Interaction of IGF-I and 1α,25(OH)2D3 on receptor expression and growth stimulation in rat growth plate chondrocytes

Kidney International, Vol. 53 (1998), pp. 1152–1161

Interaction of IGF-I and 1a,25(OH)2D3 on receptor expression and growth stimulation in rat growth plate chondrocytes G¨ UNTER KLAUS, LUTZ WEBER, JULIAN RODR´ IGUEZ, PORFIRIO FERN´ ANDEZ, THOMAS KLEIN, UGEL, EBERHARD RITZ, and OTTO MEHLS J. GRULICH-HENN, ULRIKE H¨ Department of Pediatrics, University of Marburg, Marburg, and Departments of Pediatrics and Internal Medicine, University of Heidelberg, Heidelberg, Germany

Interaction of IGF-I and 1a,25(OH)2D3 on receptor expression and growth stimulation in rat growth plate chondrocytes. Growth plate cartilage cells express receptors for, and are affected by both IGF-I and 1a,25(OH)2D3. The studies were undertaken to investigate interaction between these two hormone systems, that is, (i) to study effects of 1a,25(OH)2D3 on IGF-type 1 receptors (IGFIR), on IGF-I stimulated cell replication, colony formation, and on alkaline phosphatase activity (AP), and conversely, (ii) to study the effect of IGF-I on vitamin D receptor (VDR) expression on 1a,25(OH)2D3 stimulated growth parameters and on AP activity. Freshly isolated rat tibial chondrocytes were grown in monolayer cultures, (serum-free) or in agarose stabilized suspension cultures (0.1% FCS). Vitamin D receptor and IGFIR were visualized by immunostaining with the monoclonal antibody (mAb) 9A7g and mAb aIR3, respectively, and quantitated by RT-PCR for mRNA and by Scatchard analysis using [3H]-1,25(OH)2D3 and [125I]-aIR3. Cell proliferation was measured by [3H]-thymidine incorporation, growth curves in monolayer cultures, and by colony formation in agarose-stabilized suspension cultures. IGF-I dose-dependently increased [3H]-thymidine incorporation. 1a,25(OH)2D3, but not 1b,25(OH)2D3 was stimulatory at low (10212 M) and slightly inhibitory at high (1028 M) concentrations. The effect of IGF-I was additive to that of 1a,25(OH)2D3 [IGF-I 60 ng/ml, 181 6 12.7; 1a,25(OH)2D3 10212 M, 181 6 9.8%, IGF-I 1 1a,25(OH)2D3, 247 6 16.7%; P , 0.05 by ANOVA] and specifically obliterated by polyclonal IGF-I antibody (AB-1). Interaction could also be confirmed in suspension cultures. IGFIR mRNA and [125I]-aIR3 binding was increased by low (10212 M) but not by high (1028 M) concentrations of 1a,25(OH)2D3. Homologous up-upregulation by IGF-I (60 ng/ml) was specifically inhibited by AB-1 and markedly amplified by coincubation with 1a,25(OH)2D3 (10212 M). Immunostaining with aIR3 showed specific IGFIR expression in rat growth cartilage, but not liver tissue. Stimulation of chondrocytes with 1a,25(OH)2D3 or IGF-I suggested some increase of receptor expression in single cells, but the predominant effect was increased recruitment of receptor positive cells. Vitamin D receptor expression was markedly stimulated (fourfold) by IGF-I (60 ng/ml), but not IGF-II and inhibited by actinomycin D. This study shows that IGF-I and 1a,25(OH)2D3 mutually up-regulate their respective receptors in growth plate chondrocytes. In parallel, they have additive effects on cell proliferation and colony formation suggesting independent effector pathways.

