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Defined Carboxy-Terminal Fragments of Insulin-Like Growth Factor (IGF) Binding Protein-2 Exert Similar Mitogenic Activity on Cultured Rat Growth Plate Chondrocytes as IGF-I

University Children’s Hospital (D.K., A.V.D.P., S.C., U.H., J.O., B.T.), 69120 Heidelberg, Germany; Center of Pharmacology (L.S.), Hannover Medical School, 30625 Hannover, Germany; Kolling Institute of Medical Research (B.S.), University of Sydney, Royal North Shore Hospital, 2006 Sydney, Australia; and Research Unit Genetics and Biometry (A.H.), Research Institute for the Biology of Farm Animals, 18196 Dummerstorf, Germany

Address all correspondence and requests for reprints to: Daniela Kiepe, M.D., University Children’s Hospital, Im Neuenheimer Feld 153, 69120 Heidelberg, Germany. E-mail: daniela.kiepe{at}med.uni-heidelberg.de.

	Abstract

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The IGF/IGF binding protein (IGFBP) system is an important component in the hormonal regulation of longitudinal growth. Evidence from in vitro studies indicates that IGFBPs may have IGF-independent effects. We analyzed the biological activity of intact IGFBP-2 and defined carboxy-terminal IGFBP-2 fragments isolated from human hemofiltrate in two cell culture systems of the growth plate: rat growth plate chondrocytes in primary culture and the mesenchymal chondrogenic cell line RCJ3.1C5.18. The IGFBP-2 fragments IGFBP-2^167–279, IGFBP-2^167–289, and IGFBP-2^104–289 exerted a strong (2- to 3-fold) mitogenic effect on growth plate chondrocytes, which was comparable with IGF-I in equimolar concentrations (7.8 nM) but was not mediated through the type 1 IGF receptor. In a dose-response experiment, the most effective concentration of IGFBP-2^104–289 for the stimulation of cell proliferation was 10 nM. This biological activity of IGFBP-2 fragments was associated with cell membrane binding, demonstrated by Western blot analysis of fractionated cell lysates and immunohistochemistry. Whereas intact IGFBP-2 did not modulate chondrocyte proliferation, partially reduced (by dithiothreitol) full-length IGFBP-2 stimulated cell proliferation to a comparable extent (3.4-fold) as carboxy-terminal IGFBP-2 fragments. The mitogenic activity of these IGFBP-2 fragments and of partially reduced full-length IGFBP-2 was mediated through the use of the MAPK/ERK 1/2. These data imply a novel role of naturally occurring IGFBP-2 fragments for the endocrine and paracrine/autocrine regulation of longitudinal growth.

	Introduction

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THE GH/IGF AXIS IS an important component in the hormonal regulation of longitudinal growth. The biological activity of the IGFs within tissues and the circulation is modified by six IGF binding proteins (IGFBP). These proteins have individual and specific expression patterns and exert different functions including stimulation or inhibition of IGF bioactivity as well as IGF-independent actions (1). In the postnatal period, IGFBP-2 is the second most abundant IGFBP in the circulation and is present in various biological fluids (2, 3). In the intact growth plate of mice, exclusively IGFBP-2 mRNA, in conjunction with IGF-I and IGF-II mRNA was detectable in the proliferative and prehypertrophic zone by in situ hybridization, suggesting a functional role of IGFBP-2 for the regulation of longitudinal growth (4). In rat growth plate chondrocytes in primary culture, IGFBP-2 mRNA is expressed under baseline conditions, and IGFBP-2 protein is the second most abundant IGFBP in conditioned cell culture medium of chondrocytes (5). In in vitro studies, IGFBP-2 exerts inhibitory as well as stimulatory effects on cell proliferation, which are cell type specific (2, 3, 6). For example in growth plate chondrocytes in primary culture, intact IGFBP-2 inhibits IGF-I-induced cell proliferation (6). On the other hand, increased expression of IGFBP-2 is associated with enhanced growth of tumor cells (1). Transgenic mice overexpressing IGFBP-2 displayed significantly reduced gain of body weight, suggesting that excess IGFBP-2 can negatively affect somatic growth (7). IGFBP-2 has been demonstrated to exert specific effects on bone because IGFBP-2 transgenic mice have reduced bone size and mass but not density (8).

