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Department of Pediatrics, University of Kentucky College of Medicine (J.L.F., K.M.T.), Lexington, Kentucky 40536; Bios-Chile (C.G-N), Santiago, Chile; and the Department of Pediatrics, Duke University Medical Center (C.K.R., D.M.S.), Durham, North Carolina 27710
Address all correspondence and requests for reprints to: John L. Fowlkes, M.D., Department of Pediatrics, Division of Endocrinology, University of Kentucky College of Medicine, J462 Kentucky Clinic, 740 South Limestone Avenue, Lexington, KY 40536-0284.
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Abstract |
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10-fold
less affinity. Consistent with these regions being involved in
proteinase inhibition, heparin completely reverses their inhibitory
effects on 125I-IGFBP-4 proteolysis. Together, these data
demonstrate that IGFBP-3, -5, and -6 can function as IGF-reversible
inhibitors of IGFBP-4 proteolysis, likely through homologous, highly
basic, heparin-binding domains contained within the conserved
thyroglobulin type-1 motif present in the C-termini of these IGFBPs. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Analysis of this C-terminal region of IGFBP-3 reveals that all of the known six IGFBPs share some degree of homology in this domain, in that they all contain a domain consensus pattern consistent with a thyroglobulin type-1 motif (13). However, only IGFBP-3, -5, and -6 possess a cryptic heparin-binding consensus sequence within this region. Herein, we have determined whether IGFBP-5 and/or IGFBP-6, the two IGFBPs with the greatest degree of homology with this highly basic domain of IGFBP-3, might inhibit IGFBP-4 degradation, and we have explored potential mechanisms involved in their inhibitory activities.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MC3T3-E1 cell culture and conditioned media
Stock cultures of MC3T3-E1 osteoblasts were maintained in
-MEM containing 10% (vol/vol) FBS, penicillin (100 U/ml), and
streptomycin (100 U/ml), as previously described (15). Stock cultures
were subcultured every three days. Cells from four 150-cm2
flasks were used to a seed a 2-liter Nunc cell factory and were grown
to 80–90% confluence. To prepare conditioned media, the cells were
washed three times with Dulbecco’s PBS; then 1.5 liters of serum-free
medium (DMEM/F12), containing antibiotics, were added to
the cell factory for 48 h. This medium was collected, and
serum-containing medium was added back to the cells for 48 h. This
process was repeated until 6 liters of conditioned media were
obtained. The conditioned media were concentrated 7-fold using
Centricon-30 concentrators (Amicon, Beverly, MA).
Degradation of 125I-IGFBP-4 by MC3T3-E1-conditioned
media
125I-rhIGFBP-4 proteinase assays, using cell-free
conditioned media, were performed as described previously (5, 11).
Briefly, 50-µl samples of MC3T3-E1 cell-free conditioned media were
incubated with 125I-IGFBP-4 (10,000 cpm; 1 ng) at 37
C for 18–24 h. Proteolytic degradation of 125I-IGFBP-4 was
terminated by the addition of an equal volume of 2x nonreducing sample
buffer (16), followed by heating at 100 C for 3 min. Samples were
separated under nonreducing conditions on 15% SDS-polyacrylamide gels,
dried under vacuum, and autoradiographed to visualize intact and
degraded 125I-IGFBP-4 fragments. For inhibition studies,
various concentrations of rhIGFBP-3, rhIGFBP-4, rhIGFBP-5, rhIGFBP-6,
rhIGF-I, rhIGF-II, synthetic peptides, and/or heparin were preincubated
with MC3T3-E1-conditioned media for 3 h at 37 C before and during
the in vitro 125I-rhIGFBP-4 protease assay.
Preparation of synthetic IGFBP peptides
Peptides consisting of amino acid sequences, contained in the
highly conserved thyroglobulin type-1 motif of IGFBP-3, -4, -5, and -6,
were produced by solid-phase peptide synthesis using
9-fluorenylmethoxy-carbonyl chemistry. The sequences are as follows: 1)
213CDKKGFYKKKQCRPSKGR230 from hIGFBP-3; 2)
183CDRNGNFHPKQCHPALDG200 from hIGFBP-4; 3)
199CDRKGFYKRKQCKPSRGR216 from hIGFBP-5; and 4)
166CDHRGFYRKRQCRSSQGQ183 from hIGFBP-6. All
peptides were purified by HPLC and were demonstrated to be 
95%
pure. Sequence verification was performed by electrospray mass
spectrometry. The internal cysteine in all peptides was
acetylmethylated. Synthetic peptides were tested for their ability to
alter 125I-rhIGFBP-4 degradation by MC3T3-E1-conditioned
media, as described above.
