propeptide-mediated regulation of procollagen synthesis in imr

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 266, No. 5, Issue of February 15, pp. 2983-2987,1991 Printed in U. S. A.

Propeptide-mediated Regulation of Procollagen Synthesis in IMR-90 Human Lung Fibroblast Cell Cultures EVIDENCE FOR TRANSCRIPTIONAL CONTROL*

(Received for publication, August 20, 1990)

Catherine H. WuS, Cherie M. Walton, and George Y. Wu From the Department of Medicine, Division of Gastroenterology-Liver Disease, University of Connecticut School of Medicine, Farmington,-Connecticut 06030

We have demonstrated previously that the carboxyl- and amino-terminal propeptides of type I procollagen can inhibit procollagen synthesis by specifically de- creasing procollagen mRNA levels. The objective of the present experiments was to determine the mechanism by which propeptides cause these pretranslational effects.

IMR-90 fibroblasts were exposed to medium containing carboxyl-terminal propeptide of type I procollagen, and nuclear run-off assays were performed by hybridization to a specific a1 chain type I procollagen cDNA probe. Specific type I procollagen transcription rates were found to be decreased by 50% in the presence of 75 nM carboxyl-terminal propeptide compared with control (untreated) cells. Total cellular transcription rates as well as &actin mRNA rates were not affected significantly by any concentration of carboxyl-terminal propeptide.

Propeptide radiolabeled with 12’1 was found to be taken up by cultured cells. Furthermore, exogenous carboxyl-terminal propeptide levels increased in the cytosolic compartment and eventually reached a steady-state level of 18 2 2 pmol/g cell protein by 30 min. Of particular interest was the finding that levels of radiolabeled carboxyl-terminal propeptide were also detected in the nuclear fraction and increased with time, reaching a plateau after 60 min of incubation. Incubation of nuclei from IMR-90 cells in medium containing varying concentrations of carboxyl-terminal propeptide resulted in nuclear transcription rates that were decreased by 40% compared with untreated controls. &Actin nuclear message levels remained unchanged under identical conditions. We conclude that carboxyl-terminal propeptide of type I procollagen can be internalized and become associated with the nuclear compartment. This suggests a feedback regulatory role on procollagen synthesis by a direct effect on procollagen gene transcription.

* This work was supported in part by United States Public Health Service Grant DK-37982 (to C. H. W.), by Research Career Devel- opment Award CA-01110 (to G. Y. W.), and by a grant from the Alcoholic Beverage Medical Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “uduertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed: Dept. of Medicine, Div. of Gastroenterology-Hepatology, University of Connecticut School of Medicine, Rm. AM-044,253 Farmington Ave., Farmington, C T 06030.

Regulation of collagen synthesis is important in the control of the formation of normal extracellular matrices as well as the pathological environments that occur in fibrotic diseases. It has been shown previously that in fibrotic states type I and I11 collagens are quantitatively the most important (1-3). In late stages type I collagen is found to be the major component of broad fibrous bands. Type I collagen is synthesized in a precursor form, procollagen, that is larger than the final product because of the presence of extension propeptides on the amino and carboxyl termini of the molecule (4). During or shortly after secretion from the cell the propeptides of type I procollagen are cleaved from both ends of the collagen molecule (5). It has been shown previously that procollagen propeptides can exert an inhibitory effect on procollagen synthesis in vitro (6-8). We have examined this process further and reported previously that the effect of lower concentrations of propeptide occurred at a pretranslational level (9). The objective of the work described below was to determine whether the observed inhibitory effect occurred at a transcriptional level and to determine the mechanism by which carboxyl-terminal propeptide exerts this effect.