Proteolysis of IGFBPs in the circulation and tissues is an essential mechanism to regulate the bioavailability and half-life of IGFs. The presence of active IGFBP-2 fragments with partial IGF-binding activity was first described in human milk (9). IGFBP-2 fragments, which exhibited molecular masses of 12.7 and 12.9 kDa and started with N-terminal amino acids identified as Gly169 and Gly167, were isolated by Ständker et al. (10) from human hemofiltrate. In consecutive studies, a peptide library generated from human hemofiltrate was immunologically screened and 18 different IGFBP-2 fragments were isolated and characterized (11). All tested fragments retained low IGF-binding affinity and exhibited the integrin-binding sequence. The most abundant fragment IGFBP-2^167–279 showed 10% of IGF-II binding affinity, compared with recombinant human IGFBP-2 (11).

Because of the potential importance of IGFBP-2 for the regulation of longitudinal growth and the known IGF-independent effects of fragmented IGFBPs in other cell culture systems (12, 13), the aim of this study was to investigate the mitogenic effect of intact and fragmented forms of IGFBP-2 in two cell culture models of the growth plate, rat growth plate chondrocytes in primary culture and the mesenchymal RCJ3.1C5.18 cell line. RCJ cells, derived from fetal rat calvaria (14, 15), do not express IGF-I; therefore, the action of this hormone can be studied without interference from endogenous IGFs (16). We analyzed the biological activity of three defined carboxy-terminal fragments, IGFBP-2^167–279, IGFBP-2^167–289, and IGFBP-2^104–289, which were isolated from human hemofiltrate (11), in comparison with intact IGFBP-2 and IGF-I. Furthermore, we sought to identify the mechanisms by which intact and fragmented IGFBP-2 exert their different biological effects on growth plate chondrocytes. We focused on the MAPK/ERK 1/2 and focal adhesion kinase (FAK) intracellular signaling pathways because previous data in other cell culture models had shown that IGFBP-2 interacts with various integrin receptors; these paths signal preferably through the MAPK/ERK 1/2 and FAK pathways (17).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents
Recombinant human IGF-I was purchased from Bachem (Heidelberg, Germany), U0126 from Promega (Mannheim, Germany). [³H]thymidine (40 Ci/mmol) and enhanced chemiluminescence reagents were obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). PBS, HEPES, penicillin-streptomycin, Ham’s F-12, and DMEM were obtained from Seromed Biochrom KG (Berlin, Germany). BSA fraction V was purchased from Sigma-Aldrich Chemicals (Deisenhofen, Germany). MEM was purchased from cc Pro (Neustadt, Germany), fetal calf serum from PAA Laboratories (Pasching, Austria), and MEM nonessential amino acids (100 times) from Invitrogen Life Technologies (Karlsruhe, Germany). Clostridium collagenase (EC 3.4.24.3), deoxyribonuclease I (EC 3.1.21.1), and trypan blue were from Roche Diagnostics GmbH (Mannheim, Germany). Dithiothreitol (DTT) was purchased from Invitrogen Life Technologies. The monoclonal antimouse antibody directed against type 1 IGF receptor (catalog no. GR11L) was obtained from Calbiochem (Darmstadt, Germany). The antibodies directed against phosphorylated ERK 1/2 (catalog no. 9101), ERK 1/2 (catalog no. 9102), phosphorylated FAK (catalog no. 3284), FAK (catalog no. 3285), and the horseradish peroxidase-conjugated [antirabbit (catalog no. 7074) and antimouse (catalog no. 7076)] antibodies were from Cell Signaling Technology (Frankfurt am Main, Germany). The antibodies against Na-K-ATPase (catalog no. 05269) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; catalog no. EB06377) were from Upstate Cell Signaling Solutions (Charlottesville, VA) and Everest Biotech (Oxfordshire, UK), respectively. Polyclonal antihuman IGFBP-2 antibody (catalog no. PAB-IGFBP2–1) and intact human IGFBP-2 (catalog no. BP2BU020) were obtained from GroPep (Adelaide, Australia). According to the manufacturers, these antibodies do not recognize human IGFBP-1, -3, -4, -5, or -6.

Purification of IGFBP-2 fragments
IGFBP-2 fragments (20 kDa IGFBP-2^104–289, 14 kDa IGFBP-2^167–289, 12.8 kDa IGFBP-2^167–279) were purified from the hemofiltrate of patients with end-stage renal disease as follows: isolation of IGFBP-2 fragments was guided by immunoblot screening in fractions of a peptide library established from 10,000 liters of hemofiltrate obtained from patients suffering from end-stage renal disease as described previously (11). In brief, immediately after blood filtration using ultrafilters with a specified cutoff of 20 kDa, 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 batchwise eluted by means of a pH gradient. Each derived pH pool eluate was further separated by reversed-phase chromatography resulting in a total of 350 different peptide containing fractions, which were analyzed for the presence of naturally occurring IGFBP-2 fragments by Western blotting (11). IGFBP immunoreactive fractions were further purified to homogeneity by different analytical cation-exchange and reversed phase (RP-C4, -C5, -C18 media) chromatographical steps.

