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Am J Physiol Endocrinol Metab 290: E363-E371, 2006. First published October 4, 2005; doi:10.1152/ajpendo.00363.2005
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Differential expression of IGF system components in proliferating vs. differentiating growth plate chondrocytes: the functional role of IGFBP-5

Daniela Kiepe,* Sonia Ciarmatori,* Anke Haarmann, and Burkhard Tönshoff

University Children's Hospital, Heidelberg, Germany

Submitted 4 August 2005 ; accepted in final form 23 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The growth plate is an important target tissue for insulin-like growth factors (IGFs), but little is known about the regulation of the IGF system during the developmental sequence of chondrocytes. We therefore examined the expression profile of IGF system components in proliferating vs. differentiating growth plate chondrocytes by use of two cell culture models of the growth cartilage. In rat growth plate chondrocytes in primary culture, IGF-I expression increased twofold during the process of differentiation. IGF-binding protein-3 (IGFBP-3) expression showed a biphasic pattern of with a twofold increase at the onset of differentiation and a downregulation in late differentiating chondrocytes to 25% of baseline levels; the expression patterns of IGFBP-2, -4 and -6 were not dependent on the developmental stage. In IGF- and IGFBP-3-deficient RCJ3.1C5.18 (RCJ) mesenchymal chondrogenic cells, IGFBP-2 and -6 synthesis declined by 50% during differentiation. IGFBP-5 expression was markedly upregulated during the process of differentiation in both cell culture models. Although IGFBP-5 overexpression did not have an IGF-independent effect on RCJ cell differentiation, it promoted IGF-I-enhanced differentiation of these cells. A potential mechanism for this effect is the specific increase of Akt phosphorylation in IGFBP-5-overexpressing cells in the presence of IGF-I, indicating an increased activity of the phosphatidylinositol (PI) 3-kinase pathway. These data suggest that the developmental stage of the chondrocyte is an important determinant of IGF and IGFBP expression and imply a functional role for IGFBP-5 for upregulating IGF action during chondrocyte differentiation in vivo.

insulin-like growth factor I; insulin-like growth factor-binding proteins; growth plate chondrocytes in primary culture; RCJ3.1C5.18 cells

THE GROWTH PLATE IS A DYNAMIC TISSUE in which cells undergo a developmental program from resting cells to proliferation, maturation, and hypertrophy until they reach terminal differentiation and produce the mineralized matrix that supports endochondral bone formation (20, 34). Insulin-like growth factor I (IGF-I) is a potent growth factor in the growth plate, exerting its actions by both endocrine and paracrine/autocrine mechanisms. It is known from cell culture (23, 25, 27) and gene knockout experiments (35, 36, 38) that IGF-I stimulates both proliferation and differentiation of growth plate chondrocytes in vitro and in vivo. The action of the IGFs both in the circulation and in tissues is tightly regulated by a family of high-affinity IGF-binding proteins (IGFBPs). Until now, from a variety of vertebrate species ranging from mammalian to fish, six distinct IGFBPs have been identified, and all of them, except for IGFBP-1, are expressed in growth plate chondrocytes (7, 8, 18). Although structurally related, they have individual expression patterns and exert different functions, including stimulation or inhibition of IGF bioactivity as well as IGF-independent actions (15, 29). We (16, 17) have previously studied the biological activity of various intact IGFBPs in a growth cartilage model of proliferating growth plate chondrocytes in primary cultures. IGFBP-1, -2, -4, and -6 act exclusively as growth inhibitors on IGF-dependent cell proliferation, whereas the biological activity of IGFBP-3 is complex. It has an IGF-independent antiproliferative effect and also inhibits IGF-dependent chondrocyte proliferation under coincubation conditions. Under preincubation conditions, however, IGFBP-3 enhances the IGF-I responsiveness of growth plate chondrocytes by its ability to associate with the cell membrane, where it facilitates IGF-I receptor binding (17). Intact IGFBP-5, on the other hand, enhances IGF-I-driven chondrocyte proliferation, apparently by its association with the cell membrane in the COOH-terminal domain, thereby better presenting IGF-I to its receptor (16).

The IGFBPs are not only involved in the IGF-dependent and -independent regulation of cell proliferation, but they also play a role in the differentiation of various cell types. These proteins enhance or inhibit the actions of IGF in a manner that is specific for IGF-I or -II, a particular binding protein, and a specific cell type. Furthermore, IGF-independent actions of various IGFBPs are increasingly reported in various experimental models. For example, overexpression of IGFBP-5 in mouse osteosarcoma cells (30) or treatment with recombinant human IGFBP-5 of both serum-free cultures of osteoblast clones and osteoblasts derived from IGF-I knockout mice increased the activity of the osteoblast differentiation markers alkaline phosphatase (ALP) and osteocalcin in the absence of IGF-I (11, 22, 28), indicating an IGF-independent effect of IGFBP-5 on osteoblast differentiation. On the other hand, IGFBP-5 overexpression in C2 myoblasts blocked IGF-stimulated myogenesis (13), whereas treatment of L6A1 muscle cells with exogenous IGFBP-5 stimulated all aspects of the myogenic response to IGF-I (10). These contrasting data on the regulation and function of this specific IGFBP in differentiating cells indicate the need to study its role for differentiation in individual tissues separately.

