© 1995 American Heart Association, Inc.
Articles |
Porcine Aortic Smooth Muscle Cells Secrete a Serine Protease for Insulin-like Growth Factor Binding Protein-2
From the Department of Medicine, University of North Carolina School of Medicine, Chapel Hill.
Correspondence to David R. Clemmons, MD, Department of Medicine CB #7170, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7170.
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Abstract |
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Abstract Porcine aortic smooth muscle cells secrete two forms of insulin-like growth factor (IGF) binding proteins (IGFBP-2 and -4), and both forms have been shown to modulate IGF-I actions in this cell type. Recently, we showed that IGFBP-4 inhibited IGF-I action and that the cells produced a protease that cleaved IGFBP-4 into non-IGF binding fragments. After the cleavage of IGFBP-4, the cellular DNA synthesis response to IGF-I was enhanced. This study reports that these cells also secrete a protease for IGFBP-2. Like the IGFBP-4 protease, this protease is also secreted constitutively, but unlike the IGFBP-4 protease, its secretion is enhanced if the cells are serum-deprived for 24 hours before the collection of conditioned medium. The protease cleaved IGFBP-2 into 25- and 16-kD fragments, which had reduced IGF-I binding activity. Protease activity was enhanced by coincubation with IGF-I or IGF-II, and IGF-II was more potent than IGF-I. The protease is a serine protease, since its activity can be inhibited by 3,4-dichloroisocoumarin and aprotinin. It is also inhibited by EDTA, and its activity can be restored with calcium but not zinc. The heparin-binding serpins, specifically, heparin cofactor II and antithrombin III, are inhibitory. Heparin alone also had activity, and the combination of antithrombin III plus heparin caused complete inhibition. The conditioned medium also contained proteolytic activities for IGFBP-4 and -5 but it did not cleave IGFBP-1 and -3. Chromatography of the conditioned medium on heparin-sepharose indicated that the IGFBP-2 protease bound very weakly to heparin, since it was eluted with 0.2 mol/L NaCl. This contrasts with the IGFBP-5 protease activity, which required 2.0 mol/L NaCl to elute most of the activity.
1-Antichymotrypsin inhibited the IGFBP-2
protease, but it had no effect on the IGFBP-5 protease. Exposure to
other growth factors such as transforming growth factor-ß, fibroblast
growth factor, or platelet-derived growth factor did not alter protease
activity. In summary, porcine aortic smooth muscle cells secrete a
serine protease for IGFBP-2. This protease cleaves IGFBP-2 into two
fragments that have reduced IGF binding, and its activity is enhanced
by prior serum deprivation of the cells. The protease has the potential
to significantly modify IGF actions in this cell type.
Key Words: insulin-like growth factor I • atherosclerosis • proteolysis • somatomedin
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Introduction |
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The insulin-like growth factor (IGF) binding proteins (IGFBPs) are a family of proteins that bind IGFs with high affinity and modulate IGF action.1 2 Both IGF-I and -II stimulate growth of porcine aortic smooth muscle cells (pSMCs),3 and after balloon denudation injury to rat aortas, there is a marked increase in the expression of IGF-I mRNA.4 These findings suggest that IGF-I may be involved in the proliferative response to vessel wall injury. IGF binding proteins have also been shown to be localized in atherosclerotic lesions in hypercholesterolemic pigs, suggesting that they have the potential to be important factors controlling the capacity of IGF-I to stimulate pSMC growth. In previous studies, smooth muscle cells have been shown to synthesize IGFBP-2 and IGFBP-4.5 6 IGFBP-4 association with IGF-I blocks its ability to stimulate DNA synthesis on this cell type.5 These cells also secrete a protease that cleaves IGFBP-4 into two non-IGF binding fragments. IGF-I and -II binding to IGFBP-4 enhances its rate of proteolytic cleavage. Proteolytic cleavage of IGFBP-4 results in decreased binding of IGF-I and -II, and this results in a loss of the capacity of IGFBP-4 to inhibit IGF-I–stimulated cell growth. Therefore, the factors that regulate the synthesis and activity of this protease may be important in modulating pSMC responsiveness to IGF-I.
