THE JOURNAL 0 1987 by The American Society of Biological

OF BIOLOGICAL CHEMISTRY Chemists, Inc.

Vol. 262, No. 19, Issue of July 5, pp. 9397-9403, 1987 Printed in U. S. A.

Reduction of Collagen Production in Keloid Fibroblast Cultures by Ethyl-3,4-dihydroxybenzoate INHIBITION OF PROLYL HYDROXYLASE ACTIVITY AS A MECHANISM OF ACTION*

(Received for publication, September 18, 1986)

Tetsuo Sasaki, Kari MajamaaS, and Jouni Uittog From the Department of Medicine, UCLA School of Medicine, Division of Dermatology, Harbor-UCLA Medical Center, Torrance, California 90509 and Departments of Dermatology and Biochemistry and Molecular Biology, Jefferson Medical College, Philodelphia, Pennsylvania 19107

Excessive accumulation of collagen is the hallmark of several clinical conditions characterized by tissue fibrosis. Previously, 3,4-dihydroxybenzoic acid, a structural analog of a-ketoglutarate and ascorbate, has been shown to inhibit the activity of purified prolyl 4- hydroxylase, the enzyme catalyzing the synthesis of 4- hydroxyproline during intracellular biosynthesis of procollagen. In this study a hydrophobic modification, an ethyl ester, of 3,4-dihydroxybenzoic acid was tested for its effects on collagen synthesis and prolyl hydrox- ylase activity in human skin fibroblast cultures. The results indicated that 0.4 mM ethyl-3,4-dihydroxyben- zoate markedly inhibited the synthesis of 4-hydroxy- proline in normal cell cultures apparently as a result of reduced prolyl4-hydroxylase activity, and the syn- thesis and secretion of both type I and type I11 procol- lagens were markedly reduced. Control experiments indicated that the test compound did not affect the viability, proliferation, or plating efficiency of the cells, and it had little, if any, effect on the synthesis of noncollagenous proteins. Furthermore, determinations of type I and type I11 procollagen mRNA steady-state levels by slot-blot hybridizations suggested that the inhibition of procollagen production did not occur on the pretranslational level. Thus, ethyl-3,4-dihydroxy- benzoate selectively reduced procollagen production in fibroblast cultures by inhibiting the post-translational synthesis of 4-hydroxyproline. Similar inhibition was also observed in keloid fibroblast cultures, demonstrat- ing the potential applicability of ethyl-3,4-dihydroxy- benzoate, or other structural a-ketoglutarate or ascor- bate analogs, for treatment of fibrotic diseases.

Keloids are relatively common cutaneous lesions histologi- cally characterized by an abundance of the extracellular ma- trix of connective tissue (1, 2). Recent biochemical studies have concluded that the major extracellular component of keloids is collagen, type I being the predominant genetically distinct collagen (3). Thus, keloids can be described as a

* This work was supported in part by United States Public Health Service-National Institutes of Health Grants GM-28833, AR-28450, AR-38923, and AR-35297. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Affiliate. $ Fellow of the American Heart Association, Greater Los Angeles

3 To whom correspondence should be addressed: Dept. of Derma- tology, Jefferson Medical College of Thomas Jefferson University, 1020 Locust St., “46, Philadelphia, PA 19107.

pathologic condition resulting from excessive accumulation of type I collagen in tissue. Several different mechanisms could explain the excessive deposition of collagen in the lesional areas of skin. Previous studies have indicated that many, but not all, keloid fibroblast cultures are characterized by en- hanced collagen production in uitro (3-6). This overproduc- tion of collagen in cultures, up to 4-fold higher than in the control cultures, has been directed predominantly toward the synthesis of type I procollagen (3).

The increase in the rate of procollagen synthesis is also accompanied by increased activity of prolyl 4-hydroxylase (prolyl hydroxylase), the enzyme which catalyzes the conver- sion of selected prolyl residues to 4-hydroxyproline during intracellular elaboration of procollagen polypeptides (2, 3, 7, 8). The presence of a critical number (approximately 100) of 4-hydroxyprolyl residues/pro-u chain is required for these polypeptides to fold into the triple-helical conformation char- acteristic of collagenous molecules (9-11). The triple-helical conformation, in turn, is required for secretion of procollagen to the extracellular milieu at normal rate (lo), and in the absence of 4-hydroxyproline the nonhelical polypeptides are subject to rapid degradation both in the intracellular and extracellular space (12). Thus, inhibition of the synthesis of 4-hydroxyproline would be expected to result in reduced col- lagen deposition, and a pharmacological reduction in collagen accumulation in tissues could be thought to be beneficial to the patients with keloids (13).

Prolyl hydroxylase belongs to a group of enzymes which requires ferrous ion, molecular oxygen, ascorbate, and u- ketoglutarate for its activity (14, 15). The latter compound serves as a cosubstrate, which is stoichiometrically decarbox- ylated to yield succinate and COZ, in a reaction coupled to the hydroxylation. Recent biochemical studies have demonstrated that certain structural analogs of a-ketoglutarate can serve as potent competitive inhibitors of prolyl hydroxylase (16). These analogs compete with a-ketoglutarate for the binding in the active site of the enzyme, and thus they inhibit the activity of prolyl hydroxylase (17). Recently, another group of compounds was described which are analogous both to a- ketoglutarate and ascorbate, and the inhibitory kinetics also suggested partial identity of the u-ketoglutarate and the ascor- bate binding sites of the enzyme (18). The most potent inhib- itor of this group was 3,4-dihydroxybenzoic acid which had the K; of approximately 5 p M when tested with purified prolyl hydroxylase (18). This compound, however, was found to be a poor inhibitor of colIagen production under cell culture conditions, probably because this molecule is relatively polar and may cross the cell membranes only with difficulty (19). In the present study we have examined a hydrophobic modi-

9397

9398 Inhibition of Collagen Production

fication, an ethyl ester, of 3,4-dihydroxybenzoic acid for its effect on prolyl hydroxylation in keloid fibroblast cultures. The results indicate that ethyl-3,4-dihydroxybenzoate mark- edly inhibits collagen production in keloid fibroblast cultures.

