Epidermal Growth Factor Inhibits Transcription of Type I Collagen ...

Vol. 266, No. 15, Issue of May 25, pp. 9997-10003,1991 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY (c>1991 hy The American Society for Biochemistry and Molecular Biology, Inc

Epidermal Growth Factor InhibitsTranscription of Type I Collagen Genes and Production of Type I Collagen in Cultured Human Skin Fibroblasts in the Presence and Absence of L-Ascorbic Acid2Phosphate, a Long-acting Vitamin C Derivative* (Received for puhlication, J u l y 19, 1990)

Shun-ichi KurataS and Ryu-ichiro Hatajq From the $Department of Biochemical Genetics and the §Department of Tissue I’hyciology, Medical Research Institute, Tokyo Medical and Dental University, Kandasurugadai, Chiyoda-ku, Tokyo 101, Japan

Recombinant human epidermal growth factor (EGF,is tightly regulated to maintain homeostasisof the body. 2-10 ng/ml) stimulated growth and production of nonBiosynthesis of this collagen is a highly complex process collagenous proteins, but inhibited production of col- involving transcription of the procul(I) and procu,(I) genes, lagen by 60%in cultured human skin fibroblasts. Type processing of the pre-mRNAs to mature mRNAs, and transanalysis of the collagen produced indicated that inhi- lation of these mRNAs into preproa chainsfollowed by postbition of the collagen production observed was mainly translational steps includinghydroxylation of some of the a reflection of a reduction in typeI collagen. proline and lysine residues, attachment of carbohydrate conThe accumulation of proaI(1) and proaz(I) mRNAs stituents, and secretion and processing into the mature coland the transcriptional activity of these genes were lagen molecule which contains two al(I) chains and one cu2(I) determined in human skin fibroblasts in order to inchain coiled into a triple helical structure (3, 4). Various vestigate site(s) of regulation of type I collagen production by human EGF in the absence and presence of factors and agents stimulate or inhibit some of the above L-ascorbic acid 2-phosphate (Asc 2-P), a long-acting processes to regulate the level of type I collagen in tissues and organs (5). vitamin C derivative. Among others, transforming growth factor-@(6-9), fibro(10 ng/ml)usedalonereduced the HumanEGF steady state levelsof mRNAs for proal(I) and proaz(I)genic factor (IO), and acetaldehyde(11)activate the transcripchains and transcriptional activity of these genes in tion process of the genes for prou(1) chains in cultured fibrovitro by 45%. Asc 2-P (0.2 mM) alone, on the other blastic cells andstimulatetype I collagen accumulation. hand, raised production of type I collagen and the Whereas on the other hand, tumor necrosis factor (12) and procuz(I) glucocorticoid reduce the rate of transcription of the genes steady state levelsof mRNAs for proal(I) and collagen chains as well as stimulated transcriptional (13) and/or accelerate the degradationof the transcripts (14, activity of these genes. Human EGF attenuated these 15) and thus diminish the productionof type I collagen. stimulative effects of Asc 2-P. These results indicate Another aspect of the complexity involved is the presence that human EGF regulates typeI collagen synthesis a t of tissue-specific transcriptional regulatory mechanisms of the transcriptional level in cultured fibroblasts in the presence and absence of Asc 2-P. The possibility that type I collagen genes. For example, insertion of a retrovirus of cu,(I) collagen genesin mice human EGF plays a role as a regulator of type I colla- gene intothefirstintron completely inhibits expression of these genes in various kind gen genesin vivo was discussed. of cells including skin fibroblasts and muscle cells (16). On the other hand, the insertionmerely affects the expression of cul(I) collagen genes intoothodontoblastsandosteoblasts Type I collagen is the major structural protein and is found (17). Also, 1,25-dihydroxy vitamin D diminishes transcription in large quantity in various parts of the body, especially in of the ~ ~ (collagen 1 ) gene in osteoblastic cells but not in a the skin,bones, teeth, and tendons.A decrease in the produc- fibroblast cell line (18). tion of type I collagen or production of defective molecules In the course of studies on the regulation of extracellular induces deformity or dysfunction of organsandrestrains matrix formation and cell growth, we found that EGFI predevelopment of embryos or fetuses in extreme cases(1-3). On pared from mouse submaxillary gland specifically inhibited the other hand, aberrant accumulation of type I collagen in production of type I collagen in osteoblasts (19), skin fibrotissues and organs results in organ fibrosis such as hepatic blasts (20), aortic smoothmuscle cells,’ and tooth organs(21) fibrosisin the liver, pulmonary fibrosis inthe lung, and even though the factor stimulated cell growth and production progressive systemic sclerosis (scleroderma) in the skin and of noncollagenous proteins by these cells and organs. Since also sometimes endangers thelives of affected individuals (2, ascorbic acid (vitamin C) is always present in the normal in 3 ) .These factssuggest that expressionof type I collagen genes * T h i s work was supported in part by research grants from the Ichiro Kanehara Foundation and Terumo Life Science Foundation. The costs of puhlication of this article were defrayed in part by the payment of page charges. Thisarticlemusttherefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ll To whom correspondence should he addressed. Tel.: 03.3291SJ573.