MATERIALS AND METHODS

Isolation and Purification of Procollagen Carboxyl-terminal Propep- tide-Carboxyl-terminal propeptides were obtained from chick organ cultures according to the method of Olsen et al. (10) in which freshly dissected calvaria and leg tendons from 15-day-old chick embryos were cultured for 24 h in Dulbecco’s modified Eagle’s medium containing 50 mg/ml ascorbate and 50 mg/ml @-aminopropionitrile. After removal from the tissues, the medium was made up to 10 mM phenylmethylsulfonylfluoride, 25 mM EDTA, 10 mM N-ethylmaleim- ide, 0.05 M Tris, pH 8.6. Propeptides were isolated by DEAE-Sephacel and CM-cellulose chromatography according to the method of Olsen et al. (10) and checked for purity by 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (11). For uptake studies, propeptides were labeled with carrier-free NalZ5I (Amersham C o p ) using a solid phase lactoperoxidase-glucose oxidase method (Bio-Rad).

Chick carboxyl-terminal propeptide was selected for study because of its high degree of similarity to human carboxyl-terminal propeptide (12,13) and the ease of preparing chick carboxyl-terminal propeptide compared with the human homolog. For these reasons, chick carboxyl-terminal propeptide is a convenient tool for the examination of propeptide-mediated feedback regulation of procollagen synthesis in human cells. IMR-90 fibroblasts were chosen because of the stable, well characterized procollagen synthesis and the availability of previous data on propeptide-mediated control of procollagen synthesis in these cells (9).

Cells and Cell Culture-IMR-90 fibroblasts were maintained a t 37 “C under 5% COS in Dulbecco’s modified Eagle’s medium (GIBCO) containing 10% fetal calf serum (GIBCO), 100 units of penicillin, and 0.1 mg/ml streptomycin as described previously (14). For experiments on propeptide effects, cells were incubated for 24 h in medium alone or medium containing varying concentrations of carboxyl-terminal propeptide or, in some experiments, controls consisting of 100 nM type I procollagen or 100 nM type I collagen.

2984 Transcriptional Regulation of Procollagen Synthesis Isolation of Nuclei from IMR-90 Fibroblasts-To determine

whether propeptide-mediated pretranslational inhibitory effects occurred at a transcriptional level, nuclei from propeptide-treated and control IMR-90 fibroblasts (untreated) were isolated according to the method of Stallcup and Washington (15). In brief, cells were washed with ice-cold phosphate-buffered saline, pH 7.6, pelleted, and resuspended in 0.01 M Tris, pH 7.5, buffer containing 10 mM KC1, 1.5 mM MgC12, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride and homogenized in a Dounce homogenizer. After swelling for 10 min at 4 "c, an equal volume of the same buffer containing 0.6 M sucrose and 0.5% Nonidet P-40 (v/v) was added. Cells were again homogenized. The cell lysate was centrifuged at 1000 X g, and the nuclear pellet was washed with 10 mM Tris buffer, pH 7.6, containing 25% glycerol, 1 mM dithiothreitol, and 10 mM KC1. The nuclei examined microscopically were found to be intact and free of cellular debris.

Time Course of Total Nuclear Transcription Rates-Nuclear transcription rates were determined according to the method of McKnight and Palmiter (16). In brief, the isolated nuclei were incubated at 26 "c in 10 mM Tris, pH 7.5; 2 mM MnC12; 3.6 mM dithiothreitol; 0.2 M ammonium sulfate; 0.5% sarkosyl, 1 mM each ATP, GTP, and CTP; and 50 mCi of [3H]UTP (Du Pont-New England Nuclear). A sample was removed for determination of DNA according to the method of Kapuscinski and Skoczylas (17). As a negative control, nuclei were incubated in the presence of 30 mg/ml actinomycin D. At regular intervals, samples were removed and treated with 4 mg/ml DNase I for 15 min at 26 "C to stop the reaction. To the mixture were added 40 ml of 10% sodium dodecyl sulfate, 20 ml of 0.1 M Na2EDTA, and 30 ml of 10 mg/ml proteinase K. Nuclei were incubated at 37 "C for 1 h, and then the mixture was made up to 10% trichloroacetic acid to precipitate RNA. The incorporation of [3H] UTP into RNA was determined and expressed as cpm/mg of DNA as a function of time. The time point of maximal transcription was determined, and this time point was used in all subsequent transcription experiments.