As a step toward understanding the role of IGFBPs in the control of IGF activities in the growth cartilage, we studied IGF-I and IGFBP expression in two cell culture models of the growth plate. The first model consisted of rat growth plate chondrocytes in primary culture. The developmental stages (proliferation, differentiation, and production of extracellular matrix) through which these cells progress under appropriate experimental conditions correlate with stages of chondrocyte maturation observed in the intact growth plate (4). The second growth cartilage model was the RCJ3.1C5.18 (RCJ) mesenchymal chondrogenic line of cells, which, without biochemical or oncogenic transformation, progress in culture from mesenchymal chondroprogenitor cells to differentiated chondrocytes in a sequence that mimics the phenotype of chondrocytes of the growth plate (21, 32). Furthermore, these cells do not express IGF-I; therefore the action of this hormone can be studied without interference from endogenous IGFs (32). Here, we report multiple changes in the levels of IGF-I and IGFBP expression as the chondrocytes progress from proliferating cells to mature, differentiating chondrocytes. Furthermore, we examined the functional role of IGFBP-5 on chondrocyte differentiation by transient overexpression because IGFBP-5 was the only IGFBP that was consistently upregulated during differentiation in both cell culture models.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. PBS, HEPES, penicillin-streptomycin, Ham's F-12, and DMEM were obtained from Seromed Biochrom (Berlin, Germany). BSA was purchased from Sigma-Aldrich Chemicals (Deisenhofen, Germany). Clostridium collagenase (EC 3.4.24.3 [EC] ), deoxyribonuclease [DNase I (EC 3.1.21.1 [EC] )], and trypan blue were from Roche Diagnostics (Mannheim, Germany). DMEM was purchased from CCPro (Neustadt, Germany), fetal calf serum was purchased from Paa Laboratories (Pasching, Austria), and ascorbic acid, {beta}-glycerophosphate, and dexamethasone were obtained from Sigma (Taufkirchen, Germany). Recombinant human (rh)IGF-I was supplied by Bachem (Weil am Rhein, Germany). Enhanced chemiluminescence (ECL) reagents were obtained from Amersham Pharmacia Biotech (Freiburg, Germany). The antibodies against IGFBP-5, collagen type II, and polyclonal goat antibody against osteopontin were obtained from Santa Cruz Biotechnologies (Heidelberg, Germany). The phosphorylated extracellular signal-regulated kinase (ERK)1/2, phospho (p)-ERK1/2, Akt, p-Akt, and horseradish peroxidase-conjugated (anti-rabbit and anti-mouse) antibodies were from Cell Signaling Technology (Frankfurt, Germany).

Cell culture. Epiphysial chondrocytes from 60- to 80-g Sprague-Dawley rats (Charles River, Kieslegg, Germany) were isolated and cultured as described previously (16). 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 DNase (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, always exceeded 90%. Dissociated cells were counted using a Neubauer chamber (Scheik, Hofheim, Germany).

Cells were cultured in monolayers (Nunc, Wiesbaden, Germany), as described previously, from our laboratory (4, 19). Briefly, the F-12-DMEM (1:1) medium, which contained a nominal calcium concentration of 1.2 mM, was supplemented with 10 mM HEPES, 100 µg/ml streptomycin, and 10% FCS. Cells were grown to 80–90% confluence (day 4 of culture, corresponding to proliferating chondrocytes) and then allowed to differentiate in medium supplemented with ascorbic acid (final concentration 50 µg/ml) and {beta}-glycerophosphate (final concentration 10 mM) up to 21 days of culture. Over this period of 21 days, the cells looked chondrocyte-like, that is, polygonal and closely packed, and they formed a carpet-like monolayer.

RCJ cells (kindly provided by Dr. Anna Spagnoli, Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN) were grown at 37°C in a 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, and 10–7 M dexamethasone and studied within 25 passages. Cells were plated at a density of 6 x 104 cells/well in six-well dishes for RNA extraction and protein isolation. After the cells reached confluence (day 4), the differentiation of cartilage nodules was initiated by the additional supplement of 10 mM {beta}-glycerophosphate and 50 µg/ml ascorbic acid to the medium, as already reported (32). Cells were fed at days 7 and 11 of culture and monitored over a total period of 14 days.

Alcian blue assay. Cartilage matrix deposition in cells was quantified by Alcian blue staining, which detects the glycosaminoglycans of proteoglycans. Cells were seeded at a density of 6 x 104/well in six-well dishes. After cells reached 80–90% confluence (corresponding to day 4 of culture), differentiation was initiated by adding 10 mM {beta}-glycerophosphate and 50 µg/ml ascorbic acid to the medium, as described previously (32). At four different time points (days 4, 7, 11, and 14), cells were washed in PBS and stained with 500 µl of 1% Alcian blue (Serva, Heidelberg, Germany) in 3% acetic acid for 30 min at room temperature, washed three times for 2 min in 3% acetic acid, and rinsed with distilled water. Alcian blue that was bound to the cells was dissolved in 1 ml of 1% SDS, and the absorbance was measured by spectrophotometry at 605 nm.