Key words: vitamin D, chondrocytes, colony formation, alkaline phosphatase activity, growth cartilage, receptor expression, cell proliferation. Received for publication August 11, 1997 and in revised form November 13, 1997 Accepted for publication December 2, 1997

© 1998 by the International Society of Nephrology

Maintenance of longitudinal bone growth and normal function of the growth plate cartilage cells is dependent on several paracrine and autocrine hormonal stimuli including the vitamin D system and the somatotropic hormone axis. It is well established that growth plate cells are targets for growth hormone [1, 2] and insulin-like growth factors (IGFs) [3, 4]. Growth hormone (GH)deficiency results in (i) low concentrations of circulating IGF-I, and (ii) low local (paracrine) secretion of IGF-1 by chondrocytes [3, 5]. According to the dual-effector theory proposed by Green, Morikawa and Nixon [6], endocrine secreted GH induces differentiation of prechondrocytes, whereas IGF-I (the product of paracrine secretion) stimulates clonal expansion of growth plate chondrocytes [7]. Altered metabolism of vitamin D, such as in vitamin D deficiency rickets, vitamin D-dependent rickets type I and chronic renal failure, is characterized by disturbances of the growth plate, such as disturbed mineralization and abnormal architecture, resulting in a stunted growth. Restoration of active vitamin D levels results in catch-up growth in rickets. In vitro studies clearly demonstrate that growth plate chondrocytes express the vitamin D receptor (VDR) [8, 9], and that 1a,25(OH)2D3 modulates growth cartilage cell proliferation, that is, stimulation at physiological concentrations (10212 M) and inhibition at high concentrations (1028 M) [10]. Chronic renal failure is characterized by disturbed vitamin D metabolism [11], but also by an insensitivity to the action of somatotropic hormones [12]. Despite normal levels of circulating IGF-I measured by RIA, the biological activity of IGF-I is reduced [12]. Treatment with supraphysiological doses of GH increases IGF-I levels and restores IGF-I bioactivity [13]. In parallel, GH stimulates renal 1a-hydroxylase via IGF-dependent pathways [14], demonstrating interaction between the somatotropic and calcitropic hormone systems. Thus, simultaneous disturbance in the action of IGF-I and vitamin D can be implicated in various pathologic growth conditions. This study investigates possible interactions of these two hormone systems at the level of the growth cartilage cells, using an in vitro model. METHODS Materials 1a,25(OH)2-[26,27-methyl-3H]cholecalciferol (158 Ci/mmol) was obtained from Amersham Buchler (Braunschweig, Germany); unlabeled 1a,25(OH)2D3 and 1b,25(OH)2D3, were gifts from

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Dr. Calcanis and Dr. Uskokovic (Hoffmann-La Roche, Germany and USA); [3H]-thymidine (40 Ci/mmol); hydroxyapatite, dithiothreitol, and Triton X-100, were from Sigma Chemical Co. (Munich, Germany); F-12 and DMEM medium, PBS, gentamicin, clostridium collagenase (EC 3.4.24.3), DNAse I (EC 3.1.21.1), trypan blue were from Boehringer (Mannheim, Germany). DNase I (FPLC pure) for DNA digestion before reverse transcription was from Pharmacia Biotech (Freiburg, Germany). Human recombinant IGF-I and IGF-II were gifts from Pharmacia GmbH, Erlangen, Germany; the monoclonal antibody aIR3 was purchased from Dianova (GR 11 L) as was VDR-mab 9A7g2b. Polyclonal antibody for IGF-I (AB-1) was supplied by W. Blum (Gieben, Germany). Standard low (SL) and low gel temperature (LGT) agarose was purchased from BioRad (Richmond, VA, USA). Fetal calf serum (FCS) was from Seromed (Berlin, Germany); 1,25(OH)2D3 in FCS was 1.1 3 10210 M [10]. Alpha IR3 was labeled with [125I] using standard techniques (Fa. Immundiagnostik, Bensheim, Germany). Primers and Superscript II reverse transcriptase were purchased from Gibco BRL (Eggenheim, Germany), Taq Polymerase from Pharmacia (Erlangen, Germany), and GATC Biocycle Sequencing Kit from GATC (Konstanz, Germany). Cell cultures Isolation of chondrocytes. Epiphyseal chondrocytes from 80 g Sprague-Dawley rats were isolated and cultured as described [10, 15] using a modified technique of Benya and Shaffer [16] and Lindahl et al [17]. Viability determined by the trypan blue exclusion technique, always exceeded 90%. Dissociated cells were counted using a Neubauer chamber. Monolayer cultures. Cells were cultured in 35 mm plastic dishes (Falcon Labware Oxnard, CA, USA) for proliferation assays, on glass cover slips for immunocytology, in 100 mm dishes for VDR determination, in 24 multiwell plates for AP measurements and for IGF type-1 receptor determinations as described [10, 15]. In previous studies we demonstrated that the majority of cells under these culture conditions correspond to proliferative chondrocytes in the growth plate as assessed by morphological and biochemical characteristics [10, 18]. With prolonged culture, chondrocytes continue to differentiate into hypertrophic cells and mineralize the matrix as shown previously [18]. Agarose stabilized suspension cultures. Cells were cultured in 60 mm Petri dishes (Falcon Plastics) in agarose according to Benya and Schaffer [16] as described [10, 15]. The cells were diluted with F-12/DMEM medium, containing 0.1% FCS and 0.2% BSA and were then mixed with low gel temperature (LGT) agarose to give a final cell concentration of 80,000 cells/ml in 0.5% LGT agarose. Three milliliter of F-12/DMEM medium supplemented with 0.1% FCS or 10% FCS and various concentrations of IGFs or vitamin D metabolites (or solvent control) as indicated were added on top. The medium was changed every other day. The cultures were screened for adherent cell clusters of more than three cells. No such clusters were seen at the start of culture in any experiment presented in this study. Assays of chondrocyte growth and proliferation Clonal assay. Suspension cultures were terminated by fixation in buffered formaldehyde (4%). Colonies were counted in 100 squares (2 mm grid) for each dish. A cell colony was defined as a

