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(Received for publication, May 26, 1995; and in revised form, July 25, 1995) From the
Previous studies have shown that insulin-like growth factor
(IGF)-binding protein-4 (IGFBP-4) is degraded only in the presence of
exogenous IGFs; however, we found that cation-dependent proteinase
activity present in conditioned medium of MC3T3-E1 osteoblasts degrades
Insulin-like growth factor (IGF)( Although IGFBP-4 functions as a potent
inhibitor of IGF action, the factors controlling production, secretion,
and turnover of IGFBP-4 have only recently been addressed. IGFs have
been shown to decrease concentrations of IGFBP-4 in the conditioned
media of human fibroblasts (8, 9, 10) , human
breast cell carcinoma(11) , and human decidual cells (12, 13) . However, IGF-I or IGF-II have little or no
effect on IGFBP-4 mRNA in several cell lines (10, 14) and the effect of IGF-I on IGFBP-4 levels in
conditioned media of human fibroblasts (8, 9) and
human decidual cells (15) is not blocked by a monoclonal
antibody to the type-1 IGF receptor. These observations suggest that
IGFs might decrease IGFBP-4 concentrations via posttranslational
mechanisms. Consistent with this hypothesis, recent studies from our
laboratory have demonstrated that in human and sheep fibroblasts the
addition of IGFs induces IGFBP-4 proteolysis by a proteinase(s) not yet
identified (16) . Since this original report, IGF-dependent
IGFBP-4 proteolysis has been confirmed in human
fibroblasts(17) , and similar IGF-dependent IGFBP-4 proteinase
activity has been reported in human decidual cell
cultures(15) , vascular smooth muscle cells (18) , and
human bone cells(19) . The action of the IGF-dependent IGFBP-4
proteinase(s) provides a novel mechanism through which IGFs can
increase their own bioavailability and bioactivity. In the current
study, we use the murine MC3T3-E1 osteoblast cell line to explore the
role of IGFs in the regulation and induction of IGFBP-4 proteolysis.
Herein, we demonstrate that IGFBP-3 can function in a unique role as an
inhibitor of IGFBP-4 proteolysis; however, its inhibitory effects can
be reversed by the presence of IGFs.
To induce cellular differentiation, cells were plated at an initial
density of 5
Figure 5:
Inhibition of
Figure 1:
Degradation of
Figure 2:
Inhibition of
Figure 3:
Inhibition of
Figure 4:
Schematic representation of hIGFBP-3
demonstrating the sequence of origin of rhIGFBP-3 fragments and IGFBP-3
peptides. Human IGFBP-3 is divided into three distinct domains: the
N-terminal domain 1 (amino acids 1-87; white), the
non-homologous mid-region domain 2 (amino acids 88-183, shaded), and the C-terminal domain 3 (amino acids
184-264, cross-hatched). The putative heparin-binding
domains are represented as black boxes. IGFBP-3 fragments
produced by digestion with MMP-3 are designated a-f.
Synthetic IGFBP-3 peptides are designated I-IV.
To identify the
epitope(s) in the last
Figure 6:
Reversal of the inhibitory effect of
rhIGFBP-3 on
Because IGFs
reversed the inhibitory effects of rhIGFBP-3 on
Figure 7:
Effect of IGF-I on the inhibition of
Figure 8:
Effect of heparin on the inhibition of
IGFBP-4 was first isolated from conditioned media from an
osteosarcoma cell line and has now been identified in a variety of
biological fluids and cellular conditioned media, where it functions as
a potent inhibitor of IGF action (for recent reviews, see (1, 2, 3, 4) ). Previously, we
demonstrated that IGFs can induce the proteolytic degradation of
IGFBP-4 into fragments that display little or no affinity for IGFs,
providing a mechanism by which IGFs can directly increase their own
bioavailability and/or bioactivity(16) . Subsequently, Conover et al.(17) demonstrated that the degradation of
IGFBP-4 increased IGF activity in human fibroblast cultures. How IGFs
induce IGFBP-4 proteolysis remains unclear; we (16) and others (15) have hypothesized that IGFs induce IGFBP-4 degradation by
binding to IGFBP-4, thus making it more susceptible to proteolysis,
while others have proposed that IGFs directly activate an IGFBP-4
proteinase(17) . Insights from the current studies would
suggest that neither of these hypotheses is entirely correct, and that
a more complex mechanism is involved in IGF-induced IGFBP-4
degradation. Since other studies have now demonstrated IGF-inducible
IGFBP-4 degrading proteinase activity in a number of cell
lines(1, 2, 3, 4) , including bone
cells(19) , we chose to explore IGFBP-4 proteolysis in a murine
bone cell line, MC3T3-E1 osteoblasts. Initial studies demonstrated that
the IGFBP-4-degrading proteinase(s) present in MC3T3-E1 conditioned
media cleaved the protein into two major fragments and that the
proteinase was cation-dependent, making it similar to IGFBP-4-degrading
proteinase activity reported in other cell
lines(15, 16, 17) . Nevertheless, one major
difference existed between the IGFBP-4-degrading proteinase activity
observed in MC3T3-E1 conditioned media compared with that produced by
other cell lines; no exogenous IGFs were required to induce IGFBP-4
proteolysis(1, 2, 3, 4, 15, 16, 17, 18, 19) .