Transient transfection. RCJ cells were seeded at 1 x 105 in six-well plates. After 12 h at 50% confluence, cells were transfected with the pRC-CMV expression vector containing the cDNA fragment encoding human IGFBP-5 by using Mirus Transit LT-1 as instructed by the manufacturer (PanVera, Madison, WI). In a control experiment, 7 x 104 cells were seeded in 12-well plates and transfected with different amounts of transfection reagent and plasmid. The combination that gave the best transfection efficiency (4 µg pRC-CMV, 10 µl Mirus transfection reagent) was used to perform the final experiment. Two hundred microliters of serum- and antibiotic-free medium were transferred onto a 24-well plate, and then 4 µg of plasmid were added. In a second plate, 10 µl of transfection reagent were incubated with serum- and antibiotic-free medium. The two different samples were then mixed and left at room temperature for 20 min to allow the formation of DNA-containing complexes, and subsequently the mixture was added to the cells. After 24 h of incubation at 37°C, the medium was replaced with fresh differentiating medium supplemented with ascorbic acid and {beta}-glycerophosphate and incubated for 3 more days before harvest.

Real-time RT-PCR. For analysis of the expression pattern of the IGF/IGFBP system, RCJ cells and chondrocytes in primary culture were cultured in differentiating medium. At the indicated time points, RNA was isolated using the RNeasy minicolumns (Qiagen, Hilden, Germany). One microgram of RNA was reverse transcribed using MuLV reverse transcriptase and oligo(dT)/random hexamer primers (10:1) from Applied Biosystems (Darmstadt, Germany). For quantitative analysis, real-time RT-PCR was performed using the Abi 7000 (Applied Biosystems) according to manufacturer's protocol. The following set of primers was chosen: for ALP, forward 5'-TCATCCAGGGCTCCAATGA-3', reverse 5'-CCACTTACCGGTGTGTTTCGT-3'; for collagen type II, forward 5'-GGC AACACCATCATCGAGTAC C-3', reverse 5'-CCCTATGTCCACACCAAATTC-3'; for IGF-I, forward 5'-AGGCAGTTTACCCAGGCTCCTA-3', reverse 5'-TGAGGCCGAAGT GAAAACAAA G-3'; for IGFBP-2 forward 5'-TGAAGGAACTGGCTGTGTTC-3', reverse 5'-TGCAGGGAGTAGAGATGTTCC-3'; for IGFBP-3, forward 5'-AATGCTGGG AGTGTGGAAAG-3', reverse 5'-TGTCACAGTTTGGGATGTGG-3'; for IGFBP-4, forward 5'-AACAACAGCTTCAACCCCTG-3', reverse 5'-ACTGTTTGGGGTGGAAGT TG-3'; for hIGFBP-5, forward 5'-TCAAGATCGAGAGAGACTCCCG-3', reverse 5'-TTCACTGCTTCAGCCTTCAGC; for rIGFBP-5, forward 5'-AGTCGTGTGGCGTCTACACTGA-3', reverse 5'-TTTGCTCGCCGTAGCTCTTTT-3'; for IGFBP-6, forward 5'-AACAGCGAAGGTGAACCCTGA-3', reverse 5'-CCCAAACTC CACCCCCATACTA-3'; and for rat 18S by the Primer Express program, forward 5'-AGTTGGTGGAGCGATTTGTC-3', reverse 5'-GCTGAGCCAGTTCAGTGTAGC-3'. The software provided by the company allowed the quantitative detection of fluorescence by the incorporation of the substance SYBR green into the amplification products. Amplification was performed in the presence of Universal Mastermix (PE Applied Biosystems, Darmstadt, Germany) with SYBR green to detect PCR products at the end of each amplification step, and results were analyzed as previously described (12, 18).

Western immunoblotting and immunoprecipitation. For Western immunoblot analysis, cells were dissolved in lysis buffer, and the cell extracts were treated as previously described (12, 18). The membranes were blocked either in 5% BSA or in 3% milk for 1 h at room temperature, incubated overnight with the first antibody (dilution 1:4,000 for total Akt in 3% milk; dilution 1:2,000 for p-ERK1/2 and ERK1/2 in 5% BSA; dilution 1:1,000 for p-Akt in 5% BSA; dilution 1:1,000 for osteopontin in 5% milk; dilution 1:300 for collagen type II in 5% milk), washed extensively over a period of 30 min with Tris-buffered saline with Tween 20 (TBS-T) 0.05%, and then incubated for 1 h with the secondary antibody (dilution 1:2,000 in 3% milk), followed by further washing over a period of 30 min. Immune complexes were detected using ECL (Amersham Pharmacia Biotech). Blots were exposed to X-ray film (Kodak, Stuttgart, Germany).

