 |
INTRODUCTION |
Skeletal growth is a complex phenomenon involving numerous
regulatory mechanisms that modulate the dynamic equilibrium of the
growth plate. Mesenchymally derived growth plate chondrocytes proliferate and terminally differentiate to produce a cartilage template for linear growth. Several growth factors are involved in this
process, but how this tightly ordered equilibrium is controlled is
still not well understood. Insulin-like growth factors
(IGFs)1 appear to play a
central role in chondrogenesis, but studies directed at clarifying the
precise mechanisms of IGF action have led to contradictory results and
theories (1). The somatomedin hypothesis postulates that IGF-I produced
in extraskeletal tissues is transported to the growth plate, where it
acts as an endocrine factor. The dual effector theory advocates that
IGF-I is produced in the growth plate under the stimulation of growth
hormone (GH) and acts as a paracrine/autocrine factor; GH may also act
independently, by promoting the differentiation of resting chondrocytes
to chondrocytes (1). However, to date, clinical and experimental
evidence does not definitively support either theory. Moreover, the
regulatory action of IGF-binding proteins (IGFBPs) in chondrogenesis
requires careful evaluations.
IGFBPs are part of an IGFBP/IGFBP-related-protein superfamily, of which
six proteins (termed IGFBP-1 to -6) bind IGFs with high affinity (3).
IGFBP-3 is the major circulating IGFBP present during postnatal life
(2). IGFBP-3 is GH-dependent, prolongs IGF half-life, and
carries IGF to target tissues. In addition, a direct effect of IGFBP-3
on cell proliferation that is independent of IGF binding has been
demonstrated in several cell lines, and this effect may involve
association with cell-membrane proteins (2, 4, 5). Based upon an
increasing understanding of the IGFBP system, we have recently proposed
an IGFBP regulatory hypothesis as part of an integrated model of the
GH-IGF-IGFBP-3 effects in the growth process (1). The proposed model
accounts for the effects of circulating IGFBPs, but the possibility of a direct, IGF-independent effect of IGFBP-3 on the growth plate has
yet to be addressed.
Studies directed at the evaluation of IGFBPs in chondrogenesis have
produced contradictory findings, primarily due to the lack of a good
in vitro model. In most studies, primary cultures of
chondrocytes have been used. Under these conditions, however, the
chondrocytes undergo a process that has been termed dedifferentiation (6), which is characterized by a change in shape, attachment, and a
loss of cartilage-specific markers. Despite these limitations, receptors for IGFs and GH have been identified in chondrocytes (7-11).
Some studies have reported that, in primary cultures of articular
chondrocytes, IGFBP-5 is the major IGFBP (12), whereas others have
reported that IGFBP-2 is the most abundant IGFBP in cultured
chondrocytes (13).
In the current study, we have employed a nontransformed clonal
chondrogenic cell line, RCJ3.1C5.18. The RCJ3.1C5.18 cell line is a
mesenchymal stem cell system that, without requiring biochemical or
oncogenic transformation, spontaneously and sequentially undergoes chondrocyte differentiation (14-16). We have previously reported that
RCJ3.1C5.18 cells sequentially acquire, over 2 weeks of culture, markers for chondrocytic differentiation and terminal differentiation (Table I) (14). Although our data are based upon an in vitro situation the morphology, the histochemical markers and the temporal sequential acquisition of the chondrocytic phenotype in this cell system is identical to the chondrogenesis process that occurs in
vivo. This makes our system ideal and unique for studying
chondrocyte cellular and molecular regulation, and suggests that our
findings are relevant to the in vivo process.
Using the RCJ3.1C5.18 in vitro model for chondrogenesis,
this study is aimed at: 1) evaluating the biological effects of IGFBP-3 on chondrocyte proliferation; 2) characterizing the relationship of
this effect with differentiation stage; and 3) examining the IGF-independent effect of IGFBP-3 on chondrocyte proliferation.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Human recombinant IGF-I, des-(1-3)-IGF-I
(des-IGF-I), and longR3 (LR3-IGF-I) IGF-I were
purchased from GroPep Pty. Ltd. (Adelaide, Australia). Des-(1-3)-IGF-
and LR3-IGF-I exhibit 30-100-fold reduced affinity for
IGFBP-3, but unaltered affinity for the type I IGF receptor compared
with IGFs (17, 18). IGF-I and IGF-II were kindly provided by Eli Lilly
Co. (Indianapolis, IN). Recombinant nonglycosylated human IGFBP-3 (mass ~29 kDa) expressed in Escherichia coli was
generously supplied by Celtrix Pharmaceuticals Inc. (Santa Clara, CA).