To separate the different IGFBP-2 fragments, high-resolution chromatographic media with particle sizes from 3 to 5 µm and pore sizes of 30 nm were used as described previously (11). The obtained peptides were analyzed by electrospray mass spectrometry on an API III quadrupole mass spectrometer (Sciex, PerkinElmer, Langen, Germany) with an electrospray ionization interface. The obtained molecular masses were 20,685 Da for IGFBP-2^104–289, 14,023 Da for IGFBP-2^167–289, and 12,858 Da for IGFBP-2^167–279; the mass accuracy was at least 0.02%. Control of identity and purity was obtained by: 1) sequencing on an 473 A gas-phase sequencer (Applied Biosystems, Darmstadt, Germany) by Edman degradation and chromatographical identification of phenylthiohydantoin amino acids; 2) analysis using capillary zone electrophoresis using a P/ACE system (Beckman, San Ramon, CA) with a fused silica uncoated capillary column (50 cm x 75 µm; Polymicro Technologies, Phoenix, AZ) and supported by the System Gold software under conditions described previously (11); and 3) analysis using a LaserTec RBT II MALDI-MS (Applied Biosystems). Applying these methods, the purified peptides showed purities greater than 98%. The quantitation of the purified peptides was carried out by conventional amino acid analysis (AminoQuant 1090, Series II; Hewlett Packard, Waldbronn, Germany) after acidic hydrolysis of the IGFBP peptides and quantification of the isoindol-derivatized amino acids after their analytical RP chromatographic separation. The results of the sequence analyses of the respective N-terminal sequence were as follows: IGFBP-2^104–289, 104-GASPEQVADN ... ARGVHTQRMQ-289; IGFBP-2^167–289, 167-GKXGKHHLGL ... ARGVHTQRMQ-289; and IGFBP-2^167–279, 167-GKGGKHHLGL ... HLFYNEQQE-279 (11).

Competitive binding assay
Human recombinant IGFBP-2 in a final concentration of 0.9 µg/ml was incubated over 30 min at 37 C in a total volume of 100 µl with varying concentrations of freshly prepared DTT (0 to 20 mM) in a 50 mM sodium phosphate buffer (pH 7.4) containing 0.1% Triton X-100. The incubation was ended by diluting 50 µl of the incubation solution to 1.5 ml with the same phosphate buffer containing 0.25% BSA but without DTT (incubation buffer). Each 50 µl of this dilution was used for competitive binding with 3500 cpm iodinated IGF-II tracer and varying concentrations of cold IGF-II competitor (final concentration ranging from 1.75 · 10^–13 M to 1.75 · 10^–8 M) in a total volume of 300 µl. Recombinant IGF-II (1 µg; Mediagnost, Reutlingen, Germany) was iodinated to a specific activity of 60 mCi/mg by applying the chloramine-T method (18). The reaction mixtures were incubated over 2 h at room temperature. Then 25 µl of a 1:100 dilution of an anti-IGFBP-2 antibody in incubation buffer was added and incubation was continued for another hour. Finally, 25 µl of a 1:2 dilution of a goat antirabbit serum and 1 ml of 6% PEG2000 were added, and immunoprecipitation was carried out by centrifugation at 3000 x g at 4 C for 30 min. The fluids were decanted and the radioactivity in the precipitates determined.

Cell cultures
Epiphyseal chondrocytes from 60- to 80-g Sprague Dawley rats (Charles River, Kieslegg, Germany) were isolated and cultured as described previously (19, 20). This study was approved by the Institutional Animal Care and Use Committee (35-9185.81/102/98). Pooled growth plates from four to eight animals were digested with clostridial collagenase [0.12% (wt/vol)] and bacterial deoxyribonuclease [0.02% (wt/vol)] in F-12 medium. Viability, determined after isolation and at the end of each experiment by the trypan blue exclusion technique, invariably exceeded 90%. Dissociated cells were counted using a Neubauer chamber (Scheik, Hofheim, Germany).

Cells were cultured in monolayers in 96-well plates for proliferation (Nunc, Wiesbaden, Germany) as described previously (19, 20). The F-12/DMEM (1:1) medium contained a nominal calcium concentration of 1.2 mM and was supplemented with 10 mM HEPES, 100 µg/ml streptomycin, and 10% fetal calf serum. 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. We have reported previously by mRNA and protein studies that these cells express IGF-I, IGFBP-2, -3, -4, -5, and -6 (21). IGF-I peptide concentration by RIA (22) in conditioned medium of unstimulated cells was 3.0 ± 0.45 ng/ml.