EXPERIMENTAL PROCEDURES

Materials-The two test compounds examined in this study, ethyl- 3,4-dihydroxybenzoate and 3,4-dihydroxyhenzoic acid, were pur- chased from Aldrich. The compounds were initially dissolved in absolute ethanol in 200 mM final concentration. The incubations with cells or partially purified prolyl hydroxylase contained ethanol in 0.2% final concentration, and the concentration of ethanol in controls as well as in all samples incubated with intermediate concentrations of the test compounds was adjusted to 0.2%.

Fibroblast Cultures-Skin fibroblast primary cultures were estab- lished either from normal human skin obtained from cosmetic surgery after informed consent or from biopsy specimens obtained from patients with keloids. The primary cultures were passed by trypsini- zation, and the subcultures were maintained in Dulbecco's modified Eagle's medium plus glutamine and supplemented with 10% fetal calf serum under 5% CO,, 95% air atmosphere in a humidified tissue culture incubator at 37 "C. The fibroblast cultures were studied in passages 3-9.

Collagen Assays-For determination of collagen production in fi- broblast cultures, 3.0-4.5 x lo4 cells/well were plated in 24-well tissue culture plates. At early visual confluency the incubation medium was replaced by fresh medium containing 0.1 mM ascorbic acid, 25 rg/ml 8-aminopropionitrile, 20% dialyzed fetal calf serum, and supple- mented with the test compound (19). Following a 4-h preincubation, 30 pCi of radioactive proline (~-[2,3,4,5-~H]proline, specific activity 107 Ci/mmol; Amersham Corp.) was added, and the incubations were continued for 16 h. At the end of the incubation, the medium was removed, and stock solutions of proteinase inhibitors were added to yield the final concentrations of 10 mM N-ethylmaleimide, 0.3 mM phenylmethylsulfonyl fluoride, and 20 mM disodium ethylenedia- minetetraacetate. Cells were sonicated in 1.2 ml of 50 mM Tris-HC1, pH 7.5, containing 0.4 M NaCl and the same proteinase inhibitors as above. Aliquots of the media and the cell homogenates were dialyzed against running tap water. The samples were then hydrolyzed with an equal volume of 12 N HCl for 20 h a t 120 'C . The total incorpo- ration of 3H radioactivity and the synthesis of [3H]hydroxyproline were then determined, as described elsewhere (20). Aliquots of the cell homogenate were also used for protein determination using a dye- binding assay (21).

To study the synthesis of type I and type III procollagens, fibro- blasts in 75"' tissue culture flasks were preincubated for 4 h with 0, 0.2, or 0.4 mM ethyl-3,4-dihydroxybenzoate, and 100 NCi of [3H] proline was added. After 16 h of incubation, protease inhibitors were added to the medium fraction, and newly synthesized proteins were precipitated by the addition of 176 mg/ml of ammonium sulfate (30% of saturation). The precipitate was collected by centrifugation for 60 min at 18,000 X g at 4 "C. The precipitates were dissolved in 25 mM Tris-HC1, pH 7.5, containing 2 M urea. The samples were then chromatographed on a 1.5 X 20-cm column of DEAE-cellulose as described elsewhere (22, 23). The proteins were eluted with a linear gradient from 0 to 0.22 M NaCl in 25 mM Tris-HC1, pH 7.5, containing 2 M urea (23). The recovery of 3H-labeled proteins in DEAE-cellulose chromatography varied from 76 to 94%. The radioactive protein peaks corresponding to type I and type I11 procollagens were pooled, and their [3H]hydroxyproline content was determined after dialysis and hydrolysis in 6 N HCl (20).

To test the helical stability of the newly synthesized collagens, fibroblasts were cultured on 25-cm2 tissue culture flasks with 0, 0.2, or 0.4 mM ethyl-3,4-dihydroxybenzoate for 4 h as described above. Radioactive proline, 50 &i/flask, was added, and the incubation was continued for 16 h. The medium was then removed, mixed with stock solutions of protease inhibitors (see above), and the radioactive procollagens were recovered by precipitation with 176 mg/ml of ammonium sulfate. The precipitate, recovered by centrifugation, was dissolved in 0.1 N acetic acid, pepsin (in a final concentration of 300 pg/ml) was added, and the samples were digested for 6 h at 15 "C (23). The digests were then examined by SDS-polyacrylamide gel electrophoresis using 8% polyacrylamide gels, as described previously. The radioactive polypeptides were visualized by fluorography (24).

For determination of collagen- or fibronectin-specific mRNA lev- els, the cells were cultured in 75-cn-1' tissue culture flasks with 0, 0.2,

or 0.4 mM ethyl-3,4-dihydroxybenzoate and 0.1 mM ascorbic acid for 16 h. The medium was then removed, and the cells were homogenized in buffer containing 4 M guanidinium thiocyanate, 5 mM sodium citrate, 0.5% sarkosyl, 0.1 M 2-mercaptoethanol, and 0.1% Antifoam A (Sigma) (25). Total RNA was isolated by cesium chloride density gradient centrifugation using 5.7 M Cscl cushion in SW 40.1 rotor (Beckman Instruments) for 16 h at 35,000 rpm (25). The pellet containing total RNA was rinsed with absolute ethanol, dissolved in distilled water, and reprecipitated with 70% (v/v) ethanol containing 0.4 M NaCl; the precipitate was collected by centrifugation at 18,000 X g for 30 min at -10 'C. The total RNA in the final pellet was dissolved in sterilized water, and the RNA concentration was assessed by absorbance at 260/280 nm.

For specific mRNA level determinations, varying concentrations of total RNA were dotted on nitrocellulose filters using a commercial vacuum manifold (Schleicher & Schuell) (3). RNA was immobilized on the filters by heating at 78 "C under vacuum for 90 min, and the filters were first prehybridized and then hybridized (26, 27) with human pro-al(1) collagen (28), pro-al(II1) collagen (29), or fibronec- tin (30) specific cDNA probes. The cDNA probes were radioactively labeled by nick translation with 32P-deoxyribonucleotides (31). Fol- lowing hybridizations, the filters were washed, with the stringency of the final wash consisting of 37.5 mM NaCl, 3.75 mM sodium citrate, pH 6.8, and containing 0.1% sodium dodecyl sulfate (SDS)' (27). The [azP]cDNA-mRNA hybrids were visualized by autoradiography in x- ray cassettes equipped with intensifying screens, and the mRNA levels were quantitated by scanning densitometry at 700 nm.

