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 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. 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 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 essential for collagen biosynthesis have been (BAPN) 0.078 mM-0.39 mM (Aldrich Chemical Company, inc., Milwau­ reported in skin fibroblasts: of 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 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. in the assay. The reaction was allowed to proceed for 1 hr at 37°C, then Electrophoresis was carried out at 25°C for 5 hr at 30 milliamps stopped by addition of TCA to a final concentration of 4%. The supplied by an LKB Model 2103 D.C. power source. Gels were fixed supernatant was counted. The results are expressed in trypsin equiva­ and stained overnight in 25% isopropyl alcohol, 10% acetic acid, 0.025% Lents (1 llg of trypsin yielded 6000 cpm/hour with this substrate). Coomassie Blue (Bio-Rad), then destained in 7.5% acetic acid, 5% methanol. Gels were sliced in 1 mm sections on a Mickle gel-slicer RESULTS (Brinkmann Instruments, Westbury, NY). Slices were digested with 0.5 Type-specific procollagen synthesis was determined by ml 30% H 0 for 2 hr at 56°C and counted in 10 ml Aq uasol-2. 2 2 DEAE chromatography of fibroblast culture media (Fig 1). 80% or more of the counts applied to the column were recovered in Fluorography the eluate. Procollagen I eluted near fraction 50. On SDS polyacrylamide slab gel electrophoresis this peak migrated with Polyacrylamide gels were also studied by fluorography after the an apparent molecular weight larger than 300,000; upon reduc­ manner of Laskey and Mills [21]. Gels were prepared in the manner tion it yielded 2 smaller peaks in a ratio of 1.83:1 which migrated just described, but with 10% acrylamide and 0.12% bis. Denaturation of in positions expected for pro-a1 and pro-a2 chains (Fig 2A,B). samples was at 100°C for 2 min. After destaining, gels were equilibrated Following pepsinization of procollagen I, chains which co-mi­ with PPO (New England Nuclear) in dimethylsulfoxide (16%, W /V), grated with a (I) and a chains of lathyritic rat skin collagen on dried, apposed to "pre-flashed" Kodak X-Omat XR-5 fllm for 3 to 30 1 2 SDS slab gel electrophoresis days at -70°C, and developed in trays in Kodak liquid X-ray developer. were detected at a ratio of 2.17:1 The developed gels were scanned with a Gelman ACD-5 gel scanner. (Fig 2C). Procollagen III eluted from DEAE near fractions 60-70. Following pepsinization it migrated on SDS polyacrylamide Carboxymethyl Cellulose (CMC) Column Chromatography slab gels with an apparent molecular weight greater than 300,000. Following reduction, a single peak of activity in the Pepsinized samples for CMC chromatography were equilibrated by position of a1 (I) lathyritic rat skin collagen was observed (data dialysis (1 M urea, 12 mM potassium acetate at pH 4.8) overnight at not shown). Procollagen III synthesis represented 5-14% of the 4°C with CMC buffer; 10 mg lathyritic rat skin collagen was added as procollagen synthesis observed in these studies. carrier. Following denaturation at 42°C for 30 min, the sample was No evidence for types I or III collagen or procollagen could applied to a 1.6 X 13 em column