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The abbreviations used are: EGF,epidermal growth factor; hEGF, human EGF; Asc 2-P, L-ascorbic acid 2-phosphate; DMEM-10, Dulbecco’s modified Eagle’s medium supplemented with penicillin G (50 mg/liter),dihydrostreptomycin (50 mg/liter), Fungizone (250 p g / liter), HEPES ( 3 g/liter), and 10% fetal bovine serum; SDS, sodium dodecyl sulfate; MOPS,3-(N-morpholino)propanesulfonicacid; TES, N-tris~hydroxymethyl~methyl-2-aminoethanesulfonic acid; HEPES, N-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. R. Hata, unpublished data.

9997

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Transcriptional Control of Type I Collagen Genes

vivo situation, we introduced Asc 2-P into the culture systems dialyzed against 5 mM acetic acid after addition of inhibitors for acid t o mimic the in vivo condition and foundAsc 2-P is effective proteases (pepstatin, leupeptin, and antipain; 200 pg each/sample) and used as the collagen fraction. Lyophilized collagen samples were as a long-acting vitamin C (22). separated by SDS,5% polyacrylamide gel electrophoresisinthe Herein we investigated the effects of recombinant human presence or absence of dithiothreitol as describedpreviously(19). EGF on the expression of type I collagen genesin human skin After electrophoresis, the gels were fixed and stained withCoomassie fibroblasts to clarify regulational site(s) of EGF, a type I- Brilliant Blue R-250, immersed in 1.1 M sodium salicylate solution specific and tissue-nonspecific inhibitor of collagen synthesis for 30 min (29), and thenprocessed for fluorescence autoradiography in the presence and absence of Asc 2-P. Our data provide (30) asdescribed (24). The amountof each componentwas quantified laser densitometry of the Coomassie Brilliant Blue stained gels evidence showing that EGF regulates the activity of type I by and x-ray films. collagen genes at the transcriptionallevel. Blot Hybridization-Total RNA was isolated by extraction by the guanidinium isothiocyanate method (31), and cytoplasmic RNA was isolated from the supernatant fraction of the nuclear preparation from human skin fibroblasts as described in the next section. CytoMaterials-Dulbecco's modifiedEagle'smedium, fetal bovine serum, andFungizone were purchased from GIBCO. Penicillin G and plasmic fractions were treated twice with equal volumes of phenol/ dihydrostreptomycin were generously donated by Toyo JozoCo., Ltd., chloroform/isoamylalcohol (25:24:1) in the presence of ammonium Shizuoka, Japan. Magnesium salt of Asc 2-P.5H20 was obtained acetate, and the RNAswere precipitated by addition of 2 volumes of from Wako Pure Chemical Industries, Ltd., Osaka, Japan. Recombi- ethanol andwashed three times with70% ethanol and dried inuacuo. The RNAs were dissolved in 10 mM sodium phosphate, pH 7.0, and nant human EGF was purchased from Wakunage Pharmaceutical denatured by addition of an equal volume of 37% formaldehyde and Co., Ltd., Hiroshima, Japan, and was over 99% pure as judged by SDS-polyacrylamide gel electrophoresis and high-performanceliquid incubated a t 60 "C for 7 min. The solution was chilled on ice and spotted ona nitrocellulose filter; then the spot was dried at 80 "C and chromatography. Purified Clostridium histolyticum collagenase was obtained from Amano Pharmaceutical Co., Ltd., Nagoya, Japan, and hybridized with "P-labeled Hf-677, a cloned cDNA for human further purified by gel chromatography as reported (23); absence of proal(I), or Hf-32 for proa2(I), which was labeled by the randomnonspecific proteases in the preparation wasverified as described primer method (32) using [a-"'P]dCTP as a radioactive precursor. Hybridization was performed asdescribed previously (33).For North(24). ~-[2,3-:'H]Proline was obtained from Du Pont-New England ern blot analysis, 10pg of RNA was separated by electrophoresis (2 Nuclear, and [a-"'PIATP, [a-32P]CTP, [a-32P]GTP, [cY-~'P]UTP, and V/cm) on formaldehyde gel (2.2 M formaldehyde, 0.02 M MOPS, 8 [a-"'PIdCTP were from Amersham Japan, Tokyo. The cDNA clones for human proal(I) and proa2(I)collagen chain mM sodium acetate, 1 mM EDTA, 1.2% agarose, pH 7.0), transferred to nitrocellulose paper, and hybridized (33). sequences were generously donated by Dr.Francesco Ramirez (Mount Run-off Transcription Assays-Previously published techniques Sinai School of Medicine). One cloned cDNA, Hf-677 (1.8 kilobase (11, 34, 35) with minor modification were used for the isolation of pairs), spans from amino acid residue 787 to 270 nucleotides into the nuclei and inuitro transcription. 