For immunoprecipitation, 100 µl of packed A/G agarose beads (Santa Cruz Biotechnologies) were washed three times in PBS. Fifteen microliters of beads were then incubated overnight at 4°C with anti-IGFBP-5 antibody. Two microliters of conditioned medium supplied with benzamidine (3 mM), aprotinin (10 µg/ml), leupeptin (10 µg/ml), PMSF (1 mM), and NaVO3 (1 mM) were concentrated 10 times by trichloracetic acid. Two hundred microliters of concentrated conditioned medium were incubated with 15 µl of packed beads for 6 h at 4°C. The pellet was washed three times with 10 mM Tris·HCl, pH 7.5, to remove IGFBPs nonspecifically precipitated with the antibody. The immunoprecipitated pellet was then resuspended in 20 µl of sample SDS-loading buffer plus {beta}-mercaptoethanol (63 mmol Tris·HCL, pH 6.7, 10% glycerol, 2.1% SDS, 0.005% bromophenol blue). Samples were then applied to an SDS-protein gel and analyzed by Western immunoblotting. Equal aliquots of concentrated conditioned medium were loaded onto a 12% gel for nonreducing SDS-PAGE (10% acrylamide bis) and transferred to nitrocellulose membranes. The distinct membranes were incubated overnight at 4°C with antibodies against IGFBP-5 (dilution 1:500 in 3% milk) for Western blotting, incubated for 1 h with the secondary antibody, and washed extensively over a period of 30 min with 0.05% TBS-T. For signal detection, autoradiography (X-OMAT AR film; Amersham Pharmacia Biotech) or a chemiluminescence detection system (ECL Western Blotting Detection Reagents, Amersham Pharmacia Biotech) and Hyperfilm ECL film (Kodak) were used (12).

Electron microscopy. RCJ cells were cultured, as described in Real-time RT-PCR, on chamber slides (Lab-Tek; Nalge Nunc, Wiesbaden, Germany) and fixed in 2.5% glutaraldehyde for 30 min at room temperature, as described by Akat et al. (1). Briefly, after several rinses in cold buffer (50 mM sodium cacodylate), cells were postfixed in 2% osmium tetroxide for 1 h on ice, subsequently rinsed in water, and stained overnight at 4°C in aqueous 0.5% uranyl acetate. The cells were then dehydrated in graded ethanol series and propylene oxide, followed by embedding in Epon. Ultrathin sections were obtained on a Reichert OM U3 ultramicrotome, counterstained with uranyl acetate and lead citrate, and examined with a Zeiss EM 910 electron microscope.

Statistical analysis. All experiments were performed at least three times. For measurements with the ABI 7000 system, samples were run in duplicate to account for technical and biological variability within and between experiments. Data are given as means ± SE. 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 comparisons (Student-Newman-Keuls method) or Student’s t-test, was used as appropriate. 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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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
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Growth plate chondrocytes in monolayer culture differentiate to hypertrophic chondrocytes. After isolation, growth plate chondrocytes were grown in primary culture until confluence (day 4) and then maintained in differentiating medium with ascorbic acid and {beta}-glyerophosphate for 21 days, as described above. Light microscopy revealed that proliferating cells differentiated from displayed polygonal-shaped isolated chondrocytes to hypertrophic chondrocytes with surrounded matrix over 21 days of culture (data not shown). Differentiating chondrocytes expressed typical marker molecules of chondrocytic differentiation, such as collagen type II, ALP, and osteopontin. As shown in Fig. 1A, gene expression of the early differentiation marker collagen type II increased significantly from day 4 to day 7, confirming that these cells were undergoing differentiation. In parallel, the mRNA abundance of ALP increased up to threefold (Fig. 1B). Under the same culture conditions, both collagen type II and osteopontin concentration in cell lysates increased continuously from day 4 to day 21 (Fig. 1, C and D). These combined morphological and biochemical data indicate that growth plate chondrocytes kept in primary culture over 21 days are capable of differentiating from proliferating cells to hypertrophic chondrocytes without a significant dedifferentiation to fibroblasts.



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  		Fig. 1. Collagen type II, alkaline phosphatase (ALP), and osteopontin are upregulated during differentiation of growth plate chondrocytes in primary culture. Cells were grown to 80–90% confluence (day 4 of culture defined as baseline, corresponding to proliferating chondrocytes) and then allowed to differentiate in medium supplemented with ascorbic acid (final concentration 50 µg/ml) and {beta}-glycerophosphate (final concentration 10 mM) for 21 days of culture. Cells were harvested at indicated time points, total RNA was extracted, and gene expression of collagen type II (A) or ALP (B) was determined by real-time RT-PCR. Columns represent means ± SE of 3 independent experiments. Statistics by ANOVA. *P < 0.05 vs. day 4 of culture. For protein measurements, cell lysates were subjected to Western immunoblot analysis at indicated time points. Respective membranes were probed with antibody against collagen type II (C) or osteopontin (D). Representative autoradiographs of a total of 3 independent experiments are shown.