of carboxymethyl cellulose (CM 52, be detected in the fu-st and last elution peaks from DEAE Whatman) maintained at 42°C. The sample was eluted with a 500 m1 chromatography. Each peak was analyzed by SDS polyacryl­ linear salt gradient of 0-0.16 M LiCl in CMC buffer at a flow rate of 125 amide slab gel electrophoresis and CMC chromatography be­ ml/hr. The elution proftle of rat skin carrier was monitored spectra­ photometrically at 224 "A by a model 240 Gilford Spectrophotometer fore and after pepsinization. Likewise, cell pellets were exam­ (Gilford Instruments, Oberlin, OH); 0.5 ml aliquots of 5 ml elution ined for evidence of collagen chains by these techniques and fractions were counted in 3.5 ml Aquasol-2. under the conditions of the cultures examined no type I or III collagen could be detected. "Autoradiographs" of cell pellet electrophoretograrns suggested the presence of a small amount Collagenase Digestion of chains in the position expected for B chains [231- The first

Collagenase digestion was carried out by a modification of the method of Russell, Russell and Trupin [14]. Samples for collagenase pro-I digestion were dialyzed extensively against collagenase buffer (0.33 M 6 I 12.0 Ca-acetate, 0.25 mM tris, 0.02% NaN a, pH 7.4) at 4 °C. Reaction mixtures 5 10.0 co nsisting of 100 "A of sample, 500 "A 1.4% albumin, and 100 ,\containing 4 8.0 5.6 U of highly purified bacterial collagenase (Collagenase form III, COND\JCTIVITY Advance Biofactures, Inc.), all in collagenase buffer, were kept in 1.4 cpm>

.Bn .81 2 a 1 a 2 alyzed serum was employed. Heat inactivation of the serum had no effect on synthesis using either dialyzed or undialyzed u ! l serum (data not shown). 400 A Ascorbic acid is essential for collagen synthesis; it acts as a for the essential enzymes, prolyl and lysyl hydroxyl­ ases. The effect of ascorbate on procollagen I synthesis is shown 300 in Fig 4. Without added ascorbate, synthesis was minimal; synthesis was maximal at 0.15 mM ascorbate. Reduced synthe­ cpm 200 sis at higher ascorbic concentrations may reflect known cyto­ toxic effects [24,25]. Preincubation of fibroblasts in labeling medium for various 100 times before addition of isotope is illustrated in Fig 5. Maximal synthesis was detected after a 4 hr preincubation. Twenty-four hr of preincubation resulted in synthesis similar to that without 25 50 75 100 preincubation. BAPN was added to the cultures to inhibit , an 400 B

300 pro- a 1: pro-a2 =J · 83: I

cpm 200 4

100 3 cpm x1o-3; 6 25 50 75 100 10 cells 2 400' c 300- cpm 200 r-

100- 10 20 30 40 50 60 0 u \L--,----, /o FETAL CALF SERUM FIG 3. Effect of fetal calf serum on procollagen I synthesis. Ce ll 25 50 75 100 line-CRL-1 225. Passage #7. Cells per plate at conflu ence 1.84 (± 0.28) X 106 (n=14). Labeling media-DME in varying concentrations of DISTANCE (mm) dialyzed fetal calf serum, Na ascorbate 0.15 mM, BAPN fu marate 0.39 FIG 2. Identification of type I procollagen from DEAE chromatog­ mM . Preincubation-24 hr. Labeling period-24 hr. raphy by SDS polyacryamide slab gel electrophoresis. Migration posi­ tions for lathyritic rat skin collagen chains are denoted. A, procollagen I from DEAE. B, procollagen I fo llowing reduction by 2% ,8-mercapto­ ethano l. C, procollagen I following pepsin digestion.

