0013-7227/91/1283-1603$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 3 Printed in U.S.A.
Regulation of Insulin, Epidermal Growth Factor, and Transforming Growth Factor-a Levels by Growth Factor-Degrading Enzymes* BARRY D. GEHM AND MARSHA RICH ROSNER Ben May Institute and the Department of Pharmacological and Physiological Sciences, University of Chicago, Chicago, Illinois 60637
involved in the degradation of TGFa and EGF as well as insulin, and that the degradation of TGFa, but not EGF, is mediated in part by IDE. Inhibiting the activity of these metalloproteases decreased growth factor depletion, suggesting that these enzymes play an important role in the control of extracellular growth factor levels. The existence of separate degradative pathways for EGF and TGFa may explain how the two factors exert differential effects in some systems, and degradation of TGFa by IDE would provide a possible mechanism for interaction between the insulin and TGFa/EGF signalling systems. (Endocrinology 128: 1603-1610,1991)
ABSTRACT. The mechanisms by which growth factors are degraded and the role this process plays in the regulation of cell growth are not well understood. Insulin degradation is believed to be mediated by a specific metalloprotease, insulin-degrading enzyme (IDE). We have previously shown that IDE can also degrade transforming growth factor-a (TGFa), but not epidermal growth factor (EGF), in vitro. This selectivity was surprising, since TGFa and EGF are structurally similar and bind to the same receptor with comparable affinities. Using a spectrum of protease inhibitors, we have now analyzed the degradation of TGFa, EGF, and insulin by human hepatoma HepG2 cells. The results suggest that bacitracin-sensitive metalloproteases are
R
ELATIVELY little is known about the mechanisms by which cells degrade growth factors. The degradation of insulin in a number of mammalian cells is mediated by insulin-degrading enzyme (IDE); evidence for this includes the identical breakdown products generated from labeled insulin by hepatocytes and IDE (1) and the inhibition of insulin degradation by microinjection of anti-IDE antibodies into HepG2 cells (2). IDE is a primarily cytosolic bacitracin-sensitive metalloprotease which catalyzes a series of specific proteolytic cleavages (3). Magun and co-workers (4, 5) have shown that epidermal growth factor (EGF) degradation in Rat1 fibroblasts is initiated by an analogous series of specific proteolytic cleavages and is completed in the lysosomes. The enzyme(s) responsible for the initial cleavages has not been determined. Transforming growth factor-a (TGFa) is a polypeptide growth factor, structurally similar to EGF, that binds to the human EGF receptor with an affinity comparable to that of EGF and generally elicits similar biological effects Received October 1,1990. Address all correspondence and requests for reprints to: Dr. Marsha Rosner, University of Chicago, Ben May Institute, 5841 South Maryland, Box 424, Chicago, Illinois 60637. * This work was supported by grants from the American Diabetes Association, the Juvenile Diabetes Foundation, and the University of Chicago Diabetes Research Training Center (to M.R.R.).
(6). Few studies of TGFa degradation have been published, but it appears that in some cell types TGFa and EGF are degraded by separate pathways (7, 8). We have previously reported that in vitro, purified mammalian and Drosophila IDEs are capable of hydrolyzing TGFa, but not EGF, in addition to insulin (9). Thus, the available evidence raises the possibility that TGFa and EGF are degraded in vivo by specific metalloproteases similar to IDE and, in the case of TGFa, by IDE itself. To examine the role of IDE-like metalloproteases in growth factor degradation and to determine whether IDE mediates the cellular degradation of TGFa, we compared the effects of various protease inhibitors on the degradation of labeled insulin, TGFa, and EGF in HepG2 human hepatoma cells. HepG2 cells have been shown to degrade insulin via IDE (2), although lysosomal degradation may also occur (10). Since hepatocytes secrete and respond to TGFa in vivo (11), a hepatic cell line is an appropriate choice for studying TGFa degradation. EGF, which is not degraded by IDE, was used as a control for non-IDE-mediated effects of inhibitors. Although protease inhibitors have limited specificity, it is possible to further categorize a protease by its substrate specificity. Therefore, we also compared the ability of excess insulin to block degradation of TGFa and EGF in order to assess a possible role for IDE in TGFa degradation in
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living cells. Our results suggest that metalloproteases mediate a substantial portion of the cellular degradation of EGF and TGFa as well as insulin, and that TGFa is degraded in part by IDE. These results may have implications for the cellular regulation of growth factor levels and for the interaction of the insulin and TGFa signalling systems.
Materials and Methods Materials [125I]Insulin (A14 monoiodinated receptor grade; 2200 Ci/ mmol) was purchased from New England Nuclear (Boston, MA) and Amersham (Arlington Heights, IL). [125I]EGF (1000 Ci/mmol) was purchased from New England Nuclear. Recombinant human TGFa was the generous gift of Dr. Rik Derynck of Genentech (South San Francisco, CA) and was radioiodinated with Na125I (New England Nuclear) and Enzymobeads (Bio-Rad, Richmond, CA). The radioiodinated product had a specific activity of 2000 Ci/mmol and bound to EGF receptor (A431 cells) with an affinity similar to that of commercial [125I]EGF. Porcine insulin, bacitracin, 1,10-phenanthroline, phenylmethylsulfonylfluoride, and leupeptin were purchased from Sigma (St. Louis, MO). Irreversible metalloprotease inhibitor (IrMPI; AT-chloroacetyl-D,L-AT-hydroxyphenylalanyl-alanyl-alanine amide) was purchased from Enzyme Systems Products (Livermore, CA). Partially purified rat liver IDE was prepared by Dr. J. Victor Garcia, as previously described (9). Cell culture All cells were grown in a gassed (5.5% CO2) humidified 37 C incubator. HepG2 cells were grown on polyornithine-coated plates in Dulbecco's Modified Eagle's Medium (DME) supplemented with Earle's salts, nonessential amino acids, Na pyruvate, and 10% fetal calf serum. A431 cells were grown on uncoated plates in DME plus 10% fetal calf serum. Buffers Buffer E contained DME, 1 mg/ml ovalbumin, and 50 mM HEPES, pH 7.5. Buffer K (12) contained 100 mM HEPES (pH 8.0), 120 mM NaCl, 5 mM KC1, 1.2 mM MgSO4, 8 mM glucose, and 10 mg/ml BSA. Cellular growth factor degradation HepG2 cells grown to near confluence were incubated in buffer E or buffer K for I h before the addition of 0.1 nM 125Ilabeled insulin, TGFa, or EGF. Compared to buffer E, buffer K produced enhanced degradation of insulin and TGFa, but little degradation of EGF. In experiments requiring identical conditions for all three substrates, buffer E was employed; otherwise, buffer K was used with insulin and TGFa, and buffer E with EGF. In time-course experiments, the cells were incubated with substrate in 60-mm dishes containing 3 ml buffer, and 500-/tl aliquots were withdrawn at each time point. Fixed time experiments were performed in six-well plates containing 1.2 ml buffer/well; 500-^1 aliquots were taken at the beginning
Endo«1991 Voll28«No3
and end of the assay. Durations of fixed time incubations were chosen to produce optimal levels of degradation (25-40%) in control cells; typical durations were 1 h for insulin, 1.5-2.5 h for TGFa, and 2.5-3 h for EGF. All incubations were performed at 37 C. Insulin degradation was measured by trichloroacetic acid (TCA) precipitation of intact [125I] insulin, as previously described (13). TGFa and EGF were measured by binding to EGF receptor, as described below. To correct for loss of [125I] EGF by HepG2 cell association (see Results), [125I]EGF degradation was calculated by the formula: % degradation = % decrease in A431 binding - % HepG2-associated. EGF receptor binding assay Intact [125I]EGF and [125I]TGFa were quantified by binding to EGF receptor in a variation of the method of Wattenberg et al. (14). A431 cells, which overexpress EGF receptor, were grown to confluence in 24-well plates and preincubated for 212 h at 37 C in DME plus 0.1% CR-ITS (Collaborative Research, Waltham, MA). Aliquots from EGF and TGFa degradation assays were divided between paired wells and incubated for 2-4 h on ice. The cells were washed seven times with cold PBS and lysed with 0.1 N NaOH. Bound 125I was measured in an LKB-Wallac RiaGamma 1274 7-counter.