3'-end-untranslated region of the proal(I) mRNA (25); and Hf-32 Nuclear Isolation-Cells (0.5-2 X lo7) incubated with human EGF (2.2 kilobase pairs) begins a t about the middle of the helical portion (10 ng/ml) and/or Asc 2-P (0.2 mM) for 4 days were isolated from of the proa,(I) chain and extends to the middle portion of the C- 100-mm dishes (3-10) by treatment with trypsin (0.1%) at 37 "C for terminal propeptide (26): p-actin cDNA anda plasmid, pBR322,were 15 min. After a brief centrifugation (600 X g, 5 min), the cell pellet also used as an internal standard and for a measurement of the was suspended in 0.5 ml of lysis buffer (composed of 40 mM Tris/ background value. DNase I and RNase inhibitor (RNasin)were from HCl, p H 7.5, containing 37 mM KC1,12 mM MgC12, and 30% sucrose) Promega (Madison, WI), andnitrocellulose paper was obtained from and transferred to a ice-chilled Dounce homogenizer. Next, the cell Schleicher & Schuell. suspension was slowly homogenized 10 times with a tight (type A) Cell Culture-Human forearm skin fibroblastswere obtained from pestleafteraddition of 50 pl of the lysisbuffer containing 5.5% normal adults as described (27) andused a t population doublinglevels Nonidet P-40. The homogenate was thentransferredto a 1.5-ml of 10-30. All the experiments described here were repeated using at Eppendorf tube and centrifuged a t 500 X g and 2 "C for 5 min. The least two different cell lines. The cells were plated onto 16-, 35-, or supernatant was removed and the pellet was resuspended in 0.5 ml 100-mm dishes and grown in DMEM-10for 1-2 weeks until apparent of wash buffer (composed of 20 mM Tris/HCl, pH 8.3, containing 10 confluence was obtained. Then the cells weretreated with0-1250 ng/ mM MgCl,, 1 mM MnCl,, 140 mM KC1, and 20% glycerol). The ml of recombinant human EGF and/or 0-0.2 mM Asc 2-P for 8 h to suspension was centrifuged a t 500 X g and 2 "C for 5 min, the resulting 14 days. The medium was changed twice aweek and on the day before supernatant was removed, and 0.1 ml of the wash buffer was added labeling with radioactive materials. to the pellet. After the total volume of the resultant suspension had Metabolic Labeling and Quantification of Protein Production-All been measured, an aliquot of it was saved for the determination of the cells were incubated in 0.5 ml (16-mm dish),1.0 ml (35-mm dish), DNAcontent,andtheremainder was transferredto aCryotube or 3.0 ml (100-mm dish) of fresh DMEM-10 containing ['Hlproline (Nunc) and stored in liquid nitrogen until used. The first and the or absence of second supernatantswere combined and also stored inliquid nitrogen (10-100 pCi) and 0.2 mM Asc 2-P in the presence varying amounts of human EGF for the last 2 h when the cells were and used for the isolation of cytoplasmic RNA as described above. treated with the factors up to 24 h or the last 24 h when the cells I n Vitro Transcriptionand Isolation of Labeled RNA-Onewere treated with the factorsfor 1 day or longer. The combined cells hundred microliters of nuclear suspension was thawed on ice and and medium were processed for the determination of collagen and mixed with 50 pl of elongationbuffer (20 mM Tris/HCl, pH 8.0, noncollagenous protein productions using nonspecific protease-free containing 10 mM MgC12,600 mM KCl, 10 mM dithiothreitol), 10 ~1 collagenase as described previously (23, 24). of nucleotide mixture (30 p~ concentration of each of ATP, CTP, Relative rate of collagen production was calculated assuming that GTP, and UTP), 5 pl of RNasin (25 units/pl), and 40 ~1 of [a-:"P] collagen has an iminoacid content 5.4 times higher than thatof other N T P (100 pCi each of [a-"PIATP, [a-"PICTP, [a-:"P]GTP, and [ a proteins (23). "'PIUTP) and incubated a t 30 "C for 3-30 min. Four units of DNase Quantification of DNA Content-After the culture, the medium I (1unit/pl, RNase free) and4 pl of 0.2 M MgC12 were added, and the was removed, and the cell layer was rinsed with 1-2 ml of0.05 M mixture was incubated at 30 "C for 10 min. The digestion was stopped Tris-HC1, 0.11 M NaC1, pH 7.4, and processed for the measurement by addition of 10 p1 of stop buffer (0.1 M Tris/HCl, pH7.5, containing of DNA content by apreviouslydescribedmodification (19) of a 10% sodium sarkosyl, 0.1 M EDTA, and protease K,a t 1 mg/ml) and fluorometric method (28). incubated for 30 min at 42 "C. RNA was extracted twice with equal Preparation of Collagen Components and SDS-Polyacrylamide Slab volumes of phenol/chloroform/isoamyl alcohol (25:24:1) and then Gel Electrophoresis-After labeling of the cells with ['Hlproline for precipitated by addition of 1 ml of ethanol to the supernatant after the last 24 h, the medium and cell layer were collected and sonicated addition of200 pl of 4 M ammonium acetate. The precipitate was on ice. Procollagen was precipitated by the addition of ammonium washed 15 times with 70% ethanol to remove free nucleotides. The sulfate (176 mg/ml) in the presence of protease inhibitors (EDTA,25 precipitate was dissolved in 10 mM Tris/HCl buffer containing 1 mM mM; N-ethylmaleimide, 10 mM; phenylmethylsulfonyl fluoride, 1 EDTA. An aliquot was saved for determination of the rate of incormM). The precipitate was dissolved in 0.5 M acetic acid and treated poration of [a-"'P]NTP into RNA, and the remainder was used for with pepsin (100 pg/ml) for 6 h a t 5 "C (24). Pepsin was inactivated hybridization. Hybridization-Twenty micrograms of plasmid DNA containing by increasing the pH of the solution to pH 8, and the solution was EXPERIMENTALPROCEDURES