peak was studied in additional detail. Despite the a ddition of protease inhibitors, no counts could be retained in samples from 3 this peak during dialysis either against collagenase buffer, wa­ ter, or 0.5% acetic acid. Material precipitated from a first peak cpm x1o-3/ M }2S0 after the addition of carrier collagen with 1.4 (NH4 4 2 accounted for 1.5% of the counts in the peak, and had a 106 cells hydroxyproline proline ratio of 0.63. Thus macromolecular col­ lagenous material equal to about 0.5% of that in the procollagen I peak may be found in the forepeak. The effect of dialyzed, heat inactivated fetal calf serum on procollagen I synthesis is seen in Fig 3. Maximal recovery of procollagen I occurred at 10% fetal calf serum with reduction at higher levels. (Overall synthesis of collagenase-digestible ma­ terial, however, increased from 0 to 50% serum concentration. 0.2 0.4 0.6 Increased labeling of material which migrated outside the pro­ collagen I position on the DEAE chromatograph including mM SODIUM ASCORBATE products of procollagen I conversion and other procollagen FIG 4. Effect of sodium ascorbate on procollagen I synthesis. Cell types appeared to account for this increase.) Labeling of pro­ line-CRL-1225. Passage #17. Cells per plate at confluence 2.19 (± collagen I was 40% greater when employing dialyzed as com­ 0.24) x 106 (n=12). Labeling media-DME. 10% dialyzed fetal calf pared to undialyzed 10% fetal calf serum. This result is probably serum, varying concenb·ations of ascorbate, BAPN fumarate 0.39 mM . related to dilution of the intracellular proline pool when undi- Prein"ubation-24 hr. Labeling period-24 hr. 428 FREIBERGER ET AL Vol. 75, No.5 14 TABLE I. R esults of hydroxyproline determinations 3 hypro 4 hypro Fraction (n) 12 4 hypro proline Procollagen I (15) 0.008 ± 0.005 (SD) 0.77 ± 0.07 Pepsinized pro I (6) 0.015 ± 0.006 0.88 ± 0.02 10 Medium (6) 0.007 ± 0.002 0.39 ± 0.02 Cell P ellet: 3 0.5 N HAc-soluble (10) 0.017 ± 0.006 0.05 ± 0.01 cpm x 10- I 8 0.5 N HAc-insoluble 0.08 ± 0.04 0.012 ± 0.004 (8) 10 6 cells 6 distributed as follows: medium, 65-70%; soluble pellet, 17-21 %; insoluble pellet, 10-14%. By collagenase assay, collagen con­ 4 tained 60-70% of the labeled proline in the medium, 11-16% in the soluble pellet, and 2-6% in the insoluble pellet. Similar 2 fractions could be calculated from the results of amino acid analysis (see Table I) . Of the newly synthesized collagen, 93- 96% was found in the medium, 3.5-4.1% in the soluble pellet, and 1.5-2.1% in the insoluble pellet. 4 8 12 16 20 24 The effects of cell density on collagen synthesis are summa­ rized in Table II. Cells were plated at 5 initial concentrations, TIME (hrs) and labeled when the highest plating concentrations had re­ FIG 5. Effect of preincubation time on procollagen I synthesis. Cell sulted in confluent growth. Cultures seeded with 200,000 or Line-CRL-1229. Passage #12. Cells per plate at confluence 1.21 (± 400,000 cells per plate yielded similar numbers of cells, and 0.17) x 10° (n=10) . Labeling media-DME, 10% dialyzed fetal calf their results were pooled for statistical purposes. The total serum, Na ascorbate 0.15 mM , BAPN fumarate 0.39 mM. Preincuba­ incorporation of counts into protein increased tion-varied. Labeling period-24 hr. as cell density increased, but the labeling per cell decreased. Collagen produc­ tion per cell was independent of cell density. As a result, 5 collagen contained 25% of all counts, and comprised 5.8% of all protein, at a cell density of 0.86 X 106/plate, while the corre­ sponding values were 47% and 14% at 2.3 X 106 cells/plate. Recoverable procollagen I accounted for 9% of all counts in the 4 medium (2% of the protein) at the lowest density, and 29% (7%) at the highest, while at the same cell densities it accounted for 24% and 44%, respectively, of the collagen in the medium. 