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*
Division of Pediatric Nephrology, University Children's Hospital
Heidelberg, Heidelberg, Germany
Department of Medicine, Veterans Affairs Medical Center and University of
Washington, Seattle, Washington
J. L. Pettis Veterans Administration Medical Center and Loma Linda
University, Loma Linda, California
Lower Saxony Institute for Peptide Research, Hannover, Germany.
Correspondence to Dr. Burkhard Tönshoff, Division of Pediatric Nephrology, University Children's Hospital, Im Neuenheimer Feld 150, 69120 Heidelberg, Germany. Phone: 49-6221-562311; Fax: 49-6221-564203; E-mail: Burkhard_Toenshoff{at}med.uni-heidelberg.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, we (6) and others (7,8) demonstrated that IGFBP-4 contributes to the increased IGF-binding capacity of uremic serum. Serum levels of immunoreactive IGFBP-4 are increased fourfold in children and adults with CRF, compared with control values; this increase is attributable to elevated levels of both intact and fragmented IGFBP-4 (6,7,8). On a molar basis, serum IGFBP-4 is the second most abundant IGFBP in the serum of children with preterminal CRF (6). However, immunoreactive IGFBP-5 levels in CRF serum are normal, and the majority of IGFBP-5 is fragmented (6,7). Correlation analyses of growth parameters in clinical studies of children with CRF demonstrated that immunoreactive IGFBP-4 levels were inversely correlated with standardized height, consistent with the role of IGFBP-4 as another inhibitor of the biologic action of IGF on growth plate cartilage (6). However, serum IGFBP-5 levels were positively correlated with both standardized height and height velocity among children with CRF, consistent with a potential stimulatory role of this IGFBP on longitudinal growth (6,7).
Although it seems, on the basis of data from the clinical studies, that IGFBP-4 and -5 may have contrasting effects on longitudinal growth among uremic children, in vitro studies have not been conducted to delineate the respective roles of IGFBP-4 and -5 in normal cartilage growth. Studies with cultured normal osteoblasts (9) and other cells (10,11,12), however, have demonstrated that IGFBP-4 acts exclusively as an inhibitor of IGF action and that IGFBP-5 can either potentiate IGF-stimulated effects (12,13,14,15,16) or stimulate osteoblast activity via an IGF-independent pathway (13,16,17). Moreover, IGFBP-5 may also inhibit IGF action in other cell types, depending on experimental conditions (18,19).
Because of the potential importance of IGFBP-4 and IGFBP-5 in cartilage physiologic processes, the aim of this study was to investigate the proliferative effects of intact and fragmented forms of IGFBP-4 and IGFBP-5 on cultured growth plate chondrocytes, which express the type I IGF receptor (IGFIR) (20). Intact recombinant IGFBP-4 (21) and IGFBP-5 (22), a recombinant amino-terminal IGFBP-5 fragment (15), and defined amino-terminal and carboxy-terminal IGFBP-4 and -5 fragments isolated from hemofiltrates from patients with end-stage renal disease (23,24) were investigated. The bioactivity of these IGFBP-4 and IGFBP-5 fragments from end-stage renal disease serum, which might play a role in the growth failure of children with CRF, had not previously been directly tested. Furthermore, we sought to identify the mechanisms by which IGFBP-4 and IGFBP-5 exert their differential biologic effects on growth plate chondrocytes. Our results demonstrate that chondrocyte growth depends on the contrasting functions of IGFBP-4 and IGFBP-5, with respect to their cell surface binding capacities and their subsequent interactions with IGF-I.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The amino-terminal fragment IGFBP-41-122, the carboxy-terminal fragment IGFBP-4136-237, and the carboxy-terminal fragment IGFBP-5144-252 were purified from hemofiltrates from patients with end-stage renal failure, as follows. Isolation of IGFBP-4 and -5 fragments was guided by immunoblot screening of fractions of a peptide library established from 10,000 L of hemofiltrates obtained from patients with CRF, as described previously (23,24). In brief, immediately after blood filtration using ultrafilters with a specified cutoff of 20 kD, the filtrate was routinely chilled to 4°C and adjusted to pH 3, to prevent bacterial growth and proteolysis. For the first separation step, the ultrafiltrate was applied to a strong cation-exchange column [Fractogel TSK SP 650(M); Merck, Darmstadt, Germany] and peptides were eluted batchwise by means of a pH gradient. Each derived pH-pool eluate was further separated by reverse-phase chromatography, resulting in a total of 350 peptide-containing fractions, which were analyzed for the presence of naturally occurring IGFBP-4 and -5 fragments by Western blotting (23,24). Immunoreactive IGFBP fractions were further purified to homogeneity by analytical cation-exchange and reverse-phase chromatography and were analyzed by electrospray mass spectrometry and conventional sequence analysis, as described previously (23,24).