In addition to secretion of IGFBP-4, smooth muscle cells also secrete IGFBP-2. This is the major form of IGFBP in pSMC-conditioned medium, and there is at least 10-fold more intact IGFBP-2 than IGFBP-4 after 24 hours. IGFBP-2 has been shown to potentiate the effect of IGF-I on pSMC replication.7 We have previously noted fragments of IGFBP-2 in smooth muscle cell–conditioned medium but had noted no significant change in the amount of IGFBP-2 fragments that were present in conditioned medium that was obtained during the first 24 hours after the removal of serum from the cultures.5 However, in cultures collected 72 hours after the removal of serum, we had noted a significant decrease in the amount of intact IGFBP-2 and an increase in IGFBP-2 fragments, suggesting that proteolytic cleavage may have occurred. These studies were undertaken to determine if pSMCs secrete a protease that cleaves IGFBP-2, to classify the type of protease activity, and to determine some of the factors that regulate its activity.
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Materials and Methods |
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pSMCs were isolated from normal pig aortas obtained from animals that were between 3 weeks and 6 months of age.8 These cells were cultured in DMEM (GIBCO). The medium was supplemented with 12 mmol/L glutamine (GIBCO) and 10% fetal bovine serum (GIBCO). The cultures were maintained in 10-cm tissue culture dishes (No. 3001, Falcon Labware Division, Becton Dickinson), passaged weekly by using a split ratio of 1:4 in 0.14% trypsin and 0.3% EDTA (GIBCO), and replated at a density of 10 000 cells per square centimeter. If the purpose of the experiment was to measure proteolysis, the cells were replated in 24-well plates (Falcon 3047). If the purpose was to quantify changes in DNA synthesis and to determine proteolysis, 96-well plates (Falcon 3004) were used. In both types of experiments, cells were plated at 8000 cells per square centimeter. For collection of conditioned medium, the cells underwent one medium change after 3 days, and then on the third or fourth day after the medium change, they were rinsed twice with serum-free DMEM and exposed to 1.0 mL of fresh serum-free DMEM. The conditioned media were collected after 24-, 48-, or 72-hour incubations at 37°C. In other experiments, the cells were exposed to serum-free medium for 24 hours, the medium was aspirated, and a fresh serum-free DMEM was added for an additional 24-hour collection. Conditioned medium was centrifuged to remove cellular debris at 2000g for 10 minutes and then stored at -20°C until assay. The protease activity was shown to be stable under these storage conditions for periods as long as 5 months.
To quantify proteolysis, aliquots of conditioned media, between 15 and 30 µL, were mixed with Tris to a final concentration of 0.05 mol/L, pH 7.2, supplemented with 4 mmol/L CaCl2 and various test substances, such as IGF-I and IGF-II (50 ng/mL) or protease inhibitors. The final incubation volume was 60 µL. The incubation mixtures were maintained at 37°C for 16 to 24 hours, and the reaction was stopped by freezing. In some experiments, cell number was determined at the end of the incubation period to determine if the observed effect was due to an increase in cell number or a change in culture density. Cell number was determined by removing the cells from the plate with 0.1% trypsin and 0.02% EDTA and then counted in a particle data counter (model ZBI, Coulter). An aliquot of each mixture (25 µL) was loaded onto a sodium dodecyl sulfate (SDS)–polyacrylamide gel (12.5%), and the products were separated by electrophoresis under nonreducing conditions. In some experiments, pure bovine IGFBP-2 (500 ng/mL) was added to the incubation mixture, and the ability of the protease to cleave exogenously added IGFBP-2 was assessed. After the incubation, the degree of proteolysis was assessed by either ligand blotting or