Transcriptional Control of Type I Collagen Genes

9999

pronl(I), proocn(I),8-actin, and pBR322 sequences were treated with I h R I , and the DNA was denatured by addition of 1 mM EDTA and NaOH to a final concentration of 0.1 M. The DNA solution was spotted onto anitrocellulose filter after neutralizationof the solution by addition of an equal volume of neutralization buffer (0.5 M Tris/ HCI, 3 M NaC1, pH 7.0). The filter was dried at 80 "C and treated with 10 ml of prehybridization buffer (10 mM TES, pH7.4, containing 0.2% SDS, 10 mM EDTA, 0.3 M NaC1, and polyadenylic acid, at 100 pg/ml) at 72 "C for 2 h. Radiolabeled RNA solution was treated at 65 "C for 10 min, chilled on ice, and mixed with 7 ml of prehybridization buffer containing salmon sperm DNA(100 pg/ml) and hybridized with the DNAs spotted on the nitrocellulose filter for 3 days at 70 "C. The hybridized filters were washed three times with 15 mM sodium citrate buffer, pH 6.8, containing 15mM NaCl and 0.1% SDS at 70 "C, and the amount of hybridization was determined by counting trf the hybridized radioactivity.

were obvious after 16 h of incubation and were maintained after 14 days (Fig. 2 and data not shown). Effects of Co-presence of Human EGF and Asc 2-P on Growth and Protein Production of Human Skin FibroblastsAsc 2-P, like L-ascorbic acid, stimulates growth of fibroblasts and smoothmuscle cells and productionof proteins including collagen (20, 22, 36). As concentrations ofAsc 2-P higher than 0.2 mM showed effects on thecellular metabolism similar to those of the 0.2 mM concentration (22), 0.2 mM was used for all thefollowing experiments. Co-presence of human EGF and Asc 2-P in the culture medium of the fibroblasts for 3 days stimulated growth of the cells to a higher degree than theseparateaddition of eitherfactor (Figs. 2, A and B), suggesting that the growth stimulations by the two factors are mediated by independent mechanisms. A similar effect RESULTSANDDISCUSSION was observed when the factors were present for 3-14 days (Fig. 2B and data not shown). Effects of Human EGF on Cell Growth of andProtein Production of noncollagenous proteins per dish was also Production by Human Skin Fibroblasts-The presence of human EGFin the medium of apparently confluent human skin activated additively by the presence of both factors. These fibroblast cultures stimulated growth of the cells (Fig. 1 A ) effects maybe partly a reflection of an increasein cell number, and production of noncollagenous proteins per dish (Fig. lB, because productions of noncollagenous proteinsperDNA N C P ) , but not production of collagen; in fact, the latterwas were constant irrespective of a kind of added growth stimuinhibited by 60% (Fig. 1B) even though DNA per dish was lators (data not shown). On the other hand, the inhibitory collagen production was partly attenincreased (Fig. lA). Thus, the relative rate of collagen pro- effect of human EGF on duction was decreased from 7% (control) to 2% of the total uated by the co-presence of Asc 2-P,resultingin avalue protein produced after a 5-day exposure to human EGF (Fig. between the values obtained by the addition of each factor alone (Fig. 2, C and D).Inhibition of collagen production by IC). human EGF was obvious a t 16 h of exposure and reached The maximumeffect on thecell growth and on the producthus 4 tion of proteins by the cells was observed at 10 ng/ml (1.7 maximum after 4 days of incubation (Fig. 2, C and D); nM) of human EGF, and theeffects were less obvious with a days of incubation were used for all thefollowing experiments. further increase in concentration; thus 10 ng/ml was used for Itis known thattransforming growth factor a hasquite similar physiological activities to human EGF ( 5 ) ;thus we all the following experiments. treated the cells with transforming growth factor 01. It inhibSimilar effects on cellular metabolism were observed when mouse EGF was added instead of human EGF (20), but the ited productionof collagen but stimulatedgrowth of the cells.') Collagen Types Produced by Human SkinFibroblasts in the dose dependence was different. This difference might reflect a difference in binding specificity between human EGF and Presence or Absence of Human EGF andlorAsc 2-P-When mouse EGF for human EGF receptors. The effects of human collagen components were analysed by SDS, 5% polyacrylEGF on cell growth and relative rate of collagen production amide gel electrophoresis followed by densitometry of the fluorograms, the cells were shown to produce types I, I11 (disulfide bonded y component), andV collagens (Fig. 3A) in A DNA the ratioof 90:6:2. By treatment with human EGF, production

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FIG. 1. Effect of increasing human EGF concentrations on the metabolism of human skin fibroblasts in culture. Cells at apparent confluence (-4 pg DNA/35-mm dish) were further cultured for 5 days in the absence or presence of increasing concentrationsof human EGF (hEGF).A, growth of the cells, as determined by DNA content(micrograms/dish). B , production of collagen (0)and of noncollagenous proteins (NCP, 0 )were det.ermined by measurement of radioactivities incorporated into collagenase-sensitive and collagenase-resistantproteins(disintegrations/dish), respectively. Data of human are percentages of the control (cells cultured in the absence EGF). C, relative rate of collagen production to total protein product ion. Data are means f S.D. of quadruplicate assays, eachemploying two dishes.

