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Department of Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599
Address all correspondence and requests for reprints to: David R. Clemmons, M.D., Department of Medicine, CB 7170, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7170.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-thrombin (0.0008 NIH U/ml) cleaved IGFBP-5 into
24-, 23-, and 20-kDa non-IGF-I-binding fragments. Cleavage occurred at
a physiologically relevant thrombin concentration. The effect was
specific for IGFBP-5, as other forms of IGFBPs, e.g.
IGFBP-1, IGFBP-2, and IGFBP-4 were not cleaved by thrombin. Although
IGFBP-3 was cleaved by thrombin, this effect required a 50-fold greater
thrombin concentration. [35S]Methionine labeling followed
by immunoprecipitation confirmed that IGFBP-5 that was constitutively
synthesized by pSMC cultures was also degraded by thrombin into 24-,
23-, and 20-kDa fragments. The binding of IGF-I to IGFBP-5 partially
inhibited IGFBP-5 degradation by thrombin, and an IGF analog that does
not bind to IGFBP-5 had no effect. Thrombin did not account for the
serine protease activity that had been shown previously to be present
in pSMC-conditioned medium. This was proven by showing that 1) no
immunoreactive thrombin could be detected in the pSMC-conditioned
medium; 2) the IGFBP-5 fragments that were generated by thrombin showed
three cleavage sites (Arg192-Ala193,
Arg156-Ile157, and
Lys120-His121), whereas the serine protease in
conditioned medium cleaves IGFBP-5 at a different site; and 3) hirudin
had no effect on IGFBP-5 cleavage by the protease in pSMC medium;
however, it inhibited IGFBP-5 degradation by thrombin. To determine the
physiological significance of IGFBP-5 cleavage, the effect of an
IGFBP-5 mutant that is resistant to cleavage by the pSMC protease and
has been shown to inhibit IGF-I actions in pSMC was determined. This
mutant inhibited IGF-I-stimulated DNA synthesis, but if thrombin was
added simultaneously, IGF-I was fully active. In summary, physiological
concentrations of thrombin degrade IGFBP-5. Degradation can be blocked
by hirudin and is partially inhibited by IGF-I binding. Generation of
active thrombin in vessel walls may be a physiologically relevant
mechanism for controlling IGF-I bioactivity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGFBPs have been reported to be proteolytically degraded by a variety of serine and matrix metalloproteases (2, 3, 4, 5, 6). As the IGFBP fragments that are generated bind IGF-I weakly or not at all, proteolysis is believed to play an important role in controlling the bioavailability of IGF-I to receptors at the cellular level (2, 3, 4, 5). IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, and IGFBP-6 have been shown to undergo proteolytic cleavage (2, 3, 4, 5, 6, 7). Cultured human dermal fibroblasts, osteoblasts, and porcine aortic smooth muscle cells (pSMC) secrete a protease that cleaves IGFBP-5 into a 22-kDa fragment (2, 6, 8). Proteolysis also markedly reduces the affinity of IGFBP-5 for IGF-I (2, 8). In pSMC-conditioned medium, proteolysis is so extensive that no intact IGFBP-5 can be detected after 24 h (8). Proteolysis also modifies the cellular response to IGF-I. If a sufficient amount of intact IGFBP-5 is present in extracellular fluids, it inhibits IGF-I activity (8). In contrast, if proteolysis is occurring, IGFBP-5 can potentiate the actions of IGF-I (9). Therefore, some proteolysis of IGFBP-5 may be required for cells that constitutively synthesize this form of IGFBP to respond optimally to IGF-I. Additionally, the 22-kDa IGFBP-5 fragment that is generated by proteolysis has been shown to potentiate the mitogenic effect of IGF-I or -II on cultured osteoblasts (10). This fragment has some mitogenic activity in the absence of IGF-I, suggesting that it may function to stimulate growth by an independent mechanism (11).