Recombinant human IGFBP-2 was purchased from Austral Biologicals (San
Ramon, CA). Recombinant human IGFBP-5 was purchased from GroPep. Human recombinant insulin was purchased from Sigma. A rabbit polyclonal antibody against human IGFBP-3,
IGFBP-3g1, was generated and characterized in our laboratory (19). A polyclonal antibody against
human IGFBP-2 that weakly recognizes IGFBP-3, called
HEC 1, was
generated and characterized in our laboratory (20). In rats,
HEC 1 has been characterized to be specific for IGFBP-2 (12). A polyclonal
antibody against rat IGFBP-4 was previously produced and characterized
in our laboratory (12, 21). IGFBP-3E. coli was
iodinated by a modification of the chloramine-T technique (5).
Iodinated glycosylated IGFBP-3 was generously provided by Diagnostic
Systems Laboratories Inc. (Webster, TX). Fetal bovine serum,
-minimal essential medium, and sodium pyruvate were purchased from
Life Technologies, Inc. Dexamethasone and
-glycerophosphate were
obtained from Sigma. Ascorbic acid was obtained from Wako Pure
Biochemicals Industries, Ltd. (Osaka, Japan).
Cell Culture--
RCJ3.1C5.18 cells, generously donated by Dr.
Jane E. Aubin (University of Toronto, Toronto, Ontario, Canada), were
grown in
-minimal essential medium supplemented with 15%
heat-inactivated fetal bovine serum, 10
7
M dexamethasone, and 2 mM sodium pyruvate.
Cells were plated at a density of 6 × 104 cells/well
in six-well dishes. After reaching confluence (4 days), fresh growth
medium supplemented with 50 µg/ml ascorbic acid and 10 mM
-glycerophosphate was added. Differentiating cells were fed again
with supplemented medium at days 7 and 10 of culture. Cultures were
monitored over a total period of 14 days. We have previously shown that
RCJ3.1C5.18 cells grown in this manner maintain their differentiated
chondrocytic phenotype (14). As shown in Table
I, cells sequentially acquire at 7 days
of culture markers of chondrocytic differentiation (type II collagen
and proteoglycans synthesis) and progressively acquire at 10 and 14 days of culture markers of terminal differentiation (type X collagen
and alkaline phosphatase activity).
Analysis of IGFBPs in Conditioned Media and Cell
Lysates--
Cell lysates were obtained from cells cultured for 4, 7, 10, and 14 days, and conditioned media were obtained from cells
cultured similarly but incubated for 24 h in serum-free media. For
preparation of cell lysates, cells were solubilized for 30 min at
4 °C in lysis buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 10% glycerol)
containing a mixture of protease inhibitors (Roche Molecular
Biochemicals, Mannheim, Germany) including 1 mM
phenylmethylsulfonyl fluoride. Cell lysates were cleared by
centrifugation and protein concentrations were determined by the Lowry
assay (Bio-Rad). Conditioned media (100 µl), cell lysates (10 µg of
proteins), or rat serum (2 µl) were subjected to Western ligand blot
analysis (WLB); a mixture of 125I-IGF-I and
125I-IGF-II (1.5 × 106 cpm of each) was
used as described previously (19, 22). Cell lysates (10 µg of
proteins) were subjected to immunoprecipitation as described previously
(19, 23) using
HEC 1 polyclonal antibody (20) or a polyclonal
antibody against rat IGFBP-4 (12, 21). Immunoprecipitates were
dissociated in SDS sample buffer, boiled, and centrifuged. Supernatants
electrophoresed in SDS-PAGE were subjected to WLB analysis, a mixture
of 125I-IGF-I and 125I-IGF-II (1.5 × 106 cpm of each) was used as described previously (19,
22).
Northern Blotting and Ribonuclease Protection Assays--
Total
RNA was obtained from cells cultured for 4, 7, 10, and 14 days
incubated in serum-free medium for 24 h. Total RNA was extracted
from cultured cells, as described by the manufacturer, using RNeasy
columns (Qiagen Inc., Santa Clarita, CA) and quantified by
spectrophotometric analysis. Northern blotting analysis was performed
as described previously (12) using described previously rat IGFBP-3 and
rat IGFBP-5 cDNA probes (24, 25). RNase protection assays were
conducted with 25-µg aliquots of total RNA using the Ambion RPA-II
kit (Ambion Inc., Austin, TX) according to the supplier's protocol.
The rat IGF-I and IGF-II probes used to generate antisense RNA probes
have been described previously (26, 27); a
-actin probe was included
as an internal control.