immunoblotting. For ligand blotting, the proteins were transferred to Immobilon-P filters (pore size, 0.45 µm; Millipore), and the filters were exposed to [125I]IGF-I or [125I]IGF-II (500 000 cpm). The transfer and probing buffers were as previously described.9 10 The radiolabeled IGFBP-2 was then determined by autoradiography using Kodak AR film (Eastman Kodak). [125I]IGF-I or [125I]IGF-II (specific activities, 125 and 90 µCi/µg, respectively) were prepared as previously described.11 When immunoblotting was used to assess proteolytic cleavage, intact IGFBP-2 was separated from IGFBP-2 fragments by SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to Immobilon-P. The filters were immunoblotted by using a previously described method.12 The anti–IGFBP-2 antiserum was used in a 1:2000 final dilution and is specific for IGFBP-2. Its cross-reactivity for IGFBP-1, -3, -4, and -5 is <0.5%. The products were detected by using an alkaline phosphatase–conjugated goat anti-rabbit IgG (Sigma) using the manufacturer's recommendations. Although the antibody was prepared with bovine IGFBP-2, it can detect 0.5 ng of porcine IGFBP-2.13 The proteolytic fragments that were generated either from endogenously synthesized porcine IGFBP-2 or exogenously added bovine IGFBP-2 were identical in size. For quantification of band intensity, photographic negatives were scanned with a GS-300 scanning densitometer (Hoefer Scientific Instruments).
In additional experiments, conditioned media were collected after exposure of cells to various growth factors, including porcine transforming growth factor-ß (TGF-ß, R & D Systems, Inc), recombinant human fibroblast growth factor (basic FGF, a gift from Synergen Inc), platelet-derived growth factor (BB isoform [PDGF-BB]), or epidermal growth factor (EGF, Intergin). Each of these growth factors was added at the concentration listed, and conditioned medium was collected after 24 hours, as described previously.5 In other experiments, DMEM that was deficient in leucine, isoleucine, and valine was added, and conditioned medium was collected after 24 hours.
In some experiments, proteolysis was quantified by using [125I]IGFBP-2 as the substrate. Pure bovine IGFBP-2 was iodinated to a specific activity of 51 µCi/µg and purified as described previously.14 [125I]IGFBP-2 (100 000 cpm per tube) was incubated with conditioned medium and in 0.05 mol/L Tris, pH 7.2, for 16 hours at 37°C. The products of the reaction were analyzed by SDS-PAGE, followed by autoradiography as previously described.
To determine the effects of protease inhibitors, they were added, at
the concentrations listed, to aliquots of conditioned media containing
IGF-II (50 ng/mL), and the incubation continued for 24 hours at 37°C.
The products were separated by SDS-PAGE (12.5%) and immunoblotted as
described previously. The protease inhibitors that were tested were
3,4-dichloroisocoumarin (3,4-DCI), N-ethylmalealamide (NEM),
benzamidine, phenylmethylsulfonyl fluoride, aprotinin,
1,10-phenanthroline, and L-trans-epoxysuccinyl
leucylamide (E-64). The protein inhibitors included antithrombin III
(AT-III) and heparin cofactor II (HC-II) and were a gift from Frank
Church, University of North Carolina. They had been purified to
homogeneity by using previously described methods.15 16
AT-III peptides were a gift from Dr Charles Schasteen, Monsanto, Inc.
They were prepared as previously described16 and tested at
concentrations of 120 µg/mL.17 A 14–amino acid peptide
corresponding to the active site of 1-antichymotrypsin
and an 18–amino acid peptide containing residues 221 to 238 of IGFBP-5
were synthesized and purified as described previously.18
To determine whether protease inhibitors were present in the
24-hour conditioned media, mixing experiments using the 24- and 72-hour
conditioned media were conducted wherein increasing concentrations of
24-hour media were mixed with 72-hour media and protease activity was
determined using [125I]IGFBP-2.
