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Endocrinology Vol. 138, No. 10 4390-4400
Copyright © 1997 by The Endocrine Society

ARTICLES

Insulin-Like Growth Factors I and II Are Autocrine Factors in Stimulating Proteoglycan Synthesis, a Marker of Differentiated Chondrocytes, Acting through Their Respective Receptors on a Clonal Human Chondrosarcoma-Derived Chondrocyte Cell Line, HCS-2/81

Masaharu Takigawa, Tokutaro Okawa, Hai-Ou Pan, Chiharu Aoki, Kojiro Takahashi, Jing-De Zue, Fujio Suzuki and Akihiro Kinoshita

Department of Biochemistry and Molecular Dentistry (M.T., K.T.) and Biochemical Research Center (C.A.), Okayama University Dental School, Okayama 700, Japan; Department of Orthopaedic Surgery (T.O.), Osaka City University Medical School, Osaka 545, Japan; Departments of Biochemistry,(H.-O.P., F.S.) and Pedodontics (A.K.), Osaka University Faculty of Dentistry, Suita, Osaka 565, Japan; and Department of Medical Oncology (J.Z.), Glasgow University, Glasgow G61 1BD, United Kingdom

Address all correspondence and requests for reprints to: Prof. Masaharu Takigawa, D.D.S., Ph.D., Department of Biochemistry and Molecular Dentistry, Okayama University Dental School, Okayama 700, Japan. E-mail: takigawa{at}dent.okayama-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Both insulin-like growth factor (IGF)-I and IGF-II increased the synthesis of cartilage-type, large proteoglycan in a human chondrosarcoma-derived chondrocyte cell line, HCS-2/8. In contrast to the stimulatory effects of IGFs on costal chondrocytes of the young rabbit, the stimulatory effect of IGF-II on proteoglycan synthesis in HCS-2/8 cells was more potent than that of IGF-I. IGF-II, but not IGF-I, increased calcium influx into HCS-2/8 cells, and there was a close relation between the stimulation of proteoglycan synthesis and the calcium influx. [125I]IGF-I bound to HCS-2/8 cells, and this binding was competitively inhibited by low concentrations of unlabeled IGF-I, higher concentrations of IGF-II, and much higher concentrations of insulin. [125I]IGF-II also bound to the cells, and its binding was competitively inhibited by IGF-II and slightly inhibited by higher concentrations of IGF-I and much higher concentrations of insulin. When radioligand-receptor complexes were separated by SDS-PAGE and subjected to autoradiography, two major bands at 260 and 130 kDa were observed, which correspond to the IGF type II receptor (IGF-IIR) and the {alpha} subunit of the IGF type I receptor (IGF-IR), indicating the presence of both receptors. When confluent cultures of HCS-2/8 cells were maintained in serum-free medium, proteoglycan synthesis did not decrease unless the medium was repeatedly replaced. Conditioned medium of HCS-2/8 cells stimulated the HCS-2/8 cells to synthesize proteoglycans. RIA revealed that the cells produced both IGF-II and IGF-I. Transcripts of messenger RNAs of both IGF-I and IGF-II and both IGF-IR and IGF-IIR also were detectable by Northern analysis. Both anti-IGF-IR antibody and anti-IGF-II antibody inhibited proteoglycan synthesis. Mannose-6-phosphate, which is known to bind to IGF-IIR, stimulated proteoglycan synthesis, potentiated IGF-II-stimulated proteoglycan synthesis, and enhanced the binding affinity for IGF-II but not for IGF-I. Even in the presence of anti-IGF-IR antibody, IGF-II and mannose-6-phosphate stimulated proteoglycan synthesis in the cells. [Leu27]IGF-II, an IGF-II analogue with high affinity only for IGF-IIR, strongly stimulated proteoglycan synthesis in HCS-2/8 cells but [Arg54, Arg55]IGF-II, which binds to only IGF-IR, also stimulated proteoglycan synthesis in the cells. These findings indicate that IGF-I and IGF-II act as autocrine differentiation factors for this chondrocytic permanent cell line, HCS-2/8, mainly via respective receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factors (IGFs)-I and -II, originally identified in human serum, are two single-chain polypeptides that share a high degree of homology with polypeptide structure of proinsulin (1, 2). The best-recognized biological action of IGF-I is promotion of long bone growth, which is thought to be caused primarily by stimulation of growth plate chondrocyte proliferation and/or differentiation (1, 2, 3). Although IGF-I has been believed to mediate the action of GH on cartilage and bone, according to the somatomedin theory (1, 2), the findings that IGF-I transcripts existed in cartilage and increased by GH also suggest that IGF-I, locally produced, play a role in the mediation of the skeletal action of GH (4). However, it is still unclear whether the local action of IGF-I on chondrocytes is autocrine, because there is controversy with respect to the production and gene expression of IGF-I in chondrocytes (4, 5, 6, 7).

In contrast to IGF-I, the role of IGF-II is less clear. It is thought to be important in fetal growth (1, 2, 3). IGF-II production in the liver, which is its primary site of production, and its serum level in rodents decline rapidly after birth and are replaced by IGF-I, which is GH-dependent (1, 2). A high level of gene expression of IGF-II was detected in growth plate chondrocytes in embryonic (7) and postnatal rodents (6, 7), and its production was observed using articular chondrocytes in secondary culture (5). It stimulates clonal growth of human fetal chondrocytes (8), and multiplication-stimulating activity (rat IGF-II) stimulated DNA and proteoglycan synthesis in growth plate chondrocytes in culture (2, 3, 9). These findings suggest that locally produced IGF-II may act on chondrocytes. However, even if so, it is not clear whether the action is autocrine or paracrine, because much IGF-II is present in bone matrix (10).

There are two types of IGF receptors (3, 11, 12). The type I IGF receptor (IGF-IR) generally binds IGF-I with a higher affinity than IGF-II and interacts weakly with insulin. The type II IGF receptor (IGF-IIR) preferentially binds IGF-II. The presence of both IGF receptors on most cells and the cross-reactivity of ligands for binding to these receptors make it difficult to determine which receptor mediates a particular biologic response. Although signal transduction via IGF-IR has been extensively investigated (12), few studies have been done on the signal transduction pathway via IGF-IIR, and the action of IGF-II was suggested to be mediated by IGF-IR (11). On the other hand, Nishimoto et al. (13) showed that G protein was involved in signal transduction of the growth stimulatory action of IGF-II on BALB/c3T3 fibroblasts through their IGF-IIR. However, the results remain difficult to interpret unless they are extended to other bioresponses and other types of cells. Recently, Yu et al. (14) reported that the level of IGF-IIR transcript increased during the cartilage formation period of endochondral bone formation after implantation of demineralized bone matrix. Therefore, chondrocyte lineage cells may be a good model for this purpose because their phenotypes are highly specific.