A number of enzymes, including the serine proteases, have been reported to degrade IGFBP-5 (2, 6, 8, 11, 12, 13, 14). The activities of some of these proteases can be inhibited by serpins, such as antithrombin III or heparin cofactor II (2). Thrombin is a serine protease that has been shown to be present in the extracellular matrix (ECM) of human tissues and in macrophages (15). It is also detectable in injured vessel walls (15, 16, 17, 18, 19). In addition, thrombin has been shown to stimulate fibroblast (20) and smooth muscle cell (21, 22) replication, and its proteolytic activity is required for this effect. Although several growth factors may function together with thrombin (23, 24), it is a potent stimulant of SMC DNA synthesis when combined with IGF-I (21). The mechanisms by which thrombin potentiates the effects of IGF-I have not been defined; however, it has been shown to minimally increase IGF-I receptor number. More importantly, those investigations also showed that immunoneutralization of the IGF-I activity that was secreted by SMC resulted in significant attenuation of the DNA synthesis response to thrombin, suggesting that IGF-I receptor occupancy is a necessary component of the thrombin response (21). It has been proposed, but not proven, that there may be postreceptor cooperativity between the signal transduction pathways that are activated by these two mitogens. Other possibilities by which thrombin might amplify the effect of IGF-I, such as proteolysis of IGFBP-5, have not been excluded. Because of the presence of thrombin in the pericellular environment and its potential importance as a smooth muscle cell mitogen, we determined whether physiologically relevant concentrations of thrombin could cleave IGFBP-5 and whether cleavage resulted in a change in the cellular response to IGF-I.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-thrombin was a gift from Dr. Haifeng Wu at the Department of
Medicine, University of North Carolina (Chapel Hill, NC) (29). It was
proven to be homogeneous by SDS-PAGE. Its specific activity for
fibrinogen was 2000 NIH U/mg protein. The antiserum against human
prothrombin was purchased from ICN Biomedicals (Costa Mesa, CA). Human
IGFBP-5 was prepared as described previously (2). An IGFBP-5 mutant,
K138N-K139N-IGFBP-5, that was resistant to proteolysis by pSMC protease
was also prepared using in vitro mutagenesis as described
previously (8).
Analysis of proteolytic activity
Fifty nanograms of pure human intact IGFBP-5 were incubated with
thrombin (0.0008–0.1 NIH U/ml) for the indicated periods of time at 37
C in 30 µl assay buffer containing 100 mM Tris, 4
mM CaCl2, and 0.01% protease-free BSA, pH 8.2.
The reaction products were then analyzed by SDS-PAGE (12.5% gel) with
immunoblotting. At the concentrations tested, thrombin does not cleave
IGF-I as determined by SDS-PAGE with silver staining. To test the
activity of hirudin, it was added to the same buffer at different
concentrations between 0.05–0.2 U/ml. IGF-I and des(1, 2, 3)-IGF-I were
tested for inhibition of proteolytic activity by incubating 50 ng of
each peptide with 0.1 NIH U/ml thrombin and 50 ng IGFBP-5. In some
experiments, thrombin was incubated with 50 ng K138N-K139N-IGFBP-5.
Conditioned medium from pSMC cultures was also used as a source of
protease. Ten microliters of conditioned medium were incubated with 50
ng IGFBP-5 in the presence or absence of hirudin. To determine the
specificity of thrombin, pure IGFBP-1, -2, -3, and -4 (50 ng) were each
incubated with thrombin (0.1 NIH U/ml) as previously described. After
an 8-h incubation, they were immunoblotted with their respective
antisera using dilutions previously reported (25, 26, 27). To determine the
rate of IGFBP-5 degradation, IGFBP-5 cleavage was analyzed at different
time points as described above, except that the intact protein and
fragment band intensities were analyzed by immunoblotting with
chemiluminescence. Band intensity was determined by scanning
densitometry (GS-300, Hoeffer, San Francisco, CA) and compared with an
IGFBP-5 standard to calculate the amount of peptide that was
degraded.
Western ligand blotting and immunoblotting
After incubation with thrombin- or pSMC-conditioned medium,
IGFBP-5 and its fragments were separated using 12.5%
SDS-polyacrylamide gels. The separated proteins were then transferred
to filters (0.45-mm pore size; Immobilon PSQ, Millipore, Bedford, MA),
and the membranes were probed for IGF-I-binding activity by incubating
the filters with [125I]IGF-I as previously described (9).
For immunoblotting, the Immobilon filters were prepared as described
for ligand blotting, except that they were exposed to a 1:1000 dilution
of polyclonal antiserum that had been prepared for human IGFBP-1, -2,
-3, -4, or 5. They were rinsed and incubated with antiguinea pig
IgG-alkaline phosphatase conjugate (Sigma Chemical Co.) (5, 9). The
bands were visualized with the Protoblot immunoblotting reagents, using
the technique recommended by the manufacturer (Promega Biotech,
Madison, WI).