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FIG. 2. Effects of human EGF and Asc 2-P on cellular metabolism of human skin fibroblasts. Cells at apparent confluence were furtherincubatedwithDMEM-10 for 8 hto 7 days in the presence (0) or absence (" ) of 10 ng/ml of hEGF, in the presence of 0.2 mM Asc 2 - P ( 0 )or in the presence of both hEGF and Asc 2-P ((3). In A and C all the cells were incubated with fresh DMEM-10 supplemented with ["Hlproline and 0.2 mM Asc 2 - P for the last 2 h. In B and D all the cells were incubated with freshDMEM-10 supplemented with ["Hlproline and 0.2 mM Asc 2-P for the last 24 h. A and B, cell growth determined by DNA content. C and D , relative rates of collagen production. Data are the means of quadruplicate assays,each employingtwodishes. Standarddeviations were less than the size of symbols used.

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FIG. 3 . Effects of human EGF and Asc 2-Pon the collagen components producedby human skin fibroblasts.The cellswere cultured as d e s c r i l d in the legend o l I:ig. 2 . Tritiated collagen was prepared as desrril)ed under "l*;xperimental Procedures" and loaded (each sample corresponding to the preparationfrom 0.6 pg of cellular IINA), separated hy SIIS. 5f';, polyacrylamide gel electrophoresis in the presence (+/)'/"/') or absence (-1)"T) of dithiothreitol and visualized t)v Iluorographv ( A ) or hv staining with Coomassie Hrilliant lilue ( H ) .

of t,ype I collagen was specifically inhibited by 80%. Ry t h e treatment of Asc 2-P production of all the collagen component,s were increased, even t.hough increase in cell number was corrected. In the presence of both human EGF and Asc 2-P in the cukure medium of t h e cells, production of type I collagen became a value between the values obtained by t h e addition of each factor alone (Fig.3 A ) . Accumulation of type I collagen in t h e Asc 2-P supplemented cultures and evidence of its inhibit.ion by the co-presence of human EGF were also visualized by staining of t h e gels with Coomassie Brilliant Blue after SDS-polyacrylamide gel electrophoresis (Fig. 3 R ) . Densitometry of the Coomassie Brilliant Blue-stained gels showed that. the respect,ive densit,iesof n l ( I )and @,(I) chains were 19 f 5% and 19 k 5% of those of the control by t h e 146 f 7% presence of human EGF in t,he culture medium, and 161 f 18%, by the presence of Asc 2-P, and 69 f 10% a n d 58 k 10%, by the co-presence of both factors. These results indicat,e that human EGF and Asc 2-P competitively regulate production and accumulation of t.ype I collagen. The I~ve1.sof Messengm RNA for protr,(I) and pron,(I) Collagen Chains in Control and Human EGF- andlor Asc 2P-treated Cells-We furtherinvestigatedthesteadystate levels of mRNAs for theprotrl(I)a n d pron2(I)collagen chains in order to clarify whether or not production of pron,(I) a n d pron,(I) chains is regulated by the levels of their mRNAs. Northern-blot analysis showed that the steady state level of mRNAs for protrI(I)and pron2(I) chains were decreased by the presenceof human EGF,as observed by reduced densities of RNA hands (Fig. 4A ). On the other hand, Asc 2-P increased t h e levels of mRNAs for both chains; and the co-presenceof human EGF and Asc 2-P attenuated steady state levels of mRNAs for both protr chains. The Northern blot analysis also showedthebindingspecificit,ies of the cDNA clones. Quantitative data were ohtained by dot-blot analysis. Counting of the spots showed that the respective levelsof protu,(I) and pron,(I) mRNAs were 69 k 2% and 70 f 5% of those of

FIG. 4. Hybridization assays of human fibroblast RNAs. Total RNAwasisolated from humanskinlibrothstsculturedwith DMEM-10 in the presence or ahsenre of I O ng/ml of h u m a n R(;F (hb,Y;F)and/or 0.2 mM Asc 2-1' fnr 4 clays. A. Northern blot analysis. Approximate numbers olnucleotides in the m R S A s are indicntrd. It. dot-hlot analvsis. One and a hnlf to 8 p g o f t he R N A wrre npplird t o nitrocellulose filters, and the filters were then hytwidized with ratliolabeled cDNAs for a human procr,(l), prnuJI),o r ij-nctin sequence. Figures indicate counts/min ol the spots of the highest RSA cnnrrntrations.

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the control by the presence of human EGF in the culture medium; 131 f 4 % a n d 127 f fir;, hv the presence of Asc 2P; a n d 97 f 2% and 93 k 5% by the co-presence of human E G F a n dAsc 2-P. Counts of &actin were essentially the same irrespective of the treatments of the cells with growth stimulators (Fig. 4H). Relationship between Production I ~ c c l sof Collagrn ('hains and Steady State LRvels of Their Messcngrr RNAs- I'roduction of n l ( I ) and n J I ) chains was calculated by use of the relative amountof each collagen chain ohtainedhy densitometry of the fluorogram after SDS-polyacrylamide gel electrophoresis (Fig. 3A ) and the relative rateof collagen product ion to the production of total proteins. A linear relationship was observed when the steady state levelsof mRNAs for p r o t r , ( 1 ) and pron,(I) collagen chains (Fig. 4 R ) were plotted against production levels of their collagen chains (Fig. 5 ) . The correc r , ( l ) was lationcoefficient for n l ( I ) was 0.90 andthatfor 0.95, suggesting t,hat production of t r l ( I ) and t u , ( I ) collagen of their mRNAs under chains is mainly regulated by the levels our culture conditions. Extrapolation of the two lines does not give lines through