IGFBP-3 Proteolysis--
IGFBP-3 proteolysis was assessed in
conditioned media using IGFBP-3 Western immunoblotting analysis (WIB)
and an IGFBP-3 protease assay as described previously (28). Cells
cultured for 4, 7, 10, and 14 days were washed with PBS and treated
with recombinant human IGFBP-3 (30 nM) with or without
IGF-I (15 nM) or des-(1-3)-IGF-I (15 nM) in
serum-free media for 24 h. For IGFBP-3 WIB analysis, 100 µl
of conditioned media were processed, filters were probed with
IGFBP-3g1 polyclonal antibody (19), and enhanced chemiluminescence (ECL) (PerkinElmer Life Sciences) reagents were used. For IGFBP-3 protease activity assay, 100 µl of conditioned media were incubated with glycosylated 125I-IGFBP-3 (30,000 cpm). Densitometric
analysis was carried out using a GS700 Imaging Densitometer
(Bio-Rad).
Measurement of Cell Proliferation--
After 4, 7, 10, and 14 days of culture, cells were washed with PBS and changed to serum-free
medium for 4 h. Cells were then incubated with specified peptides
and [3H]thymidine (0.8 µCi/ml) in serum-free medium for
18 h. Incubations were terminated by washing with ice-cold PBS.
Incorporation of [3H]thymidine into DNA was determined as
uptake of radioactivity in trichloroacetic acid-precipitable material,
as described previously (29). Cells at days 4, 7, 10, and 14 of culture
were incubated in serum-free medium for 24 h; cell monolayers were
then trypsinized and cells counted in a hemocytometer.
Cell-surface IGFBP-3 Binding--
Affinity cross-linking of
IGFBP-3 to cell monolayers was performed as described by Massagué
et al. (30) with some modifications. Equilibrated cells were
incubated with 125I-IGFBP-3 (700,000 cpm) for 4 h at
4 °C with or without the indicated amount of cold peptides.
125I-IGFBP-3 ligand-receptor complexes were cross-linked
using 27 mM disuccinimidyl suberate (DSS) for 15 min at
4 °C. Cross-linking reaction mixture was aspirated and quenched by
addition of 10 mM Tris-containing detachment buffer. The
excess of Tris quenched the unreacted DSS in solution; samples were
processed as rapidly as possible to rapidly achieve the complete
quenching of residual DSS internalized into the cell compartments by
adding SDS-sample buffer. Cells were detached, lysed, and cell debris
removed by centrifugation. Supernatants were subjected to
immunoprecipitation using
-IGFBP-3g1 antibody as reported previously
(19, 23). Immunoprecipitates were dissociated by SDS-sample buffer,
boiled, centrifuged, and supernatants electrophoresed in SDS-PAGE under reducing and nonreducing conditions. Gels were dried and exposed to film.
Statistical Analysis--
Data are presented as means ± S.D. The statistical differences between means were assessed by
unpaired Student's t test or analysis of variance.
Statistical significance was set at p < 0.05.
 |
RESULTS |
Analysis of IGFBPs in Conditioned Media and in Cell
Lysates--
To determine the IGFBPs produced by RCJ3.1C5.18 cells,
conditioned media and cell lysates were obtained after 4, 7, 10, and 14 days of culture and subjected to WLB analysis. As shown in Fig.
1, the predominant IGF-labeled bands in
conditioned media and cell lysate were 32-29 and 24 kDa (Fig. 1,
lanes 1-4 and 5-8). To characterize the IGFBPs,
cell lysates were subjected to immunoprecipitation with specific
antisera against rat IGFBP-2 (
HEC 1) and rat IGFBP-4. In Fig. 1,
lane 11 is the WLB of the cell lysate. Immunoprecipitation with pre-immune rabbit serum showed the absence of any nonspecific bands (Fig. 1, lanes 13 and 17). The
antiserum against rat IGFBP-2 immunoprecipitated the 32-kDa band (Fig.
1, lanes 14 and 15) and the antiserum against rat
IGFBP-4 immunoprecipitated the 24-kDa band (Fig. 1, lane
18). The 29-kDa band identified by WLB was not immunoprecipitated
by any of the antibodies employed and may, therefore, correspond to
IGFBP-5. Northern blotting analysis was carried out, and
IGFBP-5 mRNA was identified in RCJ3.1C5.18 cells (data not
shown).

View larger version (68K):
[in this window]
[in a new window]
|
Fig. 1.