 
Gene expression of IGF-I and IGFBP-5 is upregulated during chondrocyte differentiation, whereas IGFBP-3 shows a biphasic expression pattern. To analyze the expression profile of IGF system components during the process of chondrocyte differentiation, mRNA was extracted on days 4, 7, 14, and 21 of culture, and IGF-I and IGFBP-2 to -6 gene expression was analyzed by quantitative RT-PCR. IGF-I gene expression steadily increased during chondrocyte differentiation up to 2.5-fold (Fig. 2A). We did not analyze IGF-II expression in these cells because chondrocytes used in this study were derived from juvenile rats, which produce only low amounts of IGF-II during postnatal development. IGFBP-3 gene expression peaked on day 7 of culture (twofold), turned back to baseline levels at day 14, and even dropped to 25% of baseline at day 21 of culture (Fig. 2B). By contrast, IGFBP-5 gene expression steadily and markedly increased up to 22-fold during chondrocyte differentiation (Fig. 2C). The expression pattern of IGFBP-2, -4, and -6 in differentiating chondrocytes did not change significantly compared with proliferating cells (Table 1).



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  		Fig. 2. Gene expression of IGF-I and IGF-binding protein (IGFBP)-5 is upregulated during chondrocyte differentiation, whereas IGFBP-3 shows a biphasic expression pattern. Primary growth plate chondrocytes were cultured as described in Fig. 1 and harvested at indicated time points. Total RNA was extracted, and gene expressions of IGF-I (A), IGFBP-3 (B), and IGFBP-5 (C) were determined by real-time RT-PCR. Bars represent means ± SE of 3 independent experiments. Statistics by ANOVA. *P < 0.05 vs. day 4 of culture.

 

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  		Table 1. Steady-state mRNA abundance of IGFBP-2, -4, and -6 in rat growth plate chondrocytes in primary culture during proliferation (day 4 of culture) vs. differentiation (days 7–21)

 
RCJ cells differentiate from hypertrophic chondrocytes in monolayer culture. The mesenchymal RCJ3.1C5.18 cell line is capable of spontaneously differentiating from displayed cuboidal-shaped isolated chondrocytes to cartilage nodules over 14 days of culture in differentiating medium in the presence of dexamethasone (32). As described in MATERIALS AND METHODS, differentiation of RCJ cells was promoted by incubating the cells with {beta}-glycerophosphate and ascorbic acid from day 4 of culture. By light microscopy, differentiating cells could be distinguished from proliferating cells by their tendency to become extensively nodulated (21). By electron microscopy, differentiating chondrocytes from day 7 of culture onward had a significantly lower cell nucleus-to-cytoplasm ratio than proliferating cells (Fig. 3). Concomitantly, the respective gene expression of the differentiation markers collagen type II and ALP was enhanced approximately threefold on day 7 of culture compared with baseline (day 4; data not shown).



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  		Fig. 3. Cell nucleus-to-cytoplasm ratio decreases in differentiating RCJ cells. RCJ cells were grown to 80–90% confluence and then allowed to differentiate in medium supplemented with ascorbic acid (final concentration 50 µg/ml) and {beta}-glycerophosphate (final concentration 10 mM) for ≤14 days. At indicated time points, area of cytoplasm and corresponding cell nucleus were measured, and a ratio was calculated. Bars represent means ± SE of 16 independent measurements. Statistics by ANOVA. *P < 0.05 vs. day 4 of culture.

 
Differentiating RCJ cells markedly upregulate IGFBP-5 and moderately downregulate IGFBP-2 and -6 synthesis. Because the mesenchymal RCJ cell line does not express the IGFs or IGFBP-3 at any stage of differentiation (32), we analyzed IGFBP-2, -4, -5, and -6 gene expression during cell maturation. IGFBP-2 and -6 gene expression in differentiating cells decreased significantly to ~50% of baseline levels (Fig. 4 A and D). IGFBP-4 gene expression transiently decreased by 40% at day 7 of culture but returned to baseline levels on days 11 and 14 (Fig. 4B). By contrast, IGFBP-5 gene expression was markedly stimulated during the process of differentiation with a peak at day 11 (Fig. 4C), consistent with our observation of chondrocytes in primary culture (Fig. 2C). Taken together, differentiating RCJ cells markedly upregulate IGFBP-5 synthesis and moderately downregulate IGFBP-2 and -6 synthesis.



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  		Fig. 4. Differentiating RCJ cells markedly upregulate IGFBP-5 and moderately downregulate IGFBP-2 and -6 synthesis. RCJ cells were cultured as described in MATERIALS AND METHODS and harvested at indicated time points. Total RNA was extracted, and gene expressions of IGFBP-2 (A), -4 (B), -5 (C), and -6 (D) were determined by real-time RT-PCR. Bars represent means ± SE of 6 independent experiments. Statistics by ANOVA. *P < 0.05 vs. day 4 of culture.