Chondrocytes are unique cells, in that they have many differentiated markers such as large cartilage-type proteoglycans (aggrecan) and collagen types II, IX, X, and XI (15, 16, 17). Among them, the ability to synthesize proteoglycan is one of the most important markers of chondrocytes (16, 17, 18, 19). However, for a long time, it was difficult to culture chondrocytes without losing this ability (18). Previously, we established two immortal clonal cell lines, HCS-2/8 and HCS-2/A, from a well-differentiated type of human chondrosarcoma (20, 21, 22). HCS-2/8, in particular, is the first permanent cell line resembling normal chondrocytes, in that they synthesize aggrecan, collagen types II, IX, and XI, and integrins found in chondrocytes, and they show the same responses to various vitamins and growth factors as normal chondrocytes (20, 22, 23, 24, 25, 26). Therefore, studies on why the cell line maintains the chondrocyte phenotype in long-term cultures should greatly increase our understanding of the molecular and cellular mechanisms that control the differentiation of chondrocytes and/or the genesis of chondrosarcomas in humans.

In the present study, using the clonal chondrocytic permanent cell line HCS-2/8 without any contamination of other types of cells, we found that both IGF-I and IGF-II act as autocrine factors in the maintenance of high proteoglycan synthesis activity. We also investigated IGF receptor systems in this cell line and found that IGF-I and IGF-II act through their respective receptors, in stimulating proteoglycan synthesis. It also was found that Ca influx may be involved in IGF-IIR-mediated stimulation of proteoglycan synthesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant human IGF-I was kindly supplied by Fujisawa Pharmaceuticals (Osaka, Japan), and synthetic human IGF-I was kindly provided by Dr. H. Funakoshi (Department of Pharmacology, Kyoto University, Kyoto, Japan). Both preparations of IGF had the same potency in stimulating proteoglycan synthesis in rabbit costal chondrocytes in primary culture. Recombinant human IGF-II was kindly provided by Wakunaga Pharmaceuticals (Hiroshima, Japan). [Leu27]IGF-II and [Arg54, Arg55]IGF-II were kindly supplied by Dr. K. Sakano (Molecular Biology Research Laboratory, Daichi Pharmaceutical Co., Tokyo, Japan). IGF-I RIA kit, Amerlex-M (donkey antirabbit IgG), [125I]-labeled IGF-I (~2,000 Ci/mmol) and IGF-II (~2,000 Ci/mmol), and [{gamma}-32P]ATP were from Amersham International (Aylesbury, UK). [35S]Sulfuric acid (carrier free) was from Japan Atomic Energy Institute (Tokyo, Japan). Anti-IGF-I receptor monoclonal antibody ({alpha}IR-3) (27, 28) and the 40-mer oligoprobes of human IGF-I, IGF-II, IGF-IR, and IGF-IIR were purchased from Oncogene Science, Inc. (Manhasset, NY). Rabbit antihuman IGF-II IgG was from Austral Biologicals (San Ramon, CA). All plastic culture dishes and plates were from Falcon (Oxnard, CA). FBS was from Gibco (Grand Island, NY).

Cell cultures
Unless otherwise specified, HCS-2/8 cells were inoculated at a density of 2 x 104 cells/cm2 into 96-, 48-, or 24-multiwell plates, 35-mm-diameter dishes, or 90-mm-diameter dishes and grown in Eagle’s MEM supplemented with 20% FBS and 60 µg/ml kanamycin at 37 C under 5% CO2 in air. Rabbit costal chondrocytes were isolated from growth cartilage of the ribs of young male New Zealand rabbits, weighing 300–500 g, as described previously (16, 19). The isolated cells were inoculated at a density of 2 x 104 cells/cm2 and grown in MEM containing 10% FBS and 60 µg/ml kanamycin. The medium was replaced every 3 days.

Determination of the rate of proteoglycan synthesis
Proteoglycan synthesis was determined by measuring the incorporation of [35S]sulfuric acid into glycosaminoglycans, as previously described (16, 17). Briefly, the cells were labeled with 2–5 µCi/ml [35S]sulfuric acid for an appropriate amount of time. After labeling, the cultures were digested with Pronase E, and the radioactivity in the material precipitated with cetylpyridinium chloride was measured in a scintillation counter.

Determination of relative hydrodynamic sizes of proteoglycan monomers
The hydrodynamic sizes of newly synthesized radiolabeled proteoglycans were investigated by Sepharose CL-2B column chromatography under dissociative conditions (29, 30). Briefly, the cells were labeled with 30 µCi/ml of [35S]sulfate in a mixture of Gey’s solution and Hanks’ solution (9:1, vol/vol) for 3 h. Proteoglycans were extracted from cell layers for 24 h at 4 C with a solution of 4 M guanidine-HCl containing 5 mM benzamidine-HCl, 0.1 M 6-aminohexanoic acid, 10 mM sodium-EDTA, and 60 mM Tris-HCl (pH 8.0). Portions (0.3 ml) of the proteoglycan extracts were layered on Sepharose-2B column and eluted with the same buffer. Flow rate was 12 ml/h. The proteoglycans in each fraction (2 ml) were digested with Pronase E (1 mg/ml), precipitated with cetylpyridinium chloride, and the radioactivity of each precipitate was counted (19).

Calcium incorporation assay
HCS-2/8 cells were plated at a density of 1.6 x 105 cells/well in 35-mm glass-bottom microwells (MatTek Corp., Ashland, MA) and grown to confluence. After incubation for 24 h in DMEM containing 10% FBS, incorporation of calcium into the cells was measured with an FES 300 system (Scholaratic, Osaka, Japan) in the presence of 5 µM Fura-2AM.

Protein determination
Protein was determined by the method of Lowry et al. (31) with BSA as a standard.