Characterization of IGFBPs in conditioned
media and cell lysates. Conditioned media and cell lysates were
obtained from cells cultured for 4, 7, 10, and 14 days (D)
and subjected to WLB analysis (lanes 1-10). For
conditioned media, cells were incubated for 24 h in serum-free
medium. Lanes 1-4, cell lysates obtained at the indicated
culture time point; lanes 5-8, the corresponding
conditioned media; lane 10, normal rat serum
(NRS). Cell lysate (CL, lane 11) was
subjected to immunoprecipitation using preimmune rabbit serum
(PI, lanes 13 and 17), or HEC
polyclonal antibody against IGFBP-2 ( BP-2, lanes 14 and
15), or a polyclonal antibody against IGFBP-4 ( BP-4,
lane 18) and then to WLB analysis.
|
|
The 43-39-kDa doublet that corresponds to intact IGFBP-3 in rat serum
(Fig. 1, lane 10) was not detected, even when cells were
treated with increasing doses (1-15 nM) of IGF-I. To
determine if the absence of intact IGFBP-3 was the result of specific
conditioned media protease activity, conditioned media was subjected to
protease assay. No IGFBP-3 proteolysis was detectable in conditioned
media of chondrocytes at any time point of the culture (data not shown).
Northern Blotting Analysis for IGFBP-3 and Ribonuclease Protection
Assay for IGFs--
We determined that no IGFBP-3 mRNA was present
in RCJ3.1C5.18 cells at any stage of differentiation by Northern
blotting analysis (Fig. 2, lanes
2-4). RNase protection assays revealed that no IGF-I or IGF-II
mRNA was expressed in RCJ3.1C5.18 cells at any stage of
differentiation (Fig. 3, lanes
4-7 and lanes 8-12). The fact that no IGFBP-3, IGF-I,
and IGF-II mRNAs were present in RCJ3.1C5.18 chondrogenic cells at
any time point of the cell culture makes this an ideal system for
studying the effects of IGFBP-3 and IGFs without interference from
endogenous peptides.

View larger version (88K):
[in this window]
[in a new window]
|
Fig. 2.
IGFBP-3 Northern blotting analysis.
Total RNA was obtained from cells cultured for 4 days (lane
2), 10 days (lane 3), or 14 days (lane 4)
incubated in serum-free medium for last 24 h. Total RNA was
extracted and Northern blotting analysis was performed using rat
IGFBP-3 cDNA probe. Lane 1 represents total RNA
extracted from rat liver. The 28 S ribosomal RNA bands are shown to
demonstrate equal RNAs loads in the different lanes.
|
|

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 3.
Ribonuclease protection assays for IGF-I and
IGF-II. Total cellular RNA was extracted from cells cultured for
4, 7, 10, and 14 days incubated in serum-free medium for the last
24 h. mRNA expression of IGF-I and IGF-II was determined by
RNase protection assay. A -actin probe was included in all the
samples as an internal control. Lanes 1-7 show the RNase
protection analysis using rat IGF-I probe. Lane 1, IGF-I and
-actin riboprobes; lane 2, digested probes; lane
3, RNA obtained from rat liver; lanes 4-7, samples
obtained at the indicated different time points of culture,
respectively: at 4 days (lane 5), 10 days (lane
6), and 14 days (lane 7). Lanes 8-14 show
the RNase protection analysis using rat IGF-II probe: lane
8, 4 days; lane 9, 7 days; lane 10, 10 days;
lanes 11 and 12, 14 days. Lane 14,
IGF-II and -actin riboprobes; lane 13, digested
probes.
|
|
Antiproliferative Action of IGFBP-3 in RCJ3.1C5.18 Chondrogenic
Cells: IGF-independent and IGF-dependent Effects--
We
next assessed the IGF-dependent and IGF-independent
antiproliferative actions of IGFBP-3 in RCJ3.1C5.18 chondrogenic cells and the relationship to cell differentiation stage. IGF-I, IGF-II, des-(1-3)-IGF-I, LR3-IGF-I, and insulin, promoted, in a
dose-dependent manner (1-15 nM), cell
proliferation at all stages of differentiation, as determined by
[3H]thymidine incorporation; at 15 nM, IGFs
and IGF analogs typically stimulated thymidine incorporation by
10-fold. IGFBP-3 inhibited DNA synthesis in progenitor (4 days of
culture) and early differentiated (7 days) chondrocytes stimulated by
insulin, des-(1-3)-IGF-I, and LR3-IGF-I by ~80% (Fig.