 
Overexpression of IGFBP-5 promotes IGF-I-enhanced differentiation of RCJ cells. Because IGFBP-5 was the only IGFBP that was consistently upregulated during differentiation in both chondrocyte cell culture models, we decided to evaluate a possible functional role of IGFBP-5 on chondrocyte differentiation. RCJ cells were transfected at day 4 with either a control vector (pRc/CMV) or a vector containing human IGFBP-5 cDNA, kindly provided by Dr. M. E. Gleave (Vancouver, Canada). On day 6, vector control and IGFBP-5-transfected cells were serum deprived for 12 h and then treated with or without IGF-I (100 ng/ml) for an additional 12 h. IGFBP-5 immunoblots from immunoprecipitated conditioned medium revealed a strong 33-kDa band in IGFBP-5 transfectants compared with control, indicating that more protein was secreted from IGFBP-5-transfected cells (Fig. 5). Incubation of control cells with exogenous IGF-I also increased IGFBP-5 concentration in conditioned cell culture medium, albeit to a lower extent than IGFBP-5 transfection (Fig. 5).



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  		Fig. 5. Overexpression of IGFBP-5 in RCJ cells. RCJ cells were transfected with empty vector (C) or pRC/CMV full-length human IGFBP-5 cDNA. After 24 h, medium was replaced by completed differentiating medium supplemented with ascorbic acid (50 µg/ml) and {beta}-glycerophosphate (10 mM). After 48 h, control cells were serum starved for 12 h and incubated with 100 ng/ml IGF-I for an additional 12 h. Conditioned cell culture medium was collected and IGFBP-5 protein concentration determined by immunoprecipitation and Western immunoblot analysis. Representative autoradiograph of a total of 3 experiments is shown.

 
To determine the effects of IGFBP-5 overexpression on RCJ cell differentiation, we quantified by real-time RT-PCR the mRNA abundance of collagen type II, a marker of early chondrocyte differentiation. Furthermore, we measured proteoglycan synthesis as a marker of mid-to-late chondrocyte differentiation. IGFBP-5 transfection alone did not affect these differentiation markers (Fig. 6, A and B). Incubation of control cells with IGF-I moderately stimulated both collagen type II gene expression and proteoglycan synthesis, and this effect of IGF-I was clearly more pronounced in IGFBP-5 transfectants (Fig. 6, A and B). These data indicate that overexpression of IGFBP-5 promotes the IGF-I-enhanced differentiation of RCJ cells but IGFBP-5 does not promote chondrocyte differentiation on its own.



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  		Fig. 6. Overexpression of IGFBP-5 promotes the IGF-I-enhanced differentiation of RCJ cells. A: mRNA expression of the differentiation marker collagen type II was assessed by real-time RT-PCR. B: accumulation of proteoglycans was estimated by Alcian blue assay. Each experiment was performed ≥3 times. Bars represent means ± SE. Statistics by ANOVA. *P < 0.05 vs. control (C) without IGF-I; #P < 0.05 vs. control with IGF-I.

 
To elucidate the mechanism by which IGFBP-5 promotes IGF-I-enhanced differentiation of RCJ cells, we investigated the activation state of the PI 3-kinase pathway, the signaling cascade mainly involved in the regulation of chondrocyte differentiation (5). As expected, IGF-I stimulated the phosphorylation of Akt, although the concentration of total Akt in the cell lysate of RCJ cells did not change (Fig. 7A). This IGF-I-stimulated phosphorylation of Akt was enhanced in cells overexpressing IGFBP-5, which suggests that IGFBP-5 promotes the activation of this particular pathway. Also, the phosphorylation of ERK1/2, a major component of the mitogen-activated protein kinase (MAPK)/ERK1/2 cascade, was increased in response to IGF-I (Fig. 7B). However, this increase was blunted in cells overexpressing IGFBP-5. Taken together, IGFBP-5 promotes the IGF-I-enhanced differentiation of RCJ cells by increased Akt phosphorylation, indicating an increased activity of the PI 3-kinase pathway. This effect was specific, because another IGF-I-related signaling pathway, the MAPK/ERK1/2 cascade, was rather downregulated by overexpression of IGFBP-5.



