(Received for publication, July 12, 1995; and in revised form, August 30, 1995)
From the
Osteoblast-like cells secrete insulin-like growth factor (IGF)
binding protein-5 (IGFBP-5), which may act to enhance IGF-stimulated
osteoblast function. We recently demonstrated that carboxyl-truncated
IGFBP-5 (IGFBP-5) binds to the osteoblast surface
and stimulates mitogenesis by a pathway that is independent of IGF
action. The present study was conducted to determine the mechanism of
osteoblast binding of IGFBP-5, beginning with the assumption that cell
surface glycosaminoglycans may mediate the binding of this heparin
binding protein. Intact
I-IGFBP-5 and
I-IGFBP-5
exhibited one-site
binding to mouse osteoblast monolayers with dissociation constants of
28 and 6 nM for intact
I-IGFBP-5 and
I-IGFBP-5
, respectively. Osteoblast
binding of intact
I-IGFBP-5 was inhibited by low heparin
concentrations, while
I-IGFBP-5
binding was stimulated by heparin. Treatment of cells with heparinase
or chlorate to decrease surface glycosaminoglycan density failed to
reduce the binding of either form of IGFBP-5. In contrast, pretreatment
of cells with IGFBP-5 caused down-regulation of
I-IGFBP-5
binding. Cross-linking studies revealed that both intact
I-IGFBP-5 and
I-IGFBP-5
bind to proteins in Triton extracts of osteoblast membranes,
which were absent in osteoblast-derived matrix. Purification of
membrane extracts by IGFBP-5 affinity chromatography revealed a 420-kDa
band on reduced SDS-polyacrylamide gels. While the membrane protein
internalized both forms of IGFBP-5, heparin treatment inhibited the
internalization of intact
I-IGFBP-5 but stimulated
I-IGFBP-5
internalization. These
data indicate that IGFBP-5 binds to and is internalized by an
osteoblast membrane protein, which does not appear to be a
proteoglycan. Glycosaminoglycans, however, modulate the binding and
internalization of IGFBP-5 in a way that may preferentially favor the
intracellular accumulation of the carboxyl-truncated form.
Recently, we demonstrated that a carboxyl-truncated form of
IGFBP-5, ()derived from human osteoblast-like cells,
enhanced the mitogenic action of IGF-I in cultured mouse
osteoblasts(1, 2) . In addition to its IGF-enhancing
action, we also found that human recombinant carboxyl-truncated
IGFBP-5
could bind to osteoblast monolayers and
stimulate mitogenesis without exogenous IGF-I(3) , similar to
the effects of native carboxyl-truncated IGFBP-5(2) . While it
was proposed that this intrinsic mitogenic activity was mediated by
cell surface binding, the mechanism of IGFBP-5 binding to osteoblasts
was not determined.
IGFBP-5 is one of several IGFBPs that bind heparin(4) , most likely due to the presence of one or more putative heparin binding domains(5, 6) . Heparin and other sulfated glycosaminoglycans (GAGs) decrease the binding of IGF-I to IGFBP-5 (7) and inhibit the degradation of intact IGFBP-5(8) . The latter effect, reminiscent of the protection afforded by heparin on fibroblast growth factor degradation(9) , may result from a heparin-induced altered conformation that prevents enzymatic proteolysis. Heparin also modifies the binding and internalization of fibroblast growth factor in cultured cells(10, 11) , acting in competition with the GAG moieties located on cell surface proteoglycans.
Because IGFBP-5 binds to heparin and possibly to GAG-containing proteoglycans located on osteoblast surfaces or within osteoblast-derived extracellular matrix (ECM), we examined whether heparin modifies the binding of IGFBP-5 in primary cultures of normal mouse osteoblasts. This report characterizes the binding properties of two forms of IGFBP-5 and establishes the importance of a high molecular weight osteoblast membrane protein in osteoblastic cells that binds IGFBP-5.
Figure 3:
Competitive binding of glycosaminoglycans
with intact I-IGFBP-5 to mouse osteoblasts. Osteoblast
monolayers maintained in serum-free medium were exposed to intact
I-IGFBP-5 with increasing concentrations of heparan
sulfate, dermatan sulfate, chondrointin sulfate-A, or HSP.
Cell-associated radioactivity was determined as described in Fig. 1.