In fact, addition of IGF-I to MC3T3-E1 conditioned media had no effect
on We
have demonstrated that MC3T3-E1 cells secrete immunoreactive IGFBP-2,
-4, and -5(26) , while IGFBP-3 is not produced by these cells (26) and the cells produce no IGFBP-3 mRNA(29) . Based
on the observation that MC3T3-E1 conditioned media contains little or
no IGFBP-3, while other cell lines displaying IGF-dependent IGFBP-4
proteolysis do produce
IGFBP-3(15, 16, 17, 18, 19) ,
we postulated that IGFBP-3 might function as an inhibitor of IGFBP-4
proteolysis. Indeed, the addition of rhIGFBP-3 to conditioned media
significantly inhibited the constitutive degradation of Similar to the effects of intact
IGFBP-3 and inhibitory IGFBP-3 fragments, two peptides inhibited
IGFBP-4 proteolysis, while two other peptides did not. The feature
common to both inhibitory peptides was that each contained one of the
two highly basic, putative heparin-binding domains present in
IGFBP-3(2, 25) . Further analysis revealed that
peptide IV, which contains a long heparin-binding motif, was The mechanism
by which IGFs reverse the inhibitory effects of IGFBP-3 on IGFBP-4
degradation is currently unknown; however, one explanation is that
binding of IGFs to IGFBP-3 results in a conformational change in the
binding protein, making the putative heparin-binding domains less
available to inhibit IGFBP-4 proteinase activity. Several lines of
investigation support this hypothesis. For instance, in vitro IGFBP-3 binds to cell surfaces and/or cell matrix via
glycosaminoglycans and possibly through specific IGFBP-3 cell-surface
receptors (see (4) for review). How IGFBP-3 binds to cell
monolayers is currently unclear; however, Bar et al.(35) have shown that binding of IGFBP-3 to endothelial
cells involves the putative heparin-binding motif contained in the C
terminus of IGFBP-3 and in peptide IV used in our study. Despite its
association with cell monolayers, IGFBP-3 can be released from cell
surfaces when bound to IGFs(4) . Together, these data suggest
that unsaturated IGFBP-3 may present certain epitopes (i.e. the putative heparin-binding domains) on its surface, which
interact with a variety of molecules such as glycosaminoglycans and
cell-surface receptors; however, once bound to IGFs, IGFBP-3 may
undergo a conformational change and lose its affinity for these
molecules. In the same context, binding of IGFs to IGFBP-3 may alter
its conformation in such a way that inhibitory epitopes present in the
molecule are no longer available to inhibit the IGFBP-4 proteinase. In contrast to recent reports by Gockerman and Clemmons (36) and Arai et al.(37) demonstrating that
heparin inhibits IGFBP-2 proteinase activity produced by porcine aortic
smooth muscle cells and IGFBP-5 proteinase activity produced by human
fibroblasts, respectively, our data suggest that heparin has little or
no effect on IGFBP-4 degradation by MC3T3-E1 conditioned media.
Nevertheless, heparin significantly reversed the inhibitory effects of
IGFBP-3 and peptide IV on IGFBP-3 has been shown to both enhance and
inhibit IGF activity in vitro (reviewed in (1, 2, 3, 4) ). Our data would
suggest that this dichotomy may be explained partially by the finding
that IGFBP-3 inhibits IGFBP-4 proteolysis, thus decreasing IGF
activity. However, when bound to IGFs, and possibly glycosaminoglycans,
IGFBP-3 loses its capability to inhibit IGFBP-4 proteolysis, thus
enhancing IGF activity by facilitating the degradation of IGFBP-4. Fig. 9presents a summary of these studies and a hypothetical
scheme of how IGFBP-3 might function in vivo to regulate
IGFBP-4 proteolysis. In the absence of IGFBP-3 (and possibly other
IGFBPs containing heparin-binding motifs), IGFBP-4 is degraded
constitutively (Fig. 9, A) into non-IGF binding
fragments. In the presence of IGFBP-3, IGFBP-4 degradation is markedly
attenuated (Fig. 9, B). However, the inhibitory effects
of IGFBP-3 can be reversed via binding of IGFs to IGFBP-3 (Fig. 9, C). Similarly, if IGFBP-3 is bound to
glycosaminoglycans or cell surfaces, heparin-binding domains present in
the molecule may be less available for interaction with the proteinase (Fig. 9, D). This scenario may be altered in the event
that IGFBP-3 is degraded by proteinases such as MMPs, since these
proteinases can cleave the N-terminal IGF-binding domain from the
heparin-binding domains present in the mid-region and C-terminal tail
of the molecule(24) . In this instance, fragments containing
the heparin-binding motif(s) could inhibit proteinase activity; yet
since they bind little or no IGFs, their inhibitory effects may not be
reversible by IGFs, making them IGF-resistant IGFBP-4 proteinase
inhibitors (Fig. 9, E). Together these data suggest a
new role for IGFBP-3 as an IGFBP-4 proteinase inhibitor and they
exemplify the intricate complexities involved in the
IGF/IGFBP/IGFBP-proteinase system.
Figure 9:
Hypothetical scheme demonstrating how
IGFBP-3 may regulate IGFBP-4 proteolysis and IGF bioavailability in
vivo. For details, see text.
Volume 270,
Number 46,
Issue of November 17, 1995 pp. 27481-27488
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
I-recombinant human (rh)IGFBP-4 in the absence of IGFs.
Addition of IGF-I, IGF-II, or insulin to conditioned medium had little
affect on
I-rhIGFBP-4 proteolysis, while extraction of
IGFs resulted in only a
10% reduction in proteinase activity.
Since factors other than IGFs appeared to be involved in regulating
IGFBP-4 proteolysis, we hypothesized that IGFBP-3, an IGFBP produced by
many cell lines, but not MC3T3-E1 cells, might function as an inhibitor
of IGFBP-4 proteolysis. Addition of rhIGFBP-3 to conditioned media
inhibited
I-rhIGFBP-4 proteolysis by 90%, while IGF-I and
IGF-II reversed the inhibitory effects of rhIGFBP-3 in a dose-dependent
manner.