For experiments to characterize the specificity of the protease, 100 mL of conditioned medium was added to 100 mL of 0.05 mol/L Tris and 5 mmol/L CaCl2, pH 7.2, and applied to a heparin-sepharose column (Pharmacia LKB Biotechnology). The column was washed by using the starting buffer and then eluted with increasing NaCl concentrations in the same buffer beginning with 0.2 mol/L and followed by 0.5 and 2.0 mol/L NaCl. Ten-milliliter fractions were collected and stored at -20°C until assay. To determine specificity, pure bovine IGFBP-2, human IGFBP-4, or human IGFBP-5 (500 ng/mL) was incubated for 22 hours at 37°C with 3 to 30 µL of each column fraction, and proteolysis was determined by immunoblotting. The antisera for IGFBP-4 and -5 were prepared and used as described previously.19 In other experiments to characterize calcium dependence of the protease, conditioned medium was exposed to 10 mmol/L EDTA, and then the EDTA containing the conditioned medium mixture was processed by spin column chromatography using Sephadex G-10 and a previously described method.20 The eluates from which the EDTA had been removed were assayed for protease activity.
Bovine IGFBP-2 was purified from the conditioned medium of MDBK cells. The protein was purified to homogeneity by using phenyl-sepharose, IGF affinity, and reverse-phase high-performance liquid chromatography (C-4 column).7 It was proven to be pure by amino acid sequence analysis. Bovine IGFBP-2 antiserum was prepared as previously described.14 Pure human IGFBP-1, -3, -4, and -5 were purified as previously described.19 21 22 In experiments to determine specificity of the protease, each purified form of IGFBP (500 ng/mL) was incubated with the conditioned media as described previously. The products of the reactions were determined by immunoblotting using the respective antisera.14 19 21 Protease activity for casein was quantified by using the Quanticleave protease assay (Pierce Chemical Co). Conditioned media (10 to 50 µL) was incubated with N-succinylated casein (2.0 ng/mL) in 50 mmol/L sodium borate, pH 8.5, for 2.5 hours at 37°C in a final volume of 0.15 mL. Trinitrobenzene sulfonic acid (50 µL) was added, and the incubation continued for 20 minutes at room temperature. The change in optical density during the reaction was quantified by using an enzyme-linked immunosorbent assay plate reader at 450 nmol/L. Bovine trypsin (Sigma) was used as a standard and was added at concentrations between 12.5 and 205 µg/mL.
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Results |
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When conditioned medium that had been obtained from the cultures for 48 hours was incubated for an additional 24 hours with IGFBP-2, two fragment bands of 25 and 16 kD were detected (Fig 1
). To
determine if these fragments could bind the [125I]IGF-I
or [125I]IGF-II, they were incubated with the filters
that contained the IGFBP-2 fragments. Neither fragment bound detectable
amounts of either radiolabeled growth factor (Fig 1). The addition of
IGF-I or IGF-II to 72-hour conditioned medium followed by an additional
24-hour incubation in the absence of cells markedly increased the
amount of IGFBP-2 fragment (Fig 2A). IGF-II was more
potent than IGF-I, and minimal intact IGFBP-2 was present at the
end of the incubation period, when IGF-II was added. In contrast, the
addition of insulin had no effect on IGFBP-2 proteolysis. To confirm
that proteolysis was occurring in vivo, conditioned medium was
collected from cells that had been exposed to insulin, IGF-I, or IGF-II
for 72 hours. As was noted in vitro, IGF-I and IGF-II accelerated
proteolysis, but insulin was without effect (Fig 2A). To determine the
sensitivity of the protease to IGF-II exposure, increasing
concentrations of IGF-II were added to 72-hour conditioned medium with
IGFBP-2, and proteolysis was assessed after 24 hours. A clearly
detectable effect was noted with 5 ng/mL IGF-II, but the effect was not
maximal until 50 ng/mL was added (Fig 2B). Conditioned medium obtained
after 24 and 72 hours showed a difference in the amount of the 16-kD
IGFBP-2 fragment, particularly in the presence of IGF-II. Specifically,
conditioned medium collected after 24 hours showed minimal degradation
of IGFBP-2 (Fig 3, lane 2), whereas 72-hour conditioned
medium showed a greater amount of this fragment (Fig 2B, lane 2). This
difference was accentuated by the addition of IGF-II (Fig 2B, lane 3,
