(Received for publication, March 29, 1995)
From the
The growth potentiating effects of the insulin-like growth
factor (IGF)-I and IGF-II are modulated by a family of six insulin-like
growth factor binding proteins (IGFBPs). Despite the similarity in
amino acid sequences of the IGFBPs, their effects on the growth of bone
cells differ. Studies on the molecular mechanisms for IGFBP-4 actions
revealed that coincubation of bone cells with IGFBP-4 and I-IGF-I or
I-IGF-II decreased the binding
of both of these ligands in a dose-dependent manner. In addition,
IGFBP-4 decreased the binding of IGF-I tracer to purified type I IGF
receptor. These data in conjunction with data showing that IGFBP-4 had
no effect on cell proliferation induced by analogs of IGF-I or IGF-II,
which exhibited >100-fold reduced affinity for binding to IGFBP-4
suggest that IGFBP-4 may inhibit IGF action by preventing the binding
of ligand to its membrane receptor. In contrast to IGFBP-4, IGFBP-5
treatment increased the binding of IGF tracer to bone cells but did not
increase the binding of
I-IGF-I to type I IGF receptor.
Studies on the mechanism by which IGFBP-5 increased the binding of
I-IGF tracer to bone cells suggest that IGFBP-5 could
facilitate IGF binding by a mechanism in which IGFBP-5 has cell surface
binding sites independent of IGF receptors. These data in conjunction
with the findings that IGFBP-5 potentiated cell proliferation even in
the presence of those same IGF analogs that exhibited >200-fold
reduced affinity for binding to IGFBP-5, suggest that IGFBP-5 may in
part stimulate bone cell proliferation by an IGF-independent mechanism
involving IGFBP-5-specific cell surface binding sites.
Studies in our and other laboratories provide strong support to
the concept that insulin-like growth factors (IGFs) ()function in an autocrine/paracrine manner to regulate the
proliferative and differentiative functions of bone
cells(1, 2, 3) . The findings that support
this concept are as follows. First, IGF-I and IGF-II have been found in
bone extracts of several different species, and IGFs are the most
abundant growth factors stored in bone(4, 5) . Second,
IGFs have been shown to be one of the most abundant mitogens produced
by bone cells derived from both fetal and adult bone, and the bone cell
production of IGFs is regulated by both local and systemic effectors of
bone metabolism(1, 2, 3) . Third, the
exogenous addition of IGFs have been shown to stimulate both
proliferative and differentiative functions in bone
cells(1, 2, 3) . Fourth, there is recent
evidence that demonstrates that IGFs increase bone formation parameters in vivo in both rats and humans (6, 7, 8) .
The local activity of IGFs in bone and other tissues is dependent not only on the amount of IGFs produced but also by a family of structurally related proteins that specifically bind to IGFs. Six IGF binding proteins (designated IGFBP-1 through IGFBP-6), different from the IGF receptors, have been purified from human plasma or tissues including bone(9, 10, 11, 12, 13, 14) . In previous studies, we have shown that human bone cells in culture secrete a number of IGFBPs of which the 25-kDa IGFBP-4 is one of the major IGFBPs produced by bone cells. Studies on the biological actions of IGFBP-4 revealed that IGFBP-4 inhibits both IGF-I- and IGF-II-induced cell proliferation in embryonic chick calvaria cells and MC3T3-E1 mouse osteoblasts under all culture conditions tested(15) . In addition, IGFBP-4 has been shown to inhibit the growth of embryonic chick pelvic cartilage in vitro(16) . The inhibitory effect of IGFBP-4 on IGF-induced cell proliferation does not appear to be specific to bone cells since Cheung et al.(17) have shown that IGFBP-4 inhibits IGF-induced cell proliferation in neuroblastoma cell line, and Culouscou and Shyoab et al.(18) have shown that the colon cancer cell growth inhibitor purified from conditioned medium of the HT29 human adenocarcinoma cell line is identical to IGFBP-4.