Recombinant human IGF-I and IGF-II were purchased from Bachem (Heidelberg,
Germany); des(1-3)-IGF-I was obtained from GroPep (Adelaide, Australia).
[3H]Thymidine (25 Ci/mmol) and 125I-IGF-I (>2000
Ci/mmol protein) were obtained from Amersham Pharmacia Biotech
(Buckinghamshire, UK). Radioiodination of the IGFBP (20 to 100 µCi/µg
protein) was performed by using a standard chloramine T method. Two micrograms
of each peptide were dissolved in 75 µl of 0.25 M sodium phosphate (pH
7.5), and Na125I (0.2 mCi; Amersham, Braunschweig, Germany) and 10
µl of chloramine T (2.5 mg/ml in phosphate buffer) were added. After
incubation for 60 s, the reaction was stopped by the addition of 10 µl of
Na2S2O5 (3.2 mg/ml in phosphate buffer). Each
radiolabeled peptide was purified on Sep-Pak C18 cartridges
(Waters-Millipore, Eschborn, Germany). After application of the iodinated
sample, each cartridge was rinsed with 5 ml of 10 mM HCl, to remove unbound
material. Elution was performed by using 1 ml of 80% acetonitrile in 10 mM
HCl. The homogeneity and specific activities of the radiolabeled peptides were
controlled on a 322 HPLC system (Kontron, Neufahrn, Germany) by separation on
an analytical reverse-phase C18 column using a linear acetonitrile
gradient, with online detection of the absorbance at 214 nm and of the
radioactivity using a 
-measuring cell (J-1000; Berthold, Wildbad,
Germany).
Standard low-gel temperature agarose was purchased from BioRad (Hercules, CA); fetal calf serum (FCS), phosphate-buffered saline (PBS), Hepes, penicillin-streptomycin, Ham's F-12 medium, and Dulbecco's modified Eagle's medium (DMEM) were obtained from Seromed Biochrom KG (Berlin, Germany). Bovine serum albumin (BSA) was purchased from Sigma-Aldrich Chemicals (Deisenhofen, Germany). Clostridium collagenase (EC 3.4.24.3), DNase I (EC 3.1.21.1), and trypan blue were from Roche Diagnostics (Mannheim, Germany). AmpliTaq Gold, murine leukemia virus reverse transcriptase, and oligo(dT)16 were obtained from Perkin-Elmer (Weiterstadt, Germany). The base pair ladder was obtained from Amersham Pharmacia (Freiburg, Germany), and the 18S rRNA primer pair and competimers were purchased from Ambion (Austin, TX).
Cell Cultures
Epiphyseal chondrocytes from 60- to 80-g Sprague-Dawley rats (Charles
River, Kieslegg, Germany) were isolated and cultured as described previously
(25,26,27).
Pooled growth plates from four to eight animals were digested with clostridial
collagenase (0.12%, wt/vol) and bacterial DNase (0.02%, wt/vol) in F-12
medium. Viability, which was determined after isolation and at the end of each
experiment by using the trypan blue exclusion technique, always exceeded 90%.
Dissociated cells were counted by using a Neubauer chamber (Scheik, Hofheim,
Germany).