4, A and B). In
differentiated chondrocytes (10 days), 30 nM IGFBP-3
inhibited proliferation stimulated by insulin, des-(1-3)-IGF-I, and
LR3-IGF-I by ~40% (Fig. 4C). In terminally
differentiated chondrocytes (14 days), IGFBP-3 had no effect on DNA
synthesis stimulated by insulin, des-(1-3)-IGF-I, and
LR3-IGF-I (Fig. 4D); IGFBP-3 continued, however,
to inhibit cell proliferation stimulated by IGF-I by ~80% at all
stages of cell differentiation (Fig. 4, A-D).

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 4.
IGF-independent and IGF-dependent
effects of IGFBP-3 on DNA synthesis in RCJ3.1C5.18 chondrogenic cells:
relationship to cell differentiation stage. Cells cultured for 4, 7, 10, and 14 days were changed to serum-free medium for 4 h and
then incubated for an additional 18 h with
[3H]thymidine without and with IGFBP-3 and, respectively,
insulin, des-(1-3)-IGF-I, longR3-IGF-I, or IGF-I.
Incorporation of [3H]thymidine into DNA was determined as
uptake of radioactivity in trichloroacetic acid-precipitable material.
A, 4 days of culture; B, 7 days of culture;
C, 10 days of culture; D, 14 days of culture.
Results are expressed as percentage of the control, which was given an
arbitrary value of 100%.
|
|
During the early stages of chondrogenesis (days 4 and 7 of culture)
IGFBP-3 had a dose-dependent inhibitory effect on
des-(1-3)-IGF-I and LR3-IGF-I-stimulated proliferation,
with maximal antiproliferative effect seen at 30 nM (Fig.
5, A and B). In
terminally differentiated chondrocytes, however, IGFBP-3 retained the
ability to inhibit cell proliferation stimulated by IGF-I, but had no
effect on des-(1-3)-IGF-I cell or LR3-IGF-I-stimulated
proliferation, even at the highest dose of IGFBP-3 (Fig. 5,
C and D).

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 5.
Dose-dependent IGFBP-3 effect on
des-(1-3)-IGF-I-stimulated DNA synthesis. Cells cultured for 4, 7, 10, and 14 days were changed to serum-free medium for 4 h and
then incubated for an additional 18 h with
[3H]thymidine with IGF-I or des-(1-3)-IGF-I and
increasing doses of IGFBP-3. Incorporation of
[3H]thymidine into DNA was determined as uptake of
radioactivity in trichloroacetic acid-precipitable material.
A, 4 days of culture; B, 7 days of culture;
C, 10 days of culture; D, 14 days of culture.
Results are expressed as percentage of the control, which was given an
arbitrary value of 100%.
|
|
IGFBP-2, which has an equivalent or slightly higher affinity for
des-(1-3)-IGF-I than does IGFBP-3 (17), had no effect on des-(1-3)-IGF-I-stimulated [3H]thymidine incorporation,
but inhibited IGF-I-induced DNA synthesis (Fig.
6, A and B).

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of IGFBP-2 on
des-(1-3)-IGF-I-stimulated DNA synthesis. IGF-independent
antiproliferative effect of IGFBP-3 is specific. Cells cultured for 4 (panel A) and 7 days (panel B) were changed to
serum-free medium for 4 h and then incubated for an additional
18 h with [3H]thymidine and with the following
peptides: des-(1-3)-IGF-I; des-(1-3)-IGF-I and IGFBP-3;
des-(1-3)-IGF-I and IGFBP-2; IGF-I; IGF-I and IGFBP-3; IGF-I and
IGFBP-2. Results are expressed as percentage of the control, which was
given an arbitrary value of 100%.
|
|
IGFBP-5, which has been shown to have potential IGF-independent effects
(31-33), had no effect on insulin-stimulated DNA synthesis in early
differentiated (4 days) as well as in terminally differentiated chondrocytes (14 days). Thus, the IGF-independent effect of IGFBP-3 in
the early stages of chondrogenesis appears to be a peculiar effect of
this IGFBP. Furthermore, IGFBP-5, in contrast to IGFBP-3, inhibited
IGF-I-induced DNA synthesis at the early stage of chondrogenesis (4 days), but had no effect or even a slight enhancement of
IGF-I-stimulated proliferation in terminally differentiated
chondrocytes (14 days).