I-rhIGFBP-4 proteolysis was not inhibited by
N-terminal rhIGFBP-3 fragments that bind IGFs, but was inhibited by two
synthetic peptides corresponding to sequences contained in the
mid-region or C-terminal region of IGFBP-3. Both inhibitory peptides
contain highly basic, putative heparin-binding domains and heparin
partially reversed the inhibitory effects of rhIGFBP-3 on
I-rhIGFBP-4 proteolysis. These data demonstrate that
rhIGFBP-3 inhibits IGFBP-4-degrading proteinase activity and binding of
IGFs or glycosaminoglycans to IGFBP-3 may induce conformational changes
in the binding protein, causing disinhibition of the proteinase.
)-binding proteins
(IGFBPs) are a group of six homologous, yet distinct proteins (IGFBPs
1-6) which bind both IGF-I and IGF-II with high affinity (for
recent reviews, see (1, 2, 3, 4) ).
Among these IGFBPs, IGFBP-4 has been shown to function as an inhibitor
of IGF bioactivity(1, 2, 3, 4) . For
instance, IGFBP-4 was the only inhibitory IGFBP isolated from
conditioned media from a colon adenocarcinoma cell line, although other
IGFBPs were present in the conditioned medium(5) . Furthermore,
Malpe et al.(6) have demonstrated that treatment of
human bone cells with antisense oligonucleotides directed against
IGFBP-4 mRNA results in decreased production of IGFBP-4 and a striking
increase in cellular proliferation, suggesting that IGFBP-4 plays a
major role in regulating cellular proliferation. Since IGFBP-4 has been
purified from a number of sources (1, 2, 3, 4) and since mRNA for
IGFBP-4 has been detected in all tissues studied by Shimasaki et
al.(7) , it is likely that IGFBP-4 may serve to restrain
IGF activity in many tissues.
Materials
Recombinant human (rh)IGFBP-3 produced
in Escherichia coli (rhIGFBP-3) was kindly provided by Dr.
Christopher Maack, Celtrix Pharmaceuticals, Santa Clara,
CA(20) . rhIGFBP-4 was purchased from Austral Biologicals, San
Ramon, CA. rhIGF-I was kindly provided by Genentech Inc., South San
Francisco, CA, and rhIGF-II was generously supplied by Lilly Research
Laboratories, Indianapolis, IN. Recombinant human insulin was from Novo
Nordisk Pharmaceuticals Inc., Princeton, NJ. Polyclonal antisera to
IGF-I was a gift from Dr. Lewis Underwood, University of North
Carolina, Chapel Hill, NC, and a monoclonal antibody to rat IGF-II was
purchased from Amano Pharmaceutical Co., Ltd., Nagoya, Japan. Reagents
used for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were
purchased from Bio-Rad. Low molecular mass heparin (6 kDa) from porcine
intestinal mucosa and all proteinase inhibitors were purchased from
Sigma, with the exception of the proteinase inhibitors
3,4-dichloroisocoumarin (3,4-DCI) and L-trans-epoxysuccinyl-leucylamide-(4-guanidino)butane
(E-64), which were purchased from Boehringer Mannheim.
NaI and Hyperfilm-ECL were obtained from Amersham Corp.
Tissue culture plasticware was obtained from Corning Glass Works,
Corning, NY. Growth media and cell culture reagents were obtained from
Life Technologies, Inc.
MC3T3-E1 Cell Culture and Conditioned Media
Stock
cultures of MC3T3-E1 osteoblasts were maintained in minimal essential
medium containing 10% (v/v) fetal bovine serum, penicillin (100
units/ml), and streptomycin (100 units/ml), as described
previously(21) . Stock cultures were subcultured every 3 days. 
10
cells/well in 35-mm diameter
multiwell plastic culture dishes; cells were then grown in
-minimal essential medium/10% fetal bovine serum supplemented with
10 mM 
-glycerol phosphate and 25 µg/ml ascorbic acid.
Differentiating cultures were refed every 3 days throughout a 30-day
culture period. Under these conditions, these cells display an orderly
developmental pattern in culture; replication of preosteoblasts (days
1-10) is followed by growth arrest and the sequential expression
of mature osteoblastic characteristics, including increased alkaline
phosphatase activity (>day 10), matrix accumulation (days
14-21), osteocalcin expression (>day 21), and eventual
mineralization (>day 25)(22) . Every 2-3 days during
the 30-day culture period, selected plates were washed and incubated
for 48 h in serum-free medium (Dulbecco's modified Eagle's
medium/F-12 + 0.1% bovine serum albumin). Cell-free conditioned
media collected during these 48-h incubations were used in the
subsequent experiments.Preparation of IGF-extracted MC3T3-E1 Conditioned
Media
Endogenous IGF-I and IGF-II present in MC3T3-E1
conditioned media were removed by immunoabsorption using anti-IGF-I and
anti-IGF-II protein G-Sepharose. To prepare the immunoaffinity matrix,
18 µl of anti-IGF-I polyclonal antisera and 600 ng of an
anti-IGF-II monoclonal antibody were incubated with 150 µl of
protein G-Sepharose overnight at room temperature. The matrix was
washed extensively with 20 mM Hepes, 1 M NaCl, pH
7.4, to remove excess serum. Immunoabsorption was performed by
incubating 400-µl samples of MC3T3-E1 conditioned media with 75
µl of the immunoaffinity matrix overnight at 4 °C in a
microcentrifuge tube. The mixtures were centrifuged at 2,000 rpm for 5
min at 4 °C, and the supernatant was removed and used for I-rhIGFBP-4 proteinase assays as described below.