Transcriptional Controlof Type I Collagen Genes

10001

A Robe the origin, suggesting posttranscriptional regulations such as inhibition of translation of mRNAs and/or degradation of polypeptides also exist. Transcriptional Activity of Type I Collagen Genes-Because we found that human EGF and Asc 2-P regulate mRNAlevels for pronl(I) and pron2(I) chains, we further investigated the effects of these factors on the transcriptional activityof type I collagen genes by the nuclear run-off assay. Preliminary experiments showed that incubation time of the nuclei with radioactive nucleotides affected the production ratio of protrl(I) and prons(I) mRNA precursors, so we first MQF tested the incorporation rate of radioactive nucleotides into *e. Asc2-P RNAs. Incorporationrates of radioactivenucleotidesinto cpm 197 a7 58 143) RNAs were linear until 10 min of incubation a t 30 "C,and the rates gradually decreased thereafter when the nuclei isolated from either control or growth stimulant-treated cells were used (data not shown). Thus a 10-min incubation was I used for the following experiments. Incorporation of radioactive nucleotides intoRNAsforpronl(I)andpron2(I)sequences was decreased when the nuclei isolated from human 4 EGF-treated cells were used but was increased when those from Asc 2-P-treated cellswere examined, as judged from counts of the spots. Intermediate valueswere obtained when both human EGF and Asc 2-P were present in the culture medium of the fibroblasts(Fig. 6A). Transcriptional activities were calculated by use of specific binding counts from three independent culture experiments(Fig. 6R). Percent inhibition of transcription varied from 35 to 55% depending on cultures but thepresence of human EGFin the culturemedium of the of cells significantly (45 k 10%) inhibitedtranscription pronl(I) and procu2(I)collagen genes (Fig. 6R). The ratio of 0 Control hEGF M(3F AscP-P pron,(I) to procu2(I) sequences was 1.9 f 0.2 irrespective of + Asc2-P the treatment of the cells (Fig. GB), which is quite similar to the ratio [uI(I)/ns(I)= 21 of mRNAs and the product proteins Fir;. 6. Hybridization of mRNA precursors labeled during i n vitro transcriptioninnucleiderived from control and in type I collagen. EGF- and/or Asc 2-P-treated cells with cDNAs for A linear relationship was obtained when the incorporation human procrl(I),proa2(I), 8-actin (an internal standard). and pRHR22 rate of radioactive nucleotidesintopronl(I)andpron2(I) (background). A , i n vitro transcription was carried out f o r I o rnln sequences was plotted against cytoplasmic RNAlevelsfor in thepresence of [n-~'T]NTl'as descril~eclunder "F:xperimental Procedures"withnucleiderived from control rells and from rrlls pronl(I) and pron,(I)collagen chains (Fig. 7). General Discussion-Recombinant human EGFspecifically cultured in the presence of hEGF or Asc 2-1, or both. FikqJres indicate inhibited the production and accumulationof type I collagen counts/min of respective spots minus counts/min o f pHR322 exrept for pHR322 whereoriginalcounts were shown. H . transrriptional in cultured human skin fibroblasts (Fig. 3). This inhibition activity was measured as described ahove. and specific. bintlinp for cannot be attributed to cytotoxicity of human EGF because pron,(I) and prouJl) was calculatedfrom the following eqt~ation: the factor stimulated productionof noncollagenous proteins, N - (' growth of the cells (Figs. 1 and 2), and accumulation and specific hinding = I j - C" production of hyaluronic acid,' another major extracellular matrix component produced by the cells. Human ECF also where N = counts/min of procr,(l) or p r o d l ) spot. ,j = rounts/min reduced the steady state levels of mRNA for both pronl(I) of/j-actin (internal standard). and( ' = counts/min o f pHIU'L'L (t~nrklevels of ground). Data arethemean ? S.D. of nuclei prepared from three and procr2(I) chains (Fig. 4), in additiontothe transcription of these genes (Fig. 6, A and R).On the other independent cultures. hand, Asc 2-P stimulated production and accumulation of type I collagen (Figs. 2 and 3 and Ref. 22) as does ascorbic suggest that Asc 2-P regulates production of t-ype I collagen acid in human skin fibroblasts (20). We chose to use Asc 2-P by the same means. The addition of human EGF to the Asc instead of ascorbic acid because of its stability under normal 2-P-supplemented culture medium of human skin fibroblasts attenuated production of t,-ype I collagen (Fig. 3 ) , steady state culture conditions. 4), Asc 2-P is chemically produced and its presencein our body levels of the mRNAs for pronl(I) and pronJl) chains (Fig. and transcriptional activity of those genes (Fig. 6, A and H ). is not yet reported, but it does liberate free ascorbic acid in vivo (37) and functions as a cofactor for collagen production The amountsof n l ( I )and nJI) collagen chains correlatedwell like ascorbic acid in culture (22). ThusAsc 2-P is a biological with the steady state levels of mRNAs for these chains (Fig. substance even though it may not be a physiological one. Asc 5 ) ,and mRNAlevels of pronl(I) andpro(r.JI)chains correlated 2-P increased the steady state levels of mRNA for type I wellwit.h the transcriptional rates of precursors for these collagen chains and elevated the transcriptional rates of type mRNAs (Fig. 7). Asc 2-P and human KGF also stimulated I collagen genes (Figs. 4, A and R, and 6, A and R ) . Ascorbic growth of the cells and an increase in the amount of DNA, acid was earlier shown to increase the level of translatable but the changes in the levels of mRNAs and transcriptional in DNA. because the type I collagen mRNAs and transcriptional activityof t-ype I activityare not duetotheincrease collagen genes in cultured fibroblasts (38, 39). These results amounts of DNA used for theassay were equalized: and