RCJ3.1C5.18 cells (kindly provided by Dr. Anna Spagnoli, Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN) were grown at 37 C in humidified 5% CO₂ atmosphere in MEM (with Earle’s salts) supplemented with 1 mM N-acetyl-L-glutamine, 10 mM HEPES, 100 U/ml penicillin-streptomycin, 2 mM sodium pyruvate, 15% heat-inactivated fetal bovine serum, 10^–7 M dexamethasone and studied within 25 passages. Cell viability was tested with dimethylthiazoldiphenyltetra-zoliumbromide assay. Cells were cultured in monolayers in 96-well plates for proliferation (Nunc) as described previously (19). We (21) and others (16) have previously reported based on mRNA studies that show that these cells do not express IGF-I, IGF-II, IGFBP-1, and IGFBP-3 but do express IGFBP-2, IGFBP-4, IGFBP-5, and IGFBP-6. IGF-I peptide concentration in a conditioned medium of unstimulated cells was below the detection limit of the RIA.

[³H]thymidine assay
Incorporation of [³H]thymidine into DNA was determined in 96-well plate cultures as uptake of radioactivity in trichloroacetic acid-precipitable material as described previously (19). Before the experiment, cells were synchronized in cell cycle by starving in serum-free F-12/DMEM for 24 h. The medium was changed to F-12/DMEM with 0.2% BSA, inhibitor, type 1 IGF receptor antibody, and hormones or solvents were added as indicated for 48 h. For the last 4 h, cultures were coincubated with 2 µCi/well of [³H]thymidine (1 µl in a dilution of 1:10 from a stock solution of 40 Ci/mmol). Subsequently, cells were rinsed twice with PBS and extracted with sodium hydroxide (1 M). Before counting, the extract was mixed with scintillation fluid.

Western immunoblotting
RCJ3.1C5.18 cells were incubated with the indicated IGFBPs, scraped in 50 µl ice-cold lysis buffer containing a cocktail of proteinase and phosphatase inhibitors, and cell extracts treated as previously reported (23). The protein concentration in the cell lysate was determined via a standard bicinchoninic assay protocol (Bio-Rad Laboratories, Inc., Munich, Germany). Electrophoresis of 20 µg of protein was performed on a 12% separating sodium dodecyl sulfate-polyacrylamide gel for 45 min at 200 V. Separated proteins were transferred for 90 min at 105 V to a polyvinyl difluoride membrane (Millipore, Eschborn, Germany). After blocking with 5% BSA or 3% nonfat dry milk (Roth, Karlsruhe, Germany) in Tris-buffered saline with 0.05% (vol/vol) Tween 20 (TBST) for 1 h at room temperature, membranes were incubated overnight with the first antibody [dilution 1:2000 for phosphorylated (p)-ERK 1/2, ERK 1/2, FAK, p-FAK, and IGFBP-2] in TBST containing 5% BSA. After extensive washing over a period of 30 min with TBST, the membranes were incubated for 1 h with the secondary antibody (dilution 1:2000) in TBST containing 3% nonfat dry milk, followed by further washing over a period of 30 min. The protein bands were visualized using a chemiluminescent detection system and Hyperfilm enhanced chemiluminescence film (Amersham Pharmacia Biotech) according to the manufacturer’s directions.

Membrane preparation
Membranes were prepared as described previously (24). In brief, tissues were homogenized in a lysis buffer (10 mM Tris, 2 mM phenylmethysulfonyl fluoride, 1 IU/ml aprotinin) for 1 min at 10,000 rpm on ice using a cell homogenizer (Micro-Ultrasonic cell disrupter; Kimble/Kontes, Vineland, NJ)]. Cell debris and nuclei were separated by centrifugation (1000 x g, 10 min at 4 C). The supernatant containing plasma membranes and the cytosolic fraction were separated by centrifugation for 30 min at 21,000 x g at 4 C. The protein concentrations in the cell lysate were determined via standard bicinchoninic assay protocol (Bio-Rad Laboratories). Electrophoresis of 20 µg of protein was performed according to the description in the Western immunoblotting section. The membranes with the isolated plasma membrane proteins were blocked with TBST containing 3% nonfat dry milk for 30 min and were incubated with the antibody to Na-K-ATPase (dilution 1:5000 in TBST containing 3% nonfat dry milk) for 30 min. The membranes with the cytosolic proteins were blocked with TBST containing 5% BSA for 50 min and were incubated with the antibody against GAPDH (blocked with TBST containing 5% BSA at a dilution of 1:5000) for 5 min. The protein bands were visualized according to the description in the Western immunoblotting section.