Assay of Prolyl Hydroxylase-For prolyl hydroxylase assay, fibro- blasts were cultured with or without ethyl-3,4-dihydroxybenzoate on 75-cm2 tissue culture flasks under conditions described above. A t the end of a 16-h incubation, medium was removed, and the cells were rinsed with Hanks' balanced salt solution and scraped with a rubber policeman in 1.0 ml of 20 mM Tris-HC1, pH 7.5, containing 0.2 M NaCl, 50 p~ dithiothreitol, 10 pg/ml soybean trypsin inhibitor, and 0.01% Triton X-100 (32). The cell suspension was homogenized with a Teflon-glass tissue homogenizer, stirred gently at 4 "C for 60 min, and centrifuged at 4 "C at 30,000 X g for 30 min. Aliquots of the supernatant were dialyzed for 3 h with two changes against 20 mM Tris-HC1, pH 7.5, containing 0.2 M NaC1. Prolyl hydroxylase activity was then determined by incubation with [3H]proline-labeled proto- collagen (unhydroxylated collagen), prepared from 17-day-old chick embryo tendons, as described elsewhere (9). The enzyme preparations were tested in five different concentrations to ensure that the activity determinations were performed on the linear range of the assay. The enzyme activity was assayed in the presence of the following cofactors in final concentrations: 2 mM ascorbic acid, 0.08 mM FeSO,, and 0.5 mM a-ketoglutaric acid (33). The enzyme incubations were performed at 37 "C for 180 min, and the reaction was terminated by the addition of an equal volume of 12 N HC1. The tubes were sealed, hydrolyzed, and assayed for [3H]hydroxyproline (20).

In some experiments, prolyl hydroxylase was extracted from fibro- blasts incubated on 150-cmZ tissue culture flasks, and the effects of 0.4 mM ethyl-3,4-dihydroxybenzoate or 3,4-dihydroxybenzoic acid on the enzyme activity in uitro were examined.

Other Assays-For assay of the synthesis of noncollagenous pro- teins, fibroblasts were cultured on 24-well tissue culture plates, as described above. After a 4-h preincubation with or without 0.4 mM ethyl-3,4-dihydroxybenzoate, radioactive tryptophan (~-[G-~H]tryp- tophan, specific activity 3.7 Ci/mmol; Amersham Corp.), 10 &/well, was added; the incubation was then continued for 16 h. The incor- poration of [3H)tryptophan into macromolecules precipitable with 10% trichloroacetic acid was then determined. The amount of cell protein in the same cultures was determined as above (21), and the incorporation of 3H radioactivity was expressed as cpm/pg cell pro- tein.

To assay the effect of the test compounds on cell proliferation, fibroblasts in 96-well microtiter plates were incubated with or without 0.4 mM ethyl-3,4-dihydroxybenzoate for 72 h. Radioactive thymidine ([methyL3H]thymidine, specific activity 84 Ci/mmol; Amersham Corp.), 10 &i/well, was added, and the incubations were continued for 6 h at 37 'C. The cells were harvested with an automated multi- sample cell harvester (PHD, Cambridge Technology, Inc., Boston). The radioactive macromolecules were collected on glass-fiber filters and counted by liquid scintillation counting.

To assay the plating efficiency of fibroblasts, 2 X lo5 fibroblasts were plated on 25-cm2 tissue culture flasks in medium containing

The abbreviation used is: SDS, sodium dodecyl sulfate.

Inhibition of Collagen Production 9399

10% fetal calf serum and with or without 0.4 mM ethyl-3,4-dihydrox- ybenzoate (34). After a 4-h incubation, the medium was removed, the cells were rinsed with Hanks’ balanced salt solution, and detached by brief trypsinization. The number of cells attached to the flasks was then determined by counting in a hemocytometer.

To determine the viability of the cells incubated in the presence of 0.4 mM ethyl-3,4-dihydrosybenzoate, a trypan blue exclusion test was performed as described previously (35).

The statistical significance of the differences was calculated by Student’s t test.

RESULTS

Inhibition of Prolyl Hydroxylation in Keloid Fibroblast Cul- tures-In the first set of experiments, a keloid fibroblast cell line was incubated with [3H]proline in the presence of 0.2 or 0.4 mM ethyl-3,4-dihydroxybenzoate, and the synthesis of [3H]hydroxyproline was assayed as a marker of collagen pro- duction. The results indicated a marked inhibition in the synthesis of [3H]hydroxyproline (Table I). In the same exper- iment, the cells were also incubated with the parent com- pound, 3,4-dihydroxybenzoic acid, in the corresponding con- centrations. The parent compound did not inhibit prolyl hydroxylation at 0.2 mM concentration and showed only an 11% inhibition at 0.4 mM concentration, similar to findings in a previous report (19) (Table I).

In subsequent studies, seven different keloid fibroblast lines were incubated in the presence of 0.4 mM ethyl-3,4-dihydrox- ybenzoate, and the effect on collagen production was assessed as the ratio of newly synthesized [3H]hydroxyproline/total incorporation of 3H radloactivity into protein. Incubation of keloid cell cultures with the test compound resulted in a marked inhibition of the synthesis of [3H]hydroxyproline (Fig. I). Specifically, the relative collagen synthesis in cultures incubated with 0.4 mM ethyl-3,4-dihydroxybenzoate was re- duced to 26.1 & 10.5% (mean & S.D.) of the controls incubated without inhibitor. At the same time, a similar reduction in the synthesis of [3H]hydroxyproline was observed in normal fibroblast cultures, the values in cultures incubated with 0.4 mM ethyl-3,4-dihydroxybenzoate being 28.5 k 8.8% (mean & S.D.; n = 6) of the controls (Fig. 1). As noted in Fig. 1, there is considerable variability in the relative synthesis of [3H] hydroxyproline both in the control and keloid fibroblast cul-

TABLE I Effects of 3,4-dihydroxybenzoic acid and its ethyl ester derivative ethyl-3,4-dihydroxybenzoate on the synthesis of hydroxyproline in

keloid fibroblast cultures Keloid fibroblasts were cultured in 24-well tissue culture plates a t

early confluency. The cultures were incubated with 30 pCi of [3H] proline for 16 h, and the synthesis of [3H]hydroxyproline in medium and cell fraction was determined separately. E-3.4-DHB. ethvl-3.4- dihydroxybenzoate 3,4-DHB, 3,4-dihydroxybenzoic acid.