compared with Fig 3A, lane 4). Since the 72-hour cultures had
significantly more time to allow the protease to accumulate, additional
cultures were serum-deprived for 24 hours; then media were changed, and
fresh medium was collected for an additional 24 hours. There was an
increase in the amount of proteolytic activity in medium from cultures
that had been serum-deprived compared with medium obtained from
cultures that had not been serum-deprived (Fig 3A, lane 2 compared with
lane 6). The addition of IGF-II accentuated this difference (Fig 3A,
lane 4 compared with lane 8). When the experiment was repeated three
times, the intensity of the 16-kD fragment had increased by 51±11% in
the absence of IGF-II and by 122±21% with IGF-II
(Table). This change was not due to a change in cell
number, since cell numbers for each treatment condition at the time the
medium was collected were equivalent. Therefore, the effect seen in the
72-hour medium was not due solely to the accumulation of protease over
time but was at least partially due to the effect of serum deprivation.
In contrast to IGFBP-2, the 24-hour medium collected from cultures that
had not been serum-deprived degraded IGFBP-4, and 24 hours of serum
deprivation before collection of the medium resulted in only a modest
increase in IGFBP-4 proteolysis5 (data not shown). Since
increasing the time in culture could also allow an increase in culture
density, the effect of density on release of the protease was
determined. Media obtained from dense cultures had more protease
activity compared with cultures that were 
90% confluent (Fig 3B).
When the results of three experiments were analyzed by scanning
densitometry, the 16-kD band intensity was increased 63% in serum-free
medium and 82% with IGF-II in the high density cultures (Table). These
changes were similar to the 71% increase in cell number. Taken
together, the results support the conclusion that the effect of serum
deprivation observed in Fig 3A is not due to changes in culture
density.
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To determine the variables that might be responsible for the serum deprivation effect, DMEM that was deficient in three essential amino acids was added, and conditioned media were collected after 24 hours. The amount of proteolytic activity in conditioned medium after collection under these circumstances was equal to that collected when complete DMEM was used, suggesting that the effect of serum deprivation was not due to essential amino acid depletion (data not shown). To exclude the possibility that the effect of serum deprivation was due to a generalized increase in proteolytic activity, 24- and 72-hour conditioned media were assayed for their capacity to cleave casein. There was no difference in the amount of proteolytic activity for the casein substrate in either 24- or 72-hour media, and both were equivalent to 12.5 µg/mL of trypsin.
To determine if a protease inhibitor was released during the first 24 hours of incubation and if this accounted for the difference in serum-starved and non–serum-starved cells, increasing concentrations of 24-hour conditioned media were mixed with 72-hour conditioned media, and their effect on IGFBP-2 proteolysis was assessed by using [125I]IGFBP-2 as a substrate. There was a progressive decrease in the amount of proteolytic activity as the ratio of 24- to 72-hour media was increased, indicating that no easily detectable inhibitor was present in the 24-hour conditioned medium and that the effect of serum deprivation is probably to increase the total amount of IGFBP-2 protease released rather than decreasing the amount of an inhibitor (data not shown).
To classify the protease, a variety of protease inhibitors were tested.
3,4-DCI, EDTA, 1,10-phenanthroline, and aprotinin were potent
inhibitors. Benzamidine also gave detectable inhibition, but E-64 and
NEM had no activity (Fig 4). Since the protease was
inhibited by both serine and metalloprotease inhibitors, we wished to
determine the effects of zinc, a known activator of
metalloproteases. EDTA (10 mmol/L) was used to remove calcium
from the conditioned medium, and subsequently the EDTA was removed
by using spin column chromatography. As shown in Fig 5, removing calcium from the conditioned medium resulted
in the loss of detection of proteolytic activity for IGFBP-2. In
contrast, if 5 mmol/L CaCl2 was added back to the
conditioned medium, there was full restoration of proteolytic activity.