Cell growth was also assessed directly by cell counting. Treatment of
RCJ3.1C5.18 cells with des-(1-3)-IGF-I (15 nM) and IGFBP-3 (30 nM) at day 4 of culture decreased cell number by 39%,
compared with cells treated with des-(1-3)-IGF-I alone. At day 14 of
culture, however, IGFBP-3 had no effect on des-(1-3)-IGF-I-stimulated
proliferation. IGFBP-3 had a similar effect on cell proliferation
induced by LR3-IGF-I. At day 4 of culture, cells treated
with LR3-IGF-I (15 nM) and IGFBP-3 (30 nM) showed a 34% decrease of cell number compared with
cells treated with LR3-IGF-I alone. No IGFBP-3 effect
was seen on cells treated with LR3-IGF-I at day 14 of
culture, although IGFBP-3 continued to inhibit IGF-I-induced
proliferation by 45%.
Association of IGFBP-3 with Cell-surface Proteins--
We
performed IGFBP-3 WIB analysis to detect the amount of exogenously
added human recombinant IGFBP-3 (~29 kDa) presents in conditioned
media obtained from cells after 24 h incubation with IGFBP-3 alone
or with IGFBP-3 plus IGF-I or des-(1-3)-IGF-I. The amount of IGFBP-3
was decreased when cells were incubated with IGFBP-3 alone or with
des(1-3)-IGF-I (Fig. 7, lanes 1, 2, 4, 5, 7, 8, 10, and 11 ) as compared with
cells incubated with IGFBP-3 plus IGF-I (Fig. 7, lanes
3, 6, 9, and 12). This decrease
in exogenously added IGFBP-3 detectable in the conditioned media was
not due to inhibition of IGFBP-3 proteolysis in the presence of IGF-I, since no increase in IGFBP-3 fragments was detected by WIB analysis (Fig. 7) or by an IGFBP-3 protease assay (data not shown). These observations suggested the possibility that IGFBP-3 was associating with cell membranes, and that IGF-I, but not des-(1-3)-IGF-I, was
promoting dissociation of IGFBP-3 from the cell surface into the
conditioned media.

View larger version (78K):
[in this window]
[in a new window]
|
Fig. 7.
Western immunoblotting analysis for
exogenously added human recombinant IGFBP-3. Cells cultured for 4 days (lanes 1-3), 7 days (lanes 4-6),
10 days (lanes 7-9), and 14 days (lanes 10-12)
were treated with recombinant human IGFBP-3 with or without IGF-I or
des-(1-3)-IGF-I in serum-free media for 24 h. Conditioned media
were subjected to WLB analysis using -IGFBP-3g1 antibody.
Lanes 1, 4, 7, and 10 cells
incubated with IGFBP-3 alone; lanes 2, 5,
8, and 11, cells treated with IGFBP-3 and
des-(1-3)-IGF-I; lanes 3, 6, 9, and
12, cells incubated with IGFBP-3 and IGF-I; lane
13, human recombinant IGFBP-3 (30 nM); lane
14, serum pooled from pregnant women (2 µl).
|
|
To investigate binding of IGFBP-3 to cell-surface proteins,
125I-IGFBP-3 was cross-linked to RCJ3.1C5.18 cell
monolayers, immunoprecipitated by IGFBP-3 antibody, and the
coimmunoprecipitated proteins analyzed by SDS-PAGE. As shown in Fig.
8, a prominent ~50-kDa radiolabeled band was identified in the immunoprecipitate (Fig. 8, lanes
3-5 and lanes 8-9); presumably an ~21-kDa protein
cross-linked to the 29-kDa iodinated recombinant IGFBP-3. This
~50-kDa band was not present when 125I-IGFBP-3 tracer was
cross-linked in the absence of cells (Fig. 8, lanes 1 and
2). The appearance of the ~50-kDa band was inhibited by
coincubation of cells with unlabeled (cold) IGF-I (Fig. 8, lanes
6 and 7) or with unlabeled IGFBP-3 (Fig. 8, lanes
11 and 12). A predominant ~50-kDa band was also
detected when samples obtained by cross-linking
125I-IGFBP-3 to cell monolayers and immunoprecipitation
with IGFBP-3 antibody were electrophoresed in SDS-PAGE under
reducing conditions, confirming that the cross-linked complex
represents specific binding of 125I-IGFBP-3 to the cell
surface, rather than nonspecific adherence. The ~50-kDa
125I-IGFBP-3 monolayer affinity cross-linked band was
detectable at day 7 as well as at day 14 of cell culture.

View larger version (76K):
[in this window]
[in a new window]
|
Fig. 8.
Cell-surface IGFBP-3 binding. Affinity
cross-linking of IGFBP-3 to cell monolayers. Cells were incubated with
125I-IGFBP-3 without (lanes 3, 4,
8, and 9) or with unlabeled (cold) IGF-I
(lane 7) or IGFBP-3 (lanes 11 and 12).