Figure 1:
Binding of intact I-IGFBP-5 and
I-IGFBP-5
to mouse osteoblasts. Confluent monolayers maintained in
serum-free medium were exposed to intact
I-IGFBP-5 (A) or
I-IGFBP-5
(B) with increasing concentrations of unlabeled IGFBP-5 for 2
h at 4 °C. The cells were washed and solubilized, and the
cell-associated radioactivity was determined. Insets depict
the Scatchard analysis for each.
Competition binding assays reveal that both intact I-IGFBP-5 and
I-IGFBP-5
specifically bind to
osteoblast monolayers (Fig. 1). Maximum binding averaged 24
fmol/10
cells for intact
I-IGFBP-5 and 4.5
fmol/10
cells for
I-IGFBP-5
; half-maximal
displacements occurred with 120 and 30 nM of unlabeled binding
protein for intact and carboxyl-truncated IGFBP-5 binding,
respectively. Scatchard analysis revealed a best fit for one-site
binding for both forms of IGFBP-5; the K
for
intact IGFBP-5 binding was 28 nM (Fig. 1A),
and the K
for IGFBP-5
binding was 6 nM (Fig. 1B). It is
assumed that IGFBP-5 binding is specific for osteoblast-like cells
since these studies were performed with primary cultures that were used
within 7 days of plating to minimize growth of potential contaminating
cell types, such as fibroblasts.
Because IGFBP-5 is a known heparin
binding protein, heparin-like molecules on the cell surface or within
the ECM could modify osteoblast binding of IGFBP-5 similar to their
action with other heparin binding proteins, such as fibroblast growth
factor(10) . To evaluate this possibility, heparin was used as
a competitor for IGFBP-5 binding to osteoblast monolayers (Fig. 2). Heparin at low concentrations inhibited the binding of
intact I-IGFBP-5 with half-maximal inhibition occurring
at 0.3 µg/ml (Fig. 2A). In contrast, osteoblast
binding of
I-IGFBP-5
was not
inhibited until heparin concentrations exceeded 30 µg/ml. Notably,
heparin concentrations in the range of 0.3-3 µg/ml enhanced
IGFBP-5
binding up to a maximum of 26% above
control values (Fig. 2B). Competitive binding studies
of intact
I-IGFBP-5 with heparan sulfate, dermatan
sulfate, chondroitin sulfate-A, and mouse-derived soluble heparan
sulfate proteoglycan (HSP) were compared to heparin's inhibition
of IGFBP-5 binding to determine whether differences in sulfation of the
GAG moieties affected the interaction of GAG side-chains with intact
IGFBP-5 (Fig. 3). The relative inhibition of
I-IGFBP-5 binding correlated with the degree of GAG
sulfation: heparin > heparan sulfate > dermatan sulfate. Neither
chondroitin sulfate-A nor HSP inhibited
I-IGFBP-5 binding
at concentrations of 0.1-3 µg/ml. While the results with the
soluble HSP suggest that this particular proteoglycan and its protein
backbone does not compete for IGFBP-5 binding, it is possible that the
protein structure of other proteoglycans may be competitive.
Figure 2:
Competitive binding of heparin with intact I-IGFBP-5 and
I-IGFBP-5
to mouse osteoblasts.
Osteoblast monolayers maintained in serum-free medium were exposed to
intact
I-IGFBP-5 (A) or
I-IGFBP-5
(B) with
increasing concentrations of heparin for 2 h at 4 °C.
Cell-associated radioactivity was determined as described in Fig. 1.
To
determine whether the contrasting effects of heparin on intact and
carboxyl-truncated IGFBP-5 binding were related to possible differences
in heparin binding affinity, both forms of IGFBP-5 were immobilized
onto nitrocellulose and tested for their ability to bind
[H]heparin(13) . As shown in Fig. 4A, intact IGFBP-5 bound 4-5-fold more
[
H]heparin than IGFBP-5
.
The finding that intact IGFBP-5 has a higher affinity for heparin by
this method was verified by studies with heparin-agarose
chromatography. Intact
I-IGFBP-5 eluted from the
heparin-agarose column with 0.8 M NaCl while
I-IGFBP-5
eluted from the column
with 0.24 M NaCl (data not shown). This difference in heparin
affinity may be explained on the basis of intact IGFBP-5 having a more
effective heparin binding domain compared to the carboxyl-truncated
form. For example, a putative heparin binding domain within intact
IGFBP-5 is predicted to exist within 201-218 amino acid residues,
in which a cluster of basic amino acids with the known heparin binding
motif, XBBBXXBX, is found, where B is a basic amino
acid(5) . In contrast, IGFBP-5
does not
contain a known heparin binding motif, although there is a cluster of
basic amino acids in the 133-143 region. To evaluate the relative
heparin affinities of these basic regions of IGFBP-5, the synthetic
peptides IGFBP-5
and IGFBP-5
were immobilized onto nitrocellulose, and
[
H]heparin binding was quantitated as described
by Baird et al.(13) (Fig. 4B). Under
these conditions IGFBP-5
consistently bound
more heparin than IGFBP-5
, although the latter
consistently displayed low level heparin binding (background < 200
cpm). While it is unknown whether there were differences in peptide
affinity for nitrocellulose, the findings are consistent with the
notion that the 201-218 region of IGFBP-5 contains a bona fide
heparin binding domain, and its presence may help explain the higher
heparin affinity of the intact form of IGFBP-5.