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cluster of cells with matrix stained by alcian blue as described [10, 15, 17]. [3H]-thymidine assay. Incorporation of [3H]-thymidine into DNA was determined as radioactivity in TCA-precipitable material as described previously [10]. Prior to the start of experiments cells were synchronized by maintaining them under serum free conditions for 24 hours. Medium was changed (0.2% BSA) and hormones or solvent added as indicated for 6 to 48 hours. Two mCi of [3H]-thymidine were added for the last four hours. Cells were counted in parallel cultures. Reverse transcriptase-polymerase chain reaction (RT-PCR) RNA preparation. Total RNA from cultured cells was isolated by the RNA-Cleant method (Angewandte Gentechnologie Systeme, Heidelberg, Germany) following the instructions of the manufacturers, and quantitated by measuring the absorbency at 260 nm. Reverse transcriptase (RT)-polymerase chain reaction (PCR) amplification of vitamin D receptor (VDR) and insulin-like growth factor type-1 receptor (IGFIR). RT-PCR was performed as described [19]. RT and PCR primers were deduced from rat VDR [20] and IGFIR sequence [21]. After DNase digestion, 2 mg of total RNA were reverse transcribed into cDNA using specific primers for VDR (R1: 59-CCGAACACCTCCAGCACAAG-39) and IGFIR (R1: 59-CTCGGCGTTGGGGATGTTG-39). After cDNA synthesis, excess primers were removed and PCR amplification was performed using the cDNA template with the specific primers pairs (VDR: F1: 59-GCCCACCACAAGACCTAT-39 and R2: 59-CCTTTTGGATGCTGTAACTG-39; IGFIR: F1: 59-TCCACATCCTGCTCATCTCC-39 and R2: 59-TGCCTTCCCACACACACTTG-39). The amplification profile consisted of denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds for all primers after a five-minute denaturation step at 94°C in a Perkin Elmer Gene Amp PCR System 9600. The amplified products (32 cycles, 297 bp for VDR and 30 cycles, 490 bp for IGFIR) were detected by electrophoresis in a 2% agarose gel and visualized by ethidium bromide staining and ultraviolet transillumination. As a control, b-actin mRNA was also reverse transcribed and the resulting cDNA amplified using specific primers [19] and following the same protocol resulting in a PCR product of 405 bp. Control reactions performed by omitting reverse transcriptase or template RNA showed no reaction product. The cycle products were within the linear logarithmic phase of the amplification curves. The optical densities of the PCR products were analyzed by a commercial available computed program (Bio-1D V.96.; Vilber Lourmat, France). Results were normalized for the density of b-actin. The PCR products were verified by sequencing. Assay of 1 a,25(OH)2D3 receptor and IGF type-1 receptor expression KTED extracts. Cell extracts were prepared as described elsewhere [10, 15]. In brief, cell suspensions (1 3 107 cells/ml) were homogenized in 0.4 M KTED buffer (0.4 M KCl, 10 mM Tris HCl, 1.5 mM EDTA, 2 mM dithiothreitol, 10 mM sodium molybdate, pH 7.4). A purified fraction was prepared by centrifugation at 205,000 g for 30 minutes. The supernatant was used for binding studies. Saturation analysis according to Scatchard [22] was carried out as described [10, 23].