125I-IGFBP-3 ligand-receptor complexes were cross-linked
with disuccinimidyl suberate. Supernatant were subjected to
immunoprecipitation using -IGFBP-3g1 antibody. Immunoprecipitates
were subjected to SDS-PAGE analysis, and gels were dried and exposed to
film. Lanes 1 and 2, tracer
cross-linked in the absence of cells and subjected to
immunoprecipitation. The arrow indicates cross-linked
complex of 125I-IGFBP-3 and cell surface-associated
protein.
|
|
 |
DISCUSSION |
In this study, we have demonstrated that IGFBP-3 has an
antiproliferative effect on RCJ3.1C5.18 cells, which, under in
vitro conditions, progress through the differentiation steps
observed in growth-plate chondrocytes in vivo. The
mechanisms involved in this growth-inhibitory effect of IGFBP-3 are
clearly related to the differentiation process. During the early stage
of RCJ3.1C5.18 differentiation, IGFBP-3 action appears to be
independent of its ability to bind IGF. This conclusion is supported by
the ability of nanomolar concentrations of IGFBP-3 to inhibit
stimulation of DNA synthesis and cell proliferation by insulin,
des-(1-3)-IGF-I, LR3-IGF-I, and IGF-I. In terminally
differentiated cells, on the other hand, the inhibitory effects of
IGFBP-3 become exclusively IGF-dependent. This transition
is marked by the failure of IGFBP-3 to inhibit des-(1-3)-IGF-I,
LR3-IGF-I, and insulin-induced DNA synthesis at day 10 and
day 14 of culture, despite continued inhibition of IGF-I stimulation.
To determine whether the IGF-independent effect of IGFBP-3 was
specific, we used IGFBP-2, which has a similar affinity for IGF-I, and
the IGF-I analog des-(1-3)-IGF-I (17), and IGFBP-5, which has been
shown to have potential IGF-independent effects (31-33). IGFBP-2 had
no effect on cell growth stimulated by des-IGF-I, but was capable of
inhibiting IGF-I-stimulated cell proliferation. IGFBP-5 had no effect
on insulin-stimulated DNA synthesis in early differentiated as well as
in terminally differentiated chondrocytes. Although we cannot exclude
that other IGFBPs or IGFBP-related proteins can have an IGF-independent
effect on chondrogenesis, this effect seems to be peculiar to IGFBP-3
and is not demonstrated by other IGFBPs which have similar affinities
for IGF and IGF analogs or have been shown to have IGF-independent
effects. Interestingly, IGFBP-5, in contrast to IGFBP-3, inhibited
IGF-I-induced DNA synthesis at the early stage of chondrogenesis, but
had no effect or even a slight enhancement of IGF-I-stimulated
proliferation in terminally differentiated chondrocytes. Enhancement of
IGF proliferative effect by IGFBP-5 has been demonstrated in other
systems, like osteoblasts, and it has been speculated that inhibition
of IGFBP-5 binding to extracellular matrix can facilitate the delivery
of IGF to IGF-I receptor (31-33).
IGFBP-3 binds to a membrane-associated protein located on chondrocytes,
and this binding can be totally ablated by IGF-I, presumably due to
dissociation of IGFBP-3 from the cell membrane. Binding of
125I-IGFBP-3 can also be competed by excess of unlabeled
IGFBP-3. This specific cell surface-associated protein has a mass of
~21 kDa (after subtracting 29 kDa of iodinated IGFBP-3).
Chondrogenesis is a complex phenomenon that results from the ordered
and sequential proliferation and differentiation of chondrocytes in the
growth plate. The GH-IGF system appears to play a crucial role in
chondrogenesis, but the relative contributions of GH and IGF to the
process have not been resolved (1). In RCJ3.1C5.18 chondrocytes, IGFs
and, interestingly, insulin, stimulate DNA synthesis and cell
proliferation at nanomolar concentrations. The present study reports an
IGF-independent antiproliferative effect of IGFBP-3 in the regulation
of normal cell growth in a system that mimics the in vivo
process of chondrogenesis. This finding suggests that this IGFBP-3
effect is not only important in the control of cancer and fibroblast
cell line growth, as reported previously (4, 5, 34), but also in
physiological conditions, such as chondrogenesis. We have demonstrated
that IGFBP-3 has an IGF-independent antiproliferative effect on
chondroprogenitors and early differentiated chondrocytes. The decrease
of chondroprogenitors leads to modulation of the number of cells that
will undergo terminal differentiation. Similar findings have been
reported for other signaling pathways such as the one initiated by the
fibroblast growth factor receptor-3 (FGFR-3) (34-36). In humans,
activating mutations of FGFR-3 lead to the most common forms of
chondrodysplasia, including achondroplasia (35, 36). Conversely, in the
mouse, null mutations in FGFR-3 cause skeletal overgrowth (37). These observations indicate that the normal function of FGFR-3 signaling is
to inhibit chondrocyte proliferation, although the mechanism is still
unclear. The gradually unfolding story of chondrogenesis is just
beginning, and we are starting to understand the effects of these
peptides; however, the mechanisms have yet to be characterized.