Binding studies
Cell layers of HCS-2/8 cells at confluency were washed with PBS twice and incubated with binding buffer [serum-free DMEM containing 0.2% BSA and 15 mM HEPES, pH 7.2] containing [125I]-IGF-I or [125I]-IGF-II with or without unlabeled ligands for 4 h at 15 C. Under this condition, the bindings of both ligands were saturable. Cultures were then washed five times with cold binding buffer and solubilized in 1.0 N NaOH. The radioactivity in the solution was counted in a {gamma} counter. Nonspecific bindings of [125I]-IGF-I and [125I]-IGF-II, determined by using more than 100,000-fold unlabeled ligands, were less than 3% of total bindings.

Covalent attachment of [125I]-IGF-II to receptors
Cross-linking of [125I]-IGF-II to HCS-2/8 cells was carried out as described previously (32, 33). Briefly, HCS-2/8 cells were incubated with [125I]-IGF-II with or without unlabeled IGF-I, IGF-II, or insulin for 4 h at 15 C; the cells were washed with cold PBS 5 times and incubated with a solution containing 0.1 mM disuccinimidyl suberate and protease inhibitors for 20 min at 4 C. The cross-linking reaction was stopped by washing the cell layers with PBS. The cells were then scraped into sample buffer for SDS-PAGE [14 mM Tris-HCl (pH 6.8), 10% (vol/vol) glycerol, and 3% (wt/vol) SDS], denatured by heat treatment for 5 min at 100 C, and subjected to SDS-PAGE and autoradiography.

Assays of IGFs in conditioned medium (CM)
HCS-2/8 cells and rabbit costal chondrocytes were grown in 100-mm-diameter dishes to confluence. The cells were then washed with serum-free DMEM and incubated in 10 ml of medium per dish. After 48 h of culture of these cells, CM was collected and centrifuged at 500 x g for 5 min. The supernatant was acidified with a final concentration of 0.25 M HCl at 4 C for 30 min at room temperature to separate IGFs from their binding protein and passed through Sep-Pak C18 (Waters, Milford, MA). IGFs adsorbed on Sep-Pak C18 was washed with 20 ml of 4% acetic acid, eluted with 4 ml methanol, and then lyophilized until assay. The recovery was more than 90%. IGF-I was assayed by an RIA using an assay kit (Amersham). IGF-II also was assayed by a modified method of the RIA for IGF-I, using rabbit antihuman IGF-II IgG. One assay tube contained 10 ng of the anti-IGF-II, 4,000 cpm [125I]-IGF-II, and 0.15–2.5 ng IGF-II as standards in 0.2 ml binding buffer. Under this condition, about 30% binding was obtained. After 48 h of incubation at 4 C, Amerlex-M was added, and unbound [125I]-IGF-II was removed by centrifugation.

RNA Isolation and Northern blot analysis
RNA was isolated from HCS-2/8 cells at different growth stages by the single-step method described (25). Messenger RNA (mRNA) was purified using oligo-dT30 beads (Takara, Tokyo, Japan), according to the instruction manual. Ten micrograms of total RNA or mRNA were used for Northern blotting, as described previously (25). The 40-mer oligonucleotide probes of IGF-I, IGF-II, IGF-RI, and IGF-RII were labeled with [{gamma}-32P]ATP using T4 nucleotide kinase.

Statistical analysis
Unless otherwise specified, all experiments were repeated at least twice, and similar results were obtained in the repeated experiments. Statistical analysis was performed by one-way ANOVA. Data are expressed as the mean ± SD. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of IGF-I and IGF-II on morphology of HCS-2/8 cells
When HCS-2/8 cells were grown in MEM, containing 20% FBS, and reached confluence, they showed a polygonal shape that is typical of chondrocytes (Fig. 1AGo). When HCS-2/8 cells were grown in the presence of 100 ng/ml IGF-I and 10 ng/ml IGF-II for 4 days, the cells became spherical (Fig. 1Go, B and C) and secreted much matrix. When stained with toluidine blue, this matrix showed strong metachromasia (data not shown), suggesting accumulation of proteoglycans.



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  		Figure 1. Effects of IGF-I and IGF-II on the morphology of HCS-2/8 cells. HCS-2/8 cells were inoculated at a density of 16 x 104 cells per 35-mm dish. When reaching subconfluence, IGF-1 (B) and IGF-II (C) were added to the cultures at concentrations of 100 ng/ml and 10 ng/ml, respectively. PBS was added to control cultures (A). The medium was changed 2 and 4 days later, and IGFs were added at the time of the medium change. Photomicrographs were taken 5 days after the first addition of IGFs (magnification, x85).

 
Effects of IGF-I and IGF-II on proteoglycan synthesis
The incorporation of [35S]sulfate into proteoglycans is known to be a biochemical marker of the differentiated phenotype of chondrocytes. Figure 2Go shows the effects of IGF-I and IGF-II on incorporation of [35S]sulfate into proteoglycans in HCS-2/8 cells and rabbit costal chondrocytes in primary culture. IGF-I increased proteoglycan synthesis in HCS-2/8 cells dose-dependently; the level was 130% that of control at a concentration of 100 ng/ml and reached 150% that of control at concentrations of 300-1000 ng/ml. IGF-I also increased proteoglycan synthesis in rabbit costal chondrocytes dose-dependently. However, there was a big difference between these cells. Rabbit chondrocytes responded to IGF-I better than HCS-2/8 cells. Its effect was observable at a concentration of 1 ng/ml and reached a plateau at a concentration of 100 ng/ml. IGF-II also increased proteoglycan synthesis in both cells dose-dependently. In contrast to IGF-I, HCS-2/8 cells responded to IGF-II better than rabbit costal chondrocytes; its effect on HCS-2/8 cells was observable at a concentration of 1 ng/ml and reached a peak at a concentration of 300 ng/ml, whereas its effect on rabbit chondrocytes was observable at concentrations of more than 100 ng/ml.