. - .

TmQt [3H]Hydroxyproline synthesized compound . “”

Medium Cells mM dpm x Sb dpm X %b

Control 8.17 & 0.06 100.0 0.65 2 0.05 100.0 E-3,4-DHB 0.2 4.97 & 0.40’ 60.9 0.43 f 0.02d 66.6 E-3,4-DHB 0.4 1.60 f0.14‘ 19.5 0.24 +0.02f 37.8 3,4-DHB 0.2 8.34 k 0.7Y 102.0 0.69 k 0.01’ 107.1 3,4-DHB 0.4 7.28 f 0.468 89.1 0.81 f 0.06B 124.8

Values are dpm of [3H]hydroxyproline/pg of cell protein (mean &

* Calculated as percent of the control incubated with 0.2% ethanol

The values are significantly different (p < 0.02) from the control. The values are significantly different (p < 0.05) from the control.

e The values are significantly different (p < 0.001) from the control. ’The values are significantly different (p < 0.01) from the control. #The values are not significantly different (p > 0.1) from the

S.E. of three parallel determinations).

used as solvent for the inhibitors.

control.

KELOIDS

- ~ 0 0.4

E - 3.4 - DHB (mM)

FIG. 1. Inhibition of collagen production by 0.4 mM ethyl- 3.4-dihydroxybenzoate in control and keloid fibroblast cul- tures. Seven keloid and six normal control fibroblast cultures in 24- well tissue culture plates were incubated with and without ethyl-3,4- dihydroxybenzoate, as indicated under “Experimental Procedures.” After a 4-h preincubation, the cultures were labeled with [3H]proline, and the synthesis of [3H]hydroxyproline (PHIHYPRO) both in cell and medium fractions as well as the incorporation of total 3H radio- activity were determined. The individual values represent mean8 of three parallel determinations.

tures incubated without the inhibitor. This variability prob- ably reflects the known heterogeneity of fibroblast popula- tions with respect to their procollagen synthesis (see Ref. 2).

Demonstration of Reduced Prolyl Hydroxylase Activity-To examine the mechanisms leading to reduced synthesis of hydroxyproline in cultures incubated with ethyl-3,4-dihydrox- ybenzoate, prolyl hydroxylase was extracted from the cells and the activity determined by in vitro incubation with bio- logically prepared protocollagen, unhydroxylated [3H]proline- labeled collagen, as substrate. The results indicated that the activity of prolyl hydroxylase in cultures incubated with 0.4 mM ethyl-3,4-dihydroxybenzoate was markedly reduced, and intermediate values were observed in cultures incubated in the presence of 0.2 mM concentration of the test compound (Fig. 2). These results then suggest inactivation of prolyl hydroxylase, and the reduction in prolyl hydroxylation by ethyl-3,4-dihydroxybenzoate noted in cell culture is appar- ently a result of reduced activity of this enzyme.

To examine the mechanism of the inhibition of prolyl hydroxylase activity in further detail, the enzyme from keloid fibroblast cultures was extracted, and its activity was deter- mined in vitro in the presence of varying concentrations of the inhibitor using protocollagen as substrate. Incubation of the enzyme in the presence of 3,4-dihydrobenzoic acid or ethyl-3,4-dihydroxybenzoate, both in 0.4 mM concentration, resulted in a marked inhibition of the prolyl hydroxylase activity in vitro (Table 11). Thus, ethyl-3,4-dihydroxybenzoate appears to be an inhibitor of prolyl hydroxylase in uitro, but it is unclear from these experiments whether its conversion into the parent compound by cleavage of the ester bond is required for the activity, since 3,4-dihydroxybenzoic acid was an equally effective inhibitor.

Consequences of Reduced Hydroxyproline Synthesis-As in- dicated above, the presence of 4-hydroxyproline is critical for normal synthesis of triple-helical procollagen molecules. It was of interest, therefore, to measure the stability of the triple-helical conformation of procollagen synthesized in the presence of ethyl-3,4-dihydroxybenzoate. For this purpose, newly synthesized [3H]proline-labeled macromolecules were subjected to limited proteolytic digestion with pepsin utilizing

9400 Inhibition of Collagen Production .”

- 30-

I

J

e 0 t-

n

a

; 20- 01 a >. I I - R Y

x

0 ‘0- 0

P

E-3,4-DHB (mM)

FIG. 2. Inhibition of prolyl hydroxylase activity in cell cul- tures incubated with ethyl-3,4-dihydroxybenmate. Two keloid and one normal control fibroblast lines were incubated in 75-cm2 tissue culture flasks with varying concentrations of ethyl-3,lt-dihy- droxybenzoate for 16 h. At the end of the incubation, prolyl hydrox- ylase was isolated from the cells and its activity determined by in vitro incubation with biologically prepared protocollagen (unhydrox- ylated type I collagen) as substrate. The values are expressed as [’HI hydroxyproline (p’H/HYPRO) synthesized with [’Hlproline-labeled protocollagen (percent of total radioactivity; mean f S.E. of three parallel cultures).

TABLE I1 Inhibition of prolyl hydroxylase activity in vitro by 3,4-DHB and i t s

ethyl ester derivative ethyl-3,4-dihydroxybenzoate Prolyl hydroxylase was extracted from three 150-cm2 flasks of

keloid fibroblasts in early confluency, as indicated under “Experi- mental Procedures.” Aliquots of the enzyme preparation were assayed for prolyl hydroxylase activity by incubation with [’Hlproline-labeled unhydroxylated collagen as substrate, with or without the inhibitors.