Figure 4:
[H]Heparin binding
to IGFBP-5 and IGFBP-5 peptides. [
H]Heparin
binding to intact IGFBP-5 and IGFBP-5
(A) and IGFBP-5
and
IGFBP-5
(B) was determined following
the adsorption of the binding proteins to nitrocellulose filters as
described under ``Experimental Procedures.'' After washing
the filters to remove unbound [
H]heparin,
specific binding of [
H]heparin onto the filters
was determined by subtracting background from total
cpm.
While these data
suggest that the major differences in heparin inhibition of intact and
carboxyl-truncated I-IGFBP-5 binding to osteoblasts are
related to their different heparin binding affinities, they do not rule
out the possibility that cell surface GAGs are also involved in binding
IGFBP-5. To evaluate whether GAGs mediate the binding of IGFBP-5 to the
osteoblast surface, confluent monolayers were pretreated with
heparinase to remove GAG moieties from osteoblast-surface heparan
sulfate proteoglycans before performing binding studies with
I-IGFBP-5. As shown in Table 1, pretreatment with
heparinase did not alter osteoblast binding of either intact or
carboxyl-truncated
I-IGFBP-5, suggesting that cell
surface GAGs are not the mediators of this interaction. To verify that
the heparinase treatment removed cell surface GAGs, heparinase
treatments were performed on cells prelabeled with
[
S]sulfate. Approximately 20% (22 ± 3%)
of the cell-associated
S-labeled material was released
into the medium. An additional 52 ± 4% of the cell-associated
S-labeled material was released by trypsin digestion,
presumably from chondroitin sulfate and/or dermatan sulfate. In
addition, 26 ± 4% of the cell-associated
S-labeled
material was resistant to both heparinase and trypsin digestion. Thus,
even though heparan sulfate proteoglycans may account for less than
one-fourth of the total cell surface GAG content, this should be enough
to alter binding of IGFBP-5 if GAGs were the principal binding site.
To examine whether sulfation of cell surface proteoglycans are
important in mediating IGFBP-5 binding, I-IGFBP-5 binding
studies were performed in osteoblasts that were grown in the presence
of sodium chlorate to prevent the sulfation of GAG
side-chains(10, 28) . As shown in Table 2,
chlorate treatment resulted in a 70% reduction in sulfate
incorporation, without altering the cellular binding of intact
I-IGFBP-5. Similar results were obtained with
I-IGFBP-5
binding (data not shown).
While these data strongly suggest that proteoglycans are not the
primary binding site, they do not completely exclude this possibility
owing to the variable resistance of heparan sulfate to chlorate
treatment.
To further evaluate the mechanism of IGFBP-5 binding to
osteoblasts, affinity labeling of both forms of I-IGFBP-5
to osteoblast monolayers was performed using the bifunctional
cross-linking agent DSS. As shown in Fig. 5, both intact and
carboxyl-truncated
I-IGFBP-5 bound to a 420-kDa
Triton-extractable membrane protein (450-kDa band minus 30-kDa
IGFBP-5). Affinity labeling was greater for intact than for
carboxyl-truncated IGFBP-5, and neither form cross-linked to ECM
proteins deposited by osteoblasts (lane 3). When the cells
were pretreated with either heparinase or chondroitinase, no decrease
in affinity labeling of the 420-kDa protein was evident when compared
to untreated cells (Fig. 6). While these results suggest that
both forms of IGFBP-5 may bind to the same membrane protein, it is
possible that the molecular weight assignments for each are not truly
identical and that more than one membrane protein may be present for
each form of IGFBP-5.