Results HepG2 cells sequester EGF, but not TGFa or insulin Growth factor and insulin degradation were assayed as loss of labeled substrate from the incubation medium and assessed by binding (for TGFa and EGF) or TCA precipitation (for insulin). These procedures have been widely used to quantitate degradation of these ligands (9). To correct for loss of labeled substrate or product from the medium by cell association, it was necessary to determine whether significant amounts of labeled material were being retained by the cells. Little or no loss of label from the medium was observed during incubations of HepG2 cells with [125I]TGFa or [125I]insulin (Fig. 1). In contrast, during incubations with [125I]EGF, the concentration of 125I label in the medium decreased substantially. These measurements do not reflect degradation of the ligands, since intact proteins were not separated from labeled breakdown products. Measurements of cell-associated radioactivity (data not shown) confirmed the greater sequestration of [125I] EGF. Typically 15-25% of [125I]EGF label became cell associated during assays, but only 1-2% of [125I]TGFa and [125I]insulin did so. Washing with 0.2 M acetic acid in 0.5 M NaCl, a well established method of releasing surface-bound insulin and EGF (15, 16), removed 40% of cell-associated [125I]insulin and 15% of [125I]TGFa and [125I]EGF (data not shown), indicating that the majority of cell-associated label was internalized. The difference between EGF and TGFa sequestration may result in part from different receptor affinities in this cell type (data not shown).
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REGULATION OF GROWTH FACTOR LEVELS BY PROTEASES
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FIG. 1. Removal of 126I label from the medium by HepG2 cells. HepG2 cells were incubated with 12BI-labeled insulin (•), TGFa (A), and EGF (•), as described in Materials and Methods. Aliquots were removed at the indicated times and counted for 126I counts per min. Results are expressed as a percentage of the initial concentration of 125I in the medium.
After a 1-h incubation at 37 C, cell-associated [125I] EGF was 85% intact, as judged by precipitation with TCA and phosphotungstic acid (data not shown). Since the cell-associated material was primarily undegraded, we corrrected for cell association when calculating EGF degradation. Some of the inhibitors tested altered [125I] EGF cell association, but none produced significant loss of [125I]TGFa or [125I]insulin from the medium.
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HepG2 cells degrade insulin, TGFa, and EGF HepG2 cells degraded substantial fractions (30-50%) of the extracellular insulin, TGFa, and EGF in a relatively short time (2-2.5 h), suggesting that degradation can significantly affect extracellular growth factors levels (Fig. 2). Growth factor-degrading enzymes, thus, may play an important role in regulating extracellular concentrations of their substrates. Little or no growth factor degradation was produced by conditioned reaction buffer in the absence of cells (data not shown), indicating that degradation is not mediated by extracellular proteases. Bacitracin inhibits depletion of extracellular EGF and TGFa as well as insulin The antibiotic bacitracin is a well known inhibitor of IDE (17) and has been reported to inhibit degradation of glucagon and calcitonin (18, 19), but is not a general metalloprotease inhibitor. We, therefore, examined its effect on growth factor degradation in HepG2 cells. Bacitracin (1 mM) inhibited [I25I] insulin degradation almost completely (Fig. 2A) and inhibited degradation of labeled TGFa and EGF approximately 50% (Fig. 2, B and C). Bacitracin also increased sequestration of [125I]EGF, which tended to obscure its inhibition of [125I]EGF degradation in early experiments in which no correction for loss of label by sequestration was made. High (10 mM) concentrations of bacitracin were cytotoxic, as judged by
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TIME (min)
FIG. 2. Time courses of growth factor degradation with or without bacitracin. HepG2 cells were preincubated for 1 h in buffer E in the presence (•, A, and • ) or absence (O, A, and D) of 1 mM bacitracin. At time zero, 125I-labeled insulin (A), TGFa (B), or EGF (C) was added. At the indicated times, aliquots were removed and assayed for degradation, as described in Materials and Methods. A, [125I] Insulin. For this assay, SDS were typically less than 1%. B, [125I]TGFa. Results are shown as the mean ± SD for duplicate determinations. C, [125I]EGF. Results are shown as the mean ± SD for duplicate determinations.
the appearance of numerous vacuoles in the cells after a few hours of incubation. The effects of bacitracin concentrations up to 3 mM on insulin, TGFa, and EGF degradation in intact HepG2 cells are illustrated in
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Fig. 3. Some interexperimental variablity was observed with all three, but the ranges of measured IC5Os overlapped for the three substrates (insulin, 0.1-0.7 mM; TGFa and EGF, 0.3-1 mM). In comparison, in vitro hydrolysis of [125I]insulin and [125I]TGFa by rat liver IDE was more bacitracin sensitive (IC5o, 30 /xM for both; data not shown). This difference may reflect limited access of bacitracin to the intracellular site(s) of growth factor degradation and/or partial degradation of bacitracin by the HepG2 cells. These factors may also account for some of the variable sensitivity that was observed in cellular degradation. In sum, the effective inhibitory concentrations of bacitracin were comparable for all three substrates. Metalloprotea.se inhibitors decrease degradation of insulin, TGFa, and EGF Phenanthroline, a chelator of transition metal ions and thus an inhibitor of metalloproteases, has previously been shown to inhibit IDE and insulin degradation in L6 cells (12). Phenanthroline (1 mM) almost completely inhibited [125I] insulin degradation by HepG2 cells (Fig. 4). At the same concentration, degradation of [125I]TGFa and [125I]EGF was inhibited 40% and 70%, respectively. Thus, the degree of inhibition by phenanthroline is similar to that produced by bacitracin. As with bacitracin, the precise IC50 for phenanthroline varied somewhat from experiment to experiment, but phenanthroline consistently inhibited insulin degradation more than that of TGFa or EGF. In vitro hydrolysis of [125I]insulin and [125I]TGFa by rat liver IDE showed equal sensitivity to phenanthroline (IC50, 300 /*M; data not shown), indicating that the differential inhibition of their cellular degradation was not due to differential sensitivity of IDE with different substrates. As a chelator, phenanthroline's 120
1001
if
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101
[BACITRACIN] (mM)
FIG. 3. Effect of bacitracin concentration on growth factor degradation. HepG2 cells were incubated with 125I-labeled insulin (•), TGFa (A), and EGF (•), as described in Materials and Methods. The indicated concentrations of bacitracin were added 1 h before the addition of substrate. Results are shown as the mean ± SD for duplicate determinations and are expressed as a percentage of the control without bacitracin.