Test compound enzyme added

Amount Of [3H]Hydroxyproline synthesized

Pl dpm’ %b

None 0 177 f 33 None 10.2% ethanol) 400 13.268 f 329 100.0 E-3.4-DHB’ 400 2I148 f 25d 15.1 3,4-DHB’ 400 3,265 f 404d 23.6 Values are expressed as dpm of [1H]hydroxyproline synthesized

with 1 x lo5 cpm of radioactive substrate during a 3-h incubation in vitro (mean f S.E. of three parallel determinations).

Calculated as percent of the controls incubated with 0.2% ethanol used as solvent for the inhibitors after subtraction of 177 dpm from the values.

‘Test compounds were added in 0.4 mM final concentration. E- 3,4-DHB, ethyl-3,4-dihydroxybenzoate 3,4-DHB, 3,4-dihydroxyben- zoic acid.

The values are significantly different (p < 0.001) from the control incubated with ethanol alone but not different from each other.

conditions under which the triple-helical collagen domain of procollagen molecules resists proteolysis while nonhelical a- chains are digested into small fragments. Examination of the digests by SDS-polyacrylamide gel electrophoresis revealed in control cultures the presence of intact al(1) and a2(I) chains of type I collagen (Fig. 3). In cultures incubated in the pres- ence of 0.4 mM ethyl-3,4-dihydroxybenzoate, very little radio- activity was detected in the position of a-chains. Although the total synthesis of [‘H]proline-labeled type I and type 111

dye front -

0 0.2 0.4 E-3.4-DHB (mM1

FIG. 3. Demonstration of reduced stability of newly synthe- sized type I procollagen synthesized in the presence of ethyl- 3,4-dihydroxybenzoate. Normal control fibroblasts in 25-cm’ tis- sue culture flasks were preincubated for 4 h with 0, 0.2, or 0.4 mM ethyl-3,4-dihydroxybenzoate. [‘HIProline was then added, incuba- tions were continued for 16 h, and the stability of the newly synthe- sized procollagen molecules was determined by limited pepsin prote- olysis, as indicated under “Experimental Procedures.” The samples were electrophoresed on SDS-polyacrylamide gel electrophoresis us- ing 8% polyacrylamide gels. The figure represents fluorograms of [3H] proline-labeled polypeptides, the electrophoretic mobilities of a l (1) and a2(I) of type I collagen polypeptides as well as of bromphenol blue (dye front) being indicated.

procollagen in the presence of 0.4 mM ethyl-3,4-dihydroxy- benzoate was markedly reduced (see below), essentially no procollagen molecules with stable triple-helical conformation, which would resist pepsin proteolysis, were present in these cultures. In cultures incubated with 0.2 mM ethyl-3,4-dihy- droxybenzoate, an intermediate quantity of radioactive poly- peptides in the migration position of a-chains was noted, suggesting that a critical number of prolyl residues in some of the molecules were sufficiently hydroxylated to permit pro-a chains to fold into a stable triple helix (Fig. 3).

Since the synthesis of triple-helical type I collagen was markedly inhibited by ethyl-3,4-dihydroxybenzoate, as judged by the SDS-polyacrylamide gel electrophoresis of pepsin- resistant a-chains (see Fig. 3), it was of interest to study whether the synthesis and secretion of intact procollagens type I and type I11 were equally inhibited. For this purpose, cells were incubated with 0, 0.2, or 0.4 mM ethyl-3,4-dihy- droxybenzoate, and the newly synthesized procollagen mole- cules were recovered by precipitation with 30% ammonium sulfate. The radioactive proteins were then subjected to DEAE-cellulose chromatography utilizing conditions which allow separation of intact type I and type I11 procollagens, as shown in control cell cultures (Fig. 4). In contrast, very little radioactivity was detected in the corresponding peaks in sam- ples incubated with either 0.2 or 0.4 mM ethyl-3,4-dihydrox- ybenzoate. For quantitation of type I and type I11 procolla- gens, the radioactivity in the corresponding peaks was pooled, and [”Hlhydroxyproline was determined as a specific marker of procollagen. Quantitation of [‘H]hydroxyproline indicated that the synthesis and secretion of both type I and type I11 procollagens were markedly inhibited by 0.2 or 0.4 mM ethyl- 3,4-dihydroxybenzoate (Table 111). Thus, the results indicated that a consequence of the inhibition of prolyl hydroxylation during intracellular biosynthesis of procollagens is a marked reduction in the amount of both type I and type I11 procolla- gens recovered in the extracellular space.

Demonstration That Ethyl-3,4-dihydroxybenzoate Does Not

Inhibition of Collagen Production

PROCOLLAGEN TYPE I .

yl

b 3.0 TYPE m PROCOLLAGEN

I

IO 20 30 4 0 50 60 FRACTION NO.

FIG. 4. DEAE-cellulose chromatography of ['Hlproline-la- beled medium proteins synthesized with or without 0.4 mM (ethyl-3.4-dihydroxybenzoate). Normal control fibroblast cul- tures in 75-cm2 tissue culture flasks were incubated with or without the test compound, as indicated under "Experimental Procedures." The newly synthesized procollagens were recovered from the medium fraction by precipitation with 30% ammonium sulfate and chromat- ographed on DEAE-cellulose chromatography. The proteins initially bound to the column were eluted with a linear gradient of 0-0.22 M NaCl between fractions 11 and 56. The remaining radioactivity was eluted by a stepwise addition of 1 M NaC1. Fractions of 3 ml were collected, and the radioactivity in the fractions was determined. The recovery of total radioactivity in control cultures was 76%, whereas the corresponding value in the cultures incubated with 0.4 mM ethyl- 3,4-dihydroxybenzoate was 94%. The elution positions of type I and 111 procollagens synthesized in control fibroblast cultures are indi- cated by arrows.