Cells were cultured in monolayers in 96-well plates for proliferation assays and in 24-well plates (Nunc, Wiesbaden, Germany) for binding studies, as described previously (25,27,28). F-12 medium/DMEM (1:1) contained a nominal calcium concentration of 1.2 mM, and the medium was supplemented with 10 mM Hepes, 100 µg/ml streptomycin, and 10% FCS. In previous studies using the same culture system, we demonstrated that the majority of cells after the first passage expressed typical markers for proliferative growth plate chondrocytes. Peptide hormones were dissolved in PBS and added every day unless indicated otherwise.
For agarose-stabilized suspension cultures, cells were cultured in agarose in 35-mm dishes (Nunc), as described previously (25,26,27,28). One milliliter of F-12 medium/DMEM containing 0.2% BSA, 0.1% FCS, and hormones or solvents as indicated was added to the cell suspension (40,000 cells/ml in 0.5% low-gel temperature agarose).
The medium was changed every second day, and cells were cultured for 2 wk. Cultures were screened for clusters of more than three cells. No such clusters were observed at the start of culture in any experiment. Suspension cultures were terminated, after 12 d, by fixation in 4% buffered formaldehyde and methanol. Colonies were counted under the microscope, in 100 squares (2-mm grid) for each dish. A cell colony was defined as a cluster of three or more cells with matrix stained by Alcian blue, as described previously (25,27,28).
[3H]Thymidine Assays
The incorporation of [3H]thymidine into DNA was determined, in
96-well plate cultures, as the uptake of radioactivity in TCA-precipitable
material, as described previously
(25,28).
Before experiments, cells were synchronized in the cell cycle by starving in
serum-free F-12 medium/DMEM for 24 h. The medium was changed to F-12
medium/DMEM with 0.2% BSA, and hormones or solvents were added, as indicated,
for 48 h. For the last 4 h, cultures were coincubated with 2 µCi of
[3H]thymidine. In the preincubation experiments, cells were exposed
to the respective IGFBP at 37°C for the indicated times and were washed
three times with serum-free F-12 medium/DMEM, followed by incubation with
IGF-I for 24 h. Subsequently, cells were rinsed twice with PBS and extracted
with sodium hydroxide (1 M). Before counting, the extract was mixed with
scintillation fluid.
Effects of IGFBP on 125I-IGF-I Binding to Chondrocytes in
Monolayer Cultures
Binding studies were performed according to the method described by Andress
and Birnbaum (13). Briefly,
confluent chondrocytes were rinsed twice with serum-free F-12 medium/DMEM
containing 1% BSA (pH 7.4). In the coincubation experiments, various
concentrations of IGFBP were added and incubated with 125I-IGF-I
(10,000 cpm), in the absence or presence of unlabeled IGF-I (250 ng/ml), at
4°C for 3 h. In the preincubation experiments, cultures were incubated
with the respective IGFBP at 37°C for 3 h, followed by intensive washing
with cold serum-free F-12 medium/DMEM containing 1% BSA (pH 7.4) and
incubation with 125I-IGF-I (10,000 cpm), in the absence or presence
of unlabeled IGF-I (250 ng/ml), at 4°C for 3 h. The labeled medium was
discarded, and the cells were rinsed five times with serum-free F-12
medium/DMEM and extracted with sodium hydroxide. Levels of cell
surface-associated 125I-IGF-I were determined by counting the cell
lysates in a -counter. Specific binding was calculated as the
difference between binding in the absence and in the presence of excess
unlabeled IGF-I (250 ng/ml). Nonspecific binding was consistently <7%.
125I-IGFBP Binding to Chondrocytes
For binding studies, confluent cells were rinsed twice with serum-free F-12
medium/DMEM, as described previously
(15). Each well was incubated
with 250 µl of medium and the respective 125I-IGFBP (80,000 cpm)
or vehicle, in the absence or presence of varying concentrations of the
respective unlabeled IGFBP, at 4°C for 3 h. At the end of the incubation
period, the labeled medium was discarded, and the cells were rinsed five times
with serum-free F-12 medium/DMEM. 125I-IGFBP bound to cells was
extracted with lysis buffer and counted as described previously
(15).