In contrast to IGFBP-4, studies on the biological actions of IGFBP-5, the major IGFBP stored in human bone, revealed that IGFBP-5 is a stimulator of IGF-induced mouse bone cell proliferation(19) . Similar potentiating effects of IGFBP-5 purified from U2 human osteosarcoma cell conditioned medium have been reported by Andress and Birnbaum(20, 21) . In addition, Jones et al.(22) have demonstrated that when IGFBP-5 was present in cell culture substrata, it potentiated the growth stimulatory effects of IGF-I on fibroblasts. Although Kiefer et al.(23) have demonstrated that recombinant human IGFBP-5 produced in yeast inhibited DNA synthesis in SaOS-2/B10 human osteosarcoma cells, subsequent studies from the same laboratory have shown that recombinant human IGFBP-5 stimulated DNA synthesis in rat osteoblasts(24) . Together, the findings that IGFBPs modulate IGF actions either positively or negatively and that the production of IGFBPs is regulated by various physiological agents(9, 10, 11, 12, 13, 14) support the conclusion that the balance between the stimulatory and inhibitory classes of IGFBPs will determine the degree and extent of IGF-induced cellular responses in target tissues such as bone.
Although IGFBP-4 and IGFBP-5, which share 35% sequence identity with each other, exhibit opposite effects on bone cell proliferation, very little is known about the molecular mechanisms by which these binding proteins exert their effects on bone cell proliferation. There is limited data available in other cell types for IGFBP-1 and IGFBP-3, which have been shown to both stimulate and inhibit IGF actions (25, 26, 27, 28) . Blat et al.(25) have shown that exogenous addition of IGFBP-3 caused a 100% inhibition of DNA synthesis induced by IGF-I in chick embryo fibroblasts. In contrast, preincubation of fibroblasts with IGFBP-3 prior to the addition of IGF-I resulted in the potentiation of IGF-I action(26) . While cell surface attachment of IGFBP-3, which also binds to heparin, has been suggested to be an important mechanism for the potentiation of IGF-I action by IGFBP-3, a number of other mechanisms have also been suggested(27, 28) . The current studies were undertaken to extend the mechanistic data available for the IGFBPs by characterizing the biological effects of IGFBP-4 and IGFBP-5 on MC3T3-E1 mouse osteoblasts and on normal human bone cells under identical culture conditions and by evaluating whether IGFBP-4 and IGFBP-5 modulate their actions on bone cell proliferation by different mechanisms. These data indicate that the molecular mechanisms by which IGFBP-4 and IGFBP-5 affect bone cells are different and that IGFBP-5 may bind bone cells by an IGF-independent mechanism.
Recombinant human (rh) IGFBP-5 and
carboxyl-terminal truncated 19- and 10-kDa forms of IGFBP-5 were
produced in Escherichia coli and purified by established
procedures(32) . IGFBP-4 was purified from human bone cell
conditioned medium as described previously, and the homogeneity of the
preparation was established by SDS-polyacrylamide gel electrophoresis
and amino-terminal amino acid sequence analysis (15, 33, 34) . No detectable levels of IGF-I
or IGF-II were found in the purified preparations of IGFBP-4 and
IGFBP-5. Recombinant human IGF-I and IGF-II were purchased from Austral
Biologicals (Torrance, CA). Recombinant human des-1-3-IGF-I and
des-1-6-IGF-II were kindly provided by G. L. Francis
(Co-operative Research Center for Tissue Growth and Repair, Adelaide,
Australia). Purified human IGF-I receptor was a gift from Y.
Fujita-Yamaguchi (City of Hope, Duarte, CA). Dulbecco's modified
Eagle's medium (DMEM) was purchased from Life Technologies, Inc.
Calf serum was purchased from Hyclone (Logan, UT). Bovine serum albumin
(BSA) was purchased from Fluka. [H]
Methylthymidine and
I were purchased from International
and Chemical Nuclear (Irvine, CA) and DuPont NEN. All other chemicals
used were at least reagent grade and were purchased from Sigma.