+

Transcriptional Control of Type I Collagen Genes

10002

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Acknowledgments-We are grateful to Drs. Hiroshi Nakajima and Yutaka Nagai for their continuous encouragement during the course of this work. REFERENCES

1. Prockop, D. J., and Kivirikko, K. I. (1984) New Engl. J . Med. 311,376-386 2. Krane, S.M. (1984) in Extracellular Matrix Biochemistry (Piez, K. A., and Reddi, A. H., eds) pp. 413-463, ElsevierScience Publishing Co., Inc. New York 3. Bornstein, P., and Byers, P. H. (1980) in Metabolic Control and Disease (Bondy, P. K., and Rosenberg, L. E., eds) 8th Ed., pp. 2 1 I /'/ 1089-1153, W . B. Saunders Co., Philadelphia $' I 4. Kivirikko, K. I., and Myllyla, R. (1984) in Extracellular Matrix 0 ' 1 1 2 I3 I4 51 Biochemistry (Piez, K. A., and Reddi, A. H., eds) pp. 83-118, TRANSCRIPTIONAL Elsevier Science Publishing Co., Inc., New York ACTIVITY 5. Hata, R. (1990) in Bioscience and Biotechnology of Extracellular FIG. 7. Relationship between mRNA levels for proal(I) and Matrix (Fujimoto, D., ed) pp. 165-191,287-298, IPC, Tokyo proa2(I) chains and their a-3zP-labeledmRNA precursors. 6. Ignotz, R. A., and Massagui., J. (1986) J. Biol. Chem. 261,4337Amounts of mRNAs were determined by counting of the spots (Fig. 4345 4B), and the amountof their precursors was quantified after i n uitro 7. Roberts, A. B., Sporn,M. B., Assoian, R. K., Smith, J. M., Roche, transcriptionas describedin the legend to Fig. 6B.Correlation N. S., Wakefield, L. M., Heine, U. I., Liotta, L. A,, Falanga, V., coefficient for a,(I) was 0.97 and that for a*(I)was 0.95. Kehrl, J. H., and Fauci, A. S.(1986) Proc. Natl. Acad. Sci. U. S.A. 83,4167-4171 8. Penttinen, R. P., Kobayashi, S., and Bornstein, P. (1988) Proc. mRNA levels and transcriptional activity of a constitutive Natl. Acad. Sci. U. S.A. 85, 1105-1108 component, P-actin, were constant, 106 f 6% of the control, 9. Rossi, P., Karsenty, G., Roberts, A. B., Roche, N. S., Sporn, M. B., and deCrombrugghe, B. (1988) Cell 52,405-414 in which the cells were precultured in the absenceof human EGF and/or Asc 2-P. These results suggest that production 10. Raghow, R., Gossage, D., Seyer, J . M., and Kang, A. H. (1984) J . Biol. Chem. 259,12718-12723 of type I collagen in human skin fibroblasts is mainly regu11. Brenner, D. A., and Chojkier, M. (1987) J . Biol.Chem. 262, lated by these factors by affecting transcriptional activityof 17690-17695 type I collagen genes, although modification by regulation of 12. Solis-Herruzo, J. A., Brenner, D. A,, and Chojkier, M. (1988) J . the translationof the mRNAs and/or stabilityof mRNA and Biol. Chem. 263,5841-5845 product proteins might also be present. 13. Cockayne, D., Sterling, K. M., Jr., Shull, S., Mintz, K. P., Illeyne, S., and Cutroneo, K. R. (1986) Biochemistry 25, 3202-3209 Our present study is the first example that twogrowth stimulatingfactors competitivelyregulate transcription of 14. Hamalainen, L., Oikarinen, J., andKivirikko, K. I. (1985) J . Biol. Chem. 260,720-725 type I collagen genes. R., Gossage, D., and Kang, A. H. (1986) J. Biol. Chem. Many experiments on in uitro collagen metabolism have 15. Raghow, 261,4677-4684 been reported, and usually ascorbic acid is only added at the 16. Harbers, K., Kuehn,M., Delius, H., and Jaenisch,R. (1984) Proc. time of labeling with radioactive precursors and it is not Natl. Acad. Sci. U. S. A. 81, 1504-1508 present beforehand. But under normal in vivo conditions, a 17. Kratochwil, K., von der Mark, K., Kollar, E. J., Jaenisch, R., Mooslehner, K., Schwarz, M., Haase, K., Gmachl, I., and Harcertain amount of ascorbic acid is always present. Thus the bers, K. (1989) Cell 57, 807-816 culture system containing Asc 2-P may be more favorable to 18. Lichtler, A., Stover, M. L., Angilly, J., Kream, B., and Rowe, D. study the regulation of collagen metabolism in uiuo. W . (1989) J. Biol. Chem. 264,3072-3077 In this context it is interesting that human EGF attenuatedHata, R., Hori, H., Nagai, Y., Tanaka, S., Kondo, M., Hiramatsu, 19. thestimulative effects of Asc 2-P on collagen synthesis. M., Utsumi, N., and Kumegawa, M. (1984) Endocrinology 115, Human EGF is associated with human blood platelets (40), 867-876 suggesting that human EGF functions asa regulator of col- 20. Hata, R., Sunada,H., Arai, K., Sato, T., Ninomiya, Y., Nagai, Y., and Senoo, H. (1988) Eur. J . Biochem. 173 261-267 lagen synthesisinadditionto a stimulator of growthfor various cells in uiuo, for example at the sitesof inflammation 21. Hata, R., Bassem, C., Bringas, Jr., P., Hsu, M-Y., and Slavkin, H. C. (1990) Cell Biol. Znt. Rep. 74,509-519 where platelets aggregate and degranulate. Hata, R., and Senoo, H. (1989) J . Cell. Physiol. 138,8-16 22. Transforming growth factor a also inhibits production of 23. Peterkofsky,B., and Diegelmann, R. (1971) Biochemistry 10, type I collagen,' suggesting it alsoplays a rolein the regulation 988-994 of type I collagen production. 24. Hata, R., Ninomiya, Y., Nagai, Y., andTsukada, Y. (1980) Type I collagen genes are very complex and are composed Biochemistry 19, 169-176 of multiple regions with promoter and enhancer activities as 25. Chu, M.-L., Myers, J. C., Bernard, P., Ding, J.-F., and Ramirez, F. (1982) Nucleic Acid Res. 10, 5925-5934 well as silencer activities (41-44). And also the presence of tissue-specific transcriptional regulatory mechanisms of type 26. Myers, J. C., Chu, M.-L., Faro, S.H., Clark, W . J., Prockop, D. J., and Ramirez, F. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, I collagen genes are reported (16-18, 45). It is not known 3516-3520 whether human EGF and Asc 2-P, or perhaps mediators of 27. Ninomiya, Y., Hata, R., Nagai, Y., Tajima, S., Nishikawa, T., and thesefactors,interact with thesame cis-regulatory DNA Hatano, H. (1982) Biomed. Res. 3, 70-82 element or the same trans-acting factoror whether EGF and 28. Kissane, J. M., and Robins, R. (1958) J . Bid. Chem. 233, 184188 Asc 2-P act independently at different sites of the regulatory elements of the genes. However EGF inhibits expression of 29. Chamberlain, J. P. (1979) Anal. Biochem. 98, 132-135 30. Laskey, R. A,, and Mills, A. D. (1975) Eur. J . Biochem. 56, 335both proal(I) and proa2(I) collagengenes in a tissue non341 specific manner (19-21). Thus, the present culture system 31. Maniatis, T., Fritsch, E. F., and Sambrook, J . (1982) inMolecular should be useful to clarify the ubiquitous constitutional regCloning: A Laboratory Manual, pp. 187-196, Cold Spring Harulatory mechanisms controlling typeI collagen genes includbor Laboratory, Cold Spring Harbor, NY ing cis-regulatory elements and trans-acting factors that in32. Feinberg, A. P., andVogelstein, B. A. (1983) Anal. Biochem. 132, 6-13 teract with these DNA regions.