TABLE 111 Inhibition of the synthesi of type I and type III procollngens by

ethyl-3,I-dihydroxybenzoate Fibroblast cultures in 75-cmZ tissue culture flasks were preincu-

bated with varying concentrations of E-3,4-DHB and then labeled with 100 pCi of [3H]proline for 16 h. The 3H-labeled proteins in the medium fractions were precipitated with ammonium sulfate, and type I and type 111 procollagens were separated by DEAE-cellulose chro- matography, as shown in Fig. 4. E-3,4-DHB, ethyl-3,4-dihydroxyben- zoate.

Test [SH]Hydroxyproline synthesized compound 'On'' Type I" Type 111" Totalb

mM dpm/pg protein Control 69.44 1.41 70.85 E-3,4-DHB 0.2 0.72 0.07 0.78 E-3,4-DHB 0.4 0.12 0.15 0.27

a Values are expressed as dpm of [3H]hydroxyproline in the corre- sponding peaks recovered by DEAE-cellulose chromatography and corrected for the cell protein content of the corresponding cultures.

*Values are dpm of [3H]hydroxyproline in type I plus type I11 procollagen peaks.

Affect Type I and Type III Procollagen or Fibronectin mRNA Steady-state Levels-To examine the possibility that ethyl- 3,4-dihydroxybenzoate might affect procollagen production on the transcriptional level, the steady-state abundance of al(1) and al(1II) procollagen mRNAs was determined in fibroblast cultures incubated with and without the test com- pound. Slot-blot hybridizations with human sequence specific cDNA probes under hybridization and washing conditions which exclude cross-hybridizations between type I and type 111 procollagen cDNA probes and the corresponding mRNAs (27) were utilized in these studies. The results indicated that there was no difference in al(1) and al(II1) procollagen mRNA levels in cultures incubated in the presence of 0, 0.2, or 0.4 mM ethyl-3,4-dihydroxybenzoate (Fig. 5 and Table IV). For comparison, the levels of fibronectin mRNA, a noncollag-

0 0.2 0.4

9401

Amount of RNA ( y g )

- 4.0

- 2.0 - 1.0

- 0.5

- 0.25

- 0.125

E-3.4-DHB (mM) FIG. 5. Demonstration that E-3.4-DHB does not affect the

steady-state abundance of type I and I11 procollagen or fibro- nectin mRNAs. Normal control fibroblast cultures were incubated in 75-cm' tissue culture flasks with varying concentrations of ethyl- 3,4-dihydroxybenzoate (E-3,4-DHB) for 16 h. Total RNA was isolated by cesium chloride density gradient centrifugation, and type I and 111 procollagen and fibronectin mRNA levels were determined by slot blot hybridizations with human sequence specific cDNA probes. The figure represents autoradiograms of [32P]cDNA-mRNA hybrids with type I11 procollagen cDNA probe; similar results were obtained with type I procollagen and fibronectin cDNA probes. The steady-state levels of mRNA were quantitated by scanning densitometry at 700 nm (see Table IV).

enous glycoprotein synthesized by the fibroblasts, was also determined. Again, no difference in fibronectin mRNA steady-state level was noted in cultures incubated with vary- ing concentrations of the test compound (Table IV). These observations suggest that ethyl-3,4-dihydroxybenzoate does not affect procollagen gene expression on pretranslational level and further support the notion that the inhibition is a post-translational event at the level of prolyl hydroxylation.

Control Experiments-To examine the possibility that ethyl-3,4-dihydroxybenzoate might exert its effect on collagen production through generalized inhibition of protein synthesis or by being toxic to the fibroblasts, several control experi- ments were performed. First, the incorporation of [3H]thy- midine into the cells cultured with or without 0.4 mM ethyl- 3,4-dihydroxybenzoate was determined. In these experiments, both keloid and normal fibroblast cultures were tested. The results indicated that there was no inhibition of ['Hlthymi- dine incorporation in cultures incubated with 0.4 mM ethyl- 3,4-dihydroxybenzoate. Specifically, the incorporation of ['HI thymidine in normal fibroblast cultures incubated with 0 or 0.4 mM ethyl-3,4-dihydroxybenzoate was 1.77 +. 0.46 and 1.89 f 0.20 x lo3 cpm/2 x lo3 cells, respectively (mean f S.D. of 3-4 parallel cultures). The corresponding values in keloid cultures without and with the test compound were 1.66 f 0.12 and 2.12 f 0.34 X IO3 cpm, respectively.

Second, the viability of the cells incubated with and without the test compound in 0.4 mM concentration was examined by trypan blue exclusion test. Results indicated that the viability of normal fibroblasts incubated with and without the test compound was 95.5 & 2.3 and 94.6 +. 1.2%, respectively (mean f S.D. of four parallel determinations). The corresponding values in keloid fibroblast cultures incubated with and without the test compound were 98.1 +. 0.5 and 96.0 f 3.6%, respec- tively.

Third, the effect of ethyl-3,4-dihydroxybenzoate on plating

9402 Inhibition of Collagen Production

TABLE IV Steady-state levels of al(I) of type Zprocohgen, al(III) of type IIIprocollugen, and fibronectin messenger RNAs in

cultured human skin fibroblasts incubated with ethyl-3,4-dihydroxybenzoate (E-3,4-DHB)

Concentration of Messenger RNA levels* E-3,4-DHBE al(I) al(II1) Fibronectin

mM units/& % of controld units/& % of controld units/& % of controt‘ 0 556 f 54 100.0 677 f 12 100.0 210 f 9 100.0 0.2 589 f 29 105.9 681 f 44 100.6 213 f 23 101.4 0.4 569 f 56 102.3 630 f 21 93.1 209 f 12 99.5

“Fibroblasts, cultured in 75-cm2 tissue culture flasks, were incubated for 16 h at 37 “C with ethyl-3,4- dihydroxybenzoate in final concentrations indicated. All cultures, including the controls, contained 0.2% ethanol used as solvent for ethyl-3,4-dihydroxybenzoate.