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120 • — INSULIN — A — TGF-alpha — • — EGF
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FIG. 4. Effect of 1,10-phenanthroline on growth factor degradation. HepG2 cells were incubated with 125I-labeled insulin (•), TGFa (A), and EGF (•), as described in Materials and Methods, except that buffer K was used for all three substrates. The indicated concentrations of phenanthroline were added 1 h before the substrates. Before A431 binding assay, phenanthroline was titrated by the addition of FeSO^ to a 0.1-mM excess [based on Fe(phen)32+ stoichiometry] to prevent cell detachment. Results are shown as the mean ± SD for duplicate determinations and are expressed as a percentage of the control without phenanthroline.
effectiveness can be altered by intracellular concentrations of metal ions; furthermore, it can affect many cellular processes and enzymes other than metalloproteases. For example, 1 mM phenanthroline also decreased [125I]EGF sequestration 40%. For these reasons it is difficult to explain the relative sensitivities of the three substrates purely in terms of metalloprotease inhibition, but the effects of phenanthroline are consistent with degradation by metalloproteases. To avoid the nonspecific effects of chelators, we also employed IrMPI. This compound covalently modifies the active site of metalloproteases, causing loss of activity (20). Kayalar and Wong (12) reported that IrMPI partially inhibits insulin degradation by intact L6 cells, L6 cell cytosolic extracts, and immunoprecipitated IDE. We observed partial inhibition of labeled insulin, TGFa, and EGF degradation by HepG2 cells treated with IrMPI, and the dose-response curves for [125I] insulin and [125I] TGFa degradation were similar (IC50, 0.5 mM for insulin and 0.2-0.5 mM for TGFa; Fig. 5). In a separate experiment using a single concentration, 2 mM IrMPI decreased [125I]EGF degradation by HepG2 cells 70%. IrMPI (0.5 mM) inhibited in vitro degradation of insulin by partially purified rat liver IDE by 75% (data not shown), compared to 30% inhibition of insulin degradation by HepG2 cells. As with bacitracin, the lower potency of the inhibitor against cellular compared to in vitro degradation may represent limited access of the inhibitor to the relevant enzymes and/or degradation of the inhibitor by HepG2 cells. The effects of phenanthroline and IrMPI suggest that
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TGFa and EGF, but not insulin, in lysosomes. None of the three factors appears to be degraded by serine proteases.
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Excess insulin inhibits TGFa degradation
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FIG. 5. Effect of irMPI on degradation of insulin and TGFa. HepG2 cells were incubated with 125I-labeled insulin (•) and TGFa (A), as described in Materials and Methods. The indicated concentrations of IrMPI were added as dimethylsulfoxide solutions 1 h before the substrates. All assays contained a final dimethylsulfoxide concentration of 2%. Results are shown as the mean ± SD for duplicate determinations and are expressed as a percentage of the control without inhibitor. TABLE 1. Effect of protease inhibitors on growth factor degradation Degradation (% of control) Inhibitor None NH4C1 Leupeptin PMSF
Cone.
10 mM 1 mM
Insulin 100 106.4 ± 2.5 97.0 ± 9.9 101.5 ± 0.6
TGFa
EGF
100 100 51.5 ± 6.7 44.2 ± 1.3 99.5 ± 13.1 108.1 ± 6.4 85.0 ± 8.0 115.6 ± 4.8
HepG2 cells were incubated with 125I-labeled growth factors as described in Materials and Methods. The indicated inhibitors were added 1 h before the substrates. Results are shown as the mean ± SE and expressed as a percentage of the control without inhibitor.
metalloproteases are responsible for a significant fraction of EGF and TGFa degradation as well as insulin degradation in HepG2 cells.
Because all of the IDE inhibitors tested inhibited degradation of EGF, insulin, and TGFa with similar IC50s, it was not possible to resolve the role of IDE in TGFa degradation by their use. A more specific agent is excess unlabeled insulin, which as a competitive substrate inhibits the IDE hydrolysis of both [125I] insulin and [125I]TGFa in vitro (9). When added to HepG2 cells, unlabeled insulin inhibited [125I] insulin degradation completely and [125I]TGFa degradation by 50% (Fig. 6). With both substrates, the IC50 was 100 nM, comparable to that obtained in vitro. In contrast, cellular [125I]EGF degradation was unaffected by insulin. Inhibition of TGFa degradation by insulin was observed in the presence of a large excess of BSA (150 /xu), indicating that the inhibition is not a nonspecific protein effect. Unlabeled TGFa inhibits [125I] insulin degradation in vitro (9), but the analogous cellular experiment was not attempted because of the large amount of TGFa that would be required. The ability of insulin to compete selectively for TGFa degradation, but not EGF degradation, in HepG2 cells is consistent with the idea that insulin and TGFa are degraded in part by a common enzyme. The specificity and potency both in vitro and in cells suggest that IDE is the enzyme responsible. Protease inhibitors do not inhibit receptor binding Because cellular growth factor degradation is presumably receptor mediated, inhibitors of receptor-ligand binding could decrease the rate of degradation without
Other protease inhibitors Lysosomes are the major site of intracellular protein degradation and have been implicated in the degradation of insulin, EGF, and TGFa (7, 21, 22). We used NH4C1 to examine the role of lysosomes in growth factor degradation in HepG2 cells. NH4C1 was chosen in preference to chloroquine, another widely used lysosomotropic agent, because the latter has been shown to inhibit nonlysosomal insulin degradation in hepatocytes (23). As shown in Table 1, 10 mM NH4C1 inhibited HepG2 cell degradation of labeled TGFa and EGF by 50%, but had no effect on [125I]insulin degradation. Like bacitracin, NH4CI increased cell-associated [125I]EGF. Leupeptin, an inhibitor of cysteine and serine proteases, has also been used to inhibit lysosomal proteolysis (23, 24); neither it nor phenylmethylsulfonylfluoride, an inhibitor of serine proteases, had any significant effect on degradation of labeled insulin, TGFa, or EGF. Based on these observations, HepG2 cells appear to degrade 50% of
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FIG. 6. Competition of growth factor degradation with unlabeled insulin. HepG2 cells were incubated with 125I-labeled insulin (•), TGFa (A), and EGF (•), as described in Materials and Methods. The indicated concentrations of unlabeled porcine insulin were added 1 h before the addition of substrate. Results are shown as the mean ± SD for duplicate determinations and are expressed as a percentage of the control without insulin.
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affecting protease activities. Bacitracin increased HepG2 cell-associated [125I]insulin (not shown), indicating that it does not inhibit insulin binding. Phenanthroline had no effect HepG2 cell insulin binding (not shown). Neither insulin nor any of the inhibitors used in this study affected the binding of labeled TGFa or EGF to A431 cells (not shown), indicating that they do not interfere with binding to the EGF receptor. These results indicate that the protease inhibitors tested act at a step after receptor binding, presumably by inhibiting growth factor-degrading enzymes. Discussion Our results indicate that the degradation of the growth factors EGF and TGFa by cultured HepG2 cells is mediated in part by nonlysosomal metalloproteases with inhibitor profiles similar to IDE. Furthermore, degradation of TGFa, but not EGF, was inhibited 50% by saturating concentrations of insulin, a competitive IDE substrate, suggesting that IDE is responsible for a substantial portion of TGFa degradation in HepG2 cells. The balance of TGFa degradation may occur via the same pathway(s) that degrades EGF, but it is difficult to test this idea by competition in intact cells, since the two proteins compete for binding to the same receptor. Previous reports on the effects of protease inhibitors on cellular insulin and EGF degradation suggest that these degradative processes are complex. Some investigators have reported little or no effect of lysosomal inhibitors on insulin degradation (12, 23, 25), consistent with the absence of any effect of NH4C1 or leupeptin in our system, while others have reported substantial inhibition (26, 27). These discrepancies may be due to methodological differences and/or differences in cell types. For example, Gansler et al. (28) have shown that various cell types differ substantially in sensitivity of insulin degradation to bacitracin, suggesting that insulin may be processed by different pathways in different cell types. Similarly, McLain and Olefsky (10) have reported the presence of two pathways for handling internalized insulin in HepG2 cells, one operating at high (17 nM) concentrations of extracellular insulin, the other at low (0.17 nM) concentrations. Only the low concentration pathway was sensitive to chloroquine. Two pathways for processing EGF in Swiss/3T3 cells have also been reported (29). It is likely that similar heterogeneity and multiplicity of degradation pathways exist for other growth factors as well. The existence of separate pathways for the degradation of EGF and TGFa is supported by the work of Korc and co-workers (7), who report that T3M4 and ASPC-1 cells degrade TGFa rapidly, but EGF slowly. Degradation of both substrates in both cell lines is inhibited by the