Degradation of
I-IGFBP-4 by MC3T3-E1
CM
I-rhIGFBP-4 proteinase assays using cell-free
conditioned media were performed as described previously, with minor
modifications(16) . rhIGFBP-4 was labeled with
Na
I using the chloramine-T method to a specific activity
of
50 µCi/µg of protein. To detect IGFBP-4 protease
activity present in conditioned media, samples of cell-free conditioned
medium (50 µl unless indicated otherwise) were incubated with
I-IGFBP-4 (25,000 cpm;
1 ng of rhIGFBP-4) at 37
°C for 0-72 h. Proteolytic degradation of
I-IGFBP-4 was terminated by the addition of an equal
volume of 2
non-reducing sample buffer(23) , followed
by heating at 100 °C for 3 min. Samples and prestained molecular
weight markers were then electrophoresed through 15% SDS-polyacrylamide
gels, dried under vacuum, and exposed to x-ray film to visualize intact
and degraded I-IGFBP-4 fragments. In certain experiments,
IGFBP-4 proteinase activity was characterized further in the presence
of metal-dependent proteinase inhibitors: EDTA (10 mM) and
1,10-phenanthroline (1 mM); serine proteinase inhibitors:
aprotinin (2.5 µg/ml), phenylmethylsulfonylfluoride (10
mM), and 3,4-DCI (100 µM); or the cysteine
proteinase inhibitor: E-64 (10 µM). In addition, various
concentrations of rhIGF-I, rhIGF-II, intact rhIGFBP-3, rhIGFBP-3
fragments produced by matrix metalloproteinase-3 (MMP-3) (as described
below), N-terminal IGFBP-3 fragments that bind IGFs, synthetic peptides
corresponding to sequences present in hIGFBP-3, and/or heparin were
preincubated with MC3T3-E1 conditioned media for 3 h at 37 °C prior
to in vitro
I-rhIGFBP-4 protease assay.
Preparation of IGFBP-3 Fragments Produced by
MMP-3
Sixty µg of rhIGFBP-3 was digested by 200 ng of MMP-3
(kindly provided by Dr. Hideaki Nagase, University of Kansas Medical
Center, Kansas City, KS) in a total volume of 60 µl of 50 mM Tris, pH 7.5, 0.15 M NaCl, 10 mM
CaCl
, 0.02% NaN
, 0.05% Brij 35 (TNC buffer) for
8 h at 37 °C(24) . The digestion was stopped by the
addition of EDTA (final concentration: 10 mM), and the
digestion products were analyzed on 15% SDS gels under reducing
conditions. Seven rhIGFBP-3 fragments, designated
``a-f,'' were identified as described elsewhere (24) (see Fig. 5). To separate rhIGFBP-3 fragments
produced by MMP-3 digestion, the digestion mixture was incubated with 1
ml of heparin-Sepharose (Sigma) overnight at 4 °C. The matrix was
washed with 50 mM Tris-HCl, pH 7.4, and heparin-bound
fragments were eluted in 50 mM Tris-HCl, pH 7.4, containing 1 M NaCl. All fractions were analyzed by SDS-PAGE and stained
with Coomassie Blue. The wash fractions (i.e. 50 mM Tris-HCl, pH 7.4) contained only the smallest IGFBP-3 fragments
(fragments e and f) produced by MMP-3, which correspond to the first
100-110 N-terminal amino acids of IGFBP-3. Fragments a-d
bound to the heparin column, and all four fragments were eluted with 50
mM Tris-HCl, pH 7.4, containing 1 M NaCl. The
inability of fragments e and f to bind heparin-Sepharose was
anticipated, since their sequences do not contain either of the two
putative heparin-binding sites present in
IGFBP-3(2, 25) . In covalent cross-linking studies,
fragments e and f bound specifically I-rhIGF-I and
I-rhIGF-II with IC
values of
8.9 and
3.0 nmol/liter, respectively. (
)These findings are
consistent with a previous report demonstrating that an 88-amino acid
N-terminal mutant of IGFBP-3 bound I-IGF-I (20) .
Fragments a-f together or fragments e and f together were tested
for their abilities to alter
I-rhIGFBP-4 degradation by
MC3T3-E1 conditioned media as described above.
I-rhIGFBP-4
proteolysis by IGFBP-3 synthetic peptides.
I-rhIGFBP-4
was incubated with unconditioned media (lane 1) or MC3T3-E1
conditioned media (50 µl; lanes 2-7) in the absence (lane 2) or presence (lanes 3-7) of increasing
concentrations of peptide II (panel A and
), peptide IV (panel B and 
), or both peptides II and IV (panel
C and 
) as described under ``Experimental
Procedures.'' The final concentration for each peptide is as
follows: lane 3, 10 µM; lane 4, 20
µM; lane 5, 50 µM; lane 6,
100 µM; lane 7, 200 µM. The
percentage of proteolysis was calculated from densitometric data from
two to four separate experiments as described under ``Experimental
Procedures.''
Preparation of Synthetic hIGFBP-3
Peptides
Peptides based on amino acid sequences contained in
both the non-homologous mid-region of hIGFBP-3 (peptides I, II, and
III) and in the highly conserved C terminus (peptide IV) (see Fig. 5below) were produced by solid phase peptide synthesis
using 9-fluorenylmethoxycarbonyl chemistry. The sequences are as
follows: SRLRAYLLPAPPAP
(peptide I),
KKGHAKDSQRYKVDYESQS
(peptide II),
TDTQNFSSESKRETEY
(peptide III), and
DKKGFYKKKQLRPSKGR
(peptide IV). All
peptides were purified on a Vydac C-8 HPLC column using a Gilson
automated HPLC system, and were shown to be >98% pure. Sequence
verification was performed by electrospray mass spectrometry. All
peptides were synthesized with an additional N-terminal cysteine for
use in thiol-coupling reactions. The internal cysteine in peptide IV
was acetylmethylated because it is normally involved in a disulfide
bond. All synthetic peptides were tested for their abilities to alter
I-rhIGFBP-4 degradation by MC3T3-E1 conditioned media as
described above.