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  		Fig. 7. IGFBP-5 promotes IGF-I-enhanced differentiation of RCJ cells by increased Akt phosphorylation, a major component of the phosphatidylinositol (PI) 3-kinase pathway, whereas the phosphorylation of extracellular signal-related kinase (ERK1/2), a major component of the MAPK/ERK1/2 cascade, in response to IGF-I is blunted in cells overexpressing IGFBP-5. RCJ cells were cultured and transfected as described above. After 48 h, cells were serum deprived for 12 h and incubated with or without 100 ng/ml IGF-I for an additional hour. Cell lysates were subjected to Western immunoblotting, and membranes were probed with specific antibodies against total and phosphorylated (p)-Akt (A) and against total and p-ERK1/2 (B). Representative blots of ≥3 independent experiments are shown. C: bars represent means ± SE of 3 independent experiments. Statistics by ANOVA. *P < 0.05 vs. control without IGF-I.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We observed that endogenous IGF-I synthesis is upregulated twofold in differentiating, compared with proliferating chondrocytes, and that exogenous IGF-I enhances differentiation of RCJ cells; the latter observation is in line with data in the chondrocyte cell line ATDC5 (26). Furthermore, other investigators (27, 31) detected in situ hybridization of IGF-I mainly in cells of the hypertrophic zone of the postnatal growth plate, and only a few cells expressing IGF-I were detected in the resting and proliferative zones. These data imply a functional role for IGF-I in hypertrophic chondrocytes. Indeed, it was shown in IGF-I null mice that chondrocyte numbers and proliferation are normal despite a 35% reduction in the rate of long bone growth (36). However, the IGF-I null terminal hypertrophic chondrocytes, which form the scaffold where long bone growth extends, were reduced in linear dimension by 30%, accounting for most of their decreased longitudinal growth. These in vitro and in vivo data suggest that IGF-I plays an important role for chondrocyte differentiation and hypertrophy.

We observed in both cell culture models that growth plate chondrocytes markedly upregulate IGFBP-5 synthesis during the process of differentiation. One potential mechanism for this observation could be the upregulation of sonic hedgehog (Shh), a key signal protein in early embryological patterning of limb bud development, which is also upregulated in differentiating chondrocytes in the postnatal growth plate (37). At least during chicken embryogenesis, IGFBP-5 is subject to regulation by Shh (2). To elucidate the functional relevance of IGFBP-5 during differentiation, RCJ cells were transfected with human IGFBP-5 cDNA. IGFBP-5 overexpression in RCJ cells in the absence of IGF-I did not enhance chondrocyte differentiation; however, it promoted the process of IGF-I-enhanced differentiation of RCJ cells. This observation differs from the IGF-independent stimulatory role of IGFBP-5 on osteoblast differentiation. Overexpression of IGFBP-5 in mouse osteosarcoma cells (30) or treatment with recombinant human IGFBP-5 of both serum-free cultures of osteoblast clones and osteoblasts derived from IGF-I knockout mice increased the activity of osteoblast differentiation markers ALP and osteocalcin in the absence of IGF-I (11, 22, 28). On the other hand, the knockdown of IGFBP-5 in human osteosarcoma cells resulted in a significant decrease in the bone differentiation parameter ALP activity but had little effect on basal or IGF-I-induced proliferation (39). The role of IGFBP-5 on bone metabolism in vivo is not entirely clear. Although systemic administration of rhIGFBP-5, either alone or in combination with IGF-I, increased bone formation parameters in normal mice (28), transgenic mice overexpressing IGFBP-5 in the bone microenvironment had a transient decrease in trabecular bone volume, impaired osteoblastic function, and osteopenia (9). The effect of IGFBP-5 overexpression or knockout on growth plate morphology in vivo has not yet been investigated.

Contrasting effects of IGFBP-5 on cell differentiation have also been observed in various muscle cell culture models. In C2 myoblasts, IGFBP-5 overexpression blocked IGF-stimulated myogenesis, indicating that sequestration of IGFs in the extracellular matrix could be a possible mechanism of action (13). In contrast, treatment of L6A1 muscle cells with exogenous IGFBP-5 stimulated all aspects of the myogenic response to IGF-I (10). Hence, the effect of IGFBP-5 on cell differentiation depends on the cell type, the presence of extracellular matrix proteins that are capable of binding to IGFBP-5, the mode of IGFBP-5 administration, and the presence of IGF-I.

We also sought to determine the mechanism by which IGFBP-5 promotes IGF-I-enhanced growth plate chondrocyte differentiation. We observed that IGFBP-5 promotes IGF-I-enhanced differentiation of RCJ cells by increasing Akt phosphorylation, indicating an increased activity of the PI 3-kinase pathway. This effect was specific because another IGF-I-related signaling pathway, the MAPK/ERK1/2 cascade, was rather downregulated by overexpression of IGFBP-5. This differential effect of IGFBP-5 could be due to a cross talk of IGFBP-5 receptor-related intracellular signaling molecules with the PI 3-kinase pathway and/or the MAPK/ERK1/2, which, however, remains to be defined (3). For example, it has been shown in cultured bovine fibroblasts that IGFBP-3, a binding protein with some similarities to IGFBP-5, potentiates IGF action through the PI 3-kinase pathway, and that this is associated with an alteration in protein kinase B/Akt sensitivity (6). Another possibility is that IGFBP-5 binds to extracellular matrix proteins, such as laminin and fibronectin, which are produced by differentiating chondrocytes (33). It has been shown that IGFBP-5 associated with extracellular matrix (ECM) has a lower affinity for IGF-I (14). Delivery of IGF-I from reservoirs of immobilized IGF-I bound to low-affinity IGFBP-5 in ECM environments could facilitate the binding of ligand to the type 1 IGF receptor on the cell surface. By this mechanism, IGFBP-5 could accelerate the IGF-I-mediated differentiation process without interfering with any components of these intracellular signaling pathways. Future studies will analyze more precisely the mechanism by which IGFBP-5 overexpression promotes IGF-I-enhanced RCJ cell differentiation.