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lysosomotropic compound methylamine. In contrast, methylamine inhibits the degradation of EGF, but not TGFa, in RL95-2 cells (8). Their results do not necessarily indicate that TGFa is degraded by IDE in these cell lines, but do support the idea that EGF and TGFa can be degraded by different routes. Separate degradative pathways may explain how these similar ligands, which both bind to the same receptor with comparable affinities, display different potencies in some systems (30-33). An internalized polypeptide hormone may be disposed of in several possible ways. It may be degraded by a specific protease, the products of which may be released or further degraded in lysosomes. It may be degraded in lysosomes without prior action by a specific protease. It may even be released intact, as has been reported for some insulin internalized by adipocytes (26, 34). The present experiments do not address the release of intact growth factors, which would not alter their extracellular concentrations, but such a process may account for the absence of significant insulin and TGFa sequestration even in the presence of inhibitors of degradation. The inability of lysosomal inhibitors to affect insulin degradation in HepG2 cells suggests that the products of IDE-catalyzed proteolysis are released from the cells without further processing in lysosomes. This is consistent with the results of Duckworth et al. (1), who showed that hepatocytes release insulin degradation products similar or identical to those produced in vitro by IDE. In contrast, some degradation of TGFa and EGF appears to occur in the lysosomes, based on the inhibitory effect of NH4C1. This lysosomal degradation may occur subsequent to or in parallel with degradation by specific proteases.The inhibition of EGF degradation by phenanthroline, bacitracin, and IrMPI, but not insulin, suggests that EGF degradation is mediated in part by an EGFdegrading enzyme similar to, but distinct from, IDE. The human and Drosophila IDEs show sequence similarities to E. coli protease III, but appear to be distinct from the thermolysin superfamily of metalloproteases (35, 36). The similarity of the EGF-degrading enzyme's inhibitory profile to that of the IDE raises the possibility that there is a family of specific growth factor proteases that are evolutionarily related to each other and to protease III. Several lines of evidence suggest that growth factordegrading enzymes such as IDE may be involved in the regulation of growth and development. Stoppelli et al. (37) have shown that expression of IDE is developmentally regulated in Drosophila. Couch and Strittmatter (38), using the rat skeletal muscle cell line L6 as well as rat primary muscle cells, demonstrated that myoblast fusion could be blocked by specific inhibitors of endogenous metalloprotease activity. Subsequently, Kayalar and Wong (12) showed a correlation between inhibition of IDE and inhibition of morphological and biochemical
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REGULATION OF GROWTH FACTOR LEVELS BY PROTEASES differentiation in the same cell line, and suggested that degradation of insulin by IDE is required for muscle cell differentiation. Our results raise the possibility that the inhibition of differentiation may result from inhibiting degradation of T G F a or EGF or other growth factors by IDE or related enzymes. In any case, the inhibitors' effects on differentiation indicate a possible role for IDE or other growth factor-degrading enzymes in the control of mitosis and differentiation. The decreased rate of depletion of growth factors from the medium caused by IDE inhibitors raises the possibility that IDE and/or IDE-like enzymes may play a significant role in clearance of growth factors from blood and extracellular fluid. Regulation of the activity and/or expression of such enzymes could provide a way for a cell to vary the rate of growth factor degradation and thereby alter or maintain the extracellular concentration. In such a model the degradation of T G F a by IDE is especially provocative, since it provides a possible route for interaction between the insulin and T G F a - E G F signal transduction systems. Furthermore, separate pathways for degradation of T G F a and EGF could result in faster degradation of EGF than T G F a in some cell types, which may account for the differences in relative potency that have been reported for these two growth factors (30-33).
9. 10. 11. 12.
13. 14. 15. 16. 17. 18.
19.
Acknowledgments 20. We thank J. V. Garcia for rat liver IDE; C. Kayalar, R. Perlman, W.-L. Kuo, and E. van Melle for helpful discussion and advice; and D. Zeleznik for assistance in preparation of the manuscript.
References 1. Duckworth WC, Hamel FG, Peavy DE, Liepniks JJ, Ryan MP, Hermodson MA, Frank BH 1988 Degradation products of insulin generated by hepatocytes and by insulin protease. J Biol Chem 263:1826-1833 2. Shii K, Roth RA 1986 Inhibition of insulin degradation by hepatoma cells after microinjection of monoclonal antibodies to a specific cytosolic protease. Proc Natl Acad Sci USA 83:4147-4151 3. Duckworth WC, Garcia JV, Liepnieks JJ, Hermodson MA, Frank BH, Rosner MR 1989 Drosphila insulin degrading enzyme and rat skeletal muscle insulin protease cleave insulin at similar sites. Biochemistry 28:2471-2477 4. Matrisian LM, Planck SR, Magun BE 1984 Intracellular processing of epidermal growth factor. I. Acidification of 125I-epidermal growth factor in intracellular organelles. J Biol Chem 259:3047-3052 5. Planck SR, Finch JS, Magun BE 1984 Intracellular processing of epidermal growth factor. II. Intracellular cleavage of the COOHterminal region of 125I-epidermal growth factor. J Biol Chem 259:3053-3057 6. Derynck R 1988 TGF-alpha. Cell 54:593-595 7. Korc M, Finman JE 1989 Attenuated processing of epidermal growth factor in the face of marked degradation of transforming growth factor-a. J Biol Chem 264:14990-14999 8. Korc M, Haussler CA, Trookman NS 1987 Divergent effects of
21. 22. 23. 24.
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27. 28. 29. 30.
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epidermal growth factor and transforming growth factors on a human endometrial carcinoma cell line. Cancer Res 47:4909-4914 Garcia JV, Gehm BD, Rosner MR 1989 An evolutionarily conserved enzyme degrades transforming growth factor-alpha as well as insulin. J Cell Biol 109:1301-1307 McLain DA, Olefsky JM 1988 Evidence for two independent pathways of insulin-receptor internalization in hepatocytes and hepatoma cells. Diabetes 37:806-815 Mead JE, Fausto N 1989 Transforming growth factor a may be a physiological regulator of liver regeneration by means of an autocrine mechanism. Proc Natl Acad Sci USA 86:1558-1562 Kayalar C, Wong WT 1989 Metalloendoprotease inhibitors which block the differentiation of L6 myoblasts inhibit insulin degradation by the endogenous insulin-degrading enzyme. J Biol Chem 264:8928-8934 Garcia JV, Fenton BW, Rosner MR 1988 Isolation and characterization of an insulin-degrading enzyme from Drosophila melanogaster. Biochemistry 27:4237-4244 Wattenberg EV, McNeil PM, Fujiki H, Rosner MR 1989 Palytoxin down-modulates the epidermal growth factor receptor through a sodium-dependent pathway. J Biol Chem 264:213-219 Caro JF, Muller G, Glennon JA 1982 Insulin processing by the liver. J Biol Chem 257:8459-8466 Haigler HT, Maxfield FR, Willingham MC, Pastan 11980 Dansylcadaverine inhibits internalization of 125I-epidermal growth factor in BALB 3T3 cells. J Biol Chem 255:1239-1241 Roth RA, Mesirow ML, Cassell DJ, Yokono K, Baba S 1985 Characterization of an insulin degrading enzyme from cultured human lymphocytes. Diabetes Res Clin Pract 1:31-39 Desbuquois B, Krug F, Cuatrecasas P 1974 Inhibitors of glucagon inactivation: effect