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Assay of IGF type-1 receptor expression. Specific binding of [125I]-labeled monoclonal antibody aIR3 was utilized to quantitate the level of IGFIR expression. Saturation analysis with [125I]-aIR3 was carried out as described [24]. Chondrocytes were grown to subconfluence in 24-well tissue plates. After two washes in F-12/DMEM 1/1 (vol/vol) (0.2% BSA, 1.2 mmol/liter nominal calcium concentration), increasing concentrations (0.1 to 1.5 nM) of [125I]-aIR3 alone or with 100-fold molar excess of unlabeled aIR3 were incubated with the cells for 60 minutes at 4°C. After three washes with ice-cold F-12/DMEM, the cell associated monoclonal antibody (mAb) [125I]-aIR3 was eluted with 1 N NaOH. Glacial acetic acid was used for neutralization. [125I] was determined in both the cell lysate and the supernatant. The radioactivity was measured in a liquid scintillation counter. Cell counts were performed in parallel cultures. Immunocytochemistry Immunocytochemical demonstration of vitamin D receptor. The VDR-specific rat mab 9A7g, which was raised against chicken intestinal VDR [25], was used for immunocytochemistry as described [10, 18]. Immunostaining was applied using the labeled avidin-biotin technique as described [26]. In brief, chondrocytes grown on glass cover slips were fixed in 3.7% paraformaldehyde (4°C, 10 min) followed by methanol (4°C, 4 min) and acetone 1% H2O2 (4°C, 4 min) and incubated with the primary antibody, diluted 1:1 in PBS, 0.05% BSA, 0.05% Triton X-100, pH 7. Visualization was obtained with DAB. Immunocytochemical demonstration of insulin-like growth factor type-1 receptor (IGFIR). Immunohistochemical and cytochemical demonstration of the IGFIR was performed with a modification of the method of Raile et al [27] and Bentham et al [28]. Cryostat sections of freshly isolated tibial growth plates from rabbits (500 g) or rats (80 g) and rat liver (two-month-old animals) or chondrocytes grown on glass cover slips were fixed in 3.7% paraformaldehyde (4°C, 10 min) followed by methanol (4°C, 4 min) and acetone 1% H2O2 (1°C, 1 min), and incubated with the monoclonal antibody aIR3 at final dilution of 1:100. Further processing was as described [27]. Statistics Data are given as mean 6 SD. Statistical analysis was carried out using ANOVA or the Mann-Whitney-U-test as appropriate by Sigma-Stat statistical software (Fa. Jandel, Germany). RESULTS Regulation of IGF-I type-1 receptor expression in chondrocytes by 1a,25(OH)2D3 IGFIR expression was independently assessed by detection of IGFIR mRNA by RT-PCR, immune staining, and binding studies using unlabeled and [125I]-labeled aIR3 (Table 1). Primary cultures of growth plate chondrocytes expressed IGFIR mRNA (Fig. 1A). Amplification of the specific mRNA using primers generated a 490 bp fragment. The monoclonal antibody aIR3 reacted with rat and rabbit growth plate chondrocytes in a specific manner, whereas rat liver was not stained (not shown). Figure 2 shows immune staining of chondrocyte monolayer cultures with aIR3 under basal conditions and after incubation with IGF-I (60 ng/ml) or 1a,25(OH)2D3 (10212 M) for 24 hours. Staining was markedly stimulated by 1a,25(OH)2D3, by IGF-I, and most expressed by IGF-I plus 1a,25(OH)2D3. The staining was not only more intense

Table 1. Regulation of IGF type-1 receptor: Specificity

Effector added

Specific bound [125I]-aIR3 molecules/cell

Solvent IGF-I (48 hr) IGF-I (last 30 min) IGF-I 1 AB-1 IgG (anti-mouse)

89,900 6 7500 243,900 6 23,200a 38,800 6 12,300a 113,000 6 6570b 121,000 6 11,900