Figure 5:
Affinity labeling of intact I-IGFBP-5 and
I-IGFBP-5
to mouse osteoblast
monolayers and ECM. Confluent monolayer cultures of mouse osteoblasts
maintained in serum-free medium were exposed to binding buffer
containing intact
I-IGFBP-5 or
I-IGFBP-5
in the absence or
presence of unlabeled IGFBP-5 for 2 h at 4 °C. After rinsing with
binding buffer, the cells were incubated in BSA-free binding buffer
containing DSS (final concentration, 0.135 mM) for 15 min at 4
°C. The cells were rinsed, detached, and solubilized with buffer
containing 1% Triton X-100 and protease inhibitors as described under
``Experimental Procedures.'' Following centrifugation, the
supernatant was mixed with electrophoresis sample buffer containing
2-mercaptoethanol and separated on a 4-10% SDS-polyacrylamide
gel. Affinity labeling of intact
I-IGFBP-5 to
osteoblast-derived ECM (lane 3), prepared as described under
``Experimental Procedures,'' was performed under the same
conditions as for the monolayers. Intact
I-IGFBP-5 (lanes 1-3) or
I-IGFBP-5
(lanes 4 and 5) cross-linked to osteoblast monolayers without (lanes 1 and 4) or with (lanes 2 and 5)
unlabeled intact IGFBP-5 (lane 2) or
IGFBP-5
(lane 5). Molecular mass
standards in kDa are on the left.
Figure 6:
Effect of heparinase and chondroitinase
on affinity labeling of intact I-IGFBP-5 to osteoblast
cells. Confluent monolayers of mouse osteoblasts maintained in
serum-free medium containing 0.1% BSA were pretreated with heparinase
(10 units/ml) or chondroitinase (1 units/ml) for 1 h at 37 °C. The
cells were rinsed and then incubated in binding buffer containing
intact
I-IGFBP-5 for 2 h at 4 °C before being
cross-linked with DSS and analyzed by SDS-polyacrylamide gel
electrophoresis as described in Fig. 5.
To determine whether a similar sized membrane protein could be isolated from mouse osteoblasts, Triton X-100 extracts of mouse osteoblast membranes were applied to an IGFBP-5 affinity column. Following extensive washing of the column, protein eluting from the column was identified as a single 420-kDa band on silver stain of a reduced 5% SDS-polyacrylamide gel (Fig. 7). Because of its position at the top of the gel, the molecular weight assignment may be an underestimate.
Figure 7: IGFBP-5 affinity purification of a 420-kDa membrane binding protein from mouse osteoblasts. Triton X-100 extracts of osteoblast membranes were applied to an IGFBP-5 affinity column as described under ``Experimental Procedures.'' After extensive washing of the column, the membrane protein was eluted with 10 mM sodium acetate, pH 5.0, 1.5 M NaCl, 0.2% Triton X-100, 1 mM PMSF, concentrated with a Centricon-30 filter, and separated through a 5% SDS-polyacrylamide gel and silver stained. Numbers on the left represent the molecular mass markers in kDa.
To assess whether the osteoblast binding site for
IGFBP-5 was capable of being down-regulated, cells were incubated with
IGFBP-5 for various times, and cell-associated IGFBP-5 was removed with
2 M NaCl and 20 mM sodium acetate washes before
quantifying surface binding of I-IGFBP-5. Maximal
specific binding was 22 fmol/10
cells, indicating that the
washing conditions did not alter normal binding. As shown in Fig. 8, binding of intact
I-IGFBP-5 and
I-IGFBP-5
to osteoblast monolayers
was reduced to 40 and 35% of maximal binding after 1 and 2 h of
preincubation with IGFBP-5, respectively. The inhibitory effect was
also seen with a more prolonged exposure time (18 h). This is felt to
represent down-regulation of the membrane binding site rather than
competition with residual IGFBP-5, since the cells had been extensively
washed with 2 M NaCl and 20 mM sodium acetate to
remove cell surface IGFBP-5 before performing the
I-IGFBP-5 binding studies. Additional cultures labeled
with
I-IGFBP-5 confirmed that the NaCl-acetate washes
remove 89% of
I-IGFBP-5 bound at 4 °C (data not
shown). Because proteoglycans have not been shown to down-regulate in
response to a ligand, these data further support the notion that the
membrane protein is not a proteoglycan.