For binding studies with suspended cells, chondrocytes were trypsinized and washed once with PBS and serum-free F-12 medium/DMEM. The cell number was adjusted to 320,000 cells/200 µl, as described previously (29). The respective unlabeled IGFBP in serum-free F-12 medium/DMEM or vehicle was added, and the cells were incubated with 125I-IGFBP (80,000 cpm) or vehicle at 4°C for 3 h. After the incubation period, the samples were centrifuged at 2000 rpm at 4°C for 1 min. The supernatant was carefully removed, and the cells were washed twice with serum-free F-12 medium/DMEM. The cell pellets were transferred into scintillation vials and counted. Specific binding was calculated as the difference between binding in the absence and in the presence of excess unlabeled IGFBP (1000 ng/ml).
Reverse Transcription-PCR
Chondrocytes were grown in monolayers until subconfluent. Before the
beginning of the experiments, cells were synchronized by maintenance under
serum-free conditions for 12 h. The medium was changed, and IGF-I and intact
IGFBP-5 (at the indicated concentrations) were added for 24 h. Total RNA from
cultured cells was isolated by the RNeasy method (Qiagen, Hilden, Germany),
following the instructions provided by the manufacturer, and was quantified by
measurement of the OD at 260 nm or by densitometric comparison with a set of
standards (GS 700 imaging densitometer; Bio-Rad Laboratories, Munich,
Germany). Multiplex reverse transcription-PCR for the determination of IGFIR
mRNA abundance was performed according to the user manual provided by
Perkin-Elmer. Reverse transcription and PCR primers (obtained from MWG
Biotech, Ebersberg, Germany) were deduced from rat IGFIR sequences
(30). One microgram of total
RNA was reverse-transcribed into cDNA using oligo(dT)/hexamer primers in a
ratio of 10:1. After cDNA synthesis, multiplex PCR amplification was performed
by using the cDNA template with the target primer pair (IGFIR F1,
5'-AGCCCATGTGTGAGAAGACC-3'; R2,
5'-CGCACACGCCTTTGTAGTAG-3') and a mixture of 18S rRNA
primers/competimers (Ambion), for single-tube coamplification of the specific
product and its 18S rRNA standard. The amplification profile consisted of
denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and
extension at 72°C for 30 s, after an initial 10-min preheating at 94°C
for enzyme activation, in a Perkin-Elmer GeneAmp PCR System 2400. The
amplified products (30 cycles, 215 bp for IGFIR and 488 bp for 18S rRNA) were
detected by electrophoresis in 2% agarose gels and were observed by ethidium
bromide staining and ultraviolet transillumination. Control reactions, which
were performed by omitting reverse transcriptase or template RNA, demonstrated
no reaction product. The cycle products were within the linear-logarithmic
phase of the amplification curves. The OD of the PCR products were analyzed by
using a commercially available computer program (Bio-1D, version 96; Vilber
Lourmat, Marne-La-Vallee France). Results were normalized with respect to the
density of the multiplexed 18S rRNA product.
Statistical Analyses
Data are presented as mean ± SEM. All data were examined for normal
and nongaussian distribution by using the Kolmogorov-Smirnov test. For
comparisons among normally distributed groups, oneway ANOVA, followed by
pairwise multiple comparisons (Student-Newman-Keuls method), was used. For
non-normally distributed data, the nonparametric Kruskal-Wallis test, followed
by an allpairwise multiple comparison (Dunnett's method), was used. P
< 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because levels of both intact IGFBP-4 and low-molecular weight IGFBP-4 fragments are increased in CRF serum (6,7,8), it was also of interest to study the biologic effects of IGFBP-4 fragments on growth plate chondrocytes. Amino-terminal (positions 1-122) and carboxy-terminal (positions 136-237) IGFBP-4 fragments isolated from hemofiltrates from patients with end-stage renal disease were studied. IGFBP-41-122 did not modify colony formation in the absence of exogenous IGF-I but inhibited IGF-I-mediated cell proliferation by 25%; higher concentrations of IGFBP-41-122 did not exhibit stronger inhibitory effects (Figure 1B). In contrast, IGFBP-4136-237 did not modify basal or IGF-I-driven colony formation (Figure 1C).