Subconfluent chondrocytes, synchronized by preculture in serumfree medium for 24 hr [and by implication in the absence of 1a,25(OH)2D3] were cultured in F-12/DMEM 1/1 medium, 1.2 mmol nominal calcium concentration with 0.2% BSA. IGF-I (60 ng/ml), AB-1 (1.25 mg/ml) or both were added for the entire 48-hr culture period. Specific binding [125I]-aIR3 was determined as described in the Methods section. As a negative control, an unspecific anti-mouse IgG (1.25 mg/ml) was used. AB-1 did not affect basal [125I]-aIR3 binding (data not given). The data given in the Table are from one representative experiment out of three. a P , 0.05 vs. solvent control; b P , 0.05 vs. IGF-I; statistics by ANOVA (N 5 4).

in single cells, but also a higher proportion of cells were positive for aIR3 [control 13.2 6 1.2%, 1a,25(OH)2D3 29.8 6 2.9%, IGF-I 29.3 6 0.7% and 1a,25(OH)2D3 1 IGF-I to 37.3 6 2.5%, P , 0.05 versus control and 1a,25(OH)2D3 alone or IGF-I alone versus 1a,25(OH)2D3 1 IGF-I]. Maximum specific binding of [125I]-aIR3 increased nearly threefold upon preincubation for 48 hours with IGF-I (60 ng/ml) and by preincubation with 10212 M, but not with 1028 M 1a,25(OH)2D3. Coincubation with both hormones (10212 M 1a,25(OH)2D3 and 60 ng/ml IGF-I) further enhanced [125I]-aIR3 binding. There was no change in apparent receptor affinity (Fig. 3). In contrast, IGFIR mRNA was slightly stimulated only by 1a,25(OH)2D3 at 10212 M after 24 hours incubation (Fig. 1A). The homologous increase of [125I]-aIR3 binding was prevented by specifically binding IGF-I with AB-1, while the controls (AB-1 alone and third party antimouse IgG) were not affected. Addition of IGF-I (60 ng/ml) to the culture 30 minutes prior to the assay significantly reduced [125I]-aIR3 binding (Table 1). After 48 hours incubation with IGF-I (60 ng/ml) specific [125I]-aIR3 binding was increased (Table 1). IGF-I in the supernatant at basal conditions (0.2% BSA) was 0.16 6 0.07 ng/ml. During the 48-hour incubation period preceded by 24 hours synchronization of the cell cycle in serum-free medium, no significant increase in cell number (data not given) and no increase in protein content (initial 1.60 mg/106 cells; final 1.99 mg/106 cells, P . 0.05) were found. Vitality of cells, as assessed by Trypan blue exclusion, in this and the following experiments always exceeded 96%. Effect of 1a,25(OH)2D3 and IGF-I on chondrocyte DNA replication in monolayer cultures Incubation for 48 hours with increasing concentrations of IGF-I caused a biphasic effect on [3H]-thymidine incorporation with a maximum at 60 ng/ml (Table 2 and Fig. 4). The increase was time dependent with a maximum at 24 hours (0 hr 13,477 6 752 cpm/well, 12 hr 33,929 6 1251, 24 hr 38,130 6 989, P , 0.01, ANOVA). Coincubation of IGF-I (10 to 150 ng/ml) and 1a,25(OH)2D3 (1028 M) prevented the increased DNA synthesis induced by IGF-I, whereas coincubation with low-dose 1a,25(OH)2D3 (10212 M) had an additive effect at all concentrations tested in the experiment (Fig. 4). No additive effect of

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Fig. 1. Insulin-like growth factor type-1 receptor (IGFIR) mRNA and vitamin D receptor (VDR) mRNA analyzed by reverse transcriptase-polymerase chain reaction (RTPCR). Influence of IGF-I and 1a,25(OH)2D3. Subconfluent chondrocyte monolayer cultures were synchronized in serum-free medium for 24 hours. Medium was then changed to F12/DMEM 1/1 containing 0.2% BSA. Cells were incubated with ethanol (solvent, 0.05% final concentration), IGF-I (60 ng/ml), or 1a,25(OH)2D3 (10212 M and 1028 M) for the indicated periods. Thereafter, total RNA was prepared and mRNA levels encoding the IGFIR or VDR were quantitated by RT-PCR as described in the Methods section. (A) The increase of the IGFIR mRNA by 1a,25(OH)2D3 10212 M, but not 1028 M after 24 hours. (B) VDR mRNA was up-regulated by 1a,25(OH)2D3 10212 M, but not 1028 M after 12 hours. The RT-PCR amplification products are detected by electrophoresis in a 2% agarose gel and were visualized by ethidium bromide staining and ultraviolet transillumination (lower panel). Results are given as mean 6 SEM of the IGFIR/b-actin mRNA ratio or VDR/b-actin ratio respectively of three to four separate experiments. Statistics were by ANOVA. *P , 0.05.