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Transcriptional Control 33. Hata, R., Kurata, S., and Shinkai, H. (1988) Eur. J . Biochem. 174,231-237 34. Groudine, M., Peretz, M., and Weintraub, H. (1981) Mol. Cell. Biol. 3, 281-288 35. Wang, X-F., and Calame, K. (1985) Cell 43,659-665 36. Schwartz, E., Bienkowski, R. S. Coltoff-Schiller, B., Goldfisher, S., and Blumenfeld, 0.0.(1982) J. Cell Biol. 92, 462-470 37. Imai, Y., Usui, T., Matsuzaka, T., Yokotani, H., Mima, H., and Aramaki, Y. (1967) J p n . J .Pharmacol. 17, 317-324 38. Tajima, S., andPinnell, S. R. (1982) Biochem. Biophys. Res. Commun. 106,632-637 39. Lyons, B. L., and Schwarz, R. I. (1984) Nucleic Acids Res. 12, 2569-2579

of Type I Collagen Genes

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40. Oka, Y., and Orth, D. N.(1983) J . Clin. Invest. 72, 249-259 41. Oikarinen, J., Hatamochi, A,, Crombrugghe, and de B. (1987) J . Biol. Chem. 262,11064-11070 42. Rossouw, C. M. S., Vergeer, W. P., du Plooy, S. T., Bernard, M. P., Ramirez, F., and de Wet, W. J. (1987) J . Biol. Chem. 262, 15151-15157 43. Bornstein, P., McKay, J., Morishima, J. K., Devarayalu, S., and Gelinas, R. E. (1987) Proc. Nutl. Acud. Sci. U. S. A . 84, 88698873 44. Bornstein, P., and McKay, J . (1988) J. Biol. Chem. 263, 16031606 45. Bennett, V. D., and Adams, S. L. (1990) J. Biol. Chem. 265, 2223-2230