on glucagon-receptor interactions and glucagon-stimulated adenylate cyclase activity in liver cell membranes. Biochim Biophys Acta 343:101-120 Findlay DM, Michelangeli VP, Moseley JM, Martin TJ 1981 Calcitonin binding and degradation by two cultured human breast cancer cell lines (MCF 7 and T 47D). Biochem J 196:513-520 Rasnick D, Powers JC 1978 Active site directed irreversible inhibition of thermolysin. Biochemistry 17:4363-4369 Carpenter G, Cohen S 1976 125I-labeled human epidermal growth factor: binding, internalization, and degradation in human fibroblasts. J Cell Biol 71:159-171 Gorden PH, Carpentier J-L, Freychet P, Orci L 1980 Internalization of polypeptide hormones: mechanism, intracellular localization and significance. Diabetologia 18:263-274 Ward WF, Moss AL 1985 Effects of lysosomal inhibitors on 126Iinsulin and 125I-asialofetuin degradation by the isolated, perfused rat liver and isolated rat hepatocytes. Diabetes 34:446-451 Seglen PO, Grinde B, Solheim AE 1979 Inhibition of the lysosomal pathway of protein degradation in isolated rat hepatocytes by ammonia, methylamine, chloroquine and leupeptin. Eur J Biochem 95:215-225 Gliemann J, Sonne 0 1985 Uptake and degradation of insulin and a2-macroglobulin-trypsin complex in rat adipocytes: evidence for different pathways. Biochim Biophys Acta 845:124-130 Suzuki K, Kono T 1979 Internalization and degradation of fat cellbound insulin: separation and partial characterization of subcellular vesicles associated with iodoinsulin. J Biol Chem 254:97869794 Terris S, Hofmann C, Steiner DF 1979 Mode of uptake and degradation of 125I-labelled insulin by isolated hepatocytes and H4 hepatoma cells. Can J Biochem 57:459-468 Gansler TS, Smith RM, Jarett L 1986 Cell type-specific variability of bacitracin's effects on insulin binding and intracellular accumulation. Diabetes 35:392-397 Miskimins WK, Shimizu N 1982 Dual pathways for epidermal growth factor processing after receptor-mediated endocytosis. J Cell Physiol 112:327-338 Ibbotson KJ, Harrod J, Gowen M, D'Souza S, Smith DD, Winkler
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REGULATION OF GROWTH FACTOR LEVELS BY PROTEASES
ME, Derynck R, Mundy GR 1986 Human recombinant TGF-alpha stimulates bone resorption and inhibits formation in vitro. Proc Natl Acad Sci USA 83:2228-2232 Myrdal SE, Twardzik DR, Auersperg N 1986 Cell mediated coaction of transforming growth factors: incubation of type beta with NRK cells produces a soluble activity that prolongs the ruffling response to type alpha. J Cell Biol 102:1230-1234 Schreiber AB, Winkler ME, Derynck R 1986 TGF-alpha: a more potent angiogenic mediator than EGF. Science 232:1250-1253 Schultz GS, White M, Mitchell R, Brown G, Lynch J, Twardzik JR, Todaro GJ 1987 Epithelial wound healing enhanced by TGFalpha and vaccinia growth factor. Science 235:350-352 Marshall, S 1985 Dual pathways for the intracellular processing of
35. 36. 37. 38.
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insulin: relationship between retroendocytosis of intact hormone and the recycling of insulin receptors. J Biol Chem 260:1352413531 Affholter JA, Fried VA, Roth RA 1988 Human insulin-degrading enzyme shares structural and functional homology with E. coli protease III. Science 242:1415-1418 Kuo W-L, Gehm BD, Rosner MR 1990 Cloning and expression of the complementary DNA for a Drosophila insulin-degrading enzyme. Mol Endocrinol 4:1580-1591 Stoppelli MP, Garcia JV, Decker SJ, Rosner MR 1988 Developmental regulation of an insulin-degrading enzyme from Drosophila melanogaster. Proc Natl Acad Sci USA 85:3469-3473 Couch CB, Strittmatter WJ 1983 Rat myoblast fusion requires metalloendoprotease activity. Cell 32:257-265
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Vol. 128, No. 3 Printed in U.S.A.
Regulation of Insulin, Epidermal Growth Factor, and Transforming Growth Factor-a Levels by Growth Factor-Degrading Enzymes* BARRY D. GEHM AND MARSHA RICH ROSNER Ben May Institute and the Department of Pharmacological and Physiological Sciences, University of Chicago, Chicago, Illinois 60637
involved in the degradation of TGFa and EGF as well as insulin, and that the degradation of TGFa, but not EGF, is mediated in part by IDE. Inhibiting the activity of these metalloproteases decreased growth factor depletion, suggesting that these enzymes play an important role in the control of extracellular growth factor levels. The existence of separate degradative pathways for EGF and TGFa may explain how the two factors exert differential effects in some systems, and degradation of TGFa by IDE would provide a possible mechanism for interaction between the insulin and TGFa/EGF signalling systems. (Endocrinology 128: 1603-1610,1991)
ABSTRACT. The mechanisms by which growth factors are degraded and the role this process plays in the regulation of cell growth are not well understood. Insulin degradation is believed to be mediated by a specific metalloprotease, insulin-degrading enzyme (IDE). We have previously shown that IDE can also degrade transforming growth factor-a (TGFa), but not epidermal growth factor (EGF), in vitro. This selectivity was surprising, since TGFa and EGF are structurally similar and bind to the same receptor with comparable affinities. Using a spectrum of protease inhibitors, we have now analyzed the degradation of TGFa, EGF, and insulin by human hepatoma HepG2 cells. The results suggest that bacitracin-sensitive metalloproteases are
R
ELATIVELY little is known about the mechanisms by which cells degrade growth factors. The degradation of insulin in a number of mammalian cells is mediated by insulin-degrading enzyme (IDE); evidence for this includes the identical breakdown products generated from labeled insulin by hepatocytes and IDE (1) and the inhibition of insulin degradation by microinjection of anti-IDE antibodies into HepG2 cells (2). IDE is a primarily cytosolic bacitracin-sensitive metalloprotease which catalyzes a series of specific proteolytic cleavages (3). Magun and co-workers (4, 5) have shown that epidermal growth factor (EGF) degradation in Rat1 fibroblasts is initiated by an analogous series of specific proteolytic cleavages and is completed in the lysosomes. The enzyme(s) responsible for the initial cleavages has not been determined. Transforming growth factor-a (TGFa) is a polypeptide growth factor, structurally similar to EGF, that binds to the human EGF receptor with an affinity comparable to that of EGF and generally elicits similar biological effects Received October 1,1990. Address all correspondence and requests for reprints to: Dr. Marsha Rosner, University of Chicago, Ben May Institute, 5841 South Maryland, Box 424, Chicago, Illinois 60637. * This work was supported by grants from the American Diabetes Association, the Juvenile Diabetes Foundation, and the University of Chicago Diabetes Research Training Center (to M.R.R.).
(6). Few studies of TGFa degradation have been published, but it appears that in some cell types TGFa and EGF are degraded by separate pathways (7, 8). We have previously reported that in vitro, purified mammalian and Drosophila IDEs are capable of hydrolyzing TGFa, but not EGF, in addition to insulin (9). Thus, the available evidence raises the possibility that TGFa and EGF are degraded in vivo by specific metalloproteases similar to IDE and, in the case of TGFa, by IDE itself. To examine the role of IDE-like metalloproteases in growth factor degradation and to determine whether IDE mediates the cellular degradation of TGFa, we compared the effects of various protease inhibitors on the degradation of labeled insulin, TGFa, and EGF in HepG2 human hepatoma cells. HepG2 cells have been shown to degrade insulin via IDE (2), although lysosomal degradation may also occur (10). Since hepatocytes secrete and respond to TGFa in vivo (11), a hepatic cell line is an appropriate choice for studying TGFa degradation. EGF, which is not degraded by IDE, was used as a control for non-IDE-mediated effects of inhibitors. Although protease inhibitors have limited specificity, it is possible to further categorize a protease by its substrate specificity. Therefore, we also compared the ability of excess insulin to block degradation of TGFa and EGF in order to assess a possible role for IDE in TGFa degradation in
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REGULATION OF GROWTH FACTOR LEVELS BY PROTEASES
living cells. Our results suggest that metalloproteases mediate a substantial portion of the cellular degradation of EGF and TGFa as well as insulin, and that TGFa is degraded in part by IDE. These results may have implications for the cellular regulation of growth factor levels and for the interaction of the insulin and TGFa signalling systems.