IGF-II and 1a,25(OH)2D3 on DNA-synthesis was found despite the fact that stimulation of [3H]-thymidine incorporation by IGF-II alone (10 to 60 ng/ml, data not given) was similar to IGF-I action (Table 2). The effect of 1a,25(OH)2D3 was stereospecific, since the stereoisomer 1b,25(OH)2D3 was inactive (Table 2). Addition of the polyclonal IGF-I antibody AB-1 attenuated the effect of coincubation of IGF-I and 1a,25(OH)2D3. The AB-1 was specific for IGF-I since the stimulatory effect of the third party growth factor, that is, bFGF, was not significantly affected (Table 2). Increased rate of [3H]-thymidine incorporation was associated with increased alkaline phosphatase (AP) activity. Preincubation with 10212 M 1a,25(OH)2D3 for 48 hours increased AP activity from 18.0 6 2.1 nmol/well (BSA) to 41.3 6 2.5 nmol/well (P , 0.05), and with IGF-I (60 ng/ml) to 38.5 6 5.9 nmol/well. Concomitant incubation with 1a,25(OH)2D3 (10212 M) and IGF-I (60 ng/ml) caused a further significant increase in AP activity [48 6 3.5 nmol/well, P , 0.05 vs. IGF-I or 1a,25(OH)2D3 alone; statistics by ANOVA]. Action of 1a,25(OH)2D3 and IGF-I on colony formation in agarose-stabilized suspension cultures Colony formation was significantly stimulated by 10% FCS compared to control (0.2% BSA 1 0.1% FCS) treated cultures (Fig. 5). Both 1a,25(OH)2D3 (10212 M) and IGF-I (60 ng/ml) added to BSA 1 0.1% FCS increased the colony formation slightly above the 10% FCS positive control. Coincubation with both 1a,25(OH)2D3 (10212 M) and IGF-I (60 ng/ml) caused a highly significant increase in colony number after 21 days of culture (P , 0.01; Fig. 5). Stimulation of VDR expression by 1a,25(OH)2D3 and IGF-I Preincubation of subconfluent monolayer cultures for 24 hours with various concentrations of IGF-I (20 ng/ml and 60 ng/ml) caused a dose-dependent increase of maximal specific binding of

[3H]-1a,25(OH)2D3 (control 2424 bound molecules/cell; IGF-I 20 ng/ml 3574; IGF-I 60 ng/ml 12,000, without change of apparent receptor affinity; Table 3 and Figs. 1B and 6). In contrast, IGF-II (60 ng/ml) caused only a minimal change in maximal specific binding (135% of control; Fig. 6). The IGF-I induced increase in [3H]-1a,25(OH)2D3-binding was blocked by concomitant actinomycin D (2.5 mg/ml; Table 3). Addition of IGF-I (60 ng/ml) to the medium did not increase VDR mRNA (Fig. 1B). In contrast, incubation of subconfluent chondrocyte cultures with 1a,25(OH)2D3 10212 M, but not 1028 M for 12 hours increased VDR mRNA 1.5-fold (Fig. 1B). The effect of IGF-I and 1a,25(OH)2D3 on VDR expression was independently examined using immune staining with monoclonal antibody (mAb) 9A7g. Light and scarrous staining was seen in 0.2% BSA controls, while preincubation with IGF-I (60 ng/ml), 1a,25(OH)2D3 (10212 M) and IGF-I 1 1a,25(OH)2D3 resulted in consistent intense receptor staining (not shown). DISCUSSION The results of the present study provide evidence that the somatotropic system, that is, IGF-I and the calcium regulatory system, that is, 1a,25(OH)2D3, interact with respect to proliferation and receptor regulation in growth cartilage chondrocytes. They mutually amplify their effects in this system. Cooperation between these two hormone systems has thus far been described in osteoblasts [29, 30] but has not been recognized in growth cartilage. This finding has potential clinical implications. The main finding of the study is the documentation that 1a,25(OH)2D3 increased the effects of IGF-I on (i) IGFIR expression, (ii) DNA synthesis, (iii) cell proliferation/colony formation, and (iv) activity of AP. These additive responses were dose-dependent for IGF-I and noted only at low concentrations of 1a,25(OH)2D3 (10212 M) close to the physiological range. We emphasize that our BSA system did not contain endogenous vitamin D metabolites or binding protein [10, 31]. The presence of