Immunohistochemistry
RCJ3.1C5.18 cells were seeded onto nitric acid-treated and -washed coverslips in 60-mm dishes supplemented with MEM containing 15% fetal calf serum as described previously (25). After cells attached, the coverslips were washed in PBS, and serum-free MEM was added for a period of 24 h. Vehicle, intact IGFBP-2, or IGFBP-2^104–289 fragment was added for 1 h. The cells were washed with PBS and incubated again for 1 h with serum-free MEM. The coverslips were washed twice in PBS followed by the addition of polyclonal rabbit anti-IGFBP-2 (catalog no. 06-107; Upstate Cell Signaling Solutions) at a dilution of 1:500 from a 1 µg/µl stock in 1x PBS, 4% BSA (wt/vol). Cells were incubated with antibody for 2 h at 4 C. The coverslips were washed twice in PBS, fixed in 4% (wt/vol) paraformaldehyde for 10 min at room temperature, and blocked overnight in 5% BSA (wt/vol) in 1x PBS. On the following day, the cells were washed again with PBS and incubated with 5 µg/ml secondary antibody goat antirabbit IgG conjugated with Alexa FluorÂ546 (Invitrogen Life Technologies) at a dilution of 1:400 and incubated for 1 h at room temperature. For nuclear staining, cells were washed in PBS and incubated with 5 x 10^–7 M 4',6-diamidino-2-phenyl indole (Serva, Heidelberg, Germany) at a dilution of 1:500 for 2 min. Finally, cells were washed in PBS, mounted using gel mount with antifade (Biomedica Corp., Foster City, CA), and visualized with a DMI 4000B microscope (Leica, Wetzlar, Germany).

Statistics
Data are given as mean ± SE. The results of the thymidine incorporation assays were expressed as percent of control. The mean value of nine to 12 control experiments was designated as 100%, and the corresponding SE was calculated as percent of the mean control value. All data were examined for normal and non-Gaussian distribution by the Kolmogorov-Smirnov test. For comparison among normally distributed groups, one-way ANOVA, followed by pairwise multiple comparison (Student-Newman-Keuls method) was used. For nonnormally distributed data, the nonparametric Kruskal-Wallis test followed by an all pairwise multiple comparison (Dunnett’s method) was used. P < 0.05 was considered statistically significant.

	Results

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Defined carboxy-terminal IGFBP-2 fragments, but not intact IGFBP-2, stimulate growth plate chondrocyte proliferation
IGF-I in a concentration of 60 ng/ml (7.8 nM) stimulated proliferation of growth plate chondrocytes in primary culture by 1.8- to 2.5-fold (Fig. 1). We have shown previously that this IGF-I concentration maximally stimulates cell proliferation in this cell culture model (19). Intact IGFBP-2 had no effect on cell proliferation, whereas simultaneous exposure of cells to IGF-I and intact IGFBP-2 in equimolar concentrations reduced IGF-I-induced cell proliferation by 50% (Fig. 1A). In contrast to intact IGFBP-2, the carboxy-terminal fragment IGFBP-2^104–289 stimulated cell proliferation 3-fold in the absence of exogenous IGF-I but did not inhibit IGF-I-mediated cell proliferation (Fig. 1B). The two other IGFBP-2 fragments, IGFBP-2^167–289 and IGFBP-2^167–279, also stimulated chondrocyte proliferation in the absence of exogenous IGF-I, although to a somewhat lesser degree, and did not modify IGF-I-induced cell proliferation (Fig. 1, C and D). We compared the proliferative response of growth plate chondrocytes with IGF-I, intact IGFBP-2, and the three IGFBP-2 fragments in an additional experiment (Table 1). Comparable results were obtained as in Fig. 1; the difference in the proliferative response to IGF-I and the three IGFBP-2 fragments was not statistically significant.

View larger version (26K): [in this window] [in a new window]   FIG. 1. Defined carboxy-terminal IGFBP-2 fragments, but not intact IGFBP-2, stimulate proliferation of growth plate chondrocytes in primary culture. Effect of intact IGFBP-2 (A), IGFBP-2104–289 (B), IGFBP-2167–289 (C), and IGFBP-2167–279 (D) alone (7.8 nM) and in coincubation with equimolar concentrations of IGF-I on DNA synthesis, as assessed by [3H]thymidine incorporation assay. Subconfluent chondrocytes in primary culture were cultured in serum-free medium for 24 h. Medium was changed to F12/DMEM containing 0.2% BSA, and peptides (dissolved in PBS) or vehicle were added as indicated. [3H]thymidine incorporation was determined as described in Materials and Methods. Data are the mean ± SE expressed as percent of control. Statistics were by ANOVA of 24 parallel dishes per group. Experiments were performed at least three times. *, P < 0.05 vs. control; #, P < 0.05 vs. IGF-I.