Solid-phase heparin-binding assay
The solid-phase peptide-binding assay was performed as described
elsewhere (12). Briefly, 50-µl aliquots of various concentrations of
synthetic peptides dissolved in carbonate buffer, pH 9.6, were absorbed
onto 96-well tissue culture plates overnight at 4 C. The wells were
then saturated for 1 h at room temperature with 100 µl/well PBS,
pH 7.4, containing 3% BSA that had been denatured at 60 C for 30 min.
The plate was washed with PBS, pH 7.4, containing 0.1% Tween 20
(PBST), then incubated for 3 h at room temperature with
biotinylated heparin (bHep) diluted in PBST/0.2% BSA (final
concentration 5 µg/ml). After washing the plate, 50 µl of
streptavidin-conjugated horse radish peroxidase (Amersham), diluted
1:1000 in PBST/0.2% BSA, were added to each well and incubated for
1 h at room temperature. After a final wash, the peroxidase
substrate 3,3',5,5'-tetramethylbenzidine dihydrochloride (Sigma) was
added, and the reaction was terminated with the addition of 2
M H2SO4. The plate was read in an
automated plate reader at OD450.
Statistical analysis
Relative concentrations of intact 125I-rhIGFBP-4 and
fragments of 125I-rhIGFBP-4 were determined by scanning
densitometry (Beckman, Fullerton, CA). Graphic data were normalized to
the proteolysis of 125I-rhIGFBP-4 observed in unconditioned
media (i.e. 100% inhibition) and the proteolysis of
125I-rhIGFBP-4 observed in cell-free conditioned media
(i.e. 100% proteolysis). All data are expressed as the
mean ± SD. Statistical significance between groups
was determined by paired Student’s t test. Curve-fitting
and IC50 values were calculated using PRISM software
(GraphPad Software, San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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28-kDa) into 20- and
14-kDa fragments (Fig. 1
, A–D; lane 2). Figure 1
shows that IGFBP-5 (Fig. 1, A and B; lane 3) and IGFBP-6 (Fig. 1, C and
D; lane 3), added at a concentration of 2 µg/ml, significantly
inhibit IGFBP-4 proteolysis. This concentration was chosen because
IGFBP-3 at this concentration significantly inhibits IGFBP-4 proteolyis
by MC3T3-E1-conditioned media (11). The addition of IGF-I only
marginally reverses the effects of IGFBP-5 (Fig. 1A; lanes 4–7),
whereas it reverses, more substantially, the inhibitory effects of
IGFBP-6 (Fig. 1C; lanes 4–7). In contrast, IGF-II, when added instead
of IGF-I, seems to be more potent than IGF-I in its ability to reverse
the inhibitory effects of both IGFBP-5 (Fig. 1B; lanes 4–7) and
IGFBP-6 (Fig. 1D; lanes 4–7).
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). This region of each IGFBP is found within the
conserved thyroglobulin type-1 motif and contains two of the four
conserved cysteines (see Discussion for details). As shown
in Fig. 3, synthetic peptides, containing sequences from
IGFBP-3, -5, and -6, inhibited IGFBP-4 proteolysis in a dose-dependent
manner, with the C-terminal peptide from IGFBP-5 being the most potent
(IC50, 13 µM), whereas the IGFBP-6 peptide
was the least potent of the three inhibitory peptides
(IC50, 33 µM). In contrast to these three
peptides, the IGFBP-4 peptide demonstrated little, if any, inhibition
of IGFBP-4 proteolysis. This demonstrates: 1) that the IGFBP-4 peptide
does not contain critical structural elements necessary for inhibition
of IGFBP-4 proteolysis; and 2) that this peptide does not function
as a competitive inhibitor of IGFBP-4 protease activity in the
conditioned media, consistent with other studies showing that the
IGFBP-4 cleavage sites in human IGFBP-4 are not present within this
region (17).
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, all three peptides bound bHep in a dose-dependent
fashion. The IGFBP-5 peptide demonstrated the highest affinity
(EC50, 3.7 µg/ml of peptide; 185 ng/well), the IGFBP-3
peptide was intermediate in its affinity (EC50, 4.3 µg/ml
of peptide; 215 ng/well), and the IGFBP-6 peptide demonstrated the
lowest affinity for heparin (EC50, 49.0 µg/ml of peptide;
2450 ng/well). Using the same assay, the IGFBP-4 peptide showed little
or no affinity for heparin (data not shown). Thus, all three
highly-basic peptides, each of which contains a putative
heparin-binding domain conforming to the consensus sequence
XBBBXXBX (where X =
any amino acid; and B = a basic amino acid), not only inhibited
IGFBP-4 proteolysis, but also bound heparin (albeit with different
affinities).