Statistical Analysis
All experiments were repeated
using conditioned media from two to five different experiments.
Relative concentrations of intact I-rhIGFBP-4 and
fragments of
I-rhIGFBP-4 were determined by scanning
densitometry (Beckman). Graphic data were normalized to the proteolysis
of
I-rhIGFBP-4 observed in unconditioned media (i.e. 100% inhibition) and the proteolysis of
I-rhIGFBP-4
observed in cell-free conditioned media (i.e. 100%
proteolysis). All data are expressed as the mean ± S.E.
Statistical significance between groups was determined by paired
Student's t test. Curve-fitting and IC
values were calculated using InPlot Software (GraphPad Software,
San Diego, CA).
Characterization of IGFBP-4 Proteases in MC3T3-E1
Cultures
To determine if MC3T3-E1 osteoblasts produce
IGFBP-4-degrading proteinases, conditioned media from different time
points during differentiation were examined for their abilities to
degrade I-rhIGFBP-4. When incubated with conditioned
media from MC3T3-E1 osteoblasts, intact
I-IGFBP-4
(
28 kDa) was degraded into
20- and
14-kDa fragments (Fig. 1). By 24 h, >50% of the binding protein was degraded (p < 0.0001, n = 4). IGFBP-4 protease
activity present in MC3T3-E1 conditioned media increased progressively
as osteoblasts matured in culture; maximal proteinase activity was
detected in conditioned media from osteoblasts displaying a
differentiated phenotype. Therefore, all subsequent studies were
performed with conditioned media from >20 day cultures. To
characterize further the IGFBP-4-degrading proteinase(s) present in
MC3T3-E1 conditioned medium, samples of cell-free conditioned media
were analyzed for their ability to degrade
I-rhIGFBP-4 in
the presence or absence of various protease inhibitors. As shown in Table 1, only the cation-dependent proteinase inhibitors (EDTA
and 1,10-phenanthroline) significantly inhibited IGFBP-4-degrading
proteinase activity. Serine (phenylmethylsulfonylfluoride, aprotinin,
and 3,4-DCI) and cysteine (E-64) proteinase inhibitors had little or no
effect on degradation of
I-rhIGFBP-4. These data
suggested that the IGFBP-4-degrading proteinase in MC3T3-E1 conditioned
medium is a cation-dependent proteinase.
I-rhIGFBP-4 by MC3T3-E1 conditioned media. MC3T3-E1 media
from mature osteoblasts were incubated with
I-rhIGFBP-4
for the indicated times as described under ``Experimental
Procedures.'' Intact
I-rhIGFBP-4 (
28 kDa;
denoted by &cjs0800;) and proteolytic fragments of
I-rhIGFBP-4 (
20 and 14 kDa; denoted by
) were
separated by SDS-PAGE and detected by autoradiography. Molecular size
markers are indicated on the left.
Effects of IGFs on
When IGF-I, IGF-II or insulin were added to MC3T3-E1
conditioned media, only high dose IGF-II (500 ng/ml) induced any
significant, although modest, increase in I-rhIGFBP-4
Proteolysis
I-rhIGFBP-4
proteolysis (Table 2). Because exogenous IGFs were not required
for
I-rhIGFBP-4 degradation and exerted only marginal
effects on
I-rhIGFBP-4 degradation by MC3T3-E1
conditioned medium, we postulated that endogenously produced IGFs by
MC3T3-E1 osteoblasts might account for the constitutive degradation of
I-rhIGFBP-4. To test this hypothesis, IGF-I and IGF-II
were first immunoabsorbed from cell-free MC3T3-E1 conditioned media and
IGF-extracted conditioned media were then analyzed for their ability to
degrade
I-rhIGFBP-4. Using this method, only a 10% (p = 0.03) decrease in the degradation of
I-rhIGFBP-4 was observed. In other experiments, direct
addition of anti-IGF-II monoclonal antibodies to conditioned media
resulted in no change in
I-rhIGFBP-4 proteolysis (data
not shown). These data suggested that endogenous IGFs contributed only
minimally, if at all, to the constitutive degradation of
I-rhIGFBP-4 by MC3T3-E1 cells.
Effect of rhIGFBP-3 on
In previous studies using other cell lines, we and
others have demonstrated that the addition of IGFs to cells or
conditioned media is essential for the induction of IGFBP-4 protease
activity (reviewed in (1) -4); in contrast, our current
studies failed to demonstrate an absolute requirement for IGFs in
inducing IGFBP-4 proteolysis in MC3T3-E1 osteoblasts. To clarify this
discrepancy, we searched for differences in the IGF/IGFBP systems
between MC3T3-E1 osteoblasts and cell lines previously reported to
display only IGF-induced IGFBP-4 proteolysis. Since MC3T3-E1 cells do
not produce IGFBP-3(26) , while other cell lines exhibiting
IGF-induced IGFBP-4 proteolysis do produce
IGFBP-3(15, 16, 17, 18, 19) ,
we speculated that IGFBP-3 might function as an inhibitor of IGFBP-4
proteolysis. To determine whether exogenous IGFBP-3 inhibits the
degradation of I-rhIGFBP-4
Proteolysis
I-rhIGFBP-4 by MC3T3-E1 conditioned media,
we added rhIGFBP-3 to MC3T3-E1 conditioned media and monitored
I-rhIGFBP-4 proteolysis. IGFBP-3 inhibited the
degradation of
I-rhIGFBP-4 in a dose-dependent manner (Fig. 2). At a concentration of 2 µg/ml, rhIGFBP-3 inhibited
the degradation of
I-rhIGFBP-4 by 90 ± 0.2% (p = 0.0001, n = 4). Thus, IGFBP-3, at maximal
concentrations, was as effective an inhibitor of
I-rhIGFBP-4 proteolysis as was either of the
metal-dependent proteinase inhibitors (see Table 1).