However, neither 10 nor 100 µmol/L ZnCl2 restored
proteolytic activity, indicating that this was not a metalloprotease.
This experiment was repeated with media obtained after exposure to
1,10-phenanthroline, and the addition of 100 µmol/L ZnCl2
did not restore activity (data not shown). These results indicate that
this is a calcium-dependent serine protease and that it falls within
the same general classification as the IGFBP-3 and IGFBP-5
proteases.
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Since calcium-dependent serine proteases are often inhibited by heparin
and heparin-binding serpins, we wished to determine if these members of
the serpin family of protease inhibitors had an effect on this
protease. AT-III and HC-II were tested alone and in the presence of
heparin. Heparin has been shown to be an extremely potent inhibitor of
the IGFBP-5 protease. Heparin exposure resulted in complete inhibition
of IGFBP-2 proteolysis (Figs 4
and 6). AT-III was a very
weak inhibitor (Fig 6). HC-II also was a potent inhibitor, and the
combination of HC-II plus heparin was completely inhibitory. The
combination of AT-III and heparin also resulted in complete inhibition.
A synthetic peptide that contained the active site of
1-antichymotrypsin also had inhibitory activity
against the IGFBP-2 protease. To further assess the effect of serpins,
two peptides that contain the active site sequences of AT-III were
incubated with IGFBP-2 and the protease. Both peptides, PB-39 and
PB-145, had activity (Fig 6). A nonsense peptide, PB-221, had no
activity. This strongly suggests that the protease is a serine protease
and that it is sensitive to the effects of heparin-binding serpins.
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To determine if other growth factors would alter the amount of IGFBP-2 protease, several pSMC mitogens were incubated with the pSMC cultures for 72 hours, and then the conditioned medium was tested for protease activity. Although IGF-II exposure resulted in enhanced proteolysis, TGF-ß, insulin, basic FGF, PDGF-BB, and EGF had no significant effect on the amount of proteolytic activity. When media obtained from cultures exposed to these growth factors were analyzed by immunoblotting immediately after their collection but without an additional 24-hour incubation, only IGF-II exposure resulted in an easily detectable change (data not shown).
Because of the similarity among the IGFBP proteases and because smooth
muscle cell conditioned medium contained protease activity for both
IGFBP-2 and -4, we added pure IGFBP-1, -3, and -5 to 72-hour
conditioned medium and measured its capacity to degrade these
substrates. IGFBP-5 was cleaved by the conditioned medium, but IGFBP-1
and -3 remained intact even in the presence of exogenously added IGF-II
(Fig 7). To determine if IGFBP-2, -4, and -5 were being
cleaved by the same protease, or if each protease was unique, the
conditioned medium was fractionated over a heparin-sepharose column.
The IGFBP-5 protease had previously been shown to bind tightly to
heparin-sepharose.23 The results show that the IGFBP-2
protease bound weakly; eg, not all of the activity was extracted by the
column, and most of the activity was eluted with 0.2 mol/L NaCl (Fig 8). The 0.2 mol/L NaCl fractions containing the protease
had no activity against pure IGFBP-5 and only minimal activity for
IGFBP-4, suggesting that the IGFBP-2 protease activity was distinct
from the IGFBP-5 protease activity. In contrast, the fractions that
eluted with 2.0 mol/L NaCl activity cleaved only IGFBP-5, suggesting
that this protease had a higher affinity for heparin. Furthermore, the
IGFBP-5 protease activity was not inhibited by
1-antichymotrypsin (data not shown), whereas this
protease inhibitor did inhibit the IGFBP-2 protease (Fig 6).