We observed a biphasic pattern of IGFBP-3 expression during differentiation of rat growth plate chondrocytes in primary culture. Although IGFBP-3 gene expression was increased twofold on day 7, coincident with the onset of differentiation, it dropped to 25% of baseline in late differentiating chondrocytes. IGFBP-3 is the most abundant IGFBP in conditioned medium of growth plate chondrocytes (18) and is an inhibitory IGFBP under most experimental conditions by both IGF-dependent and -independent mechanisms (17, 32). The upregulation of IGFBP-3 in early differentiating chondrocytes on day 7 of culture might contribute, by its antiproliferative effect, to the maturation process from proliferation to differentiation. The precise functional role of this biphasic IGFBP-3 expression during differentiation will be characterized in future studies.

The expression pattern of IGF system components during differentiation in RCJ cells, which do not express both IGFs and IGFBP-3, was comparable to that in growth plate chondrocytes in primary culture regarding IGFBP-4 and -5 but somewhat different regarding IGFBP-2 and -6. Although in primary cells the expression of IGFBP-2 and -6 was not dependent on the developmental stage, these two IGFBPs were persistently downregulated in differentiating IGFBP-3-deficient RCJ cells, potentially as a compensation for the lacking IGFBP-3 expression. We (17) have shown previously that both IGFBP-2 and -6 act exclusively as inhibitory binding proteins in growth plate chondrocytes. Hence, the functional consequence of this persistent downregulation of inhibitory IGFBPs is likely an increased local IGF-I bioavailability for cell differentiation.

Other investigators have previously studied components of the IGF system in experimental models of the growth plate. Olney and Mougey (24) isolated growth plate chondrocytes from fetal cows and fractionated them on discontinuous Percoll gradients. By RT-PCR, they found that IGF-I and -II gene expressions in proliferative chondrocytes were 32- and 5-fold more abundant, respectively, than in hypertrophic chondrocytes. The gene expression of IGFBP-3, -4, and -5 were also reduced in hypertrophic chondrocytes, whereas IGFBP-2 expression was unchanged (24). These contrasting results may be due to either species differences or the different stages of development (fetal vs. postnatal). Two groups of investigators studied IGF system components in the intact growth plate. Smink et al. (31) examined IGF and IGFBP expression by in situ hybridization in the intact growth plate of normal 7-wk-old mice. Only expression of IGF-I, IGF-II, and IGFBP-2 could be detected, which were located predominantly in the hypertrophic zone. De Los Rios and Hill (7) found, by immunocytochemistry and in situ hybridization, little mRNA for IGF-I within the growth plates of fetal sheep but that mRNA for IGF-II was abundant in germinal and proliferative chondrocytes, although absent from some differentiating chondrocytes and hypertrophic cells. Immunohistochemistry for IGF-I and IGF-II showed a presence of both peptides in all chondrocyte zones, including hypertrophic cells. The precise expression pattern of the IGFBPs in the intact growth plate could not be determined in this study due to the insensitivity of in situ hybridization.

In conclusion, our data demonstrate that the developmental stage of the chondrocyte is an important determinant of IGF and IGFBP expression. IGF-I plays an important role in chondrocyte differentiation because endogenous IGF-I synthesis is upregulated in differentiating chondrocytes in primary culture, and exogenous IGF-I enhances differentiation of IGF-I-deficient RCJ cells. In both chondrocyte cell culture systems, IGFBP-5 gene expression increases markedly during differentiation. These congruent data indicate that the enhanced IGFBP-5 synthesis during chondrocyte differentiation applies to growth plate chondrocytes in general. The finding that IGFBP-5 enhances growth plate chondrocyte differentiation through an IGF-I-dependent mechanism implies a role for IGFBP-5 in upregulating IGF action during chondrocyte differentiation in vivo.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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This work was supported by a research grant from the Faculty of Medicine, University of Heidelberg, and the Eli Lilly International Foundation. D. Kiepe was the recipient of a scholarship from the Deutsche Forschungsgemeinschaft (Graduiertenkolleg Experimentelle Nieren- und Kreislaufforschung).


   ACKNOWLEDGMENTS
 
We thank Dr. Anna Spagnoli (Nashville, TN) for generously providing the RCJ3.1C5.18 cell line. We are grateful to Dr. Martin E. Gleave (Vancouver, Canada) for providing the human full-length IGFBP-5 cDNA pRc/CMV vector. We thank Nikolaus Gassler and Peter Rieger (Institute for Pathology, Heidelberg, Germany) for assistance with the electron microscopy.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. Tönshoff, University Children's Hospital, Im Neuenheimer Feld 153, 69120 Heidelberg, Germany (e-mail: Burkhard.Toenshoff{at}med.uni-heidelberg.de)

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.

* Both authors contributed equally to this study. Back


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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