Effect of Propeptides on Specific Procollagen a1 Chain Gene Tran- scription-In order to determine specific transcription rates for the genes of procollagen type I a1 chain and p-actin, IMR-90 fibroblasts were incubated at 37 "C for 24 h in medium alone, medium containing 75 nM carboxyl-terminal propeptide, or controls consisting of 100 nM type I procollagen or 100 nM type I collagen. Nuclei were isolated as described above and incubated for 30 min at 26 "C with 50 mCi of [3ZP]UTP in transcription buffer as described above, digested with 50 mg/ml proteinase K, extracted with phenol/chloroform (18), and RNA was recovered by ethanol precipitation. The ethanol precipitates were purified further by extraction with 4 M guanidine HCl. The 32P- labeled nuclear transcripts were hybridized to nitrocellulose filters containing 5 mg of immobilized cDNA probes consisting of Hf 404, a type I procollagen a1 chain probe (from D. W. Rowe, University of Connecticut, Farmington, CT), a @-actin probe (from D. Cleveland, Johns Hopkins University, Baltimore, MD), and a pBR322 probe after prehybridization at 42 "C for 2 h in 50% formamide, 5 X 0.15 M NaC1, 0.015 M sodium citrate, pH 7.0, 0.2% sodium dodecyl sulfate, 0.02 M Tris, pH 7.5, 1 mM EDTA, 4 X Denhardt's solution, 10 mg/ ml single-stranded DNA, and 20 yg/ml yeast RNA as described by Otto et al. (19). After hybridization for 48 h at 42 "C, the filters were washed and exposed to XARphotographic film (Kodak). Quantitation of hybridized RNA was accomplished by densitometric scanning of the autoradiograms and confirmed by scintillation counting of the corresponding dots. Specific hybridization of type I procollagen a1 chain nuclear transcription was determined by measuring the radioactivity hybridized to the procollagen probe minus that hybridized to the pBR322 probe and expressed as a fraction of the total input counts (20).

Detection of Internalized Propeptides-To determine whether exogenous carboxyl-terminal propeptide could be transported to intracellular sites, IMR-90 fibroblasts were incubated at 37 "C in medium containing '251-labeled carboxyl-terminal propeptide at 75 nM. At various times cells were chilled to 4 "C and washed with ice-cold saline. Surface-bound propeptide was removed by exposure of the cells to phosphate-buffered saline, pH 5.0, containing 10 mM EDTA (21). The cells were then homogenized in 10 mM Tris-HC1, pH 7.5, 1.5 mM NaC1, 10 mM KC1, 1 mM dithiothreitol, 300 mM sucrose, 0.25% Nonidet P-40 and centrifuged at 1000 X g for 10 rnin at 4 "C. The nuclear pellet was resuspended in 10 mM Hepes,' pH 7.6, con-

' The abbreviations used are: Hepes, 4-(2-hydroxyethyl)-l-pipera- zineethanesulfonic acid; SDS, sodium dodecyl sulfate.

taining 300 mM glycerol and 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride and recentrifuged. This supernatant was com- bined with the cytoplasmic supernatant, and radioactivity in the nuclear pellet, cytoplasmic and surface compartments was determined using a y-counter. Nonspecific uptake was determined in the presence of a 100-fold excess of unlabeled carboxyl-terminal propeptide, and specific distribution of '251-labeled propeptide was determined as the difference between total and nonspecific accumulation of radioactivity in each compartment. The studies were performed in triplicate, and results were expressed as pmol of propeptide/g of cell protein 5 S.D.

To determine whether the radioactivity found in the compartments represented intact or degraded propeptides, samples of each compartment were applied on a 10% SDS-polyacrylamide gel electrophoresis (11) along with standard lZ51-labeled carboxyl-terminal propeptide. The gel was dried, and an autoradiogram was obtained by exposure to XAR film.

