumn were hydrolyzed overnight at 108°C in 6 N HCl under N2; hydrolyzed fractions selected for gel electrophoresis were brought to an ammonium samples were flash-evaporated, then taken up in citrate buffer and sulfate concentration of 176 mg/ml following the addition of carrier resolved on a Beckman model 119 automatic ·amino acid analyzer and lathyritic rat skin collagen (0.2 mg); after centrifugation at 15,000 X g counted. for 30 min, the precipitates were dissolved in a cocktail composed of 3% Neutral protease activity of collagenase and of cell pellets was sodium dodecyl sulfate (SDS), 10% glycerol, 0.01% Bromphenol Blue, assayed with tritium-labeled casein. 20 ,\ labeled casein in phosphate (Fisher Scientific Co., Fairlawn, NJ) and 62.5 mM Tris at pH 6.7. buffer, pH 4, was brought to a final volume of 60 ,\ by addition of Polyacrylamide gel electrophoresis was conducted with 1.5 rom­ collagenase buffer adjusted to yield a fmal collagenase concentration of thick vertical gels [6% acrylamide, (Bio-Rad), 0.44% bis (Bio-Rad)], 11.2 U/ml, and 5 x w-om NEM or 1% albumin. For assay of the cell using a running buffer of 0.02 M glycine, 0.25 mM tris, 0.2% SDS, pH pellet a whole pellet was dialyzed against phosphate buffered saline pH 8.3. Samples containing 5000-10,000 cpm were denatured for 10-15 min 7.4 at 4°C overnight, and a volume equivalent to 80,000 cells employed at 42 °C, with or without 2% ,8-mercaptoethanol, in slab gel cocktail.
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