Fig. 2. Immunocytology of insulin-like growth factor type-1 receptor (IGFIR) in cultured growth plate chondrocytes. Primary cultures of growth plate chondrocytes were kept in serum-free medium for 24 hours. Medium was changed to F-12/DMEM 1/1 supplemented with 0.2% BSA. (A) Solvent, (B) IGF-I 60 ng/ml, (C) 1a,25(OH)2D3 10212 M, or (D) both hormones together were added for 24 hours. Cell were fixed with paraformaldehyde (3.7%) and incubated with the mAb aIR3 at a final dilution of 1:100. The bound aIR3 was visualized by the streptavidin-biotin technique. An increased percentage of stained cells in the hormone treated groups is shown. Negative control were performed using a polyclonal IgG of the same subclass (not shown). Magnification 3400.

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Fig. 3. Modulation of [125I]-aIR3-binding by 1a,25(OH)2D3, IGF-I or their combination. Symbols are: (‚) 1a,25(OH)2D3 at 10212 M 1 IGF-I 60 ng/ml; (E) 1a,25(OH)2D3 at 10212 M; (Œ) IGF-I 60 ng/ml; (F) 1a,25(OH)2D3 at 1028; (X) control. Subconfluent chondrocytes, synchronized by preculture in serum-free medium for 24 hours [and by implication in the absence of 1a,25(OH)2D3] were cultured in F12/DMEM 1/1 medium, 1.2 mmol nominal calcium concentration with 0.2% BSA. 1a,25(OH)2D3 at 10212 M or 1028 M were added for the entire 48-hour culture period. Specific binding [125I]- aIR3 was determined as described in the Methods section. 1a,25(OH)2D3 10212 M, but not 1a,25(OH)2D3 1028 M increased specific binding of [125I]-aIR3 without change in affinity. Nmax solvent control 89,800 molecules/cell; 1a,25(OH)2D3 10212 M, 257,242; 1a,25(OH)2D3 1028 M, 89,880; IGF-I, 243,960; 1a,25(OH)2D3 1 IGF-I, 459,672; Kd solvent control, 0.95 3 1029 M; 1a,25(OH)2D3 10212 M, 1.8; 1a,25(OH)2D3 1028 M, 1.8; IGF-I, 1.77; 1a,25(OH)2D3 1 IGF-I, 2.1. Table 2. Action of IGF-I, 1a,25(OH)2D3 on DNA synthesis: Specificity Effector added

[3H]-thymidine incorporation % of solvent control

Solvent IGF-I 1a,25(OH)2D3 1b,25(OH)2D3 IGF-I 1 1a,25(OH)2D3 IGF-I 1 1b,25(OH)2D3 AB-1 IGF-I 1 AB-1 IGF-I 1 1b,25(OH)2D3 1 AB-1 IGF-II IGF-II 1 1a,25(OH)2D3 bFGF bFGF 1 AB-1

100 6 6 181 6 4a 175 6 5a 105 6 7 220 6 10a,b 153 6 12a,c 106 6 3 110 6 3 109 6 7d 183 6 9a 180 6 5a,c 172 6 5a 179 6 3a

Cell cycles of subconfluent chondrocyte cultures were synchronized by incubation in serum-free medium for 24 hr. After changing the medium to F12/DMEM 1/1 with 0.2% BSA IGF-I (60 ng/ml), IGF-II (60 ng/ml), 1a,25(OH)2D3 (10212 M), 1b,25(OH)2D3 (10212 M) with or without polyclonal IGF-I antibody AB-1 (1.25 mg/ml) or bFGF (10 ng/ml) were added as indicated. [3H]-thymidine (2 mCi/well) was added for the last 4 hours of the 48-hr incubation period. Statistics by ANOVA, N 5 8 per group. a P , 0.01 vs. solvent control b P , 0.05 vs. IGF-I or Ia,25(OH)2D3 c P , 0.05 vs. IGF-I 1 Ia,25(OH)2D3 d P , 0.05 vs. IGF-I 1 1b,25(OH)2D3