Figure 1: Amino terminal protein sequence comparisons of IGFBP-5. IGFBP-5 sequence deduced from cDNA clones derived from human placental (37) and human osteosarcoma library (38) (A); previously described sequence for human bone derived 29-kDa IGFBP (19) (B); nonreduced human bone derived 29-kDa IGFBP (this study) (C); nonreduced human bone-derived 19-kDa IGFBP fragment (this study) (D); reduced and pyridyl ethylated human bone derived 29-kDa IGFBP (this study) (E). Sequences are shown in single-letter amino acid code.
Figure 2:
Effect
of IGFBP-4 and IGFBP-5 on MC3T3-E1 cell proliferation as measured by
the incorporation of [H]thymidine. Values are
expressed as a percent of the BSA-treated control (errorbars give the standard deviation of the measured cpm
divided by the control cpm). Panel A shows the effect of
IGFBP-4 on basal, IGF-I-, or des-1-3-IGF-I-induced MC3T3-E1 cell
proliferation in the absence of exogenous IGFBP-4 (openbars) and in the presence of 100 ng/ml IGFBP-4 (cross-hatchedbars). Panel B shows the
effect of IGFBP-4 concentration on cells stimulated with 3 ng/ml IGF-II (shortdash and opensquares) or
des-1-6-IGF-II (dots and opendiamonds). PanelC shows the effect of
the 29-kDa full-length and the 10-kDa rhIGFBP-5 forms on basal, IGF-I-,
or IGF-II-stimulated MC3T3-E1 cell proliferation; 29-kDa IGFBP-5 on
basal MC3T3-E1 cell proliferation (longdash and filledsquares); 10-kDa IGFBP-5 on basal cell
proliferation (longdash and filledtriangles); 29-kDa IGFBP-5 on 100 ng/ml IGF-I-induced
MC3T3-E1 cell proliferation (solidline and filledcircles); 10-kDa IGFBP-5 on 100 mg/ml
IGF-I-induced MC3T3-E1 cell proliferation (solidline and filledtriangles); 29-kDa IGFBP- 5 on 100
ng/ml IGF-II-induced MC3T3-E1 cell proliferation (shortdash and filledsquares); 10-kDa
IGFBP-5 on 100 ng/ml IGF-II-induced MC3T3-E1 cell proliferation (shortdash and filledtriangles).
[
H]thymidine incorporation in BSA-treated control
cultures ranged from 450 to 1200 cpm with standard deviation typically
less than 15%.
Figure 3:
Competition for binding of I-IGF-I or
I-IGF-II to human IGFBP-4 and
IGFBP-5 by the IGFs and their analogs. PanelA shows
competition for binding of
I-IGF-I to IGFBP-4 (opensymbols) and to IGFBP-5 (filledsymbols) by unlabeled IGF-I (solidline and circles), des-1-3-IGF-I (mediumdash and triangles), IGF-II (shortdash and squares) and des-1-6-IGF-II (dottedline and diamonds). PanelBshows the competition for binding of
I-IGF-II to IGFBP-4 (opensymbols) and
to IGFBP-5 (filledsymbols) by unlabeled IGF-I (solidline and circles),
des-1-3-IGF-I (mediumdash and triangles), IGF-II (shortdash and squares) and des-1-6-IGF-II (dottedline and diamonds). Each point represents the mean of four
replicate determinations from two
experiments.
In contrast to IGFBP-4, exposure of MC3T3-E1 mouse osteoblasts under identical culture conditions to IGFBP-5 results in an increased proliferation of cells both in the absence and in the presence of exogenously added IGFs. Fig. 2C shows that basal cell proliferation was increased by 29-kDa rhIGFBP-5 in a dose-dependent manner. At 100 ng/ml, cell proliferation was increased to 435% of vehicle-treated control (p < 0.001). In addition to stimulating basal cell proliferation, rhIGFBP-5 also stimulated MC3T3-E1 cell proliferation in the presence of 100 ng/ml IGF-I or IGF-II in a dose-dependent manner. The 19- and 10-kDa size variants of IGFBP-5 were also mitogenic when assayed on unstimulated MC3T3-E1 cells or on cells exposed to 100 ng/ml IGF-I or IGF-II (Fig. 2C). However, the 19-kDa variant was less potent than the 29-kDa protein (data not shown) while the 10-kDa variant required 1-2 orders of magnitude higher concentrations to achieve equivalent proliferation (Fig. 2C). Both rhIGFBP-5 and hBDIGFBP-5 exhibited similar potency in stimulating IGF-I-induced cell proliferation. At 10 ng/ml, rhIGFBP-5 and hBDIGFBP-5 increased IGF-I-induced cell proliferation by 92 ± 36% (p < 0.001) and 74 ± 48% (p < 0.001), respectively.