3 DISCUSSION cpm x 10-3 I 6 Human skin fibroblasts under the conditions studied in this 10 cells · report devoted approximately 85-95% of their collagen synthe­ 2 sis to . This agrees with the data of Mayne, Vail, and Miller [26]. Type I collagen is synthesized in fibroblasts in a precursor form designated procollagen I. Following secretion to the extracellular space, procollagen is converted to collagen by excision of amino- and carboxy-terminal extension peptides by specific . In human skin fibroblast culture most collagen is secreted and remains in soluble form in the medium, although under serum free conditions some type I collagen is deposited in the insoluble cell layer [27]. Under 12 24 36 48 60 72 optimal conditions determined in this study (synthesis for 24 hr or less in the presence of fetal calf serum) nearly all type I TIME (hrs) collagen was present as procollagen. Virtually no type I collagen FIG 6. Effect of labeling period on procollagen I synthesis. Cell other than procollagen could be detected in the cell pellets or line-CRL-1220. Passage #14. Cells per plate at confluence 3.34 (± in media separated by DEAE chromatography. 0.39) X 10° (n=14). Labeling media-DME, 10% dialyzed fetal calf Bashey and Jimenez reported that approximately 5% of the serum, Na ascorbate 0.15 mM, BAPN fumarate 0.39 mM. Preincuba­ hydroxyproline synthesized by human skin fibroblasts was de­ tion-4 hr. Labeling period-varied. posited in the cell layer in the presence of fetal calf serum [12]. Our studies with collagenase confirm this; 6-7% of all collagenase-digestible or hydroxyproline counts were found in essential for collagen cross-linking. Maximal procolla­ the cell pellet. Lembach et al have suggested that at least some gen I synthesis was observed with 0.078 mM ,8-aminopropion.i­ collagen in the cell layer is not type I and may be related to a trile fumarate; this is a minimal level for complete inhibition of cell attachment factor [28]. These authors have shown that the human fibroblast lysyl oxidase (unpublished data). Procollagen 3-hydroxyproline content of the cell pellet is higher than that synthesis was. inhibited at higher BAPN concentrations (21% of the medium, or of procollagen I. In our system, the 3-hypro/ at 0.39 mM) but similar inhibition was observed with equimolar 4-hypro ratio was the same in procollagen I as in the whole sodium fumarate (data not shown). medium, but was twice as high in the 0.5 N acetic acid-soluble As illustrated in Fig 6, procollagen I synthesis under the portion of the cell pellet, and 8 times as high in the insoluble culture conditions examined was linear for 18 hr. Procollagen portion (see Table I). This is consistent with our analysis of the III synthesis as assayed by DEAE column chromatography was cell pellet by polyacrylamide gel electrophoresis and "autora­ likewise linear during this interval (data not shown) . diography"; these studies indicate that very little, if any, type In confluent cultures, tritiated proline-labeled protein was I collagen is deposited in the cell pellet, but that another Nov. 1980 PROCOLLAGEN BIOSYNTHESIS IN HUMAN SKIN FIBROBLASTS 429

TABLE II. Effect of cell density on incorporation of tritiated proline into all , collagen (collagenase-digestible protein), and procollagen I

Initial Final Total cpm Total Procollagen I Procollagen I T otal Coll agen = Density density inc01;porated collagen cpm cpm in medium cpm in medium collagen cpm % of all 10 cells 6 Total Coll agen Total cpm protein cells/ plate 10 cells Total cpm 25,000 0.87 X 106 2.90 (±0.30) (s.d.) 0.750 (±0.118) 0.087 0.239 0. 251 5.8% 6 (n = 3) X 106 X 10 ±0.016 ±0.048 ±0.037 50,000 1.24 X 106 2.20 (±0.34) 0.775 (±0.118) 0.152 0.308 0.354 9.2% 6 (n = 3) X 106 X 10 ±0.025 ±0.058 ±0.032 100,000 1.60 X 106 1.98 (±0.17) 0.880 (±0.018) 0.204 0.330 0.445 12.9% 6 (n = 3) X 106 X 10 ±0.021 ±0.035 ±0.033 200,000 or 2.29 X 106 1.50 (±0.14) 0.707 (±0.070) 0.288 0.442 0.471 14.2% 400,000 X 106 X 106 ±0.025 ±0.044 ±0.016 (n = 5) species, possibly AB collagen, is present. Since AB collagen, atmosphere in the presence of 24 mM bicarbonate, tricine or particularly the B chain, is relatively rich in 3-hydroxyproline HEPES organic buffers in nontoxic doses had little effect on [23], it might account for the increased 3-hydroxyproline con­ pH. We fmd these buffers to be useful, however, in