Total RNA was isolated by CsCl density gradient centrifugation, and messenger RNA levels were determined by molecular hybridizations with human al(I), al(III), and fibronectin specific cDNA probes as indicated under “Experimental Procedures.”

e The values are expressed as densitometric absorbance units obtained by scanning densitometry at 700 nm/pg total RNA (mean f S.E. of four to six parallel determinations) and do not reflect absolute levels of type I and type I11 procollagen and fibronectin mRNAs.

Calculated as percent of the control cultures incubated without ethyl-3,4-dihydroxybenzoate.

efficiency of the fibroblasts was tested. No difference in the presence and absence of the test compound in 0.4 mM concen- tration was noted, the plating efficiency being 65.9 f 0.1 and 69.9 f 0.1% in normal fibroblast cultures and 79.5 f 0.1 and 76.5 f 0.1% in keloid cell cultures, respectively (mean f S.D.; n = 4).

Fourth, the effects of ethyl-3,4-dihydroxybenzoate on the synthesis of noncollagenous proteins were tested by incubat- ing the cells with [3H]tryptophan, an amino acid which is not present in any significant quantities in collagenous proteins; the incorporation of [3H]tryptophan was determined in pro- teins precipitable with 10% trichloroacetic acid. The results indicated that 0.4 mM ethyl-3,4-dihydroxybenzoate had little, if any, effect on the synthesis of noncollagenous proteins. Specifically, the values for [3H]tryptophan incorporation in normal and keloid fibroblast cultures in the absence of the test compound was 7.0 -+ 1.6 and 6.0 f 0.6 x 10‘ cpm/pg cell protein (mean f S.D. of three parallel incubations). The corresponding values in the same cell cultures incubated with 0.4 mM ethyl-3,4-dihydroxybenzoate were 6.3 f 0.8 and 5.9 & 0.7 X 10’ cpm/pg, respectively (statistically not significant; p > 0.1). Thus, the control experiments demonstrated that ethyl-3,4-dihydroxybenzoate in 0.4 mM concentration had no effect on cell proliferation, viability, or plating efficiency. Also, the test compound did not affect the synthesis of non- collagenous proteins, suggesting a specific effect on collagen production.

DISCUSSION

Excessive deposition of collagen is a major pathologic fea- ture in several fibrotic diseases which can affect either skin or a variety of internal organs (2, 13, 36). In many of these conditions, increase in collagen synthesis may not be the primary event of the disease process; nevertheless, the exces- sive accumulation of collagen has major consequences in terms of structure and function of the affected organs. Thus, a pharmacologic approach which could arrest collagen depo- sition in the tissues would be thought to be beneficial to the patients suffering from clinical fibrotic diseases.

Previously, several pharmacologic agents have been tested for their effects on collagen metabolism (see Refs. 13, 17, and 36). Many of these compounds have been shown to be effective in inhibiting collagen production, and some of them are currently in clinical use for treatment of fibrotic diseases. The rationale for the clinical use in most cases has been derived from studies on collagen metabolism in isolated tissues and

in cell cultures. Unfortunately, many of the compounds are not specific for collagen, and their clinical efficacy is fre- quently compromised by side effects. Consequently, there is a further need for pharmacologic agents which would display action properties specific for collagen metabolism.

Collagens are a family of closely related connective tissue proteins which have several features in common (37, 38). All collagen polypeptides undergo extensive post-translational modifications, many of which are catalyzed by specific en- zymes with stringent cofactor and cosubstrate requirements (14, 15, 37). Thus, the enzymatically mediated modification reactions, many of them unique to collagen, could serve as targets for pharmacologic modulation. Of particular interest is the reaction leading to formation of 4-hydroxyproline, an imino acid necessary for normal synthesis of triple-helical procollagen molecules (9-11,39). Inhibition of prolyl hydrox- ylation leads to reduced secretion of newly synthesized pro- collagen polypeptides, and the nonhelical pro-a-chains are subject to rapid both intra- and extracellular degradation.

In the present study, we have demonstrated that ethyl-3,4- dihydroxybenzoate is a potent inhibitor of prolyl hydroxyla- tion during intracellular biosynthesis of procollagen in human skin fibroblast cultures. As a consequence of the inhibition of prolyl hydroxylation, the secretion of type I and type I11 procollagens was markedly reduced, apparently due to com- promised stability of the procollagen triple helix. The inhibi- tion of procollagen production was shown not to result from generalized inhibition of protein synthesis or from toxicity of the test compound to the cells. Furthermore, type I and type I11 procollagen mRNA steady-state level determinations sug- gested that the inhibition was a post-transcriptional event. Further studies specifically demonstrated that the activity of prolyl hydroxylase, the enzyme catalyzing the conversion of prolyl residues to 4-hydroxyproline, was inhibited by the test compound.

Previous studies (18) have demonstrated that 3,4-dihydrox- ybenzoic acid, the parent molecule for the ethylester deriva- tive tested in this study, is a competitive analog of a-ketoglu- tarate and ascorbate, a cosubstrate and a cofactor for prolyl hydroxylase. 3,4-Dihydroxybenzoic acid is able to compete with a-ketoglutarate and ascorbate for the binding to the active site of the enzyme (18). Thus, ethyl-3,4-dihydroxyben- zoate would appear to be a relatively specific inhibitor for prolyl hydroxylation with limited effects on other enzymes or on the synthesis of noncollagenous proteins.

To demonstrate the potential feasibility of ethyl-3,4-dihy-

Inhibition of Collagen Production 9403

droxybenzoate for inhibition of collagen production in fibrotic skin diseases, several keloid cell lines were examined in this study for their response to ethyl-3,4-dihydroxybenzoate. As indicated above, keloids are cutaneous lesions characterized by excessive accumulation of type I collagen, and this accu- mulation appears to result from enhanced synthesis of colla- gen by lesional fibroblasts. The inhibition of collagen produc- tion in keloid fibroblast cultures demonstrated in this study suggests that ethyl-3,4-dihydroxybenzoate, or other structural analogs of a-ketoglutarate and ascorbate, might serve as use- ful agents to limit collagen production in uiuo.

Acknowledgments-Human sequence specific cDNA probes were kindly provided by Drs. Mon-Li Chu, Francesco Ramirez, and Darwin J . Prockop, Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Rutgers Medical School. We thank Helmi Konola for skillful technical help and Charlene D. Aranda for expert secretarial assistance.

