Antiproliferative Effects of Insulin-like Growth Factor-binding Protein-3 in Mesenchymal Chondrogenic Cell Line RCJ3.1C5.18

RELATIONSHIP TO DIFFERENTIATION STAGE*

Anna SpagnoliDagger §, Vivian HwaDagger , William A. Horton, Gregory P. Lunstrum, Charles T. Roberts Jr.Dagger , Francesco Chiarelli||, Monica TorelloDagger , and Ron G. RosenfeldDagger

From the Dagger  Department of Pediatrics, Oregon Health Sciences University, Portland, Oregon 97201, the  Research Department, Shriners Hospital for Children, Portland, Oregon 97201, and the || Department of Pediatrics, University of Chieti, 66013 Chieti, Italy

Received for publication, June 13, 2000, and in revised form, October 29, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chondrogenesis results from a complex equilibrium between chondrocyte proliferation and differentiation. Insulin-like growth factors (IGFs) have a crucial role in chondrogenesis, but their mechanisms of action are not well defined. IGF-binding protein-3 (IGFBP-3) is the major carrier for circulating IGFs in postnatal life, and has been shown to have IGF-independent effects on proliferation of several cancer cell lines. In this study, we have evaluated the IGF-independent and -dependent effects of IGFBP-3 on chondrocyte proliferation and the relationship of these effects with chondrocyte differentiation stage. We used the RCJ3.1C5.18 nontransformed mesenchymal chondrogenic cell line, which, over 2 weeks of culture, progresses through the differentiation pathway exhibited by chondrocytes in the growth plate. We demonstrated that IGFBP-3 inhibited, in a dose-dependent manner (1-30 nM), the proliferation of chondroprogenitors and early differentiated chondrocytes, stimulated by des-(1-3)-IGF-I and longR3-IGF-I (IGF-I analogs with reduced affinity for IGFBP-3), and by insulin and IGF-I. In terminally differentiated chondrocytes, IGFBP-3 retained the ability to inhibit cell proliferation stimulated by IGF-I, but had no effect on cell growth stimulated by insulin, or des-(1-3)-IGF-I or longR3IGF-I. By monolayer affinity cross-linking, we demonstrated a specific IGFBP-3-associated cell-membrane protein of ~20 kDa. We determined that IGFBP-3 has an antiproliferative effect on chondrocytes and, that this effect is related to the differentiation process. In chondroprogenitors and early differentiated chondrocytes, antiproliferative effect of IGFBP-3 is mainly IGF-independent, whereas, following terminal differentiation this effect is IGF-dependent.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, alpha IGFBP-3g1, was generated and characterized in our laboratory (19). A polyclonal antibody against human IGFBP-2 that weakly recognizes IGFBP-3, called alpha HEC 1, was generated and characterized in our laboratory (20). In rats, alpha 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, alpha -minimal essential medium, and sodium pyruvate were purchased from Life Technologies, Inc. Dexamethasone and beta -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 alpha -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 beta -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).


                              
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Table I
Chondrocyte differentiation markers sequentially acquired by RCJ3.1C5.18 cells over 2 weeks of culture (14)

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 alpha 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 beta -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 alpha 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 alpha -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (alpha 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).



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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 alpha HEC polyclonal antibody against IGFBP-2 (alpha BP-2, lanes 14 and 15), or a polyclonal antibody against IGFBP-4 (alpha 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.



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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.



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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 beta -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 beta -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 beta -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).



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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).



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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).



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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.



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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 alpha -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.



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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 alpha -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    FOOTNOTES

* This work was supported in part by a grant from the Medical Research Foundation of Oregon (to A. S.), by a grant from Pharmacia & Upjohn (to A. S.), and by National Institutes of Health Grant CA58110 (to R. G. R.). This work was presented in part at the 81st Annual Meeting of the Endocrine Society, June 12-15, 1999, San Diego, CA.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Pediatrics (NRC-5), Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97201. Tel.: 503-494-0880; Fax: 503-494-0428; E-mail: spagnoli@ohsu.edu.

Published, JBC Papers in Press, November 10, 2000, DOI 10.1074/jbc.M005088200


    ABBREVIATIONS

The abbreviations used are: IGF, insulin-like growth factor; GH, growth hormone; IGFBP, IGF-binding protein; des-IGF-I, des-(1-3)-IGF-I; LR3-IGF-I, longR3-IGF-I; WLB, Western ligand blotting; WIB, Western immunoblotting; FGFR-3, fibroblast growth factor receptor-3; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; DSS, disuccinimidyl suberate.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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