In the current study, the major IGFBPs detected by WIB analysis of
conditioned media and cell lysates have masses of 32-29 and 24 kDa. To
characterize the IGFBPs, cell lysates were subjected to
immunoprecipitation with specific antisera against rat IGFBP-2 and rat
IGFBP-4 (12, 20, 21). The antiserum against rat IGFBP-2
immunoprecipitated the 32-kDa band and the antiserum against rat
IGFBP-4 immunoprecipitated the 24-kDa band. The 29-kDa band identified
by WLB was not immunoprecipitated by any of the antibodies employed and
may, therefore, correspond to IGFBP-5. Northern blotting analysis was
carried out and IGFBP-5 mRNA was identified in RCJ3.1C5.18 cells.
In a previous study, we have reported a similar IGFBP pattern in
conditioned media from primary cultured rat articular chondrocytes (12). Different patterns of IGFBP expression, even within the same
species, have been reported in primary cultured chondrocytes (13, 38).
We attribute this inconsistency to the fact that primary cultured
chondrocytes undergo dedifferentiation, with loss of typical
chondrocyte markers and, furthermore, that cells are represented by
variable, from culture to culture, heterogeneous populations. In the
current study, employing a nontransformed cell system that in culture
maintains and sequentially expresses cartilage-specific markers,
neither IGFBP-3 protein nor mRNA was found, indicating that little,
if any, IGFBP-3 transcription and translation occurs in these cells.
This finding, associated with the absence of detectable IGF mRNAs,
makes this system ideal for the study of IGF-independent effects of
IGFBP-3, without interference from endogenous peptides.
The absence of IGFBP-3 in our chondrogenic cell system does not,
however, preclude the production and presence of IGFBP-3 in the growth
plate. Two hypotheses can be proposed: that cells other than
chondrocytes, present in the growth plate locally produce IGFBP-3,
which acts as a paracrine factor; or that IGFBP-3 can leave the
vascular space and has a direct action on the target tissue, thereby
constituting an IGFBP-3 endocrine action. In support of the first
hypothesis are the data of Wang et al. (39), showing by
in situ hybridization that IGFBP-3 mRNA in mouse embryos
is expressed only in the capillaries present in the perichondrium. The
possible validity of the second hypothesis is in the observation that
IGFBP-3 fragments can cross the capillary barrier and that IGFBP-3 and
its acid-labile subunit have been identified in synovial fluid (40,
41). A 29-kDa IGFBP-3 fragment purified from peritoneal dialysate
obtained from children with chronic renal failure has been shown to be
able to inhibit IGF-II-stimulated thymidine incorporation in a
chondrosarcoma cell line (42). This study, however, had the limitation
of employing a chondrosarcoma cell line that has not been demonstrated
to express chondrocyte differentiation markers, and only an
IGF-dependent mechanism for the IGFBP-3 effect was identified.
We have reported previously, in a breast cancer cell line, an IGFBP-3
receptor-like protein with a molecular weight similar to the specific
cell membrane-associated protein identified in RCJ3.1C5.18 cells (5).
It is of note that the current finding of a similar IGFBP-3 receptor,
as well as an IGF-independent effect of IGFBP-3, is in a nontransformed
cell system. Interestingly, the IGFBP-3 receptor was present in cells
at every differentiation stage, whereas the IGF-independent
antiproliferative effect was only evident during the early stages of
differentiation. Further studies are needed to determine whether
different post-receptor signaling mechanisms or intranuclear
localization processes for IGFBP-3 can explain this selective
differentiation-dependent, growth inhibitory effect of
IGFBP-3.
In conclusion, these studies demonstrate IGF-independent and
IGF-dependent effects of IGFBP-3 on chondrocyte
proliferation; the nature of these IGFBP-3 effects relates to the stage
of cellular differentiation. These findings provide new insight into
the biological actions of IGFBP-3, as well as the impact of cell
differentiation, and characterize a novel, informative model for the
investigation of the role of the IGF-IGFBP-3 system in the growth process.