REFERENCES

1. Murray, J. C., Pollack, S. V., and Pinnell, S. R. (1981) J. Am. Acad. Dermatol. 4,461-470

2. Abergel, R. P., and Uitto, J. (1986) in Diseases of Connectiue Tissue: The Molecular Pathology of the Extracellular Matrix (Uitto, J., and Perejda, A. J., eds) pp. 345-366, Marcel Dekker, New York

3. Abergel, R. P., Pizzurro, D., Meeker, C. A., Lask, G., Matsuoka, L. Y., Minor, R. R., Chu, M.-L., and Uitto, J . (1985) J. Inuest. Dermatol. 84, 384-390

4. Craig, D. P., Schofield, J. D., and Jackson, D. S. (1975) Br. J. Surg. 6 2 , 741-744

5. Diegelmann, R. F., Cohen, I. K., and McCoy, B. J. (1979) J. Cell. Physiol. 9 8 , 341-346

6. Russell, S. B., Russel, J. D., and Trupin, J. S. (1982) J. Biol.

7. Fleckman, P. H., Jeffrey, J. J., and Eisen, A. Z. (1973) J. Invest.

8. Trupin, J. S., Russel, S. B., and Russel, J. D. (1983) Collagen

9. Berg, R. A., and Prockop, D. J. (1973) Biochemistry 12, 3395-

10. Uitto, J., and Prockop, D. J . (1974) Biochem. Biophys. Res.

11. Jimenez, S. A., and Rosenbloom, J. (1974) Arch. Biochem. Bio-

12. Kao, W. W-Y., Prockop, D. J., and Berg, R. A. (1979) J. Biol.

13. Uitto, J., Ryhanen, L., Tan, E. M. L., Oikarinen, A. I., and

14. Kivirikko, K. I., and Myllyla, R. (1982) in Collagen in Health and

Chem. 257,9525-9531

Dermatol. 60, 46-52

Relat. Res. 3 , 13-23

3401

Commun. 60,414-423

phys. 163,459-465

Chem. 254,2234-2243

Zaragoza, E. J. (1984) Fed. Proc. 4 3 , 2815-2820

Disease (Jayson, M. I. V., and Weiss, J. B., eds) pp. 101-120, Churchill Livingstone, New York

15. Kivirikko. K. I.. and Mvllvla, R. (1985) Ann. N . Y. Acad. Sci. 460,187-201'

" .

16. Maiamaa. K.. Hanauske-Abel. H. M.. Giinzler. V.. and Kivirikko. K. I. (1984) Eur. J . Biochen. 138,' 239-245 '

17. Kivirikko, K. I., and Majamaa, K. (1985) in Ciba Foundation Symposium 114, Fibrosis, pp. 34-64, Pitman Books, Turnbridge Wells, UK

18. Majamaa, K., Giinzler, V., Hanauske-Abel, H. M., Myllyla, R., and Kivirikko, K. I. (1986) J. Biol. Chem. 261, 7819-7823

19. Majamaa, K., and Uitto, J. (1986) Clin. Res. 34, 417A (abstr.) 20. Juva, K., and Prockop, D. J. (1966) Anal. Biochem. 15,77783 21. Bradford, M. M. (1976) Anal. Biochem. 72 , 248-254 22. Lichtenstein, J. R., Byers, P. H., Smith, B. D., and Martin, G. R.

23. Uitto, J., Booth, B. A., and Polak, K. L. (1980) Biochim. Biophys.

24. Bonner, W. M., and Laskey, R. A. (1974) Eur. J. Biochem. 46,

25. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter,

26. Thomas, P. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5201-

(1975) Biochemistry 14, 1589-1594

Acta 6 2 4 , 545-561

83-92

W. J . (1979) Biochemistry 18, 5294-5299

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

5205

F. (1985) Proc. Natl. Acad. Sci. U. S. A . 8 2 , 5935-5939

F. (1982) Nucleic Acids Res. 10, 5925-5934

Ramirez, F. (1985) J. Biol. Chem. 260 , 4357-4363

Uitto, J., Perejda, A. J., Abergel, R. P., Chu, M.-L., and Ramirez,

Chu, M.-L., Myers, J. C., Bernard, M. P., Ding, J . F., and Ramirez,

Chu, M.-L., Weil, D., deWet, W., Bernard, M., Sippola, M., and

Bernard, M. P., Kolbe, M., Weil, D., Chu, M.-L. (1983) Biochem-

Maniatis, T., Jeffrey, A., and Kleid, D. G. (1975) Proc. Natl.

Booth, B. A., and Uitto, J . (1981) Biochim. Biophys. Acta 6 7 5 ,

Myllyla, R., Majamaa, K., Giinzler, V., Hanauske-Abel, H. M., and Kivirikko, K. I. (1984) J. Biol. Chem. 259 , 5403-5405

Tan, E. M. L., Ryhanen, L., and Uitto, J. (1983) J. Inuest. Dermatol. 80, 261-267

Phillips, H. J . (1973) in Tissue Culture Methods and Applications (Kruse, P. F., and Patterson, M. K., eds) pp. 406-408, Academic Press, Orlando, FL

Uitto, J., Tan, E. M. L., and Ryhanen, L. (1982) J. Inuest. Dermatol. 79, 113s-120s

Prockop, D. J., and Kivirikko, K. I. (1984) N . Engl. J . Med. 3 11,

Burgeson, R. E., and Morris, N. P. (1986) in Diseases of Connec- tive Tissue: The Molecular Pathology of the Extracellular Matrix

New York (Uitto, J., and Perejda, A. J., eds) pp. 3-28, Marcel Dekker,

Prockop, D. J., Berg, R. A., Kivirikko, K. I., and Uitto, J. (1976) in Biochemistry of Collagen (Ramachandran, G. N., and Reddi, A. H., eds) pp. 163-273, Plenum Press, New York

istry 2 4 , 2698-2704

Acad. Sci. U. S. A . 72, 1184-1188

117-122

376-386