preventing tent of the pellet. wide pH swings as C02 concentration changes (exposure to Care was taken to ensure that type I procollagen was not room air). The latter may be preferable for some studies since degraded or converted during evaluation. Collagenase and pro­ tricine shows binding of divalent cations [39]. collagen peptidases have been detected in human skin fibroblast Under optimal conditions for collagen synthesis determined cultures [27,29-31]. Protease inhibitors were added immedi­ in these studies, procollagen I synthesis was linear for 18 hr. ately to the cultures following harvest. In addition, DEAE Longer labeling times resulted in new peaks appearing on the chromatography was canied out in the presence of inhibitors. DEAE chromatogram; they may be related to procollagen The latter precaution was found to be essential for reproducible conversion or degradation, but their identity has not been yields; yields were lower in the absence of inhibitors. Our cell determined. In studies comparing type specific collagen synthe­ pellets had caseinase activity at neutral pH equivalent to 0.2 sis in different skin fibroblast lines, kinetic parameters for each J.lg of trypsin per 106 cells. cell line will need to be determined. Procollagen I synthesis was stimulated approximately 4 times Collagenase was used to estimate the ratio of collagen bio­ in the presence of 10% fetal calf serum when compared to synthesis to overall protein biosynthesis. Highly-purified clos­ serum-free controls. Narayanan and Page reported a similar tridial collagenase (Form III, Advance Biofactures, Inc.) was effect of serum on collagen synthesis in human gingival fibro­ found previously to have no protease activity against a variety blasts although even more synthesis occurred in 20% fetal calf of substrates [ 40]. We found very slight residual protease activ­ serum [32]. This difference may be related to the type of ity against tritium-labeled casein, even in the presence of 5 X fibroblast studied or may be a manifestation of differences in 10- 5 M NEM; this activity was abolished in the presence of 1% 5 serum source. Bankowski reported increased collagen synthesis BSA with NEM, 5 X 10- M. in human skin fibroblasts in the presence of calf serum. His Over a fairly wide range, fmal cell density appeared to have studies suggested that this increased collagen synthesis oc­ little or no effect on collagen biosynthesis when expressed on a curred as a result of a generalized increase in all protein syn­ per cell basis. The overall incorporation of label into protein thesis [11]. On the other hand, the data of Lembach et al fell with increasing final cell concentrations (Table II), thus the suggest a modest decrease in relative collagen synthesis under synthesis of noncollagenous proteins declined considerably as conditions of serum deprivation [28]. We found that total cell density increased. The portion of all synthesized proteins protein synthesis and total collagen synthesis increased in par­ comprised by collagen reached 14% at the higher cell density allel from serum concentrations of 0 to 50%. However, as studied; this is more than twice the fraction previously reported discussed below, total protein and collagen synthesis each were [41]. We presume that this difference is due to improvement in affected differently by alterations in cell density. each of the several conditions of culture that were studied. Ascorbic acid is essential for procollagen I synthesis and is a In confluent cultures, collagen comprised 21% of the labeled necessary cofactor for hydroxylation of proline and lysine [33]. protein in the medium, by the collagenase assay. Two indepen­ Hydroxyproline is essential for stability of the collagen triple dent estimates gave similar results: hydroxyprohne/ proline ra­ helix; lacking hydroxyproline the chains are not stable in helical tio, 26% (using our own figure, 0.77, for the ratio of hydroxypro­ form and are not secreted at·a normal rate [34]. In addition, line to proline in procollagen I); polyacrylamide gel electropho­ ascorbate may stimulate collagen synthesis