Effects of IGFBP-5 on Basal and IGF-I-Driven Growth Plate Chondrocyte
Proliferation
In contrast to IGFBP-4, exposure of growth plate chondrocytes to IGFBP-5
under identical experimental conditions increased cell proliferation, in the
absence or presence of exogenous IGF-I. In the absence of exogenous IGF-I,
incubation of cells with IGFBP-5 under serum-free conditions stimulated DNA
synthesis ([3H]thymidine uptake) in a dose-dependent manner
[IGFBP-5 (1 nM), 126 ± 7% of control value; IGFBP-5 (100 nM), 155
± 5%; P < 0.05 versus control; IGF-I (7.8 nM), 155
± 20%; P < 0.05 versus control]. Similarly, colony
formation was stimulated threefold by IGFBP-5 (10 nM), compared with control
values (results not shown). In the presence of IGF-I, IGFBP-5 clearly had a
dose-dependent and additive effect on colony formation
(Figure 2A); the combined
administration of 7.8 nM IGF-I and 15.6 nM IGFBP-5 stimulated colony formation
tenfold, compared with control values. However, coincubation of des
(1-3)-IGF-I with IGFBP-5 did not potentiate des (1-3)-IGF-I-driven cell
proliferation (Figure 2B) and
DNA synthesis [IGF-I (7.8 nM), 140 ± 7.8% of control value; P
< 0.05 versus control; IGFBP-5 (7.8 nM), 128 ± 5.9% of
control value; des(1-3)-IGF-I plus IGFBP-5 (1:1), 137 ± 7.0% of control
value; P < 0.05 versus control; des(1-3)-IGF-I plus
IGFBP-5 (1:2), 128 ± 7.5% of control value]. These data indicate that
the IGF-I-synergistic effect of IGFBP-5 in this cell culture system requires,
at least in part, binding of the peptide to the binding protein.
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To investigate whether the enhancing effect of IGFBP-5 on IGF-I-mediated DNA synthesis could be attributable to membrane association of IGFBP-5, rat growth plate chondrocytes were preincubated for 24 h with IGFBP-5 in the absence of IGF-I, followed by extensive washing. IGFBP-5 preexposure potentiated IGF-I-stimulated [3H]thymidine uptake (Figure 3A). A longer preincubation period (48 h) did not modify the potentiating effect of IGFBP-5 on IGF-I actions (results not shown). Preincubation with IGFBP-2 did not alter IGF-I-stimulated DNA synthesis under identical experimental conditions (Figure 3B).
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Because most IGFBP-5 in CRF serum is fragmented (6,7), the biologic effects of specific IGFBP-5 fragments on chondrocyte proliferation were of interest. The amino-terminal recombinant fragment IGFBP-51-169 did not affect thymidine incorporation or cell proliferation in the absence of exogenous IGF-I (Table 1 and Figure 4A), even at high concentrations (1 µM) (results not shown). However, IGF-I-mediated DNA synthesis and cell proliferation were reduced by 50% in the presence of IGFBP-51-169; higher concentrations did not exhibit stronger inhibitory effects. In contrast, the carboxy-terminal fragment IGFBP-5144-252 did not modify basal or IGF-I-stimulated DNA synthesis and cell proliferation (Table 1 and Figure 4B).
