(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 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.
Insulin-like growth factor (IGF)()-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.
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 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.
Figure 5:
Inhibition of 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.''
Figure 1:
Degradation of 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.
Figure 2:
Inhibition of 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.
Figure 3:
Inhibition of 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.''
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 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).
Figure 6:
Reversal of the inhibitory effect of
rhIGFBP-3 on 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.
Because IGFs
reversed the inhibitory effects of rhIGFBP-3 on 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.
Figure 7:
Effect of IGF-I on the inhibition of 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.
Figure 8:
Effect of heparin on the inhibition of 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.
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 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.
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 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.
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 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.
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 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.
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.