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  		Figure 2. Effects of IGF-I and IGF-II on proteoglycan synthesis in HCS-2/8 cells. HCS-2/8 cells ({circ}, •) and rabbit costal chondrocytes ({square}, {blacksquare}) were inoculated at a density of 4 x 104 cells and 3 x 104 cells/well, respectively, in a 24-well multiwell plate. When they reached confluence, the medium was replaced by serum-free DMEM. After 24 h, the cells were fed with fresh DMEM containing IGF-I ({circ}, {square}) and IGF-II (•, {blacksquare}). PBS was added to control cultures. The cells were labeled with 2 µCi/ml [35S]sulfuric acid for 17 h from 5 h after the addition of IGFs. The incorporation of [35S]sulfuric acid into glycosaminoglycans was determined as described in Materials and Methods and normalized by the protein content. Values are means of 9–16 cultures from 3 experiments, with the individual values varying less than 6% from the means.

 
To confirm that the increases in proteoglycan synthesis by IGF-I and IGF-II reflect increases in cartilage-type proteoglycans, HCS-2/8 cells were labeled with [35S]sulfate, and proteoglycans were extracted with guanidine-HCl and separated by gel filtration under dissociative conditions. As shown in Fig. 3Go, both IGF-I and IGF-II increased synthesis of cartilage-type, large proteoglycan monomers.



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  		Figure 3. Sepharose CL-2B column chromatography of proteoglycans extracted from HCS-2/8 cells. The cells were inoculated at a density of 3 x 105 cells/35-mm dish and grown to confluence. After washing with serum-free DMEM, the cells were fed with DMEM containing 500 ng/ml IGF-I or 100 ng/ml IGF-II. PBS was added to control cultures. After 18 h, 30 µCi/ml [35S]sulfuric acid was added to the cultures and incubated for 6 h. Proteoglycans were extracted with 4 M guanidine-HCl and subjected to Sepharose CL-2B column chromatography under dissociative conditions, as described in Materials and Methods.

 
Calcium incorporation into cells by IGF-I and IGF-II
IGF-II has been shown to bind IGF-IIR and then stimulate calcium influx into cells via activation of Gi-2 protein (13). Therefore, we investigated weather IGFs cause calcium influx into HCS-2/8 cells. As shown in Fig. 4Go, 10Go ng/ml of IGF-II, which significantly stimulated proteoglycan synthesis, caused calcium influx into the cells. This effect was maximal at concentrations of 100–500 ng/ml IGF-II, which caused maximal stimulation of proteoglycan synthesis. In contrast, 500 ng/ml IGF-I, which increased proteoglycan synthesis, failed to cause calcium influx into HCS-2/8 cells (Fig. 4Go).



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  		Figure 4. Effects of IGF-I and IGF-II on calcium incorporation into HCS-2/8 cells. Calcium incorporation through the cell membrane was measured as described in Materials and Methods. The indicated concentrations of IGF-I (top) and IGF-II (bottom) were added at the times indicated by vertical bars. The ratio of fluorescence (340 nm/380 nm) indicates the relative rate of incorporation at each concentration of IGFs.

 


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  		Figure 10. Effect of M-6-P on nonstimulated (A) and IGF-stimulated proteoglycan synthesis (B) in HCS-2/8 cells. HCS-2/8 cells were inoculated at a density of 4 x 104 cells/well of a 24-well multiwell plate. When they reached confluence, the medium was replaced by serum-free DMEM. After 24 h, the cells were fed with fresh DMEM containing M-6-P and mannose at the concentrations indicated (A), or 10 µM M-6-P and 10 ng/ml IGF-II in combination (B). The cells were labeled with 2 µCi/ml [35S]sulfuric acid for 17 h from 5 h after the addition. Other methods were as described in Materials and Methods. Columns and bars are means and SD of 18 to 30 cultures from 3 to 5 experiments. *, P < 0.05, significantly different from the control cultures; **, P < 0.05, significantly different from the IGF-II-treated cultures.

 
Expression of IGF-IR and IGF-IIR on HCS-2/8 cells
Because IGF-I and -II stimulated proteoglycan synthesis in HCS-2/8 cells, we next investigated whether the cells have receptors for IGF-I and IGF-II. Figure 5AGo shows the results of a typical binding experiment of radiolabeled IGF-I. The addition of IGF-I decreased the binding of [125I]-IGF-I dose-dependently; the inhibition was observable at a concentration of 0.1 ng/ml and was complete at a concentration of 100 ng/ml. The addition of IGF-II also decreased the binding of [125I]-IGF-I dose-dependently; the inhibition, however, was observable at a level of 10 ng/ml and was about 60% at a concentration of 100 ng/ml. On the other hand, insulin only slightly inhibited the binding of [125I]-IGF-I. Scatchard analysis of the data indicated the presence of two classes of binding sites on HCS-2/8 cells. The apparent Kd values of the high- and low-affinity binding sites were 0.06 nM and 1.2 nM, and the numbers of these high- and low-affinity binding sites (Bmax numbers) were about 1.3 x 104 sites/cell and 6.0 x 104 sites/cell, respectively. Figure 5BGo shows the results of a typical binding experiment of radiolabeled IGF-II. The addition of IGF-II decreased the binding of [125I]-IGF-II dose-dependently; the inhibition was observable at a concentration of 10 ng/ml and was almost complete at 1000 ng/ml. On the other hand, IGF-I and insulin only slightly inhibited the binding of [125I]-IGF-II. Scatchard analysis of the data indicated the presence of two classes of binding sites on HCS-2/8 cells. The Kd values of the high- and low-affinity binding sites were 1.5 nM and 40 nM, and Bmax numbers were about 3.6 x 105 sites/cell and 1.9 x 106 sites/cell, respectively.



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  		Figure 5. Competitive inhibition of [125I]-IGF-I binding (A) and [125I]-IGF-II binding (B) by unlabeled IGF-I (•), IGF-II ({blacksquare}) and insulin ({blacktriangleup}) to HCS-2/8 cells (top). The cells were inoculated at a density of 4 x 104 cells per 16-mm-diameter multiwell plate in growth medium. When they reached confluence, they were incubated with [125I]-IGF-I (65,000 cpm/well) and [125I]-IGF-II (65,000 cpm/well), as described in Materials and Methods. Specific binding was calculated by subtracting nonspecific binding from total binding. Nonspecific bindings of [125I]-IGF-I and [125I]-IGF-II were 135 cpm/well and 143 cpm/well, respectively. Points are means for three cultures. The numbers of HCS-2/8 cells were 2.20 x 105 per well. Scatchard plot from a representative experiment (bottom).