directly [35]. resis with autoradiography, 24%. Collagenase assay and amino The activity of prolyl hydroxylase is increased in cells by acid analysis also gave very similar estimates of the collagen supplying ascorbate in addition to other cofactors for the hy­ fraction of the soluble and insoluble cell pellet. droxylase reaction (34,36,37). This apparent activation of en­ Ratios of hydroxyproline to proline determined for several zyme seems to occur because prolyl hydroxylase is tightly procollagen peaks were compared before and after pepsin diges­ bound to unhydroxylated substrate in the cell. As cofactors are tion. The respective ratios, 0.77 and 0.88 (p < 0.0025), indkate supplied, the substrate is hydroxylated, the affinity is reduced, that proline residues in procollagen in this system are more and prolyl hydroxylase becomes available for new substrate. fully hydroxylated than has been previously noted in skin This increase in available enzyme activity occurs in human skin collagen [ 42]. Increased hydroxylation of ex, (I) chains in tissue fibroblasts over 4 hr (Murad S, and Pinnell SR, unpublished culture has been noted recently [ 43); ow· observations are in results) and corresponds to the optimal preincubation period agreement with the observation, and extend it to the whole observed in these studies for new procollagen I synthesis. type I collagen molecule. pH control may be important for optimal collagen synthesis. Highly purified bacterial collagenase released all of the la­ Nigra et al studied collagen synthesis in human skin fibroblasts beled protein from the procollagen I fraction, although the and showed optimal synthesis above pH 7.6 [38]. We undertook reason for this is not clear. The enzyme digested 99.5±3.6% experiments to modify pH and found that bicarbonate levels (n=6) of the nondialyzable counts recovered from the DEAE less than 24 mM led to diminished cell growth. In a 5 % C02 procollagen I peak. The hydroxyproline/ proline ratio of this 430 FREIBERGER ET AL Vol. 75, No. 5 material was 0. 76; this ratio, lower than that (0.88) for processed 17. Goldberg B, Epstein Jr EH, Sheer CJ: Precursors of collagen collagen, reflects the presence of proline in the nonhelical secreted by cultured human fibroblasts. Proc Nat! Acad Sci USA 69:3655-3659, 1972 extensions. Whether the collagenase attacks numerous sites on 18. Uitto J, Lichtenstein JR, Bauer EA: Characterization of procolla­ the nonhelical extensions, yielding only very small peptides, or gen synthesized by matrix-free cells isolated from chick embryo attacks only a few sites, releasing a few larger species which are tendons. Biochemistry 15:4935-4942, 1976 still too small to be precipitated by 5% TCA, is not clear, but 19. James GT: Denaturation of the protease inhibitor phenylmethyl­ polyacrylamide gel electrophoresis sulfonyl fluoride in buffer. Anal Biochem 86:574-579, 1978 of the supernatant counts 20. Laemmli UK: Cleavage of structural proteins during the assembly from collagenase digests did not suggest the presence of any of the head of bacteriophage T4. Natw·e 227:680-685, 1970 homogeneous population of peptides (data not shown). 21. Laskey RA, Mills AD: Quantitative film detection of 3H and 14 C on Thus, over a rather broad range of cell densities, collagen polyacrylamide gel by fluorography. Europ J Biochem 56:335- biosynthesis per cell was constant, while synthesis of other 341, 1975 22. Peterkofsky B, Diegelmann R: Use of a mixture of proteinase-free proteins decreased progressively. The reason why other pro­ for the specific assay of radioactive collagen in the teins are "turned off' is moot, but polyacrylamide gel electro­ presence of other proteins. Biochemistry 10:988-994, 1971 phoretograms suggest that fibronectin may be the only other 23. Rhodes RK, Miller EJ: Physicochemical characterization and mo­ protein found in large amounts in the medium in our cultures; lecular organization of the collagen A and B chain. Biochemistry 17:3442-3448, 1978 reduction of fibronectin production in the face of increasing 24 . 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