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Effects of IGFBP-4 and IGFBP-5 on the Binding of IGF-I to Growth
Plate Chondrocytes
On the basis of these data, we hypothesized that IGFBP-4 and -5 modulate
IGF-I actions on chondrocytes by differentially affecting the binding of IGF-I
to its receptor. To examine this hypothesis, we investigated the effects of
IGFBP-4 and IGFBP-5 on 125I-IGF-I binding to the cell surface of
chondrocytes. When these cell binding assays were performed under IGFBP and
tracer coincubation conditions, intact IGFBP-4 and, to a lesser extent, intact
IGFBP-5 led to dose-dependent reductions in the binding of
125I-labeled IGF-I to the cell membrane
(Figure 5A), presumably because
soluble IGFBP-5 in the cell medium has a higher affinity for labeled IGF-I
than does membrane-bound IGFBP-5 and thus masks the effect of membrane-bound
IGFBP-5 on IGF-I tracer binding to the cell membrane. We therefore modified
the experimental conditions and preincubated the cells in monolayer cultures
with the respective IGFBP for 3 h, to allow potential cell membrane binding,
followed by extensive washing. Under these experimental conditions, intact
IGFBP-5 produced a dose-dependent increase in the binding of labeled IGF-I to
chondrocytes, whereas preincubation with IGFBP-4 or IGFBP-51-169
did not modify 125I-IGF-I binding
(Figure 5B).
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IGFBP-4 and IGFBP-5 Binding to Growth Plate Chondrocytes
Next, we sought to determine whether the enhanced binding of IGF-I to the
cell membrane in the presence of intact IGFBP-5 is mediated by direct binding
of IGFBP-5 to the cell membrane. In serum-free monolayer cultures, intact
IGFBP-4 and IGFBP-51-169 did not bind to the cell surface.
125I-IGFBP-5 bound to the chondrocyte cell surface, but this
binding was only 30% affected by a 2000-fold excess of unlabeled IGFBP (1000
ng/ml), presumably because of additional binding of IGFBP-5 to extracellular
matrix components (results not shown). However, when cells were suspended in
buffer solution, to avoid interference with extracellular matrix components
and to facilitate membrane binding, specific binding of intact IGFBP-5 and, to
a lesser extent, IGFBP-5144-252, which was affected by added
unlabeled IGFBP-5 but not by added unlabeled IGFBP-3, was observed
(Figure 6). In contrast, intact
IGFBP-4 and IGFBP-51-169 (80,000 cpm) did not bind to the cell
surface (IGFBP-4, 230 ± 32 cpm bound in the absence of unlabeled IGFBP;
IGFBP-51-169, 165 ± 28 cpm bound in the absence of unlabeled
IGFBP).
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Regulation of IGFIR mRNA Abundance
Increased binding of radiolabeled IGF-I to chondrocytes in the presence of
intact IGFBP-5 could be attributable to upregulation of IGFIR by IGF-I or
IGFBP-5. However, IGF-I and IGFBP-5, in the presence or absence of IGF-I (24-h
incubation), exhibited no effects on IGFIR mRNA abundance in cultured
chondrocytes, indicating that these proteins do not regulate IGFIR within 24
h, at least at the level of gene expression
(Figure 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the main IGF binding site in various IGFBP seems to be located in the amino-terminal part (33), other investigators have observed that the carboxy-terminal domain retains IGF-binding capacity (34,35). It has therefore been suggested that both domains contribute to the high-affinity binding of intact IGFBP (35,36). However, in our cell culture system with cultured chondrocytes, the biologic activity of the carboxyterminal fragment IGFBP-4136-237 was too weak for significant modulation of the action of IGF-I. The circulating IGFBP-4 fragments did not exhibit any IGF-independent effects on the proliferation of cultured growth plate chondrocytes (Figure 1).
Our finding that intact IGFBP-4 and, to a lesser extent, the amino-terminal fragment IGFBP-41-122 are inhibitors of IGF-I-mediated growth plate chondrocyte DNA synthesis and cell proliferation in vitro is consistent with our previous clinical observation of an inverse relationship between immunoreactive IGFBP-4 levels in CRF serum and standardized height (6). These in vitro and in vivo data strongly suggest that IGFBP-4 acts as an inhibitory IGFBP in the growth cartilage.