Cell culture
pSMC were isolated from thoracic aortas of 3-week-old piglets
and maintained in DMEM supplemented with glucose (4.5 g/liter),
penicillin (100 U/ml), streptomycin (100 mg/ml), 4 mM
glutamine (complete medium), and 10% FCS in 10-cm tissue culture plate
(Falcon 3001, Falcon Labware Division, Becton Dickinson, Rutherford,
NJ) at 37 C containing 5% CO2 in air. The medium was
changed every 3 days. Serum-free conditioned medium was collected after
the cultures had reached confluence.
Immunoprecipitation
To analyze the proteolytic effect of thrombin on IGFBP-5 that
was synthesized and secreted by pSMCs, cell monolayers were exposed to
thrombin and 50 µCi/ml [35S]methionine (56 Ci/mmol) for
6 h in methionine-deficient DMEM in the presence of 100 µg/ml
heparin to inhibit the endogenous IGFBP-5 protease. The medium was
incubated with an anti-IGFBP-5 or IGFBP-2 antiserum using a dilution of
1:1000. The immune complexes were precipitated with protein A-Sepharose
as previously described (30). The precipitates were analyzed by
SDS-PAGE with fluorography and autoradiography (30).
Measurement of [3H]thymidine
incorporation into pSMCs
pSMCs were plated at a density of 2 x
104/cm2 in 96-well culture plates and grown for
5 days without a change of culture medium. Cultures were rinsed once
with DMEM without FBS and incubated in 100 µl/well DMEM supplemented
with 0.2% platelet-poor plasma for 24 h in the presence of 1.0
µCi/well [3H]thymidine (SA, 35 Ci/mmol), IGF-I,
thrombin, and K138N-K139N-IGFBP-5. At the end of incubation, the plates
were placed on ice, washed with ice-cold 5% trichloroacetic acid for
10 min. Trichloroacetic acid precipitates thus formed were solubilized
by adding 0.1% SDS-0.1 N NaOH, and radioactivity was
measured with a Beckman scintillation counter (Beckman, Palo Alto, CA)
using ScintiSafe Econo 2 (Fischer Scientific, Fairlawn, NJ) as a
scintillant.
Identification of the thrombin cleavage site of IGFBP-5
Fifteen micrograms of pure IGFBP-5 were incubated with 0.2 NIH
U/ml thrombin in 140 µl 0.1 M Tris, 6 mM
CaCl2, and 0.01% BSA (pH 8.2) at 37 C for 24 h. The
products were loaded onto a reverse phase HPLC column (Vydac, Hesperia,
CA) equilibrated in 0.04% trifluoroacetic acid and H2O.
The IGFBP-5 fragments were separated using a linear gradient from
0–100% acetonitrile in 0.04% trifluoroacetic acid H2O
over 1 h. The fractions containing the immunoreactive IGFBP-5
fragments were determined by immunoblotting, and the peptides in those
fractions were subjected to NH2-terminal sequencing by
Automated Edman degradation (model 470A sequencer, Applied Biosystems,
Foster City, CA) equipped with on-line analysis using an Applied
Biosystems model 120A phenylthiohydantoin analyzer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). IGFBP-5 was sensitive to low
concentrations of thrombin, and 0.1 NIH U/ml thrombin, a concentration
that generated sufficient fibrinogen cleavage to obtain platelet
aggregation (31), resulted in nearly complete proteolysis after 8
h. A detectable effect was obtained with a concentration as low as
0.0008 U/ml. Immunoblotting with anti-IGFBP-5 antibody showed that
thrombin degradation yielded 24-, 23-, and 20-kDa fragments (Fig. 1A),
and Western ligand blotting with [125I]IGF-I demonstrated
that those fragments did not bind IGF-I (Fig. 1B).
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, 25 pmol/ml IGFBP-5 were
degraded in 2 h, and the degradation rate of IGFBP-5 by 0.01 U/ml
thrombin was estimated to be 0.22 pmol/ml IGFBP-5·min. The same
concentration of thrombin degrades 3.3 pmol/ml fibrinogen·min (32).
Although both experiments estimated the rate of degradation using the
imprecise technique of measuring changes in substrate band intensity,
the results suggest that IGFBP-5 is an excellent substrate for
thrombin.
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, direct addition of
IGF-I, but not des-IGF-I, resulted in partial inhibition of
proteolysis. When the results of four separate experiments were
analyzed by scanning densitometry, IGF-I inhibited proteolysis by
40 ± 3.8% (P < 0.01), but des-IGF-I resulted in
no change.
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).