In contrast to IGFBP-4, which did not inhibit cell proliferation induced by either des-1-3-IGF-I or des-1-6-IGF-II, IGFBP-5 potentiated MC3T3-E1 cell proliferation even in the presence of those IGF analogs that exhibited >200-fold reduced affinity for binding to IGFBP-5. Des-1-3-IGF-I and IGF-I at 100 ng/ml stimulated cell proliferation to 304 ± 70 and 225 ± 35% of vehicle treated control, respectively, which were increased further by 27 ± 22 and 20 ± 18%, respectively, upon exposure with 3 ng/ml IGFBP-5 and by 48 ± 15 (p < 0.001) and 56 ± 22% (p < 0.001), respectively, upon exposure with 30 ng/ml IGFBP-5. IGFBP-5 treatment also caused similar increases in cell proliferation induced by des-1-6-IGF-II (data not shown).
The effect of IGFBP-4 and IGFBP-5 on IGF-induced cell proliferation
was also tested using untransformed normal human bone cells in
serum-free culture. Table 1shows that basal cell proliferation
was inhibited by 43 and 41%, respectively, in human bone cells derived
from rib and skull by 100 ng/ml IGFBP-4. In addition, 10 ng/ml
IGF-II-induced cell proliferation was completely inhibited by the
exogenous addition of 100 ng/ml IGFBP-4 in human bone cells derived
from both rib and skull. In contrast to IGFBP-4 effects, exogenous
addition of 100 ng/ml IGFBP-5-stimulated basal cell proliferation by
50-80% in human bone cells derived from rib and skull in
different experiments (data not shown). This increase in cell
proliferation does not appear to be an artifact of
[H]thymidine uptake in the presence of IGFBP-5
since we obtained similar data using the incorporation of bromodeoxy
uridine (data not shown). In addition, both IGF-I- and IGF-II-induced
cell proliferation was increased by 53 ± 34 and 75 ± 11%,
respectively, in the presence of 100 ng/ml rhIGFBP-5 (Table 2).
Figure 4:
Effect of added IGFBP on the binding of I-IGF-I or
I-IGF-II to MC3T3-E1 cells. Data
are the mean of four replicate determinations from two experiments
expressed as the percent of the control determined in the absence of
IGFBP for the effect of IGFBP-4 on the binding of
I-IGF-I (opencircles) and
I-IGF-II (opensquares) and for the effect of IGFBP-5 on the binding of
I-IGF-I (filledcircles) and
I-IGF-II (filledsquares).
The ability of IGFBP-4 and IGFBP-5 to influence the
interaction between IGF-I and the type I receptor was investigated
employing IGF-I iodinated with I and a gel shift assay in
which the 350-kDa type I receptor migrated much more slowly than 25-kDa
IGFBP-4 or 29-kDa IGFBP-5. Upon affinity cross-linking, gel
electrophoresis, and autoradiography,
I label was found
in the 350-kDa IGF-I receptor, which was displaced by unlabeled IGF-I (Fig. 5). Increasing the concentration of IGFBP-4 from 2 to 20
ng/ml resulted in a decrease in the amount of
I signal
bound to type I receptor and a corresponding increase in the amount of
I signal bound to IGFBP-4. These results are consistent
with the possibility that IGFBP-4 upon binding to its ligand may modify
the affinity of the ligand such that IGFs no longer bind to the
receptors. In contrast to IGFBP-4, IGFBP-5 at 2 or 20 ng/ml had no
significant effect on the amount of
I- signal bound to
the type I receptor. In other experiments, IGFBP-5 at concentrations
greater than 100 ng/ml resulted in a decrease in the amount of
I-labeled tracer bound to the type I receptor and a
corresponding increase in the amount of
I-labeled signal
bound to IGFBP-5 (data not shown). The IGFBP-5-induced increase in
I-IGF-I binding in MC3T3-E1 cells was inhibited
completely by unlabeled IGF-I but not by insulin or
des-1-3-IGF-I, which bind to IGF-I receptor but not to IGFBP-5
(data not shown).