The mean concentration of immunoreactive IGFBP-4 in the serum of children with CRF is approximately 40 nM, and the concentration of IGFBP-5 is approximately 12 nM (6,7). It has been estimated that the concentrations of IGFBP from the 35-kD serum fraction (i.e., IGFBP-4 and IGFBP-5, in addition to others) in the interstitial fluid correspond to approximately 10% of their respective serum concentrations (37). To these concentrations, the amount of IGFBP produced by the respective tissue must be added. Unpublished data from our laboratory indicate that growth plate chondrocytes are able to synthesize IGFBP-4 and IGFBP-5. We therefore think that the concentration of IGFBP (7.8 nM) used in this study likely reflects the concentrations of IGFBP within the growth plates of children with CRF and is physiologically relevant. We chose the IGFBP concentration of 7.8 nM because it is equimolar with the maximally effective concentration of IGF-I (7.8 nM or 60 ng/ml) in the colony formation assay used in this study.
Our results with IGFBP-5 contrast with the inhibitory effects of IGFBP-4, although IGFBP-5 has only a slightly lower binding affinity for IGF-I (Kd = 3.7 nM) (33) than does IGFBP-4 (Kd = 1.2 nM) (24). Under identical experimental conditions, chondrocyte growth was stimulated by intact IGFBP-5, and this effect was enhanced by coincubation with IGF-I. Because the additive effect was absent when the non-IGFBP-binding form of IGF-I, des (1-3)-IGF-I, was used, we assumed that intact IGFBP-5 needed to bind IGF-I before the enhanced proliferative effect could be observed. Consistent with this hypothesis was our finding that the carboxy-terminal fragment with low IGF-I binding affinity, IGFBP-5144-252, did not enhance IGF-I-stimulated chondrocyte proliferation. Interestingly, although both intact IGFBP-5 and IGFBP-5144-252 were able to bind to chondrocyte surfaces (Figure 6), only the intact form recruited IGF-I for cell surface binding (Figure 5B). Such perturbations in the ambient IGF-I or IGFBP-5 concentrations did not alter IGFIR mRNA expression. Taken together, these results suggest that intact IGFBP-5 stimulates chondrocyte growth only when IGF-I is present and that IGFBP-5 may not have intrinsic (IGF-independent) effects on chondrocytes like those it exhibits in osteoblast cultures (13).
Also different in these studies was our finding that IGFBP-51-169 did not stimulate proliferation, in contrast to previous studies with osteoblasts (15). Rather, we observed that IGF-I-stimulated chondrocyte growth was inhibited by IGFBP-51-169, apparently by a mechanism similar to that of intact IGFBP-4, because neither binds to the chondrocyte surface nor increases IGF-I binding to the cells. This fidning is consistent with results from studies of recombinant amino-terminal fragments of IGFBP-5, which indicated that the entire IGFBP-5 protein contains only one high-affinity binding site for IGF, located between residues Ala40 and Ile92 (33), whereas the cell binding site comprises carboxy-terminal residues 169 to 252 (17). IGFBP-51-169 retains some binding affinity for IGF-I, thus mitigating IGF-I-driven chondrocyte proliferation, but does not bind to the cell membrane and is therefore not capable of enhancing IGF-I-mediated cell proliferation. Our findings differ from the previous observation that IGFBP-51-169 has intrinsic mitogenic activity in osteoblast-like cells (15). In those cells, IGFBP-51-169 was observed, in cross-linking studies, to bind to a 420-kD membrane protein (22) and to stimulate the phosphorylation of this putative serine kinase receptor (17). Those findings suggest that residues within the aminoterminus of IGFBP-5 are capable of binding to specific membrane proteins that may not exist in growth plate chondrocytes. Additional cross-linking studies must be performed to determine whether specialized receptors for IGFBP-5 are important for normal cartilage physiologic processes. Such studies may reveal that cartilage cells are, by design, deficient in IGFBP-5 signaling pathways that specify IGF-I-independent functions.