These molecular mass estimates were similar to those generated in
vitro (Fig. 1A
). In contrast, immunoprecipitation with an
anti-IGFBP-2 antibody showed that IGFBP-2 that was also
constitutively synthesized by pSMC was not degraded by thrombin (Fig. 4B).
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). In contrast, IGFBP-3 was
cleaved by thrombin into multiple bands ranging from 22–25 kDa. To
further analyze this effect, increasing concentrations of thrombin were
tested for activity against IGFBP-3. No cleavage was noted with 0.0008
U/ml, and a concentration of 0.1 U/ml was required to detect cleavage
that was equal to the effect of 0.0008 U/ml on IGFBP-5 (Fig. 6). Although IGF-I resulted in partial
inhibition of IGFBP-5 proteolysis of IGFBP-5, it did not affect the
cleavage of IGFBP-3 or other IGFBPs by thrombin (data not shown).
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). In contrast, addition of increasing
concentrations of hirudin to an incubation mixture containing IGFBP-5
and thrombin resulted in significant inhibition of proteolysis (Fig. 8). Hirudin (0.2 NIH U/ml) completely
inhibited the proteolytic effect of 0.1 NIH U/ml thrombin.
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) would presumably contain amino acids
1–192, and the band that migrates at 23 kDa would presumably contain
residues 1–156. The band that migrates with an estimated molecular
mass of 20 kDa presumably would contain amino acids 1–120. The
C-terminal fragments were not detected due to the fact that the
antibody was raised using amino-terminal fragments of IGFBP-5 as the
immunogen (25). When a C-terminal-specific antiserum was used to
analyze a digested sample that contained the 20-kDa band, an additional
12-kDa fragment was detected (data not shown). This fragment probably
contains residues 121–252. The proximal residues at all three cleavage
sites, e.g. K120, R156, and R192, are preceded by a proline.
Proline often precedes basic residues at thrombin cleavage sites
(35).
The cleavage site in IGFBP-5 that is used by the serine protease that
is secreted by pSMC has been determined to be residues K138 and K139
(8). As this site is different from the thrombin cleavage sites in the
IGFBP-5, an IGFBP-5 mutant, K138N-K39N, that is resistant to cleavage
by the serine protease in pSMC-conditioned medium was used to confirm
that a different cleavage site is used by thrombin. This mutant and
wild-type IGFBP-5 were incubated with thrombin (0.1 U/ml). As shown in
Fig. 9, thrombin degraded the mutant
IGFBP-5 to the same extent as it did the wild-type IGFBP-5. It
should be noted that the IGFBP-5 mutant has two amino acid residues
substituted (8), which results in an altered electrophoretic mobility.
The fragments that migrate aberrantly also contain these substitutions.
In contrast, the 20-kDa fragment does not contain these mutations, and
its mobility is unchanged. These results suggest that the altered
mobility is due to the two substituted residues.
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, the mutant inhibited
IGF-I-stimulated DNA synthesis. However, if thrombin was added, IGF-I
stimulation was restored.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cleavage of IGFBP-5 by thrombin results in the appearance of fragments that have a marked reduction in their capacity to bind to IGF-I. Although the fragments were not purified to homogeneity and tested in direct binding assays, generally when this has been examined the results correlate with a loss of binding activity, as determined by ligand blotting. Therefore, our findings suggest that the IGFBP-5 fragments that are present in tissue culture medium after thrombin digestion have reduced affinity for IGF-I. Intact IGFBP-5 has an affinity for IGF-I at least 10-fold greater than that for the IGF-I receptor (1), and intact IGFBP-5 in pSMC conditioned medium has been shown to have an inhibitory effect on the actions of IGF-I (8). Our results show that thrombin cleavage of IGFBP-5 eliminates this inhibitory effect. By cleaving IGFBP-5, small amounts of thrombin would provide for the controlled release of IGF-I to receptors, thus facilitating its mitogenic effect.
SMCs also secrete a serine protease that cleaves IGFBP-5 (8). We have determined that the cleavage sites used by that protease are different from those used by thrombin. Additional evidence that thrombin was different from that serine protease was obtained by showing that the protease in pSMC-conditioned medium was not inhibited by hirudin. Furthermore, when an IGFBP-5 mutant that was not cleaved by the serine protease that was released by SMC was used as a substrate, it was cleaved rapidly by thrombin. Taken together, these findings strongly support the conclusion that thrombin is not the serine protease that is secreted by pSMC cells in culture. As it has not been determined whether the pSMC medium protease is present in vessel walls, whereas active thrombin is detectable, it is possible that IGFBP-5 cleavage by thrombin may be physiologically important in vivo.