View larger version (38K): [in this window] [in a new window]   FIG. 2. Defined carboxy-terminal IGFBP-2 fragments, but not intact IGFBP-2, stimulate proliferation of RCJ cells. Effect of intact IGFBP-2 (A), IGFBP-2104–289 (B), IGFBP-2167–289 (C), and of IGFBP-2167–279 (D) alone (7.8 nM) and in coincubation with equimolar concentrations of IGF-I on DNA synthesis, as assessed by [3H]thymidine incorporation assay. Subconfluent RCJ cells were cultured in serum-free medium for 24 h. Medium was changed to MEM containing 0.2% BSA, and peptides (dissolved in PBS) or vehicle were added as indicated. [3H]thymidine incorporation was determined as described in Materials and Methods. Data are the mean ± SE expressed as percent of control. Statistics were by ANOVA of 24 parallel dishes per group. Experiments were performed at least three times. *, P < 0.05 vs. control; #, P < 0.05 vs. IGF-I.

View larger version (18K): [in this window] [in a new window]   FIG. 3. Subconfluent RCJ cells were cultured in serum-free medium for 24 h. Medium was changed to MEM containing 0.2% BSA, and peptides (dissolved in PBS) or vehicle were added for 48 h as indicated. [3H]thymidine incorporation was determined as described in Materials and Methods. Data are the mean ± SE expressed as the percent of control. A, IGFBP-2104–289 exerts its mitogenic effect most effectively in a 10-nM concentration. Statistics by ANOVA of 10–12 parallel dishes per group. *, P < 0.05 vs. control; #, P < 0.05 vs. IGFBP-2104–289 10 nM. B, An amino acid solution does not exert a mitogenic effect on RCJ cells. Medium was changed to MEM containing 0.2% BSA, and nonessential amino acids solution (7.8 nM), IGF-I (dissolved in PBS), or vehicle was added for 48 h as indicated. Statistics were by ANOVA of nine to 12 parallel dishes per group. *, P < 0.05 vs. control; #, P < 0.05 vs. nonessential amino acids (AS) solution (7.8 nM). C, IGFBP-2104–289 does not exert its mitogenic effect through the type 1 IGF receptor. Medium was changed to MEM containing 0.2% BSA, peptides (7.8 nM) (dissolved in PBS), anti-type 1 IGF receptor antibody (IR) (1 µg/ml), or vehicle was added for 48 h as indicated. Statistics were by ANOVA of nine to 10 parallel dishes per group. *, P < 0.05 vs. control; #, P < 0.05 vs. IGF-I.

The mitogenic effect of IGFBP-2^104–289 on RCJ cells was not mediated through the type 1 IGF receptor because blockade of this receptor with the monoclonal anti-type 1 IGF receptor antibody IR3 did not modify the effect of IGFBP-2^104–289 but abrogated as expected the mitogenic effect of IGF-I (Fig. 3C).

Cell membrane binding of intact and fragmented IGFBP-2
We next sought to investigate whether this effect of IGFBP-2 fragments on cell proliferation is associated with binding to the cell membrane. The separation of cells into defined compartments was verified by Western immunoblotting with antibodies against GAPDH as a specific marker for the cytosol and against Na-K-ATPase as a specific marker for the cell membrane. Na-K-ATPase was detected exclusively in the cell membrane fraction and GAPDH in the cytosolic fraction (Fig. 4A). After incubation of cells with intact and fragmented IGFBP-2 for 2 h, the localization of these peptides in the cell membrane, cytoplasm, and nucleus was investigated by Western immunoblotting of different cell compartments with an antibody against human IGFBP-2 (Fig. 4B). Intact IGFBP-2 (32 kDa) was detected in the plasma membrane but not in the cytosolic fraction (Fig. 4B). A comparable pattern was obtained for the fragments IGFBP-2^104–289, IGFBP-2^167–289, and IGFBP-2^167–279. In control cells, the faint 32-kDa band most likely represents IGFBP-2 from endogenous synthesis.

View larger version (15K): [in this window] [in a new window]   FIG. 4. Cell membrane binding of intact and fragmented IGFBP-2. A, The membrane (M) and cytosolic (C) fractions were isolated as described in Materials and Methods. To verify cell compartment separation, cell lysates from RCJ cells were run in parallel and probed for the cytoplasmic fraction with an antibody against GAPDH and for the plasma membrane preparation with an antibody against Na-K-ATPase. B, Western immunoblot analysis of the respective cell compartments incubated with vehicle or intact or fragmented IGFBP-2 for 2 h. The respective cell lysates of RCJ cells were run in parallel and probed with a specific antibody against IGFBP-2, as described in the Materials and Methods.