Direct Effect of Propeptides on Procollagen Gene Transcription-In order to determine whether propeptides could exert a direct effect on procollagen gene transcription, nuclei were isolated from untreated cells and exposed to buffer containing 50 mCi of [32P]UTP and 1 mM each CTP, ATP, and GTP with or without 75 nM carboxyl-terminal propeptide at 26 "C for 30 min. The nuclei were then digested with proteinase K, extracted with phenol/chloroform, and RNA precipi- tated with ethanol. The 3ZP-labeled transcripts were hybridized to nitrocellulose filters containing 5 mg of immobilized type I procollagen a1 chain (Hf 404), @-actin, and pBR322 cDNAprobes as described above. After hybridization, the filters were washed, and autoradiograms were obtained by exposure to XAR film. The data were quantitated by densitometric scanning of the dots, and specific hybridization was calculated as the radioactivity obtained with procollagen type I or 0-actin probes minus that obtained with the pBR322 control probe and expressed as a fraction of the total input counts.

RESULTS

The rate of [3H]UTP incorporation into nuclear RNA in nuclei isolated from IMR-90 fibroblasts is shown in Fig. 1. Total nuclear transcription increased steadily and then pla- teaued by 30 min of incubation. The addition of actinomycin D to nuclear run-off assays that were performed identically resulted, as expected, in 90% inhibition of transcription. Because maximal transcriptional activity occurred after 30 min of incubation, this time point was used for all subsequent nuclear run-off transcription assays.

Fig. 2 shows that treatment of cells with 75 nM carboxyl- terminal propeptide had no significant effect on the levels of total precipitable transcripts in nuclei isolated from those cells. Variation of the input counts in nuclei of control and propeptide-treated cells resulted in changes in transcription

40.

30.

20,

I O

I x +30pplml I X Aclmomycm C

I L I

IO 20 30 40 TIME (minuled

FIG. 1. The rate of ["HIUTP incorporation into nuclear RNA in nuclei isolated from IMR-90 fibroblasts. Nuclei were incubated at 26 "C for various times in transcription buffer containing 1 mM each ATP, GTP, and CTP and 50 mCi of [3H]UTP. Samples were removed for DNA quantitation, and radioactivity incorporated into trichloroacetic acid-precipitable RNA was determined as a function of time, as described under "Materials and Methods."

Transcriptional Regulation of Procollagen Synthesis 2985

I

Control

I

I n *(I) 75 nM Act. D

FIG. 2. The effects of carboxyl-terminal propeptide pretreatment of f ibroblasts on total cellular transcription rates. IMR-90 fibroblasts were incubated a t 37 "C for 24 h in medium alone or medium containing 75 nM carboxyl-terminal propeptide (pC(1)). Nuclei were isolated as described above and incubated in transcription buffer containing [3H]UTP. As a negative control, nuclei were incubated in the presence of 30 mg/ml actinomycin D (Act. D). At regular intervals, samples were removed and the reaction stopped. The mixture was made up to 10% trichloroacetic acid to precipitate RNA, and incorporation of [3H]UTP into RNA was determined and expressed as cpm/mg of DNA, as described under "Materials and Methods."

in direct proportion, indicating that the transcriptional assay was linear under these conditions (data not shown). Our previous studies (9) have shown that a 24-h incubation with carboxyl-terminal propeptide resulted in a specific decrease in steady-state type I procollagen mRNA. In order to determine whether this effect was due to transcriptional control, nuclear run-off experiments were performed at this same time point.