 
For characterization of the physicochemical properties of IGF receptors, [125I]-IGF-II was covalently attached to HCS-2/8 cells by treatment with disuccinimidyl suberate, and the labeled IGF-II-receptor complex was separated by SDS-PAGE under reducing conditions (Fig. 6Go). An autoradiogram of the gel revealed two major bands, with apparent molecular masses of 260 kDa and 130 kDa. The 130-kDa band disappeared on the addition of 30 ng/ml unlabeled IGF-I but not on the addition of the same concentration of unlabeled IGF-II (Fig. 6Go). The band, however, disappeared on the addition of high concentrations of unlabeled IGF-II and became faint on the addition of a high concentration of insulin (data not shown), indicating that this band represents the complex of [125I]-IGF-II and the {alpha}-subunit of the IGF-IR. The 260-kDa band disappeared on the addition of IGF-II and became faint on addition of IGF-I but not on addition of insulin, indicating that this band represents the complex of [125I]-IGF-II and IGF-II receptor.



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  		Figure 6. Cross-linking of IGF receptors on HCS-2/8 cells with [125I]-IGF-II. The cells were inoculated at a density of 4 x 104 cells per 16-mm-diameter multiwell plate in growth medium. When they reached confluence, they were incubated with the [125I]-IGF-II (5 x 105 cpm; 3.8 x 10-10 M) in the presence of PBS (lane 1) and 30 ng/ml of unlabeled IGF-II (lane 2), IGF-I (lane 3), and insulin (lane 4). After cross-linking, the cells were rinsed and harvested in 50 µl of sample buffer and subjected to SDS-PAGE and autoradiography.

 
To confirm the presence of IGF-IR and IGF-IIR on HCS-2/8 cells, we also investigated the gene expression of these receptors by Northern blotting. As shown in Fig. 7AGo, transcripts of both receptors were detected in confluent cultures of HCS-2/8 cells.



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  		Figure 7. Northern analysis of transcripts of IGF-IR, IGF-IIR (A), IGF-I and IGF-II (B) from HCS-2/8 cells. A, HCS-2/8 cells were inoculated at a density of 2 x 106 cells/90-cm-diameter dish. When the cells reached confluence, they were harvested for isolation of RNA. Ten micrograms of poly(A) RNA was used for Northern blotting. IGF-IR, 9 days exposure; IGF-IIR, 2 days exposure. Slight signals were also detected when 20 µg of total RNA was used. B, Ten micrograms of total RNA isolated from HCS-2/8 cells at sparse phase (lane G), subconfluent phase (lane SC), confluent phase (lane C), and overconfluent phase (lane OC) of culture were used for Northern blotting. IGF-I, 8 days exposure; IGF-II, 4 days exposure. Ethidium bromide staining revealed that equal amounts of RNA were present in each lane (data not shown).

 
Production of IGFs by HCS-2/8 cells
Table 1Go shows the effect of medium change on proteoglycan synthesis in HCS-2/8 cells cultured in serum-free medium. When the cells became confluent, the culture medium was replaced with fresh serum-free medium, and they were preincubated for 24 h. Then the cells were cultured, with or without medium change, and proteoglycan synthesis was measured at 0, 24, and 48 h. Without medium change, proteoglycan synthesis did not decrease significantly; the levels after 24 h and 48 h were 95% and 83%, respectively, of the time-zero level. When the medium change was repeated every day, proteoglycan synthesis decreased to 48% after 24 h and 36% after 48 h. When the medium was changed at 0 h, but not 24 h, the proteoglycan synthesis decreased to 42% after 48 h.


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  		Table 1. Effect of medium change on proteoglycan synthesis in HCS-2/8 cells

 
In addition, when concentrated CM of HCS-2/8 cells was added to cultures of HCS-2/8 cells, proteoglycan synthesis in the cells increased dose-dependently (Fig. 8Go). Because this activity was adsorbed to Sep-Pak C18, which has been frequently used for the purification of IGFs, we measured IGF-I and IGF-II in the CM of HCS-2/8 cells. The concentrations of IGF-I and IGF-II in the medium conditioned with the cells for 48 h were 0.31 ± 0.04 and 2.92 ± 0.53 ng/ml (mean ± SD of 3 cultures), respectively. The concentrations of IGF-I and IGF-II in primary cultures of rabbit chondrocytes were 0.14 ± 0.02 and 1.18 ± 0.18 ng/ml (mean ± SD of 3 cultures), respectively.



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  		Figure 8. Effect of CM of HCS-2/8 cells on proteoglycan synthesis in HCS-2/8 cells. HCS-2/8 cells were inoculated at a density of 4 x 104 cells/well of a 24-well multiwell plate. When they reached confluence, the medium was replaced by serum-free DMEM containing concentrated CM of HCS-2/8 cells at the final concentrations indicated. The incorporation of [35S]sulfuric acid into glycosaminoglycans was determined as described in Materials and Methods. Points and bars are means and SD for 4 cultures. The horizontal axis indicates the number of times greater than the original concentration.

 
As shown in Fig. 7BGo, Northern blot analysis also revealed that HCS-2/8 cells expressed IGF-I and IGF-II. The levels of IGF-I and IGF-II transcripts were increased as the cells became confluent and were highest in the overconfluent phase.

Role of IGF-IR and IGF-IIR in autocrine regulation of proteoglycan synthesis
Antihuman IGF-IR antibody ({alpha}IR-3) dose-dependently inhibited proteoglycan synthesis (Fig. 9Go). The maximal inhibition was observed at a concentration of 10 µg/ml and was 70 to 80%. This finding suggests that autocrine factor(s), which acts through IGF-IR, mainly supports proteoglycan synthesis in HCS-2/8 cells. On the other hand, antihuman IGF-II antibody also inhibited proteoglycan synthesis, but its maximal inhibition was only 30%, suggesting that both IGF-I and IGF-II act as autocrine factors to support proteoglycan synthesis in the cells.