Figure 5:
Effect of added IGFBP-4 and IGFBP-5 on the
binding of I-IGF-I to type I IGF receptor. Purified type
I IGF receptor was incubated with
I-IGF-I in the presence
or absence of excess of unlabeled IGF-I. Various concentrations of
purified IGFBP-4 or IGFBP-5 were added to the reaction mixture as
indicated in the figure. The samples were subjected to electrophoresis
in a 3-17% of polyacrylamide-gradient gel as described under
``Experimental Procedures.'' and
autoradiographed.
Figure 6:
Effect of added IGFBP on the binding of I-IGFBP-5 to MC3T3-E1 cells. Monolayer cultures were
exposed to
I-IGFBP-5 with increasing concentrations of
IGFBP-5 (filledcircles) and IGFBP-4 (opencircles) as described under ``Experimental
Procedures.'' Data are the mean (± S.D.) of the binding
observed in four experiments, expressed as the percent of the binding
observed without added IGFBP. Control cpm determined in the absence of
IGFBP were 12,156 (S.D.: 604; n =
4).
Similar cell binding assays
were employed to investigate the effect of heparin on the binding of I-IGFBP-5 to MC3T3-E1 cells based on the previous
evidence that IGFBP-3 binds to heparin(28) . These data
revealed that treatment of MC3T3-E1 cells with heparin decreased the
binding of
I-IGFBP-5 in a dose-dependent manner. At 2
µg/ml, the binding to MC3T3-E1 cells was decreased to 68 ±
2% of control (p < 0.001). In contrast to heparin,
treatment of MC3T3-E1 cells with fibronectin, an RGD-containing protein
resulted in an increase in the binding of
I-IGFBP-5 to
MC3T3-E1 cells (163 ± 12% of control at 5 µg/ml fibronectin, p < 0.001). This increase in binding of
I-IGFBP-5 to MC3T3-E1 cells was abolished by the addition
of 4 µg/ml unlabeled IGFBP-5 but not IGFBP-4. In addition, the
increased binding of
I-IGFBP-5 to MC3T3-E1 cells in the
presence of fibronectin was decreased to 103 ± 6% of control by
adding heparin along with fibronectin.
The effect of IGF-I and
IGF-II on IGFBP-5 binding to MC3T3-E1 cells was investigated by
conducting cell binding assays, carried out in quadruplicate, in which I-IGFBP-5 was exposed to MC3T3-E1 cells. In these studies
neither 2 µg/ml IGF-I nor 2 µg/ml IGF-II had any significant
effect on the binding of
I-IGFBP-5 to MC3T3-E1 cells (94
± 3 and 103 ± 2% binding in the presence of IGF-I and
IGF-II, respectively, compared with control). These data are consistent
with the IGF binding results with purified IGF-I receptor and suggest
that the increased binding of
I-IGF-I to MC3T3-E1 cells
in the presence of IGFBP-5 is not to IGF receptors.