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Since the IGFBP-4 protease activity eluted near the IGFBP-2 protease
activity, further studies to attempt to determine if the IGFBP-4 and
IGFBP-2 protease activities were distinct were undertaken with
[125I]IGFBP-2. Pure unlabeled IGFBP-2 inhibited the
degradation of [125I]IGFBP-2 by the IGFBP-2 protease at
50 ng/mL (Fig 9). In contrast, pure IGFBP-4 did not
inhibit [125I]IGFBP-2 proteolysis unless concentrations
of 141 or 200 ng/mL were used and the degree of inhibition was much
less than that produced by 50 ng/mL of IGFBP-2. This suggests that the
protease activities are distinct but does not exclude the possibility
that the IGFBP-2 protease simply has a much lower affinity for
IGFBP-4.
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The findings show that aortic smooth muscle cells secrete a serine protease that cleaves IGFBP-2. Binding of IGF-I or IGF-II to IGFBP-2 accelerates its cleavage into fragments with reduced binding activity, whereas insulin, which does not bind to IGFBP-2, has no effect. Importantly, coincubation with IGF-I or IGF-II accelerates proteolysis even in the absence of cells, suggesting that this is a direct effect of these peptides and does not require continuous secretion of a cofactor. These findings are similar to observations previously reported for the IGFBP-4 protease,5 22 24 but they are not similar to IGFBP-3 and -5 proteases, whose activities are not accelerated by the binding of IGF-I or -II to the substrate.25 26 27 The findings suggest that IGFBP-2 undergoes a conformational change after binding that makes it more susceptible to cleavage and that IGF-II appears to induce this change better than IGF-I. This is similar to the IGFBP-4 protease and suggests that these two activities are accounted for either by the same protease or by two distinct proteases that have similar mechanisms of action. The acceleration of proteolysis by binding of the IGFs to IGFBP-2 and IGFBP-4 could provide a rapid means of regulating the concentrations of these IGFBPs in extracellular fluids.
The protease appears to be a serine protease, since it is inhibited potently by 3,4-DCI and aprotinin but not inhibited by cysteine or aspartate inhibitors. Although it does have a requirement for the calcium, it is not a metalloprotease, since it is not activated by zinc, even when concentrations as high as 100 µmol/L are used. These characteristics are similar to the IGFBP-4 protease that is present in the smooth muscle cell conditioned medium and to the IGFBP-5 protease previously shown to be secreted by cultured human fibroblasts.
Multiple lines of evidence suggest that the IGFBP-5 protease activity
is distinct from the IGFBP-2 protease.
1-Antichymotrypsin is a good inhibitor of the IGFBP-2
protease but had no effect on IGFBP-5 proteolysis. The IGFBP-5 protease
adheres tightly to heparin-sepharose and requires 2.0 mol/L NaCl to be
eluted, whereas the IGFBP-2 and IGFBP-4 proteases elute with lower salt
concentrations. Finally, IGF-II binding to IGFBP-5 does not
accelerate proteolysis.23 In contrast, neither the
protease inhibitor experiments nor the binding to heparin-sepharose
clearly distinguishes the IGFBP-2 and -4 protease activities. The
protease inhibitors that were tested had similar activities for both
protease activities. However, competition studies showed that unlabeled
IGFBP-2 competed with [125I]IGFBP-2 as substrate for the
protease much better than did unlabeled IGFBP-4. Additionally, 24-hour
conditioned medium from nonstarved cultures rapidly degrades
IGFBP-45 but does not degrade IGFBP-2 rapidly, and at
least 24 hours of serum deprivation is required to obtain comparable
results. Taken together, these data suggest that the IGFBP-2 protease
is distinct from the IGFBP-4 protease, but at present they do not
allow us to draw a definitive conclusion.