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  		Figure 9. Effect of anti-IGF-IR and anti-IGF-II antibodies on proteoglycan synthesis in HCS-2/8 cells. HCS-2/8 cells were inoculated at a density of 1 x 104 cells/well of a 96-well multiwell plate. When they reached confluence, the medium was replaced by serum-free DMEM containing antihuman IGF-I receptor antibody ({alpha}IR-3) ({circ}) or antihuman IGF-II antibody (•) at the concentrations indicated. Rabbit IgG was added to control cultures and had no effect on proteoglycan synthesis. The cells were labeled with 2 µCi/ml of [35S]sulfuric acid for 17 h from 5 h after the addition. The incorporation of [35S]sulfuric acid into glycosaminoglycans was determined as described in Materials and Methods. Values are means of 4 cultures, with the individual values varying less than 5% from the means. In repeated experiments, maximal inhibitions by antihuman IGF-I receptor antibody ({alpha}IR-3) and antihuman IGF-II antibody were about 70–80% and about 30%, respectively.

 
To clarify which receptor is involved in the action of IGF-II, we investigated the effect of mannose-6-phosphate (M-6-P) on proteoglycan synthesis in HCS-2/8 cells. As shown in Fig. 10Go, M-6-P dose-dependently stimulated proteoglycan synthesis, and the stimulatory effect was observable at a concentration of 1 mM and reached a maximum at 3–5 mM (Fig. 10AGo). Fructose-1-phosphate also stimulated proteoglycan synthesis (data not shown), whereas mannose had no effect on proteoglycan synthesis (Fig. 10AGo). When a low concentration of M-6-P, which had no effect by itself, was added to cultures of HCS-2/8 cells in conjunction with 10 ng/ml of IGF-II, which caused a half-maximal stimulation, the M-6-P potentiated the stimulatory effect of IGF-II (Fig. 10BGo).

To clarify the mechanism of action of M-6-P, we next investigated the effect of M-6-P on the binding of [125I]-IGF-I and [125I]-IGF-II to HCS-2/8 cells. As shown in Fig. 11Go, M-6-P shifted the dose-dependent inhibition curve for [125I]-IGF-II binding to lower concentration but had no effect on that for [125I]-IGF-I binding.



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  		Figure 11. Effect of M-6-P on competitive inhibition of [125I]-IGF-I binding (A) and [125I]-IGF-II binding (B) by unlabeled ligands to HCS-2/8 cells. The cells were inoculated at a density of 4 x 104 cells per 16-mm-diameter multiwell plate in growth medium. When they reached confluence, the cells were washed with binding buffer. After preincubation with (•) or without 10 µM of M-6-P ({circ}) for 1 h, the cells were incubated with [125I]-IGF-I and [125I]-IGF-II. Other methods are described in the legend of Fig. 5Go and in Materials and Methods. The numbers of cells in HCS-2/8 cells were 2.20 x 105 per well. Values are means of 4 cultures, with the individual values varying less than 5% from the means.

 
Because anti-IGF-IR antibody blocked IGF-IR-mediated proteoglycan synthesis, we next investigated the effects of IGF-II and M-6-P on proteoglycan synthesis in the presence of anti-IGF-IR antibody (Table 2Go). Even in the presence of anti-IGF-IR antibody, IGF-II and M-6-P stimulated proteoglycan synthesis. The stimulatory effects of IGF-II and M-6-P in the presence of anti-IGF-IR were almost the same as those in the absence of the antibody, indicating that IGF-IIR mediates the actions of IGF-II and M-6-P.


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  		Table 2. Stimulation by IGF-II and M-6-P of proteoglycan synthesis in HCS-2/8 cells inhibited by anti-IGF-IR antibody

 
To confirm this further, we tested the effect of two analogs of IGF-II. [Leu27]IGF-II, with high affinity only for IGF-IIR, significantly increased proteoglycan synthesis at concentrations of more than 50 ng/ml (Fig. 12Go). These data indicate that IGF-IIR mediates the stimulatory action of IGF-II on proteoglycan synthesis in the HCS-2/8 cells. Another analogue, [Arg54, Arg55]IGF-II, which binds only IGF-IR, also increased proteoglycan synthesis (Fig. 12Go). The stimulatory effect of this analogue was significantly less than that of IGF-II, also indicating that IGF-IIR mediates the stimulatory action of IGF-II on proteoglycan synthesis in HCS-2/8 cells.



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  		Figure 12. Effects of [Leu27]IGF-II and [Arg54, Arg55]IGF-II on proteoglycan synthesis in HCS-2/8 cells. HCS-2/8 cells were inoculated at a density of 4 x 104 cells/well of a 24-well multiwell plate. When they reached confluence, the medium was replaced by serum-free DMEM. After 24 h, the cells were fed with fresh DMEM containing IGF-I ({circ}), IGF-II ({square}), [Leu27]IGF-II (•), and [Arg54,Arg55]IGF-II ({blacksquare}). The cells were labeled with 2 µCi/ml [35S]sulfuric acid for 17 h from 5 h after the addition of IGFs. The incorporation of [35S]sulfuric acid into glycosaminoglycans and the protein content were determined as described in Materials and Methods. Points and bars are means and SD of 5 to 12 cultures. a, P < 0.05, significantly different from the respective control cultures; b, P < 0.05, significantly different from the [Arg54,Arg55]IGF-II-treated cultures. In repeated experiments, the stimulation by [Leu27]IGF-II was significant (P < 0.05) at concentrations of more than 30 ng/ml, and there was no significant difference between IGF-II-treated and [Leu27]IGF-II-treated cultures.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Like previous findings using primary to tertiary cultures of growth plate chondrocytes (2, 3, 8, 9, 34, 35, 36), both IGF-I and IGF-II stimulated proteoglycan synthesis in HCS-2/8 cells. The size of major proteoglycan monomers synthesized by this cell line corresponded to that of the cartilage type of proteoglycan, the so-called aggrecan (Fig. 3Go), and both growth factors increased the synthesis of this type of proteoglycan (Figs. 2Go and 3Go). In addition, the major glycosaminoglycan chains contained within the proteoglycan monomers were about 38 kDa and mainly composed of chondroitin sulfate, especially chondroitin-6-sulfate, and the proteoglycan monomers also contained a little keratan sulfate chains (22). Moreover, the cells also strongly expressed mRNA of aggrecan core protein (23) and synthesized a single-link protein with a molecular mass of 42 kDa (22). These findings indicate that HCS-2/8 cells synthesized typical cartilage proteoglycans, and both IGF-I and IGF-II stimulated expression of this typical differentiation marker of chondrocytes. In contrast to many actions of IGFs reported previously (1) and to the actions of IGFs on rabbit costal chondrocytes, IGF-II was more potent than IGF-I in stimulating proteoglycan synthesis in HCS-2/8 cells. It has been believed that IGF-II is the principal somatomedin in the embryos and fetuses because its prenatal expression is higher than its postnatal expression (1, 2). Vetter et al. (8) also reported that IGF-II was more effective than IGF-I in stimulating clonal growth of fetal chondrocytes, whereas IGF-I was more effective than IGF-II in stimulating clonal growth of adult chondrocytes. Although the HCS-2/8 cell line has many of the differentiated functions of normal chondrocytes, it is originally derived from a human chondrosarcoma. Therefore, high responsiveness to IGF-II may be one of the onco-fetal properties of the chondrocyte lineage. IGFs are known to stimulate not only proteoglycan synthesis, but also DNA synthesis in growth cartilage cells in confluent culture (9). However, both IGFs failed to increase DNA synthesis in HCS-2/8 cells in confluent culture, although IGFs did increase DNA synthesis in HCS-2/8 cells in sparse culture (our unpublished observation). HCS-2/8 cells express the differentiated phenotype of chondrocytes as they become confluent (25), and it was suggested that the role of IGF-I in regulating the growth plate chondrocytes switched from DNA synthesis to proteoglycan synthesis as the cells maturated (2). Therefore, because HCS-2/8 cells in confluent culture are in a highly maturated stage (25), it is feasible that both IGF-I and IGF-II stimulated only proteoglycan synthesis in the cells.