Figure 8:
Down-regulation of intact I-IGFBP-5 and
I-IGFBP-5
binding to mouse
osteoblasts. Confluent cultures of mouse osteoblasts were incubated in
serum-free DMEM containing 0.1% BSA without or with 3 µg/ml of
either intact IGFBP-5 or IGFBP-5
for 1, 2, or 18
h at 37 °C. To remove surface-bound IGFBP-5, the cells were rinsed
twice with cold 2 M NaCl in 20 mM sodium acetate, pH
4.0, twice with 2 M NaCl in 20 mM HEPES, pH 7.5, and
twice with cold PBS to remove excess salt. The monolayers were labeled
and quantitated for surface binding of
I-IGFBP-5 and
I-IGFBP-5
as described in Fig. 1.
Because earlier experiments
demonstrated that low concentrations of heparin enhanced the binding of I-IGFBP-5
, internalization
experiments were performed to determine whether the cellular uptake of
IGFBP-5
was also increased by heparin. In these
experiments, cell-associated radioactivity was removed with 2 M NaCl and 20 mM sodium acetate. As shown in Fig. 9,
internalization of
I-IGFBP-5
was
stimulated by 26% with low heparin concentrations (0.3-1.0
µg/ml) and enhanced by 80 and 120% with heparin concentrations of 3
and 10 µg/ml, respectively. In contrast, internalization of intact
I-IGFBP-5 was decreased at all heparin concentrations in
a dose-dependent manner, consistent with the inhibitory effect of
heparin on the cellular binding of intact IGFBP-5 (Fig. 2).
Figure 9:
Effect of heparin on the internalization
of I-IGFBP-5
and intact
I-IGFBP-5. Confluent osteoblast monolayers maintained in
serum-free medium were incubated with
I-IGFBP5
(A) or intact
I-IGFBP-5 (B) without or with increasing
concentrations of heparin for 2 h at 37 °C. The cells were
sequentially washed with PBS, 2 M NaCl in 20 mM HEPES, pH 7.5, and 2 M NaCl in 20 mM sodium
acetate, pH 4.0, to remove cell-associated
I-IGFBP-5. The
cells were then extracted with 0.5% Triton X-100 in 0.1 M sodium phosphate, pH 8.1, to release internalized radioactivity.
Values represent the mean of triplicate cultures from a representative
experiment.
These data demonstrate that IGFBP-5 binds to a membrane
protein isolated from primary cultures of mouse osteoblast-like cells.
Although the exact cell types were not identified, it is presumed that
IGFBP-5 binding was specific for osteogenic cells since they were
primary cultures enriched for cells of the osteoblast lineage. While
the identity of the cell surface protein was not revealed by these
studies, its apparent absence from osteoblast-drived ECM suggested that
the membrane protein was not a secreted proteoglycan. This notion is
further supported by the following findings: solubilized mouse heparan
sulfate proteoglycan does not compete for IGFBP-5 binding; heparinase
treatment, to remove cell surface GAGs, and chlorate treatment, to
prevent sulfation of proteoglycans, do not alter IGFBP-5 binding;
IGFBP-5 binding is down-regulated by prior exposure to the ligand; the
protein's appearance on affinity cross-linking gels and silver
stain is not typical of the diffuse gel mobility, which characterizes
large molecular weight proteoglycans. Finally, the membrane protein may
be involved in receptor-mediated signal transduction, since earlier
studies demonstrated that IGFBP-5 directly
stimulates mitogenesis in mouse osteoblasts(3) .
Even though
osteoblast-surface GAGs do not mediate binding of IGFBP-5, GAG moieties
are important in mediating the effect of heparin on osteoblast binding
and internalization of IGFBP-5. Low concentrations of soluble heparin
inhibit the cellular binding of intact but not IGFBP-5 apparently because intact IGFBP-5 binds more heparin than
IGFBP-5
. This may be due, in part, to the heparin
binding domain within amino acids 201-218 having a higher
affinity for heparin than the 133-143 region. Conformational
differences may also play a role if, for example, carboxyl truncation
results in a tertiary structure that binds heparin with lower affinity.
Since heparin-like molecules are important in many biochemical and
cellular functions(18) , it is noteworthy that specific
GAG-induced alterations in the function of IGFBP-5 have recently been
identified(7, 8) . One particularly relevant finding
is that GAG molecules having O-sulfated iduronic regions
prevent proteolysis of intact IGFBP-5, the effect of which directly
correlates with the extent of GAG sulfation: heparin > heparan
sulfate > dermatan sulfate(8) . In the present study, the
degree of GAG sulfation was similarly correlated with the inhibition of
intact
I-IGFBP-5 binding to osteoblasts where heparin was
the most inhibitory and dermatan sulfate the least. Chondroitin
sulfate-A, which lacks O-sulfated iduronic residues, did not
affect IGFBP-5 binding in this study and failed to inhibit IGFBP-5
degradation in the study by Arai et al.(8) . Thus, one
potential effect of selected sulfated GAGs that are localized within
osteoblast-derived ECM would be to sequester intact IGFBP-5 and allow
for cellular binding of IGFBP-5
. This may be one
explanation for the preferential localization of intact but not
truncated IGFBP-5 to fibroblast-derived ECM(19) .