I-rhIGFBP-3
proteolysis by rhIGFBP-3.
I-rhIGFBP-4 was incubated with
Dulbecco's modified Eagle's medium containing 0.1% bovine
serum albumin (lane 1) or MC3T3-E1 conditioned media in the
absence (lane 2) or presence (lanes 3-8) of
intact rhIGFBP-3. rhIGFBP-3 was added in the following amounts: lane 3, 5 ng of rhIGFBP-3; lane 4, 10 ng of
rhIGFBP-3; lane 5, 25 ng of rhIGFBP-3; lane 6, 50 ng
of rhIGFBP-3; lane 7, 100 ng of rhIGFBP-3; lane 8,
500 ng of rhIGFBP-3. Intact
I-rhIGFBP-4 and proteolytic
fragments of
I-rhIGFBP-4 were separated by SDS-PAGE and
detected by autoradiography. Molecular size markers are indicated on
the left.
Determination of the Epitope(s) in IGFBP-3 Involved in
Inhibition of IGFBP-4 Proteolysis
To investigate the mechanism
through which rhIGFBP-3 inhibits IGFBP-4-degrading proteinase activity,
the effects of (a) intact rhIGFBP-3, (b) rhIGFBP-3
fragments produced by MMP-3, and (c) IGF-binding, N-terminal
rhIGFBP-3 fragments were compared for their inhibitory effects on I-rhIGFBP-4 proteolysis by MC3T3-E1 conditioned media. Fig. 3demonstrates that the addition of intact rhIGFBP-3 to
MC3T3-E1 conditioned media readily inhibited the degradation of
I-rhIGFBP-4 (IC
= 0.24 µg/ml).
When a mixture of all rhIGFBP-3 fragments produced by MMP-3 (fragments
a-f) were added to MC3T3-E1 conditioned media,
I-rhIGFBP-4 degradation was also inhibited in a
dose-dependent fashion, with
2-fold less potency than intact
rhIGFBP-3. In contrast, the addition of IGF-binding, N-terminal
fragments of IGFBP-3 (fragments e and f) had little or no effect on
I-rhIGFBP-4 proteolysis (IC
> 40.0
µg/ml), suggesting that inhibitory activity does not reside in the
first 110 amino acids present in IGFBP-3 (see Fig. 4). Since
N-terminal IGFBP-3 fragments bind IGF-I and IGF-II, sequestration of
endogenous IGFs could not explain the inhibitory effect of IGFBP-3 on
I-rhIGFBP-4 proteolysis. These observations support
earlier conclusions that endogenous IGFs contribute little to the
constitutive IGFBP-4-degrading proteinase activity in MC3T3-E1
conditioned media. Together, these data suggested that the inhibitory
domain(s) in IGFBP-3 resides in the non-homologous, mid-region of the
molecule and/or in the C-terminal domain (Fig. 4) and that
degradation of rhIGFBP-3 fails to destroy the inhibitory effects of
rhIGFBP-3 on
I-rhIGFBP-4 proteolysis.
I-rhIGFBP-4
proteolysis by rhIGFBP-3 or rhIGFBP-3 fragments. MC3T3-E1 conditioned
media (50 µl) were incubated with
I-rhIGFBP-4 in the
absence or presence of increasing concentrations of rhIGFBP-3 (
),
rhIGFBP-3 fragments a-f (
), or IGF-binding, N-terminal
rhIGFBP-3 fragments e and f () as described under
``Experimental Procedures.'' Data were obtained from
densitometric analysis of autoradiograms, and the percentage of
inhibition was calculated as described under ``Experimental
Procedures.''
150 amino acids of IGFBP-3 that inhibit
IGFBP-4 degradation, four synthetic peptides were prepared. Three of
these peptides correspond to epitopes present in the mid-region of the
IGFBP-3 molecule (peptides I, II, and III), a region that has little or
no homology with the other five IGFBPs(1) ; the fourth peptide
(peptide IV), corresponds to a region in the C-terminal portion of the
binding protein (Fig. 4). Table 3demonstrates that both
peptide II and peptide IV, when added to MC3T3-E1 conditioned media at
200 µM/liter, inhibited significantly the degradation of
I-rhIGFBP-4. In contrast, peptides I and III, used at the
same concentrations, had no discernible inhibitory activities. Fig. 5demonstrates that both peptides II and IV inhibited
I-rhIGFBP-4 degradation in a dose-dependent manner (Fig. 5, panels A and B, respectively), and
each produced a displacement curve parallel to the other peptide (Fig. 5). However, peptide IV (IC
= 25
µm) was approximately 3-fold more potent than peptide II (IC
= 74 µM) in inhibiting IGFBP-4-degrading
proteinase activity. When used together, the peptides demonstrated no
additive effect on inhibiting
I-rhIGFBP-4 proteolysis (Fig. 5, panel C).
Effects of IGFs on the Inhibitory Activity of IGFBP-3 and
IGFBP-3 Peptides on
Although exogenous IGFs had little or no effect on I-rhIGFBP-4
Proteolysis
I-rhIGFBP-4 degradation, IGFs effectively reversed the
inhibitory effects of rhIGFBP-3 (Fig. 6). When increasing
concentrations IGF-I (Fig. 6, panel A) or IGF-II (Fig. 6, panel B) were added to MC3T3-E1 conditioned
media containing
I-rhIGFBP-4 and a maximal inhibitory
dose of rhIGFBP-3 (2 µg/ml), both ligands effectively reversed the
inhibitory effect of rhIGFBP-3 on
I-rhIGFBP-4 proteolysis (Fig. 6). A 1:1 IGF-II:rhIGFBP-3 molar ratio produced a 50%
reversal of IGFBP-3's inhibitory effect, while molar ratios of
greater than 3:1 produced almost 100% reversal. IGF-I (IGF-I:IGFBP-3
IC
= 2.9:1) was approximately 3 times less potent
than IGF-II in reversing the inhibitory effects of rhIGFBP-3 on
I-rhIGFBP-4 proteolysis (Fig. 6).