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demonstrates that heparin alone
(lane 2) had no significant effect on IGFBP-4 proteolysis (compare
lanes 1 and 2). In contrast, the IGFBP-3 peptide (lane 3), the IGFBP-5
peptide (lane 5), and the IGFBP-6 peptide (lane 7) markedly inhibited
the degradation of IGFBP-4. However, when heparin (500 µg/ml) was
included in the incubation (lanes 4, 6, and 8, respectively), all three
inhibitory peptides were incapable of inhibiting IGFBP-4
proteolysis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The exact mechanisms by which IGFBP-3, IGFBP-5, and IGFBP-6 inhibit IGFBP-4 proteolysis are only partially understood. Although each IGFBP might function as a competitive inhibitor of the IGFBP-4-degrading proteinase, several lines of evidence suggest that this is unlikely. First, the IGFBP-4 proteinase activity in several cell lines has been shown to be specific to IGFBP-4 (1, 2, 3, 4); second, IGFBP-5 is degraded in MC3T3-E1 cells principally by MMPs, as is evidenced by the finding that tissue inhibitor of metalloproteinases almost completely blocks IGFBP-5 degradation. In contrast, tissue inhibitor of metalloproteinases does not inhibit IGFBP-4 proteolysis by MC3T3-E1-conditioned media, strongly suggesting that the IGFBP-4-degrading proteinase is different from those that degrade IGFBP-5 (Fowlkes and Serra, unpublished data); and third, IGFBP-6 has not been shown to be readily degraded in any biological fluids to date.
Alternatively, IGFBP-3, -5, and -6 may contain regions within the molecule that are capable of inhibiting IGFBP-4-degrading activity. In support of this hypothesis, we have shown that a highly homologous region within the C-terminal region of each of these three IGFBPs inhibits IGFBP-4 proteolysis, suggesting that this region likely plays a significant role in the inhibitory process. Though none of the IGFBPs or their synthetic peptides resemble known inhibitors of previously identified IGFBP-degrading proteases [such as the metalloproteinases (18), plasmin (19), or prostate-specific antigen (20)], this region of each IGFBP is contained within a larger consensus sequence, known as a thyroglobulin type-1 repeat, with the consensus pattern: [FYWHP]-x-P-x-C-x(3, 4)-G-x-[FYW]-x(3)-Q-C-x-(4, 10)-C-[FYW]-C-V-x(3, 4)-[SG] (the underlined sequence corresponds to the synthetic peptides; the consensus sequence was obtained from PROSITE). This motif is repeated 10 times in the N-terminal portion of the thyroglobulin molecule and single copies of the motif also are found in several other proteins, including: 1) the long form of the HLA class II associated invariant chain; 2) saxphillin, a transferrin-like protein from frog that binds the neurotoxin saxitoxin; 3) entactin (nidogen), a sulfated glycoprotein found associated with laminin in basement membranes; 4) the human pancreatic carcinoma marker proteins GA733–1 and GA733–2; 5) human testican, a testicular proteoglycan; 6) insectotoxin 12, found in scorpion venom; and 7) ECI, egg cysteine protease inhibitor, from chum salmon (from PROSITE and Refs. 21 and 22). Based on the findings of Yamashita and Konagaya (22), showing that ECI (a protein containing 74 amino acids) inhibited papain and cathepsin B, and our findings that IGFBP-3 inhibited IGFBP-4 proteolysis, Molina et al. have hypothesized that the thyroglobulin type-1 motifs may have a common function as internal, selective, and reversible proteinase inhibitors (21). This hypothesis would suggest that a number of proteins found throughout nature that contain thyroglobulin type-1 motifs might serve as regulators of a wide variety of proteolytic pathways. Interestingly, whereas this region of IGFBP-3, -5, and -6 inhibits the IGFBP-4-degrading proteinase [a cation-dependent proteinase (11)], ECI inhibits the cysteine proteinases papain and cathepsin B, but not the serine proteinases trypsin or chymotrypsin, nor the cation-dependent proteinase m-calpain (22). Together, these data suggest that these domains may demonstrate distinct actions in their abilities to inhibit certain types of proteinase(s). Furthermore, they suggest that as proteinase inhibitors, these domains may function outside their traditional pathways. For instance, IGFBP-3 has been shown in several studies to demonstrate IGF-independent actions on cell growth and proliferation (reviewed in Refs. 2 and 3). Therefore, it is possible that IGFBP-3, functioning as a proteinase inhibitor, may participate in other proteolytic pathways necessary for normal cell replication. Such interactions deserve further investigation.