The findings of this study demonstrate that purified IGFBP-4 and IGFBP-5 exhibited opposite effects on bone cell proliferation under identical culture conditions. IGFBP-4 inhibited while IGFBP-5 stimulated both basal and IGF-induced bone cell proliferation. The potentiation of cell proliferation by IGFBP-5 cannot be explained by contamination of IGFBP-5 preparation with IGFs or other proteins since both bone-derived and recombinant IGFBP-5 exhibited similar potency and since no detectable level of IGF-I or IGF-II were found in either of these preparations. The effects of IGFBP-4 and IGFBP-5 are not peculiar to MC3T3-E1 mouse osteoblasts since similar effects were seen using untransformed normal human bone cells. These two binding proteins, IGFBP-4 and IGFBP-5, also exhibited opposite effects on the binding of radiolabeled IGF ligand to bone cells in monolayer culture. Although IGFBP-4 decreased the binding of both IGF-I and IGF-II in a similar manner, IGFBP-5 treatment increased IGF-I binding much more than that of IGF-II binding to cells in monolayer culture. The reason for greater changes in IGF-I binding compared with IGF-II in the presence of IGFBP-5 is unknown. Additionally, purified IGFBP-4 decreased the binding of IGF-I to the type I receptor. In contrast, IGFBP-5 did not cause an increase in the binding of radiolabeled IGF-I to type I receptor. Although the findings that at 2 and 20 ng/ml, IGFBP-4 but not IGFBP-5 decreased the binding of IGF-I ligand to type I receptor are consistent with the possibility that the complex of IGFBP-5 and IGF-I may bind to the type I receptor, further studies are needed to test this possibility.
The findings of this study demonstrate that the
IGFBP-5-induced increase in I-IGF-I binding to bone cells
cannot be explained on the basis of increased IGF-I binding to its
receptors. In addition, IGFBP-5-induced increase in
I-IGF-I binding in MC3T3-E1 cells was inhibited
completely by unlabeled IGF-I but not by insulin or
des-1-3-IGF-I, which bind to IGF-I receptor but not to IGFBP-5.
These data suggest that IGFBP-5 binds to bone cells independent of IGF
receptors and thus increases the binding of IGF-I to bone cells. Our
results on IGFBP-5 binding to bone cells are similar to studies with
cultured mouse osteoblasts using 23-kDa IGFBP-5 by Andress and Birnbaum (21) and studies with cultured fibroblasts using intact
IGFBP-3(27) . In regard to the identification of potential
binding sites for IGFBP-5, the findings that IGFBP-5 binding to bone
cells is not affected by excess of either IGF-I or IGF-II suggest that
IGFBP-5 binds to sites independent of IGF receptors. In addition, Jones et al.(22) have shown that IGFBP-5 binds to various
extracellular matrix proteins including types III and IV collagen,
laminin, and fibronectin. Because IGFBP-5 does not contain an RGD
sequence, which has been shown to be involved in the binding of
extracelluar matrix proteins to integrin receptors(40) , the
findings of this study that fibronectin, an RGD containing protein,
increases IGFBP-5 binding to bone cells together with the previous
finding that IGFBP-5 binds to fibronectin is consistent with the
possibility that IGFBP-5 may bind to integrin receptors via binding to
RGD-containing proteins such as fibronectin.
Based on the findings of this study and other previous studies, we have developed models as shown in Fig. 7to explain the potential mechanisms by which IGFBP-4 and IGFBP-5 may modulate IGF actions in bone cells. According to these models, IGFBP-4 inhibits IGF binding to the receptor by binding to IGFs near or at the receptor binding site. The findings that IGFBP-4 inhibited the binding of radiolabeled IGF-I to purified IGF-I receptors and radiolabeled IGF-II to the IGF-II/mannose-6-phosphate receptors in H4-II-E rat hepatoma cells (35) support the conclusion that IGFBP-4 inhibits IGF-induced bone cell proliferation by preventing IGF binding to its receptors. Consistent with this model of IGFBP-4 action are the findings that IGFBP-4 had no significant effect on the mitogenic activity of des-1-3-IGF-I or des-1-6-IGF-II analog, which has >100-fold reduced affinity for IGFBP-4 than wild-type IGF-I or IGF-II. Thus, IGFBP-4 may act like soluble interleukin-1 receptor, which has been shown to inhibit interleukin-1 action by inhibiting the binding of interleukin-1 to its receptors(41) .