The
mechanism for the enhanced internalization of IGFBP-5 by heparin is unknown. While heparin increased osteoblast binding
of IGFBP-5
, this did not exceed a 26% stimulation (Fig. 2), which corresponds to less than 1 fmol/10
cells of additional IGFBP-5
internalized.
Thus, the enhanced internalization (an additional 80-120%)
induced by higher heparin concentrations (3 and 10 µg/ml) must
result from a mechanism other than enhanced binding. For example,
heparin may stimulate the internalization of the membrane
protein
IGFBP-5
complex by making the
complex more resistant to proteolysis. Irrespective of the mechanism,
heparin causes a shift in the ratio of IGFBP-5
to
intact IGFBP-5 being internalized, resulting in the preferential
intracellular accumulation of the carboxyl-truncated fragment. This
change may induce specific intracellular events that might otherwise be
absent if GAG-associated molecules were not present near the cell
surface.
Proteolytic cleavage of IGFBP-5 may be important in determining the extent of IGFBP-5 binding to osteoblasts, especially as it relates to the formation of carboxyl-truncated fragments. Medium conditioned by osteoblast-like cells contains carboxyl-truncated forms of IGFBP-5(2) , and cultured osteoblasts secrete proteases that cleave IGFBPs(20, 21) , including IGFBP-5. While some of the IGFBP-5 proteases may not be unique to osteoblasts(22, 23, 24) , their production of truncated forms of IGFBP-5 having reduced affinity for ECM (19) suggests a mechanism for enhanced cellular binding of IGFBP-5 fragments. Regulation of protease activity would be an additional mechanism for control of IGFBP-5 binding to osteoblasts, either through direct stimulation of enzyme activity (25) or through ECM-associated inhibition of protease action (8) . The ECM constituency would then be important in the cell-specific response to degradation of intact IGFBP-5, depending, in part, on the type and amount of GAG that is present in the pericellular environment.
Binding of IGFBPs to cell surfaces (26, 27, 28, 29, 30) and modulation of IGFBP binding by heparin (28) have been previously reported. While the mechanism for binding was not revealed in those studies, Oh et al. have recently demonstrated that IGFBP-3 binds to specific membrane proteins (20, 26, and 50 kDa) located on human Hs578T breast cancer cells(30) , which may be responsible for mediating the growth inhibitory effect of IGFBP-3(29) . More recently, Booth et al.(28) have shown that intact IGFBP-5 binds to microvascular bovine endothelial cells and that its cellular binding is inhibited by sulfated GAGs but not by heparinase or chlorate treatment of the cells, similar to the results of the present study with mouse osteoblasts. Taken together, these data indicate that IGFBP-3 and IGFBP-5 have specific binding sites on cell surfaces that are not proteoglycans and suggest that their cellular binding may be cell-type dependent. Identifying these membrane binding proteins will be important in determining the mechanisms of the independent actions of IGFBPs in directly stimulating (2) and inhibiting (29) cell growth and differentiated cell functions.
It is likely that IGFBP-5 has a role in normal bone cell physiology through its osteoblast stimulatory effect. IGFBP-5 has been extracted from mineralized bone matrix where it appears to sequester IGF-I and -II for potential use in bone formation(31) . IGFBP-5 production in cultured osteoblasts is stimulated by parathyroid hormone(32, 33) , which may help explain its anabolic action on bone in osteoporosis (34, 35, 36) . The bone loss induced by glucocorticoids (37) may result, in part, from the inhibition of osteoblast-derived IGFBP-5 (38) to cause decreased bone formation. While intact (31) and carboxyl-truncated IGFBP-5 (2) have been shown to enhance IGF-stimulated osteoblast mitogenesis in vitro, their effect on differentiated osteoblast functions, such as collagen synthesis, has not been reported. Nevertheless, endogenous IGFBP-5 may have a role in the anabolic action of IGF-I in experimental osteoporosis(39) , particularly if IGF-I stimulates IGFBP-5 production as well in vivo as it does in vitro(40) . Whether bioactive truncated forms of IGFBP-5 directly promote bone formation in vivo remains to be determined.