Treatment of cells with carboxyl-terminal propeptide resulted in concentration-dependent specific decreases in type I procollagen a1 chain nuclear mRNA levels in seven repeti- tive experiments of which Fig. 3 is a representative. However, @-actin gene transcription was not affected significantly under identical conditions, indicating that under these conditions the inhibitory effect of the carboxyl-terminal propeptide on type I procollagen a1 chain gene transcription was not due to a nonspecific or a generalized effect on nuclear transcription. Background nonspecific signal as determined by hybridization to pBR322 was not significant.

alW

&Actin

FIG. 3. A representative dot blot assay for type I procollagen gene t ranscr ip t ion a f te r p re t rea tment o f ce l l s wi th propeptides. IMR-90 fibroblasts were incubated a t 37 "C for 24 h in medium alone or medium containing 75 nM carboxyl-terminal propeptide (pC(1)). Nuclei were isolated and incubated for 30 min at 26 "C with 50 mCi of ["PIUTP in transcription buffer. "P-Labeled RNA was extracted and hybridized to filters containing 5 mg of immobilized cDNA probes consisting of Hf 404, a procollagen type I probe (a I f I ) ) , a @-actin probe, and a pBR322 probe after prehybridization, as described under "Materials and Methods." After hybridization the filters were washed and exposed to XAR photographic film.

0 20 4 0 60 80 100

P W ( n W

FIG. 4. Quant i ta t ion of type I procollagen (pC(1)) gene transcription af ter pretreatment of IMR-90 cells with propeptides. Dot blots of hybridized RNA were quantitated by densitometric scanning of the autoradiograms and confirmed by scintillation counting of the corresponding dots. Specific hybridization of type I procollagen nl (palfl)) chain nuclear transcription was determined by measuring the radioactivity hybridized to the procollagen probe minus that hybridized to the pBR322 probe, expressed as a fraction of the total input counts (18).

Quantitation of the dot blots is shown in Fig. 4 in which are plotted the means f S.D. of seven experiments. Carboxyl- terminal propeptide decreased procollagen gene transcription to 90% at 17.5 nM, 75% a t 35 nM, and 50% at 75 nM. The transcription of the @-actin gene remained essentially unchanged and was significantly different from that of procollagen gene transcription a t propeptide concentrations of 35 nM and above. Exposure of cells to control media containing 100 nM of either type I procollagen or collagen had no effect on either procollagen or @-actin gene transcription (data not shown).

To determine whether carboxyl-terminal propeptide was internalized by the fibroblasts, cells were incubated with "'1- labeled carboxyl-terminal propeptide, and uptake by cells was determined a t 37 "C. At various times cells were harvested, and surface-bound, cytoplasmic, and nuclear distribution of labeled carboxyl-terminal propeptide was followed by SDS- polyacrylamide gel electrophoresis and autoradiography. Fig. 5A shows a representative autoradiogram of the distribution of labeled propeptide within cellular compartments as a function of time from 15 min to 1 h. Lanes 1, 5, and 9 show that propeptide added to the incubation medium remained largely intact during the 1st h. The trimer, 100 kDa, was predomi- nant, but monomers, 31 and 33 kDa, were also present. Lanes 2, 6, and IO demonstrate that binding of propeptide to the