Materials and Methods Materials [125I]Insulin (A14 monoiodinated receptor grade; 2200 Ci/ mmol) was purchased from New England Nuclear (Boston, MA) and Amersham (Arlington Heights, IL). [125I]EGF (1000 Ci/mmol) was purchased from New England Nuclear. Recombinant human TGFa was the generous gift of Dr. Rik Derynck of Genentech (South San Francisco, CA) and was radioiodinated with Na125I (New England Nuclear) and Enzymobeads (Bio-Rad, Richmond, CA). The radioiodinated product had a specific activity of 2000 Ci/mmol and bound to EGF receptor (A431 cells) with an affinity similar to that of commercial [125I]EGF. Porcine insulin, bacitracin, 1,10-phenanthroline, phenylmethylsulfonylfluoride, and leupeptin were purchased from Sigma (St. Louis, MO). Irreversible metalloprotease inhibitor (IrMPI; AT-chloroacetyl-D,L-AT-hydroxyphenylalanyl-alanyl-alanine amide) was purchased from Enzyme Systems Products (Livermore, CA). Partially purified rat liver IDE was prepared by Dr. J. Victor Garcia, as previously described (9). Cell culture All cells were grown in a gassed (5.5% CO2) humidified 37 C incubator. HepG2 cells were grown on polyornithine-coated plates in Dulbecco's Modified Eagle's Medium (DME) supplemented with Earle's salts, nonessential amino acids, Na pyruvate, and 10% fetal calf serum. A431 cells were grown on uncoated plates in DME plus 10% fetal calf serum. Buffers Buffer E contained DME, 1 mg/ml ovalbumin, and 50 mM HEPES, pH 7.5. Buffer K (12) contained 100 mM HEPES (pH 8.0), 120 mM NaCl, 5 mM KC1, 1.2 mM MgSO4, 8 mM glucose, and 10 mg/ml BSA. Cellular growth factor degradation HepG2 cells grown to near confluence were incubated in buffer E or buffer K for I h before the addition of 0.1 nM 125Ilabeled insulin, TGFa, or EGF. Compared to buffer E, buffer K produced enhanced degradation of insulin and TGFa, but little degradation of EGF. In experiments requiring identical conditions for all three substrates, buffer E was employed; otherwise, buffer K was used with insulin and TGFa, and buffer E with EGF. In time-course experiments, the cells were incubated with substrate in 60-mm dishes containing 3 ml buffer, and 500-/tl aliquots were withdrawn at each time point. Fixed time experiments were performed in six-well plates containing 1.2 ml buffer/well; 500-^1 aliquots were taken at the beginning
Endo«1991 Voll28«No3
and end of the assay. Durations of fixed time incubations were chosen to produce optimal levels of degradation (25-40%) in control cells; typical durations were 1 h for insulin, 1.5-2.5 h for TGFa, and 2.5-3 h for EGF. All incubations were performed at 37 C. Insulin degradation was measured by trichloroacetic acid (TCA) precipitation of intact [125I] insulin, as previously described (13). TGFa and EGF were measured by binding to EGF receptor, as described below. To correct for loss of [125I] EGF by HepG2 cell association (see Results), [125I]EGF degradation was calculated by the formula: % degradation = % decrease in A431 binding - % HepG2-associated. EGF receptor binding assay Intact [125I]EGF and [125I]TGFa were quantified by binding to EGF receptor in a variation of the method of Wattenberg et al. (14). A431 cells, which overexpress EGF receptor, were grown to confluence in 24-well plates and preincubated for 212 h at 37 C in DME plus 0.1% CR-ITS (Collaborative Research, Waltham, MA). Aliquots from EGF and TGFa degradation assays were divided between paired wells and incubated for 2-4 h on ice. The cells were washed seven times with cold PBS and lysed with 0.1 N NaOH. Bound 125I was measured in an LKB-Wallac RiaGamma 1274 7-counter.
Results HepG2 cells sequester EGF, but not TGFa or insulin Growth factor and insulin degradation were assayed as loss of labeled substrate from the incubation medium and assessed by binding (for TGFa and EGF) or TCA precipitation (for insulin). These procedures have been widely used to quantitate degradation of these ligands (9). To correct for loss of labeled substrate or product from the medium by cell association, it was necessary to determine whether significant amounts of labeled material were being retained by the cells. Little or no loss of label from the medium was observed during incubations of HepG2 cells with [125I]TGFa or [125I]insulin (Fig. 1). In contrast, during incubations with [125I]EGF, the concentration of 125I label in the medium decreased substantially. These measurements do not reflect degradation of the ligands, since intact proteins were not separated from labeled breakdown products. Measurements of cell-associated radioactivity (data not shown) confirmed the greater sequestration of [125I] EGF. Typically 15-25% of [125I]EGF label became cell associated during assays, but only 1-2% of [125I]TGFa and [125I]insulin did so. Washing with 0.2 M acetic acid in 0.5 M NaCl, a well established method of releasing surface-bound insulin and EGF (15, 16), removed 40% of cell-associated [125I]insulin and 15% of [125I]TGFa and [125I]EGF (data not shown), indicating that the majority of cell-associated label was internalized. The difference between EGF and TGFa sequestration may result in part from different receptor affinities in this cell type (data not shown).
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REGULATION OF GROWTH FACTOR LEVELS BY PROTEASES
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100
5 o
1! Q
p 20
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TIME (min)
FIG. 1. Removal of 126I label from the medium by HepG2 cells. HepG2 cells were incubated with 12BI-labeled insulin (•), TGFa (A), and EGF (•), as described in Materials and Methods. Aliquots were removed at the indicated times and counted for 126I counts per min. Results are expressed as a percentage of the initial concentration of 125I in the medium.
After a 1-h incubation at 37 C, cell-associated [125I] EGF was 85% intact, as judged by precipitation with TCA and phosphotungstic acid (data not shown). Since the cell-associated material was primarily undegraded, we corrrected for cell association when calculating EGF degradation. Some of the inhibitors tested altered [125I] EGF cell association, but none produced significant loss of [125I]TGFa or [125I]insulin from the medium.
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TIME (min)
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TIME (min)
HepG2 cells degrade insulin, TGFa, and EGF HepG2 cells degraded substantial fractions (30-50%) of the extracellular insulin, TGFa, and EGF in a relatively short time (2-2.5 h), suggesting that degradation can significantly affect extracellular growth factors levels (Fig. 2). Growth factor-degrading enzymes, thus, may play an important role in regulating extracellular concentrations of their substrates. Little or no growth factor degradation was produced by conditioned reaction buffer in the absence of cells (data not shown), indicating that degradation is not mediated by extracellular proteases. Bacitracin inhibits depletion of extracellular EGF and TGFa as well as insulin The antibiotic bacitracin is a well known inhibitor of IDE (17) and has been reported to inhibit degradation of glucagon and calcitonin (18, 19), but is not a general metalloprotease inhibitor. We, therefore, examined its effect on growth factor degradation in HepG2 cells. Bacitracin (1 mM) inhibited [I25I] insulin degradation almost completely (Fig. 2A) and inhibited degradation of labeled TGFa and EGF approximately 50% (Fig. 2, B and C). Bacitracin also increased sequestration of [125I]EGF, which tended to obscure its inhibition of [125I]EGF degradation in early experiments in which no correction for loss of label by sequestration was made. High (10 mM) concentrations of bacitracin were cytotoxic, as judged by
100
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TIME (min)
FIG. 2. Time courses of growth factor degradation with or without bacitracin. HepG2 cells were preincubated for 1 h in buffer E in the presence (•, A, and • ) or absence (O, A, and D) of 1 mM bacitracin. At time zero, 125I-labeled insulin (A), TGFa (B), or EGF (C) was added. At the indicated times, aliquots were removed and assayed for degradation, as described in Materials and Methods. A, [125I] Insulin. For this assay, SDS were typically less than 1%. B, [125I]TGFa. Results are shown as the mean ± SD for duplicate determinations. C, [125I]EGF. Results are shown as the mean ± SD for duplicate determinations.