Figure 7: Proposed models for IGFBP-4 and IGFBP-5 actions in bone cells (see text for explanation). IGFBP-4 inhibits IGF binding to IGF receptors by binding IGFs near or at the receptor binding site. With regard to IGFBP-5, three alternate models are proposed. In model 1, the complex of IGFBP-5+IGF binds to IGF receptors. In model 2, IGFBP-5 binds to bone cell surface independent of IGF receptors and thus may stimulate cell proliferation via IGF independent mechanism. In model 3, IGFBP-5 binding sites in bone cell surface may recruit IGFs at the surface of IGF responsive cells, enabling the ligand to be easily captured by IGF receptors.
The molecular mechanisms by which IGFBP-5 stimulates IGF-induced bone cell proliferation appear to be more complex than those of IGFBP-4. It is possible that IGFBP-5 binds to IGF at a site distinct from that of IGFBP-4 and that the complex of IGFBP-5 and IGF binds to the receptor with higher affinity than IGFs alone. This would be analogous to a mechanism whereby the affinity of basic FGF to its receptor is increased upon binding to heparin sulfate(42) . Alternately, IGFBP-5 action may be mediated by IGFBP-5 binding to bone cells independent of IGF receptors. In this regard, IGFBP-5 may bind to extracellular matrix proteins such as fibronectin and may recruit and concentrate IGFs near IGF receptors. Based on the findings that the extracellular matrix-bound IGFBP-5 has lower affinity for the ligand than soluble IGFBP-5, Jones et al.(22) have proposed that extracellular matrix bound IGFBP-5 could facilitate delivery of IGF to IGF-I receptors. A similar mechanism of action has been proposed for IGFBP-3 by Conover and Powell(43) . In these studies, it has been proposed that soluble IGFBP-3 and IGFBP-5 may inhibit IGF actions when in high affinity states while extracellular matrix-bound IGFBPs stimulate IGF actions when their affinity is lower and approximates the affinity of the IGF-I receptor. If IGFBP-5 stimulates IGF actions primarily via increasing the delivery of IGFs to their receptors, we would then anticipate IGFBP-5 to be more active in the presence of IGF-I than in the presence of des-1-3-IGF-I, which has >200-fold reduced affinity than IGF-I for IGFBP-5. However, this was not the case. These findings together with the findings that the mitogenic activities of the maximally active concentrations of IGF-I or IGF-II were increased by treatment with IGFBP-5 are consistent with the model that IGFBP-5 may mediate some of its actions via binding to its own receptors.
The idea that IGFBPs can have intrinsic (IGF-independent) biological activity is also evident from other studies. For example, Cohen et al.(44) have shown that the growth rates of the IGFBP-3 transfected fibroblasts, when grown in insulin-containing media, were not restored to those observed in plasmid-transfected control cells, suggesting that the expression of endogenous IGFBP-3 has an inhibitory effect on cell growth, which is IGF independent. Oh et al.(45) have also recently shown evidence for inhibition of cell growth by exogenous IGFBP-3 in Hs578T human breast cancer cells via an IGF-independent mechanism. In addition, Villaudy et al.(46) have found differences in the biological effects between IGFBP-3 and IGFBP-1 and postulated that IGFBP-3 has multifunctional properties and has a function different from its known function to bind IGFs. Although these findings are consistent with the notion that IGFBPs may modulate some of their effects independent of IGFs via binding to their own binding sites/receptors, further studies involving the identification of these signal-transducing receptors and establishment of the cause and effect relationship between IGFBP binding to its potential receptor and IGFBP actions are needed to verify this mode of action for IGFBPs. The isolation of any such high affinity IGFBP receptors has been made more complicated by bone cell production of a number of abundant extracellular matrix proteins to which IGFBP-5 also binds.
In summary, we have demonstrated that 1) IGFBP-4 inhibits while IGFBP-5 stimulates IGF-induced bone cell proliferation under identical culture conditions, 2) IGFBP-4 inhibits the binding of IGF-I tracer to the type I IGF receptor and IGFBP-4 may modulate its effects on bone cell proliferation by acting like soluble interleukin-1 receptor, and 3) IGFBP-5 binds to bone cells and IGFBP-5 may modulate its effects on bone cell proliferation via an IGF-independent mechanism.