I-rhIGFBP-4 proteolysis by IGFs.
I-rhIGFBP-4 was incubated with unconditioned media (lane 1) or MC3T3-E1 conditioned media (lanes
2-8) in the absence (lane 2) or presence (lanes
3-8) of rhIGFBP-3 (2 µg/ml) and increasing
concentrations of IGF-I (panel A and
) or IGF-II (panel B and ) as described under ``Experimental
Procedures.'' The amount of IGF-I or IGF-II added per lane is as
follows: lane 4, 10 ng; lane 5, 25 ng; lane
6, 50 ng; lane 7, 100 ng; lane 8, 500 ng. The
percentage of proteolysis was calculated from densitometric data
obtained from three separate experiments as described under
``Experimental Procedures'' and is expressed as a ratio of
IGF:IGFBP-3 added to the sample.
I-rhIGFBP-4 proteolysis, IGF-I was examined for its
ability to reverse the inhibitory effects of peptides II and IV on
I-rhIGFBP-4 proteolysis by MC3T3-E1 conditioned media. As
shown in Fig. 7, IGF-I had little or no effect on reversing the
inhibitory effects of peptides II and IV on
I-rhIGFBP-4
degradation. In covalent cross-linking studies, it was demonstrated
that while
I-IGF-I and
I-IGF-II both bound
to intact rhIGFBP-3 or IGFBP-3 fragments e and f, neither radioligand
bound to peptides II and IV (data not shown). These data suggest that
IGFBP-3 inhibits IGFBP-4 proteolysis via epitopes present within the
amino acid sequences contained in peptides II and IV and that IGFs must
be able to bind to IGFBP-3 in order to reverse the inhibitory effects
of IGFBP-3 on IGFBP-4 proteolysis.
I-rhIGFBP-4 proteolysis by IGFBP-3 synthetic peptides.
I-rhIGFBP-4 was incubated with MC3T3-E1 conditioned media
in the absence (lane 1) or presence (lanes 2-9)
of various IGFBP-3 synthetic peptides (200 µM): peptide I, lanes 2 and 3; peptide II, lanes 4 and 5; peptide III, lanes 6 and 7; peptide IV, lanes 8 and 9. IGF-I (20 µg/ml) was added to lanes 3, 5, 7, and 9. Intact
I-rhIGFBP-4 and proteolytic fragments of
I-rhIGFBP-4 were separated by SDS-PAGE and detected by
autoradiography. Molecular size markers are indicated on the left.
Effect of Heparin on rhIGFBP-3 Inhibition of
As indicated in Fig. 5, peptides II and IV both contain putative heparin-binding
domains(2, 25) . Peptide II contains the sequence I-rhIGFBP-4 Proteolysis
KKGHA
which resembles a short
heparin-binding domain (BBXBX; B = basic amino
acid, and X = non-basic amino acid) and peptide IV
contains the sequence
YKKKQCRP
, which
resembles a long heparin-binding motif (XBBBXXBX)(25) . This suggested that
both of these highly basic, putative heparin-binding domains present in
IGFBP-3 could inhibit IGFBP-4-degrading activity in MC3T3-E1
conditioned media; therefore, we next examined the effect of heparin on
modulating the inhibitory effect of rhIGFBP-3 on
I-rhIGFBP-4 proteolysis. For this purpose, a submaximal
dose of rhIGFBP-3 (200 ng/ml) or of peptide IV (20 µM) was
added to MC3T3-E1 conditioned medium with or without heparin (100
µg/ml). All solutions were incubated with
I-rhIGFBP-4
and processed in parallel with conditioned media containing no
additives or heparin alone. As Fig. 8demonstrates, heparin
alone (bar 4) had no significant effect on
I-rhIGFBP-4 proteolysis. In contrast, when heparin was
added to conditioned media containing rhIGFBP-3, heparin reversed the
inhibitory effects of rhIGFBP-3 on
I-rhIGFBP-4
proteolysis by 33% (p < 0.01) (compare bars 2 and 3). Similarly, heparin almost entirely reversed the inhibitory
effects of peptide IV on
I-rhIGFBP-4 proteolysis (data
not shown).
I-rhIGFBP-4 degradation by rhIGFBP-3. MC3T3-E1
conditioned media was incubated with
I-rhIGFBP-4 in the
absence (bar 1), or the presence of rhIGFBP-3 (200 ng/ml; bars 2 and 3) and/or heparin (100 µg/ml; bars3 and 4) as described under ``Experimental
Procedures.'' The percentage of proteolysis was calculated from
densitometric data. *, p < 0.01 for comparisons of 1
versus 2 or 3 and 2 versus
3.