the appearance of numerous vacuoles in the cells after a few hours of incubation. The effects of bacitracin concentrations up to 3 mM on insulin, TGFa, and EGF degradation in intact HepG2 cells are illustrated in
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REGULATION OF GROWTH FACTOR LEVELS BY PROTEASES
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Fig. 3. Some interexperimental variablity was observed with all three, but the ranges of measured IC5Os overlapped for the three substrates (insulin, 0.1-0.7 mM; TGFa and EGF, 0.3-1 mM). In comparison, in vitro hydrolysis of [125I]insulin and [125I]TGFa by rat liver IDE was more bacitracin sensitive (IC5o, 30 /xM for both; data not shown). This difference may reflect limited access of bacitracin to the intracellular site(s) of growth factor degradation and/or partial degradation of bacitracin by the HepG2 cells. These factors may also account for some of the variable sensitivity that was observed in cellular degradation. In sum, the effective inhibitory concentrations of bacitracin were comparable for all three substrates. Metalloprotea.se inhibitors decrease degradation of insulin, TGFa, and EGF Phenanthroline, a chelator of transition metal ions and thus an inhibitor of metalloproteases, has previously been shown to inhibit IDE and insulin degradation in L6 cells (12). Phenanthroline (1 mM) almost completely inhibited [125I] insulin degradation by HepG2 cells (Fig. 4). At the same concentration, degradation of [125I]TGFa and [125I]EGF was inhibited 40% and 70%, respectively. Thus, the degree of inhibition by phenanthroline is similar to that produced by bacitracin. As with bacitracin, the precise IC50 for phenanthroline varied somewhat from experiment to experiment, but phenanthroline consistently inhibited insulin degradation more than that of TGFa or EGF. In vitro hydrolysis of [125I]insulin and [125I]TGFa by rat liver IDE showed equal sensitivity to phenanthroline (IC50, 300 /*M; data not shown), indicating that the differential inhibition of their cellular degradation was not due to differential sensitivity of IDE with different substrates. As a chelator, phenanthroline's 120
1001
if
80604020
10°
10-
101
[BACITRACIN] (mM)
FIG. 3. Effect of bacitracin concentration on growth factor degradation. HepG2 cells were incubated with 125I-labeled insulin (•), TGFa (A), and EGF (•), as described in Materials and Methods. The indicated concentrations of bacitracin were added 1 h before the addition of substrate. Results are shown as the mean ± SD for duplicate determinations and are expressed as a percentage of the control without bacitracin.
Endo • 1991 Voll28«No3
120 • — INSULIN — A — TGF-alpha — • — EGF
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it1 I5°
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HI vO
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i
1 20-
0.00
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[PHENANTHROLINE] (mM)
FIG. 4. Effect of 1,10-phenanthroline on growth factor degradation. HepG2 cells were incubated with 125I-labeled insulin (•), TGFa (A), and EGF (•), as described in Materials and Methods, except that buffer K was used for all three substrates. The indicated concentrations of phenanthroline were added 1 h before the substrates. Before A431 binding assay, phenanthroline was titrated by the addition of FeSO^ to a 0.1-mM excess [based on Fe(phen)32+ stoichiometry] to prevent cell detachment. Results are shown as the mean ± SD for duplicate determinations and are expressed as a percentage of the control without phenanthroline.
effectiveness can be altered by intracellular concentrations of metal ions; furthermore, it can affect many cellular processes and enzymes other than metalloproteases. For example, 1 mM phenanthroline also decreased [125I]EGF sequestration 40%. For these reasons it is difficult to explain the relative sensitivities of the three substrates purely in terms of metalloprotease inhibition, but the effects of phenanthroline are consistent with degradation by metalloproteases. To avoid the nonspecific effects of chelators, we also employed IrMPI. This compound covalently modifies the active site of metalloproteases, causing loss of activity (20). Kayalar and Wong (12) reported that IrMPI partially inhibits insulin degradation by intact L6 cells, L6 cell cytosolic extracts, and immunoprecipitated IDE. We observed partial inhibition of labeled insulin, TGFa, and EGF degradation by HepG2 cells treated with IrMPI, and the dose-response curves for [125I] insulin and [125I] TGFa degradation were similar (IC50, 0.5 mM for insulin and 0.2-0.5 mM for TGFa; Fig. 5). In a separate experiment using a single concentration, 2 mM IrMPI decreased [125I]EGF degradation by HepG2 cells 70%. IrMPI (0.5 mM) inhibited in vitro degradation of insulin by partially purified rat liver IDE by 75% (data not shown), compared to 30% inhibition of insulin degradation by HepG2 cells. As with bacitracin, the lower potency of the inhibitor against cellular compared to in vitro degradation may represent limited access of the inhibitor to the relevant enzymes and/or degradation of the inhibitor by HepG2 cells. The effects of phenanthroline and IrMPI suggest that
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REGULATION OF GROWTH FACTOR LEVELS BY PROTEASES
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TGFa and EGF, but not insulin, in lysosomes. None of the three factors appears to be degraded by serine proteases.
100
Excess insulin inhibits TGFa degradation
2.0
1.0
0.0
[IrMPI] (mM)
FIG. 5. Effect of irMPI on degradation of insulin and TGFa. HepG2 cells were incubated with 125I-labeled insulin (•) and TGFa (A), as described in Materials and Methods. The indicated concentrations of IrMPI were added as dimethylsulfoxide solutions 1 h before the substrates. All assays contained a final dimethylsulfoxide concentration of 2%. Results are shown as the mean ± SD for duplicate determinations and are expressed as a percentage of the control without inhibitor. TABLE 1. Effect of protease inhibitors on growth factor degradation Degradation (% of control) Inhibitor None NH4C1 Leupeptin PMSF
Cone.
10 mM 1 mM
Insulin 100 106.4 ± 2.5 97.0 ± 9.9 101.5 ± 0.6
TGFa
EGF
100 100 51.5 ± 6.7 44.2 ± 1.3 99.5 ± 13.1 108.1 ± 6.4 85.0 ± 8.0 115.6 ± 4.8
HepG2 cells were incubated with 125I-labeled growth factors as described in Materials and Methods. The indicated inhibitors were added 1 h before the substrates. Results are shown as the mean ± SE and expressed as a percentage of the control without inhibitor.
metalloproteases are responsible for a significant fraction of EGF and TGFa degradation as well as insulin degradation in HepG2 cells.