Although the thyroglobulin type-1 motif may be involved in the
inhibitory activity of the IGFBPs, other variables also seem important
in inhibiting the IGFBP-4-degrading proteinase activity produced by
MC3T3-E1 cells. This is evidenced by the finding that an IGFBP-4
peptide that contains the conserved thyroglobulin type-1 repeat motif
(see Fig. 3
) does not inhibit IGFBP-4 proteolysis. Comparison of this
region among all four IGFBPs examined reveals one major difference:
IGFBP-3, -5, and -6 contain a significant number of basic residues,
giving these regions calculated pI’s >10 (see Fig. 3), whereas the homologous region from IGFBP-4 has few basic residues
and has a calculated pI, which is essentially neutral. This
would suggest that the charge of the region contributes significantly
to its ability to inhibit proteinase activity. This is supported by
studies that demonstrate that polymeric lysine is able to inhibit, to
some extent, the degradation of IGFBP-4 by MC3T3-E1-conditioned media
(Fowlkes and Serra, unpublished data). It is noteworthy that peptide
concentrations necessary to achieve almost complete inhibition of
IGFBP-4 degradation are significantly higher than those used for intact
IGFBP-3, -5, and -6. This could occur for several reasons, including
multimerization of peptides or insufficient secondary or tertiary
structure of the peptides. Because the IGFBP-4-degrading proteinase has
not yet been purified, it is currently impossible to fully delineate
the mechanisms by which these highly-basic regions inhibit IGFBP-4
degradation.
Within the thyroglobulin type-1 repeat of IGFBP-3, -5, and -6 resides a
putative heparin-binding consensus sequence (Fig. 3). We have
demonstrated previously that IGFBP-3 contains at least two domains
capable of binding heparin and several other glycosaminoglycans;
however, the heparin-binding, C-terminal domain demonstrates 4-fold
higher affinity for heparin than does the internal heparin-binding
domain (12). In the present study, we have demonstrated that the
homologous C-terminal domains from IGFBP-5 and -6 also bind heparin,
and whereas the IGFBP-5 heparin binding domain binds heparin with the
highest affinity among the 3 peptides tested, IGFBP-6 binds heparin
with the weakest affinity. This would suggest that the two additional
basic residues in both the IGFBP-3 and the IGFBP-5 peptides, which are
distal to the heparin-binding consensus sequence (see Fig. 3), may
contribute to the higher affinities of these two peptides for heparin.
Furthermore, the lower affinity of the IGFBP-6 peptide for heparin may
help clarify why IGFBP-6 does not seem to associate with cell
monolayers (23, 24), because it may not associate readily with cell
surface or extracellular matrix glycosaminoglycans. Previously,
synthetic peptides containing the C-terminal, heparin-binding domains
from IGFBP-3, -5, and -6 have been shown to inhibit IGFBP-3 and IGFBP-5
binding to endothelial cell monolayers (23, 24). Furthermore, IGFBP-5,
the best studied IGFBP in regards to its interaction with cell surfaces
and extracellular matrix, has been shown not to associate with mouse
osteoblasts when it lacks the C-terminal, heparin-binding domain (25);
and point mutations of the basic amino acids present in the C-terminal
heparin-binding domain of IGFBP-5 have been shown to significantly
reduce its affinity for heparin (26) and extracellular matix (27).
Thus, these highly basic, heparin-binding domains located within the
thyroglobulin type-1 repeat of IGFBP-3, -5, and -6 may perform a
variety of functions in addition to inhibiting the IGFBP-4-degrading
proteinase. Taken together, these studies suggest that although all
IGFBPs sequester IGFs, their roles in regulating IGF action may be much
more complex, such that unsaturated IGFBP-3, -5, or -6 may perform very
different functions, compared with their functions when they are
partially or fully saturated with IGFs. The demonstration that IGFBP-5
and IGFBP-6 may serve similar functions to IGFBP-3 in controlling
IGFBP-4 proteolysis emphasizes the precision and the redundancy that
exist within IGF/IGFBP/IGFBP proteinase system.
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Footnotes |
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Received December 23, 1996.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
), IGFBP-5 (
), or
IGFBP-6 (X) were incubated with 50 µl MC3T3-E1 osteoblast-conditioned
medium in the presence of 125I-IGFBP-4. The degree of
125I-IGFBP-4 proteolysis was determined and inhibition
curves and IC50 were calculated, as described in
Materials and Methods. Values represent the mean ±
SD.