View larger version (27K): [in this window] [in a new window]   FIG. 5. Intact IGFBP-2 and IGFBP-2104–289 bind to the cell surface. RCJ cells were plated on coverslips placed in 60-mm plates and treated with vehicle, intact IGFBP-2 (7.8 nM), and IGFBP-2104–289 (7.8 nM) for 1 h, followed by washing with PBS and incubating again for 1 h with serum-free MEM. Cells were incubated with polyclonal rabbit anti-IGFBP-2 antibody, fixed, and incubated with secondary antibody as described in the Materials and Methods (magnification, x40). Strong immunofluorescence was observed at the surface of RCJ cells incubated with intact IGFBP-2 and a labeled anti-IGFBP-2 antibody (A and B) and IGFBP-2104–289 (D and E). Some fluorescence was also present at the surface of the nucleus (staining in blue). No fluorescence was detectable in control cells incubated with vehicle and the anti-IGFBP-2 antibody (C and F).

View larger version (20K): [in this window] [in a new window]   FIG. 6. A, Partially reduced full-length IGFBP-2 is still able to bind IGF-II. Intact IGFBP-2 was incubated with varying concentrations of DTT. Mild reducing conditions with a DTT concentration of 5.0 mM led to a partial reduction of IGFBP-2, which was still able to bind IGF-II, although to a lesser degree (66%) than untreated (intact) full-length IGFBP-2. B, Partially reduced full-length IGFBP-2 does not contain proteolytic fragments of IGFBP-2. Intact IGFBP-2 was incubated with varying concentrations of DTT, followed by immunoblot analysis using a polyclonal IGFBP-2 antibody. C, Partially reduced full-length IGFBP-2 stimulates cell proliferation to a comparable extent as equimolar IGF-I. RCJ cells were cultured until confluence, serum-starved for 12 h and stimulated with IGF-I (7.8 nM) and intact IGFBP-2 (7.8 nM) incubated with or without DTT for an additional 48 h. Control cells were cultured without IGF-I in the absence or presence of DTT (5 mM for 30 min at 37 C). [3H]thymidine incorporation into the acid-extractable pool was determined by scintillation counting and used as a measure of DNA synthesis, as described in Materials and Methods. Data are mean ± SE. Statistics were by ANOVA were for 12 parallel wells per group from three independent experiments. *, P < 0.05 vs. control.

View larger version (30K): [in this window] [in a new window]   FIG. 7. Both fragmented and partially reduced full-length IGFBP-2 stimulate the MAPK/ERK 1/2 and FAK signaling pathways. A, RCJ cells were serum starved for 12 h and incubated for 1 h with the respective IGFBP-2 peptides: control (lane 1), intact IGFBP-2 (lane 2), IGFBP-2104–289 (lane 3), IGFBP-2167–289 (lane 4), IGFBP-2167–279 (lane 5), and partially reduced full-length IGFBP-2 (lane 6). Cell lysates were subjected to Western immunoblot analysis, and the respective membranes were probed with specific antibodies against p-ERK and total ERK. Representative autoradiographs of a total of three independent experiments are shown. B, After 12 h of starvation, cells were incubated for 1 h with the respective IGFBP-2 peptides: control (lane 1), intact IGFBP-2 (lane 2), IGFBP-2104–289 (lane 3), IGFBP-2167–289 (lane 4), IGFBP-2167–279 (lane 5), partially reduced full-length IGFBP-2 (lane 6), and the respective membranes were probed with specific antibodies against p-FAK and total FAK. Representative blots of two independent experiments are shown.

View larger version (27K): [in this window] [in a new window]   FIG. 8. IGFBP-2104–289 exerts its mitogenic effect through the MAP/ERK 1/2 pathway. A, RCJ cells were cultured until confluence, serum starved for 12 h, and stimulated with IGF-I (7.8 nM), IGFBP-2104–289 (7.8 nM), and inhibitor U0126 (2.5 nM) and incubated for an additional 48 h. [3H]thymidine incorporation into the acid-extractable pool was determined by scintillation counting and used as a measure of DNA synthesis, as described in Materials and Methods. Data are mean ± SE. Statistics were by ANOVA were for 12 parallel wells per group from three independent experiments. *, P < 0.05 vs. control; #, P < 0.05 vs. IGF-I; , P < 0.05 vs. IGFBP-2104–289. B, After 12 h of starvation, RCJ cells were incubated for 1 h with IGFBP-2104–289, IGF-I, and inhibitor, and the respective membranes were probed with specific antibodies against p-ERK and total ERK. Representative blots of three independent experiments are shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In contrast to intact IGFBP-2, all three IGFBP-2 fragments investigated did not inhibit IGF-I-dependent chondrocyte proliferation. This observation is most likely due to the reduced binding affinity of these fragments for IGF-I. Via saturation binding assays using [¹²⁵I]IGF-I, Mark et al. (11) demonstrated a 150-fold reduced IGF-I binding affinity for IGFBP-2^167–279 (equilibrium rate constant = 495 nM), compared with intact IGFBP-2 (equilibrium rate constant = 3.4 nM); the binding affinities for the other two fragments were not investigated in this study.