2986 Transcriptional Regulation of Procollagen Synthesis -3

A KD x 10

- 200 r 7 U -

- 97

- 43

- 31

1 2 3 4 5 6 7 8 9 I O 1 1 1 2 13

KD x 1c3 -200

""0" * - 97

- 43

- 31

1 2 3 4 5 6 7 0 9 101112 13

FIG. 5. A, detection of internalized carboxyl-terminal propeptide. IMR-90 fibroblasts were incubated in medium containing 75 nM "'I- labeled carboxyl-terminal propeptide and cellular radioactive propeptide distribution, as described under "Materials and Methods." Sam- ples of each compartment were applied on a 10% SDS-polyacrylamide gel (ll), and an autoradiogram was obtained by exposure to XAR film. Lanes 1-4, medium, surface-bound, cytosol, and nuclei, respectively, a t 15 min; lanes 5-8, medium, surface-bound, cytosol, and nuclei, respectively, a t 30 min; lunes 9-12, medium, surface-bound, cytosol, and nuclei, respectively, a t 1 h. Lane 13 contains standard "'I-labeled carboxyl-terminal propeptide. KD indicates kilodaltons. R, extended time course and specificity of propeptide internalization. Fibroblasts were incubated for 120 and 240 min in medium containing 75 nM 1Y511-labeled carboxyl-terminal propeptide, and cellular radioactive propeptide distribution was determined as described in A. Lanes 1-4, medium, surface-bound, cytosol, and nuclei, respectively, a t 120 min; lanes 5-8, medium, surface-bound, cytosol, and nuclei, respectively, a t 240 min; lanes 9-12, medium, surface-bound, cytosol, and nuclei, respectively, a t 240 min in medium containing "'I-labeled carboxyl-terminal propeptide plus a 100-fold molar excess of unlabeled carboxyl-terminal propeptide. Lane 13 contains standard "'I- labeled carboxyl-terminal propeptide. KD indicates kilodaltons.

cell surface occurred within 15 min of exposure. Propeptide was faintly visible in the cytoplasmic fraction at this time as seen in lane 3 and was barely detectable in the nuclear fraction a t 15 min. However, lanes 3, 7, and 11 show that propeptide in the cytoplasm increased with time. Of particular interest was the finding that carboxyl-terminal propeptide levels also became measurable in the nuclear pellet by 30 min and increased with time.

Fig. 5B shows the distribution of radiolabeled propeptide at 120 and 240 min. Propeptide was clearly present in all compartments. Substantial degradation of labeled propeptide occurred as fragments were found in the cytoplasmic ( l a n e 7) and nuclear fractions (lane 8) by 240 min. This degradation was not present in the medium, suggesting that the fragmen- tation was derived from internalized, originally intact, pro-

peptide and was not due to internalization of degraded propeptide fragments. Lanes 9-12 show the distribution of propeptide after incubation of cells for 240 min with labeled propeptide with a 100-fold molar excess of unlabeled propeptide to compete for internalization sites. '2sI-Labeled propeptide associated with the cell surface decreased by 81% whereas labeled propeptide in the cytosolic and nuclear fractions became barely detectable, supporting the notion that the uptake process was specific for the carboxyl-terminal propeptide. Quantitation of the data in these autoradiograms is shown in Fig. 6 and indicates that uptake of labeled carboxyl-terminal propeptide into the cytosol was rapid and reached a plateau value of 18 f 2 pmol/g cell protein after 30 min of incubation. Accumulation of labeled carboxyl-terminal propeptide in the nuclear pellet was delayed, in comparison, reaching a steady- state value of approximately 15 pmol/g cell protein by 60 min.

Because carboxyl-terminal propeptide was found in the nuclei of propeptide-treated cells, we wondered whether the observed propeptide-mediated transcriptional inhibition could have been due to a direct effect of the propeptide on the nuclear transcriptional machinery. To test this hypothesis, isolated nuclei were incubated in the presence or absence of propeptides followed by assays of in vitro transcription of type I procollagen and /3-actin genes. At 75 nM, carboxyl- terminal propeptide inhibited type I procollagen a1 chain gene transcription by 42% (Table I). As a control for nonspecific depression of transcription, @-actin gene transcription was also measured and found to be unchanged under identical conditions.

DISCUSSION

It has been demonstrated previously that at high ( p ~ ) concentrations procollagen propeptides can inhibit procolla-

60 1,

1 5 30 6 0 120 240

Tlma (mln)

FIG. 6. Quantitation of specific intracellular distribution of '2"I-carboxyl-terminal propeptide (pC(1)) as a function of incubation time. Specific distribution was calculated as the difference between compartment radioactivity with labeled ligand alone, and labeled ligand plus a 100-fold excess of unlabeled ligand.