Because all of the IDE inhibitors tested inhibited degradation of EGF, insulin, and TGFa with similar IC50s, it was not possible to resolve the role of IDE in TGFa degradation by their use. A more specific agent is excess unlabeled insulin, which as a competitive substrate inhibits the IDE hydrolysis of both [125I] insulin and [125I]TGFa in vitro (9). When added to HepG2 cells, unlabeled insulin inhibited [125I] insulin degradation completely and [125I]TGFa degradation by 50% (Fig. 6). With both substrates, the IC50 was 100 nM, comparable to that obtained in vitro. In contrast, cellular [125I]EGF degradation was unaffected by insulin. Inhibition of TGFa degradation by insulin was observed in the presence of a large excess of BSA (150 /xu), indicating that the inhibition is not a nonspecific protein effect. Unlabeled TGFa inhibits [125I] insulin degradation in vitro (9), but the analogous cellular experiment was not attempted because of the large amount of TGFa that would be required. The ability of insulin to compete selectively for TGFa degradation, but not EGF degradation, in HepG2 cells is consistent with the idea that insulin and TGFa are degraded in part by a common enzyme. The specificity and potency both in vitro and in cells suggest that IDE is the enzyme responsible. Protease inhibitors do not inhibit receptor binding Because cellular growth factor degradation is presumably receptor mediated, inhibitors of receptor-ligand binding could decrease the rate of degradation without
Other protease inhibitors Lysosomes are the major site of intracellular protein degradation and have been implicated in the degradation of insulin, EGF, and TGFa (7, 21, 22). We used NH4C1 to examine the role of lysosomes in growth factor degradation in HepG2 cells. NH4C1 was chosen in preference to chloroquine, another widely used lysosomotropic agent, because the latter has been shown to inhibit nonlysosomal insulin degradation in hepatocytes (23). As shown in Table 1, 10 mM NH4C1 inhibited HepG2 cell degradation of labeled TGFa and EGF by 50%, but had no effect on [125I]insulin degradation. Like bacitracin, NH4CI increased cell-associated [125I]EGF. Leupeptin, an inhibitor of cysteine and serine proteases, has also been used to inhibit lysosomal proteolysis (23, 24); neither it nor phenylmethylsulfonylfluoride, an inhibitor of serine proteases, had any significant effect on degradation of labeled insulin, TGFa, or EGF. Based on these observations, HepG2 cells appear to degrade 50% of
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[INSULIN] (nM)
FIG. 6. Competition of growth factor degradation with unlabeled insulin. HepG2 cells were incubated with 125I-labeled insulin (•), TGFa (A), and EGF (•), as described in Materials and Methods. The indicated concentrations of unlabeled porcine insulin were added 1 h before the addition of substrate. Results are shown as the mean ± SD for duplicate determinations and are expressed as a percentage of the control without insulin.
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affecting protease activities. Bacitracin increased HepG2 cell-associated [125I]insulin (not shown), indicating that it does not inhibit insulin binding. Phenanthroline had no effect HepG2 cell insulin binding (not shown). Neither insulin nor any of the inhibitors used in this study affected the binding of labeled TGFa or EGF to A431 cells (not shown), indicating that they do not interfere with binding to the EGF receptor. These results indicate that the protease inhibitors tested act at a step after receptor binding, presumably by inhibiting growth factor-degrading enzymes. Discussion Our results indicate that the degradation of the growth factors EGF and TGFa by cultured HepG2 cells is mediated in part by nonlysosomal metalloproteases with inhibitor profiles similar to IDE. Furthermore, degradation of TGFa, but not EGF, was inhibited 50% by saturating concentrations of insulin, a competitive IDE substrate, suggesting that IDE is responsible for a substantial portion of TGFa degradation in HepG2 cells. The balance of TGFa degradation may occur via the same pathway(s) that degrades EGF, but it is difficult to test this idea by competition in intact cells, since the two proteins compete for binding to the same receptor. Previous reports on the effects of protease inhibitors on cellular insulin and EGF degradation suggest that these degradative processes are complex. Some investigators have reported little or no effect of lysosomal inhibitors on insulin degradation (12, 23, 25), consistent with the absence of any effect of NH4C1 or leupeptin in our system, while others have reported substantial inhibition (26, 27). These discrepancies may be due to methodological differences and/or differences in cell types. For example, Gansler et al. (28) have shown that various cell types differ substantially in sensitivity of insulin degradation to bacitracin, suggesting that insulin may be processed by different pathways in different cell types. Similarly, McLain and Olefsky (10) have reported the presence of two pathways for handling internalized insulin in HepG2 cells, one operating at high (17 nM) concentrations of extracellular insulin, the other at low (0.17 nM) concentrations. Only the low concentration pathway was sensitive to chloroquine. Two pathways for processing EGF in Swiss/3T3 cells have also been reported (29). It is likely that similar heterogeneity and multiplicity of degradation pathways exist for other growth factors as well. The existence of separate pathways for the degradation of EGF and TGFa is supported by the work of Korc and co-workers (7), who report that T3M4 and ASPC-1 cells degrade TGFa rapidly, but EGF slowly. Degradation of both substrates in both cell lines is inhibited by the
Endo • 1991 Voll28«No3
lysosomotropic compound methylamine. In contrast, methylamine inhibits the degradation of EGF, but not TGFa, in RL95-2 cells (8). Their results do not necessarily indicate that TGFa is degraded by IDE in these cell lines, but do support the idea that EGF and TGFa can be degraded by different routes. Separate degradative pathways may explain how these similar ligands, which both bind to the same receptor with comparable affinities, display different potencies in some systems (30-33). An internalized polypeptide hormone may be disposed of in several possible ways. It may be degraded by a specific protease, the products of which may be released or further degraded in lysosomes. It may be degraded in lysosomes without prior action by a specific protease. It may even be released intact, as has been reported for some insulin internalized by adipocytes (26, 34). The present experiments do not address the release of intact growth factors, which would not alter their extracellular concentrations, but such a process may account for the absence of significant insulin and TGFa sequestration even in the presence of inhibitors of degradation. The inability of lysosomal inhibitors to affect insulin degradation in HepG2 cells suggests that the products of IDE-catalyzed proteolysis are released from the cells without further processing in lysosomes. This is consistent with the results of Duckworth et al. (1), who showed that hepatocytes release insulin degradation products similar or identical to those produced in vitro by IDE. In contrast, some degradation of TGFa and EGF appears to occur in the lysosomes, based on the inhibitory effect of NH4C1. This lysosomal degradation may occur subsequent to or in parallel with degradation by specific proteases.The inhibition of EGF degradation by phenanthroline, bacitracin, and IrMPI, but not insulin, suggests that EGF degradation is mediated in part by an EGFdegrading enzyme similar to, but distinct from, IDE. The human and Drosophila IDEs show sequence similarities to E. coli protease III, but appear to be distinct from the thermolysin superfamily of metalloproteases (35, 36). The similarity of the EGF-degrading enzyme's inhibitory profile to that of the IDE raises the possibility that there is a family of specific growth factor proteases that are evolutionarily related to each other and to protease III. Several lines of evidence suggest that growth factordegrading enzymes such as IDE may be involved in the regulation of growth and development. Stoppelli et al. (37) have shown that expression of IDE is developmentally regulated in Drosophila. Couch and Strittmatter (38), using the rat skeletal muscle cell line L6 as well as rat primary muscle cells, demonstrated that myoblast fusion could be blocked by specific inhibitors of endogenous metalloprotease activity. Subsequently, Kayalar and Wong (12) showed a correlation between inhibition of IDE and inhibition of morphological and biochemical
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REGULATION OF GROWTH FACTOR LEVELS BY PROTEASES differentiation in the same cell line, and suggested that degradation of insulin by IDE is required for muscle cell differentiation. Our results raise the possibility that the inhibition of differentiation may result from inhibiting degradation of T G F a or EGF or other growth factors by IDE or related enzymes. In any case, the inhibitors' effects on differentiation indicate a possible role for IDE or other growth factor-degrading enzymes in the control of mitosis and differentiation. The decreased rate of depletion of growth factors from the medium caused by IDE inhibitors raises the possibility that IDE and/or IDE-like enzymes may play a significant role in clearance of growth factors from blood and extracellular fluid. Regulation of the activity and/or expression of such enzymes could provide a way for a cell to vary the rate of growth factor degradation and thereby alter or maintain the extracellular concentration. In such a model the degradation of T G F a by IDE is especially provocative, since it provides a possible route for interaction between the insulin and T G F a - E G F signal transduction systems. Furthermore, separate pathways for degradation of T G F a and EGF could result in faster degradation of EGF than T G F a in some cell types, which may account for the differences in relative potency that have been reported for these two growth factors (30-33).
9. 10. 11. 12.
13. 14. 15. 16. 17. 18.
19.
Acknowledgments 20. We thank J. V. Garcia for rat liver IDE; C. Kayalar, R. Perlman, W.-L. Kuo, and E. van Melle for helpful discussion and advice; and D. Zeleznik for assistance in preparation of the manuscript.
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