[125I]-IGF-I bound to HCS-2/8 cells, and its binding was replaced by low concentrations of unlabeled IGF-I, higher concentrations of IGF-II, and much higher concentrations of insulin (Fig. 5AGo), indicating that the cells had typical IGF-I binding sites (11). [125I]-IGF-II also bound to the cells, and its binding was replaced by IGF-II and by high concentrations of IGF-I and much higher concentrations of insulin, although the inhibitions were small (Fig. 5BGo), indicating that the cells had typical IGF-II binding sites (11) but that IGF-II tracer also may be binding to IGF-IR. The apparent dissociation constants of the high-affinity binding sites of both IGF-I and IGF-II were smaller than those reported previously on chondrocytes or cartilage, but this is probably because radiolabeled IGFs with high specific activity have only recently become available (2). The Bmax numbers of both IGF-I and IGF-II per cell were consistent with the respective values of growth plate chondrocytes reported by several laboratories (2). A cross-linking study, using [125I]-IGF-II, showed two major bands of 260 kDa and 130 kDa, which correspond to the complex of IGF-II and IGF-IIR and the complex of IGF-II and {alpha}-subunit of IGF-IR, respectively (Fig. 6Go) (37). In addition, Northern blot analysis revealed gene expression of IGF-IR and IGF-IIR (Fig. 7Go), the sizes of which are consistent with those reported previously (1). These findings indicate that HCS-2/8 cells express both IGF-IR and IGF-IIR at both mRNA and protein levels.

The half-maximal stimulation of proteoglycan synthesis by IGF-II was observed at about 10 ng/ml (1.33 x 10-9 M) (Fig. 2Go); this concentration was almost the same as the Kd value (1.5 x 10-9 M) shown in Fig. 5Go. In contrast, the concentration of IGF-I (100 ng/ml) that caused a half-maximal stimulation of proteoglycan synthesis (Fig. 2Go) was much higher than the Kd value (6 x 10-11 M) shown in Fig. 5Go. There are two possible explanations for this discrepancy. One is that IGF-binding proteins (IGFBPs) modulate the IGF-I action. Recently, IGFBPs have been identified and shown to modulate IGF activity (5, 38, 39, 40) and IGF binding (41). Moreover, IGF-I regulates the production of some IGFBPs by bovine chondrocytes, including IGFBP-3, which is known to inhibit IGF-I activity (38). IGF-I also dramatically increased production of IGFBP-3 in human articular chondrocytes (our unpublished observations). Therefore, it is feasible that IGFBP-3 is increased by IGF-I and that this increase, in turn, inhibits IGF-I activity. In this regard, Weber et al. (42) reported that the higher responsiveness to IGF-II than to IGF-I of ACTH-stimulated bovine adrenocortical cells is caused by increased production of binding proteins for IGF-I by ACTH. In this regard, our cross-linking study revealed that there was a specific band just below 30 kDa, suggesting the presence of IGFBP (Fig. 6Go). In addition, HCS-2/8 cells expressed at least mRNAs of IGFBP-3 and -4 (our unpublished observations). Another possibility is that an unknown modification occurs at a postreceptor level in HCS-2/8 cell line. Further investigation is required to clarify this phenomenon.

Repeated medium changes decreased proteoglycan synthesis in HCS-2/8 cells (Table 1Go), and CM of the cells stimulated proteoglycan synthesis (Fig. 8Go), which is thought to be caused by IGF-I and IGF-II. We also found that rabbit costal chondrocytes in primary culture produced IGF-I and IGF-II. Rabbit articular cartilage cells in secondary culture also had been shown to produce both IGF-I and IGF-II (5, 43), but it is very difficult to take cartilage tissue from rabbit joints without contamination of bone, so it was feared that their cultures might have been contaminated with osteoblasts and/or periosteal cells, which produce IGF-I and II (44, 45, 46) and express IGF-I and II transcripts (6, 7). Moreover, when chondrocytes were subcultured, they easily de-differentiated (18), and secondary cultures might contain dedifferentiated cells, which produce much IGF-I (47). In contrast, HCS-2/8 is an established, clonal chondrocytic cell line that maintains the chondrocyte phenotype very well, so our findings clearly showed that chondrocytes indeed produce both IGF-I and IGF-II. The concentration of IGF-I in the CM was close to the Kd value of IGF-IR (6 x 10-11 M), and the concentration of IGF-II in the CM was one order lower than the Kd value of IGF-IIR (1.5 x 10-9 M). Therefore, IGF-I may be a major contributor to the maintenance of proteoglycan synthesis in HCS-2/8 cells as an autocrine differentiation factor in this cell line. However, anti-IGF-II antibody inhibited proteoglycan synthesis by about 30% (Fig. 9Go), suggesting that IGF-II also is an autocrine differentiation factor in the cells, although its autocrine role is less important than IGF-I. The difference between responsiveness to exogenous IGF-I and exogenous IGF-II may depend on whether the factors secreted by autocrine mechanism are enough to occupy their receptors.