I-rhIGFBP-4 degradation, while a maximal dose of
IGF-II (500 ng/ml) only modestly increased the degradation of the
binding protein. This is in marked contrast to our previous report
using human and sheep fibroblast condition media, where we demonstrated
that little or no proteolysis of IGFBP-4 occurred during a prolonged
incubation (i.e. 72 h), yet with the addition of IGF-I or
IGF-II, almost complete proteolysis of IGFBP-4 was
achieved(16) . Since MC3T3-E1 osteoblasts secrete both IGF-I
and IGF-II(26, 27) , it was possible that endogenous
IGFs induced IGFBP-4 proteolysis in this cell line. In this regard,
Durham et al.(28) have very recently demonstrated
that in certain primary human bone cell lines, IGFBP-4 can be degraded
without the addition of IGFs, while in other bone cell lines the
addition of IGFs is necessary for IGFBP-4 proteolysis. They suggest
that cell lines not requiring exogenous IGFs for IGFBP-4 proteolysis
produced more IGFs than those cell lines requiring exogenous IGFs for
IGFBP-4 proteolysis. Since immunoabsorption of IGFs from conditioned
media or the addition of IGF-binding, N-terminal fragments of IGFBP-3
to conditioned media had little or no effect on inhibiting
I-rhIGFBP-4 degradation, endogenous IGFs do not appear to
contribute significantly to the constitutive degradation of
I-rhIGFBP-4 observed in MC3T3-E1 cells. Furthermore,
since many cell lines that display IGF-induced IGFBP-4 proteolysis also
produce some IGF-I and/or IGF-II, it seemed unlikely that the presence
of IGFs in MC3T3-E1 conditioned media could account for the
constitutive nature of the IGFBP-4-degrading proteinase activity.
I-rhIGFBP-4 by conditioned media from MC3T3-E1 cells in a
dose-dependent manner. This demonstrated that exogenous IGFBP-3 is an
IGFBP-4 proteinase inhibitor and also suggests that endogenous IGFBP-3
could function as a physiological inhibitor of IGFBP-4 proteolysis
since it is present in a variety of biological fluids at concentrations
5-20 times greater (30) than the IC
needed to inhibit IGFBP-4 degradation. IGFBP-3 may also be
induced in certain cell systems and function as an IGFBP-4 proteinase
inhibitor. For instance, Conover and colleagues (31) have
demonstrated that treatment of human fibroblasts with phorbol esters
induces an inhibitor(s) of IGFBP-4 proteolysis, while Albiston et
al.(32) have shown that IGFBP-3 promotor activity is
increased when transfected cells are treated with a phorbol ester.
Therefore, it is possible that the phorbol ester-induced inhibitor of
IGFBP-4 proteolysis is IGFBP-3.
3
times more potent in inhibiting IGFBP-4 proteolysis than was peptide
II, which contains a short heparin-binding site, and there was no
demonstrable synergy when both peptides were used together. This
suggests that both peptides work through a similar inhibitory
mechanism, although the specifics of the mechanism are currently
unknown and are under investigation. Preliminary data from our
laboratory demonstrate that other proteins, such as fibronectin and
vitronectin, which are commonly found in cell cultures and which
contain both long and short forms of heparin-binding motifs, do not
inhibit IGFBP-4 proteolysis by MC3T3-E1 conditioned media.
However, IGFBP-5, which contains almost the same stretch of basic
residues that is present in IGFBP-3 and peptide IV(1) , also
inhibits IGFBP-4 proteolysis, suggesting that these basic
domains are specific in their ability to inhibit IGFBP-4 proteolysis.
These highly basic regions may interact with IGFBP-4 itself, protecting
it from proteolysis; however, this mechanism seems doubtful since
heterodimerization of IGFBPs has not been reported to date. These
regions may function as competitive substrates for the IGFBP-4
proteinase; however, this seems unlikely since IGFBP-4 itself contains
no heparin-binding motifs (2) and since a recent study
demonstrated that the cleavage site in human IGFBP-4 produced by the
IGF-dependent IGFBP-4 proteinase is at
Met-Lys
(33) , a bond that is not
present in either of the inhibitory peptides. Interestingly, Nam et
al.(34) have demonstrated recently that an IGFBP-5
synthetic peptide that is
80% homologous with peptide IV
significantly inhibited the proteolysis of IGFBP-5 by a partially
purified IGFBP-5-degrading proteinase. Thus, together these data
suggest that IGFBPs containing heparin-binding domains (i.e. all IGFBPs with the notable exception of IGFBP-4; (2) )
may function as direct inhibitors of the IGFBP-4 proteinase(s).
Furthermore, the finding that IGF-I was unable to reverse the
inhibitory effects of peptides containing heparin-binding domains
suggests that fragments of IGFBP-3 that contain one or more of these
domains, but do not contain IGF-binding sites, might function as
inhibitors whose effects are not reversible by IGFs.
I-rhIGFBP-4 degradation by
MC3T3-E1 conditioned media. The divergent effects of heparin on
degradation of these various IGFBPs may simply reflect the differences
in the proteinases that are involved. However, it is also possible that
heparin alters the inhibition of these proteinases in divergent ways.
For example, heparin has been shown to enhance inhibition of thrombin
and factor Xa by antithrombin III(38) , but decrease the rate
of inhibition of neutrophil elastase by
-proteinase
inhibitor(39) , although both proteinase inhibitors are members
of the serpin family. The mechanism by which heparin interferes with
IGFBP-3's ability to inhibit IGFBP-4 proteolysis is unclear;
however, association of heparin with heparin-binding sites present in
IGFBP-3 may make them less available to inhibit the proteinase. Because
heparin has been shown to interfere with binding of IGFs to IGFBP-5 and
IGFBP-3(25) , an alternative hypothesis would be that heparin
causes dissociation of IGFs from endogenous IGFBPs, making IGFs
available for binding to exogenous rhIGFBP-3, thereby partially
mitigating the inhibitory effect of IGFBP-3 on IGFBP-4 proteolysis.
Regardless of the mechanism, heparin, and possibly other
glycosaminoglycans, may have a role similar to the IGFs in inducing
IGFBP-4 proteolysis.
)))
We thank Dr. Darryl Quarles for providing the MC3T3-E1
osteoblasts for these studies and Drs. Michael Freemark and Jan Enghild
for reviewing the manuscript and making helpful suggestions. We also
acknowledge the generous gift of rhIGFBP-3 from Dr. Christopher Maack
(Celtrix Pharmaceuticals, Santa Clara, CA).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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