TABLE I Procollagen gene transcription rates 30 min after direct exposure of

nuclei to carboxyl-terminal propeptide

Transcription rate = cpm probe - cpm pBR322

inDut cDm

Probe Treatments

Control pC(W (75 nM)

n l ( 1 ) b 27.6 -t 3.1 15.9 f 5.8

&Actin 9.4 f 2.4 10.7 & 1.8 O pC(I), type I procollagen carboxyl-terminal propeptide.

nl(I) , a cDNA probe specific for an mRNA sequence for the a1 chain of type I procollagen.

Transcriptional Regulation of Procollagen Synthesis 2987

gen synthesis at a translational level (6-8). Our previous studies have shown that at lower (nM) concentrations, administration of procollagen propeptides resulted in a specific decrease in steady-state type I procollagen mRNA levels, indicating that the inhibitory effects under these conditions were pretranslational (9). The observed decreases in procollagen mRNA levels could have been because of an inhibition of transcription or a decrease in the stability of procollagen message. Our current results from nuclear run-off assays indicate that procollagen carboxyl-terminal propeptide specifically inhibits procollagen gene transcription in a concentration-dependent manner. Transcriptional regulation of procollagen synthesis has also been found with other polypeptides such as the factor isolated from thioacetamide-induced liver fibrosis (22).

The exact manner in which propeptides exert their inhibitory effect has not yet been established completely. Our demonstration that exogenous carboxyl-terminal propeptide can be internalized by cells and a proportion associated with the nucleus shortly after administration of carboxyl-terminal propeptide to cells in culture medium supports the possibility of a feedback regulatory role. The internalization data for carboxyl-terminal propeptide are also consistent with the studies of Schlumberger et al. (23) which demonstrated im- munocytochemically that the exogenous amino-terminal propeptide of type I procollagen can also be taken up by fibroblasts. However, the possibility still exists that regulation could also occur via interaction with the plasma membrane with subsequent release of intracellular messengers, as has been shown to be the case with other peptides (24).

Our data indicate that the inhibitory effect on procollagen gene transcription obtained by direct exposure of nuclei was less than that obtained by exposure of the cells themselves to propeptide (42 uersus 50% inhibition, respectively). This may be because of the nonphysiological conditions associated with isolation of the nuclei and/or segregation of the nuclei from critical cytoplasmic components in the direct exposure experiments. Nevertheless, the finding of transcriptional inhibition of procollagen genes upon direct exposure of nuclei to carboxyl-terminal propeptide supports the hypothesis that this propeptide can exert a direct transcriptional effect under these conditions.

With increasing time, fragments of propeptides as well as intact propeptides were found in the nuclear pellet after administration of propeptides to intact cells, indicating that substantial degradation of propeptides occurs intracellularly. This raises the additional possibility that degraded fragments may also be regulatory and the presence of intact propeptides may not play an exclusive role in the regulation of collagen synthesis.

Although 24-h incubations with carboxyl-terminal propeptide were performed in the current experiments, our data on the internalization of carboxyl-terminal propeptide indicate that maximal nuclear levels were attained within 60 min. This suggests that the long (24-h) exposure to carboxyl-terminal propeptide although sufficient, may not be necessary to pro- duce transcriptional effects on procollagen synthesis.

That propeptides can regulate collagen synthesis by direct interaction with intracellular components is consistent with earlier work by Paglia et al. (6), who found that calf propeptides inhibited translation of procollagen message when propeptide was added directly to a cell-free translation system.

We speculate that a knowledge of the mechanism by which propeptides interact with the nucleus, the nuclear structures, and the portions of the propeptide molecule which are in- volved may permit design of simple peptides that could ulti- mately be useful in the control of collagen synthesis in fibrotic disease states.

Acknowledgments-We acknowledge gratefully the expert assist- ance of Rosemary Pavlick in the preparation of this manuscript. We thank Dr. David Rowe for his most helpful advice and encouragement.

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propeptide-mediated regulation of procollagen synthesis in imr

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