Antibody against IGF-IR decreased proteoglycan synthesis in the HCS-2/8 cells to about 20% (Fig. 9Go), suggesting that IGF-IR-mediated signaling is important in the retention of the cartilage phenotype of these cells. Recently, Sakano et al. (48) developed two IGF-II analogs: [Leu27]IGF-II, with high affinity for IGF-IIR but markedly reduced affinity for IGF-IR; and [Arg54, Arg55]IGF-II, with high affinity for IGF-IR but no affinity for IGF-IIR. These analogs have been used to clarify which receptor mediates the effects of IGF-I and IGF-II. For example, Burguera et al. (49) reported that glucose uptake, stimulated by IGF-II in human muscle, was not mediated by IGF-IIR because [Arg54, Arg55]IGF-II had a similar effect to that of IGF-I, whereas [Leu27]IGF-II had no effect on the tissue. Weber et al. (42) reported that IGF-II was more potent than IGF-I in stimulating cortisol secretion from cultured bovine adrenal cells but that IGF-IR mediates this effect, because [Arg54, Arg55]IGF-II was equipotent to native IGF-II, whereas [Leu27]IGF-II had no effect. On the other hand, Rosenthal et al. (37) reported that [Leu27]IGF-II at a concentration of 50 ng/ml, which interacts only with IGF-IIR, stimulated myogenin expression in muscle cells, but native IGF-II, which binds both IGF-IR and IGF-IIR, was more potent than [Leu27]IGF-II, suggesting that the role of both receptors is significant. In the case of HCS-2/8, [Leu27]IGF-II significantly increased proteoglycan synthesis at a concentration of 50 ng/ml (Fig. 12Go), which is known to interact only with IGF-IIR (37). On the other hand, [Arg54, Arg55]IGF-II also increased proteoglycan synthesis at concentrations of 200 ng/ml and was almost equipotent to native IGF-I (Fig. 12Go). These findings indicate that IGF-I and IGF-II increased proteoglycan synthesis mainly via their respective receptors, although a cross-interaction between IGF-II and IGF-IR also contributes to the stimulatory action of IGF-II on proteoglycan synthesis in HCS-2/8 cells.

IGF-II also caused calcium influx into HCS-2/8 cells in a dose-dependent manner (Fig. 4Go). The dose-dependency was almost the same as that of stimulation of proteoglycan synthesis by IGF-II, indicating a close relation between the stimulation of proteoglycan synthesis by IGF-II and the calcium influx caused by IGF-II. Because IGF-II has been shown to bind IGF-IIR and then stimulate calcium influx into 3T3 fibroblasts via activation of Gi-2 protein (13), these findings suggest that calcium influx may be involved in the IGF-IIR-mediated IGF-II stimulation of proteoglycan synthesis in HCS-2/8 cells. IGF-I did not cause calcium influx (Fig. 4Go), indicating that calcium influx may not be involved in IGF-IR-mediated proteoglycan synthesis.

High concentrations of M-6-P increased proteoglycan synthesis in HCS-2/8 cells, and a low concentration of M-6-P, which did not increase proteoglycan synthesis in the cells, potentiated the stimulation of proteoglycan synthesis by IGF-II (Fig. 10Go). M-6-P also enhanced the binding affinity of IGF-II to HCS-2/8 cells (Fig. 11Go). There are two possible explanations for this phenomenon. Because M-6-P has been shown to bind IGF-IIR at a site other than the IGF-II-binding site (50, 51, 52), binding of M-6-P to IGF-IIR might change the conformation of the receptors so that IGF-II binds to the receptors with higher affinity, resulting in enhanced responsiveness to autocrine and exogenous IGF-II. Another possibility is that M-6-P may cause release from IGF-IIR of lysosomal enzymes, which endogenously bind the M-6-P sites of IGF-IIR and inhibit IGF-II binding to the receptors, resulting in enhancement of IGF-II binding to the receptors (53, 54). Even when IGF-IR function was inhibited by anti-IGF-IR antibody, both IGF-II and M-6-P increased proteoglycan synthesis (Table 2Go). Moreover, [Leu27]IGF-II, which binds to IGF-IIR but not IGF-IR, increased proteoglycan synthesis (Fig. 12Go). All these findings also indicate that IGF-IIR is functional and that IGF-II stimulates proteoglycan synthesis in HCS-2/8 cells via IGF-IIR.

In conclusion, the present study was the first study in which a clonal chondrocytic cell line not contaminated with other types of cells was used to demonstrate that IGF-I and IGF-II act as autocrine differentiation factors for chondrocytic cells mainly via respective receptors, although a close interaction between IGF-II and IGF-IR also contribute a little to the IGF-II action. In particular, the finding that IGF-IIR stimulates cell differentiation is very important because there have been only three models in which IGF-IIR is functional, in contrast to the involvement of IGF-IR in IGF-II action: first is stimulation of proliferation of fibroblasts (13), second is induction of muscle cell differentiation (55), and third is stimulation of motility in human rhabdomyosarcoma cells (56). Because chondrocytes have many unique differentiated functions and because the HCS-2/8 cell line retains many of them, this clonal cell line should be a useful model to investigate signal transduction pathways via IGF-IIR that are related to expression of the differentiated phenotype and/or cytodifferentiation.


   Acknowledgments
 
We thank Drs. Kentaro Inui (Department of Orthopedic Surgery, Osaka City University Medical School) and Chisa Shukunami (Department of Biochemistry, Osaka University Faculty of Dentistry) for technical assistance. We also thank Ms. Etsuko Fujisawa for secretarial assistance.


   Footnotes
 
1 This work was supported in part by grants-in-aid for scientific research (to M.T. and F.S.) from the Ministry of Education, Science, Sports and Culture of Japan, and grants from the Yamada Scientific Foundation (to M.T.), the Health Science Foundation (to M.T.), and the RSK Science and Culture Foundation (to M.T.). Back

Received February 27, 1997.


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 Introduction
 Materials and Methods
 Results
 Discussion
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