On the basis of the findings that chondrocytes in culture produce IGF, it can be speculated that IGF produced by chondrocytes could have influenced the effects of exogenously added IGFBP on cell proliferation. We observed that the IGF-I concentration in serum-free medium under basal conditions was low (2.5 to 3.5 ng/ml) in this cell culture system (38). The chondrocytes used in this study were derived from mature rats, and it is known that a developmental switch from IGF-II to IGF-I occurs in rodents, resulting in shutdown of the IGF-II promoter soon after birth. It is therefore unlikely that the effect of exogenously added IGF-I is influenced by autocrine IGF-II produced by chondrocytes.
The precise characterization of the biologic effects of inhibitory and stimulatory IGFBP and their respective fragments on IGF-I-mediated chondrocyte proliferation may have implications for new therapeutic strategies. Novel peptides that exhibit remarkable specificity in binding to defined IGFBP have recently been discovered, using phage-displayed peptide libraries (39). These peptides have the potential to act as IGF displacers by both preventing IGF-I binding to specific inhibitory IGFBP and displacing IGF-I that is bound to the IGFBP, thus increasing the amount of endogenous IGF-I that is available for binding to IGF receptors (40). An alternative approach involves IGFBP-selective IGF-I variants that are unable to bind to certain inhibitory IGFBP, with preserved affinity for other IGFBP (41). The therapeutic potential of these novel peptides in disease conditions associated with upregulated inhibitory IGFBP levels, such as renal failure, is currently being investigated.
In summary, we have demonstrated that IGFBP-4 and IGFBP-5 have contrasting functions in growth plate chondrocytes. Both intact IGFBP-4 and the fragment IGFBP-41-122 have exclusive inhibitory roles in IGF-I-stimulated cells, binding IGF-I in the amino-terminal domain and preventing or reducing the binding of ligand to its signaling receptor. Intact IGFBP-5, however, stimulates chondrocyte proliferation, apparently through its association with the cell membrane in the carboxy-terminal domain, thus better presenting IGF-I to its receptor. However, if accumulated amino-terminal forms of IGFBP-5 predominate, then IGFBP-5 inhibits IGF-I-stimulated proliferation. This action of amino-terminal IGFBP-5 forms in chondrocytes contrasts with the stimulatory effects of IGFBP-5 on osteoblast activity and may be important in preserving the cartilage-to-bone developmental sequence that is necessary for normal longitudinal bone growth.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25(OH)2D3 on receptor expression and growth
stimulation in rat growth plate chondrocytes. Kidney
Int 53:1152
-1161, 1998[Medline]
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 S. Ciarmatori, D. Kiepe, A. Haarmann, U. Huegel, and B. Tonshoff Signaling mechanisms leading to regulation of proliferation and differentiation of the mesenchymal chondrogenic cell line RCJ3.1C5.18 in response to IGF-I J. Mol. Endocrinol., April 1, 2007; 38(4): 493 - 508. [Abstract] [Full Text] [PDF]
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 D. Kiepe, S. Ciarmatori, A. Haarmann, and B. Tonshoff Differential expression of IGF system components in proliferating vs. differentiating growth plate chondrocytes: the functional role of IGFBP-5 Am J Physiol Endocrinol Metab, February 1, 2006; 290(2): E363 - E371. [Abstract] [Full Text] [PDF]
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D. Kiepe, S. Ciarmatori, A. Hoeflich, E. Wolf, and B. Tonshoff Insulin-Like Growth Factor (IGF)-I Stimulates Cell Proliferation and Induces IGF Binding Protein (IGFBP)-3 and IGFBP-5 Gene Expression in Cultured Growth Plate Chondrocytes via Distinct Signaling Pathways Endocrinology, July 1, 2005; 146(7): 3096 - 3104. [Abstract] [Full Text] [PDF]
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 B. Gigante, S. Rubattu, R. Stanzione, A. Lombardi, A. Baldi, F. Baldi, and M. Volpe Contribution of Genetic Factors to Renal Lesions in the Stroke-Prone Spontaneously Hypertensive Rat Hypertension, October 1, 2003; 42(4): 702 - 706. [Abstract] [Full Text] [PDF]
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