Since this protease is a serine protease, it was reasonable to assume that certain serine protease inhibitors in the serpin family might inhibit its activity. Of those that were screened, only HC-II had significant activity. Likewise, the two synthetic peptides that contain AT-III sequences also inhibited proteolysis. This suggests that other heparin-binding serpins, such as protein C inhibitor, may also inhibit this protease. These findings suggest that naturally occurring serine protease inhibitors that inhibit the IGFBP-2 protease may exist and that these could provide an additional level of control of its activity in vivo.
We were unable to replicate the effect of serum deprivation with essential amino acid deprivation. This suggests that this effect is not the result of amino acid depletion. The effect was specific for IGFBP-2 proteolysis, since there was no difference between medium obtained from cultures after 24 hours of serum deprivation and medium from non–serum-deprived cultures in total proteolytic activity for a casein substrate. The effect of serum deprivation on IGFBP-2 proteolysis could not be prevented by exposure to growth factors such as basic FGF, TGF-ß, or PDGF-BB.
Interestingly, fragments of IGFBP-2 with size estimates similar to those reported in the present study have been detected in serum from newborn pigs that are made catabolic by fasting.28 When these pigs were starved for 24 to 48 hours, there was a decrease in intact IGFBP-2 in serum and an increase in IGFBP-2 fragments. This observation suggests that fasting in the newborn period may stimulate the induction of a similar protease that cleaves serum IGFBP-2 in serum.
Our findings raise the question as to what the main function of
this protease might be in controlling smooth muscle cell replication.
We have previously demonstrated that the effects of IGF-I on DNA
synthesis in pSMCs can be potentiated 60% by exposure to IGFBP-2.
From this, one would predict that cleavage of IGFBP-2 would result in a
reduced response of these cells to IGF-I. However, Andress and
Birnbaum29 have recently shown that fragments of IGFBP-5
can stimulate osteoblasts to increase their DNA synthesis response to
IGF-I, suggesting that the net effect of proteolysis may be more
complex.
In addition to growth, cellular migration is a major variable
contributing to the response of arterial smooth muscle cells to injury.
IGF-I has been shown to be a potent stimulant of rat aortic smooth
muscle cell migration, but the role of IGFBPs in modulating the
migration response to IGF-I is undefined.30 We recently
demonstrated that Chinese hamster ovary cells transfected with a cDNA
encoding a mutant form of IGFBP-1, in which the RGD sequence was
mutated to WGD, did not migrate comparably with cells that expressed
wild-type IGFBP-1. We also showed that IGFBP-1 binds to the
5ß1-integrin receptor and that binding to
this receptor mediated cell migration.31 Since smooth
muscle cells have been shown to contain the
5ß1-integrin receptor and other RGD
binding integrins, it is also possible that IGFBP-2, which has an RGD
sequence, could function coordinately with IGF-I or IGF-II to stimulate
pSMC migration. If this occurs, then the IGFBP-2 protease could play an
important role in altering cell migration.
The role of proteases in the development of lesions that follow vascular injury or those induced by hypercholesterolemic diets is not well defined. In the acute mechanical damage model of atherogenesis, proteases such as tissue plasminogen activator are released. These proteases can cleave extracellular matrix components, making mitogens that are sequestered in the extracellular matrix, such as FGF, available to cell surface receptors.32 Likewise, activation of the serine protease thrombin not only leads to fibrin clot formation, but it also has direct mitogenic effects on smooth muscle cells.33 34 Whether the IGFBP-2 protease is functioning coordinately with other serine proteases that are involved in the responses to vessel wall injury is unknown, but this might serve to be a modulating factor, along with factors that enhance IGFBP-2 synthesis or synthesis of the IGFs themselves.
In summary, we have defined a further potential level of control of IGF action in pSMCs. The degree to which this process might influence cell replication or migration requires further analysis, but it is an important candidate for controlling IGF action within this system.
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Acknowledgments |
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This study was supported by a grant from the National Institutes of Health (HL-26309). The authors wish to thank Leigh Elliott for her help in preparing the manuscript. We thank Alex Parker and Walker Busby for their kind assistance.
Received July 11, 1994; accepted December 1, 1994.
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References |
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