The main finding of this study was that all tested carboxy-terminal IGFBP-2 fragments had significant IGF-I-independent stimulatory activity on proliferation of both rat growth plate chondrocytes in primary culture and of RCJ cells. This congruent data indicates that the effect of IGFBP-2 fragments on chondrocyte proliferation applies to growth plate chondrocytes in general. This is the first report on the biological activity of naturally occurring defined IGFBP-2 fragments in cell culture. It is of particular importance that the effects of IGFBP-2^104–289 and IGF-I on cell proliferation were not additive, consistent with the hypothesis that both peptides use similar intracellular signaling pathways. Indeed, the mitogenic activity of carboxy-terminal IGFBP-2 fragments was mediated through activation of the MAPK/ERK 1/2, which also mediates the mitogenic effect of IGF-I in growth plate chondrocytes (23). According to our observations, binding of fragmented IGFBP-2 to the cell membrane, is involved in the mechanism of the mitogenic effect of IGFBP-2 fragments. All three IGFBP-2 fragments investigated contain an Arg-Glu-Asp motif within the residues 265–267 (32) and a heparin-binding domain (PKKLRP) present within residues 180–185 (33). It has been demonstrated that IGFBP-2 binds to the glycosaminoglycans chondroitin-4- and -6-sulfate and to the proteoglycan aggrecan (24), which are both molecular components of the chondrocyte cell membrane (34).

We observed for the first time that partially reduced full-length IGFBP-2 has the same mitogenic activity in growth plate chondrocytes as equimolar concentrations of IGF-I. We hypothesize that partial reduction by DTT exposes specific binding sites of IGFBP-2, which are responsible for mitogenic activity and are otherwise covered in folded protein. It will be the subject of future studies whether and under which conditions partial reduction of full-length IGFBP-2 is also operative in vivo.

We found that both fragmented and partially reduced full-length IGFBP-2 exert their mitogenic effects by stimulation of the MAPK/ERK 1/2 and FAK signaling pathways. Other investigators observed that IGFBP-2 promotes deadhesion and reduced proliferation of a breast cancer cell line and a Ewing sarcoma cell line, associated with dephosphorylation of FAK and the p42/44 MAPKs (35). An early event in integrin signaling is marked by alterations in the phosphorylation status of FAK, which can be triggered by Arg-Glu-Asp-containing peptides such as IGFBP-2. This pathway is involved in the regulation of cell migration, apoptosis, and cell growth (36). Hence, although the biological activity of IGFBP-2 differs in various cell culture systems, similar intracellular signaling pathways appear to be involved.

One limitation of this study is the use of a heterologous test system. The IGFBP-2 fragments were derived from human serum, whereas their mitogenic effects were examined in two cell culture systems derived from rats, human growth plate chondrocytes being not readily available; IGFBP-2 is not entirely homologous between these species. Future confirmatory experiments with human test systems are required.

In summary, the defined carboxy-terminal fragments IGFBP-2^167–279, IGFBP-2^167–289, and IGFBP-2^104–289, which occur naturally in human serum, exert a mitogenic effect on growth plate chondrocytes comparable with that of equimolar concentrations of IGF-I. This biological activity of IGFBP-2 fragments was associated with cell membrane binding. The mitogenic activity of these IGFBP-2 fragments was mediated through activation of the MAPK/ERK 1/2 signaling pathway. These data imply a novel role of naturally occurring IGFBP-2 fragments for the endocrine and paracrine/autocrine regulation of longitudinal growth.

	Acknowledgments

 
We thank A. Spagnoli for generously providing the RCJ3.1C5.18 cell line. We are grateful to Silke Mark and Ute Bloch for isolation of carboxy-terminal IGFBP-2 fragments from human hemofiltrate and Wolf-Georg Forssmann for his generous support.

	Footnotes

 
This work was supported by a research grant from the Faculty of Medicine, University of Heidelberg.

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 12, 2008

Abbreviations: DTT, Dithiothreitol; FAK, focal adhesion kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IGFBP, IGF binding protein; p, phosphorylated; RP, reversed phase; TBST, Tris-buffered saline with Tween 20.

Received October 11, 2007.

Accepted for publication May 19, 2008.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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