Thrombin has been shown by several investigators to be present in macrophages and to be localized in vessel wall ECM (15, 16, 17, 18, 19). That the thrombin that is present is activated is supported by the observation that it binds to hirudin (15). Plasminogen activator inhibitor I (PAI-I) activity is also present in smooth muscle and endothelial cell extracellular matrix, and its activity can be neutralized by binding to thrombin. In contrast, PAI-I activity is stabilized by binding to vitronectin, and thrombin binding to the vitronectin/PAI-I complex also alters PAI-I activity (36, 37, 38, 39, 40). PAI-I and vitronectin bind to IGFBP-5 and help to localize it in the SMC ECM (41). Therefore, PAI-I binding to IGFBP-5 could regulate the cleavage of IGFBP-5 by thrombin; however, cleavage in the presence of PAI-I has not been analyzed. Likewise, PAI-I/vitronectin complexes that have been shown to neutralize thrombin activity also bind to IGFBP-5 (41). Therefore, it is possible that thrombin, which has been shown to release PAI-I from the ECM, could release IGF from IGF/IGFBP-5 complexes that are associated with the ECM (41). As the capacity of SMC and fibroblasts to replicate in response to IGF-I is partially dependent upon the amount of IGF-I that is bound to IGFBP-5 in the ECM (9), this could be a mechanism for rapid release of IGF-I to receptors, resulting in potentiation of SMC DNA synthesis. That this might occur is supported by the observation of Delafontaine et al., who showed that 24 h after exposure to thrombin, IGF-I messenger RNA was markedly suppressed in pSMC, but IGF-I peptide levels in conditioned medium remained the same, suggesting that IGF-I might be being released from IGFBPs (21).
Thrombin activation of its own receptor has been shown in some experiments to be sufficient to achieve maximum SMC DNA synthesis responses (22). However, other investigators have reported that these responses are not maximal (21) and that the proteolytic action of thrombin on other proteins or thrombin stimulation of the release of autocrine growth factors is required for full cellular responsiveness (42). Our findings support the conclusion that thrombin may have actions other than cleavage of its receptor which result in stimulation of DNA synthesis. Some of the previous studies that have shown maximum effects of thrombin receptor stimulation alone using the tethered ligand peptides have used high concentrations of insulin, e.g. 0.5 x 10-6 M, in their incubation medium (22, 43). This results in a maximum stimulation of IGF receptors that is independent of binding to IGFBPs, because insulin does not bind to any of the IGFBPs. However, insulin does bind to the IGF-I receptor at these concentrations (9). This suggests that these experiments with the tethered ligand peptides and 10-6 M insulin may not mimic the pericellular environment, wherein insulin is present at concentrations of 10-10 M and almost all of the IGF-I that is present is associated with IGFBPs.
We have determined previously that if IGFBP-5 proteolysis is allowed to proceed, the SMC response to IGF-I is greater than if proteolysis is completely inhibited. This suggests that proteolytic cleavage of IGFBP-5 by thrombin may contribute to thrombin’s mitogenic effects. We have shown that the IGFBP-5 that is associated with ECM is resistant to cleavage by the serine protease that is secreted by fibroblasts and pSMC. In contrast, because thrombin can cleave ECM proteins, such as PAI-I and vitronectin, it may provide a mechanism for IGF-I release. Therefore, the potential role of IGFBP-5 proteolysis by thrombin as a component of thrombin potentiation of the mitogenic effect of IGF-I deserves further consideration.
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Acknowledgments |
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-thrombin
and helpful comments. We thank Mr. George Mosley for his help in
preparing the manuscript. We thank Ms. Christine Smith (Monsanto) for
determining the amino acid sequences of the IGFBP-5 fragments. |
Footnotes |
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Received September 5, 1997.
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), IGF-I and thrombin (0.1 U/ml;
), IGF-I with K138N-K139N-IGFBP-5 (200 ng/ml; 
), and IGF-I with
K138N-K139N-IGFBP-5 (200 ng/ml) plus thrombin (0.1 U/ml; 
). The
amount of [3H]thymidine incorporated into cells was
measured as described in Materials and Methods. The
results were expressed as the percent increase over values in control
cultures that were incubated with DMEM plus 0.2% platelet-poor plasma,
but received no other treatments. Each value is the mean of triplicate
determinations from three independent experiments.