serum as described by other authors [32, 33] was not necessary in short term experiments (48 hr) to demonstrate a stimulation of DNA synthesis by IGF-I, confirming the results of Bo ¨hme at al [34]. In contrast to Bo ¨hme et al, we saw a stimulatory effect of IGF-I in less than three days. The discrepancies are difficult to explain, but may be due to persistent effects of serum (-factors), present in the incubation medium prior to the FCS deprivation (“starvation”) step. Such prolonged actions have been demonstrated for 1a,25(OH)2D3 [35, 36] and glucocorticoids [37]. The mechanisms by which 1a,25(OH)2D3 amplified the proliferative effect of IGF-I deserves comment. IGFIR expression was not increased on the mRNA level, but markedly on the protein

Fig. 4. Modulation of [3H]-thymidine incorporation by IGF-I (–), 1 224,25(OH)2D3 (solid line) or their combination (z-z). Subconfluent chondrocyte cultures were starved by incubation in serum-free medium for 24 hours. After changing the medium to F12/DMEM 1/1 with 0.2% BSA IGF-I (0 to 150 ng/ml) with or without 1a,25(OH)2D3 (10212 to 1028 M) was added for the entire 48-hour incubation period as indicated. [3H]thymidine (2 mCi/well) was added for the last four hours. The graph shows additive effects of 1a,25(OH)2D3 (10212 M) and IGF-I (60 ng/ml). The high concentration (1028 M) of 1a,25(OH)2D3 also inhibited the effect of IGF-I alone. Statistics were by ANOVA, N 5 8 per group. *P , 0.05 versus IGF-I; #P , 0.05 versus solvent control (IGF-I 0 ng/ml).

level (binding studies) by 10212 M 1a,25(OH)2D3. On immunocytochemical studies, qualitative observations suggested increased single cell staining for aIR3, but the major effect was the presence of more aIR3-positive cells upon treatment with 1a,25(OH)2D3. In these experiments, the total number of cells did not increase within 24 to 48 hours, suggesting recruitment of IGFIR positive

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Klaus et al: IGF-1 and vitamin D in pathologic growth Table 3. Effect of actinomycin D on IGF-I induced up-regulation of vitamin D receptor (VDR) Effector added

Nmax molecules/cell

Solvent IGF-I Actinomycin D IGF-I 1 actinomycin D

2327 12287 2918 4438

Kd 10210

M

4.3 3.4 6.2 6.3

Subconfluent chondrocytes, synchronized by preculture in serum free medium for 24 hr [and by implication in the absence of 1a,25(OH)2D3] were cultured in F-12/DMEM 1/1 medium, 1.2 mmol nominal calcium concentration with 0.2% BSA and IGF-I (60 ng/ml final concentration) added for the entire 24-hr culture period. Incubation with actinomycin D (2.5 mg/ml) was performed for the first 16 hours, followed by 3 washes. Postincubation was performed in the presence or absence of IGF-I (60 ng/ml). Specific binding of [3H]-1a,25(OH)2D3 was determined in KTED whole cell extracts as described in the Methods section.

Fig. 5. Effect of 1a,25(OH)2D3, IGF-I, and their combination on colony formation. Symbols are: () 1a,25(OH)2D3 10212 M 1 IGF-I 60 mg/ml; (Œ) IGF-I 60 ng/ml; (E) 1a,25(OH)2D3 10212 M; (F) 10% FCS; (X) control. Chondrocytes were cultured in agarose stabilized suspension as described in the Methods section. F-12/DMEM 1/1 medium, 1.2 mmol nominal calcium concentration supplemented with 0.2% BSA and 0.1% FCS was changed every other day. 1a,25(OH)2D3 10212 M dissolved in ethanol or ethanol alone as solvent control or IGF-I 60 ng/ml dissolved in PBS or PBS were added. A further group was coincubated with 1a,25(OH)2D3 10212 M and IGF-I 60 ng/ml. As a positive control, 10% FCS addition to the medium was used. Cultures were terminated after 21 days by fixation in buffered formaldehyde (4%) and colonies stained with alcian blue. The colonies were counted in 100 squares of a 2 mm grid using an inverted light microscope. Cloning efficiency was calculated from colonies formed/1000 seeded cells. Data are mean 6 SD of six dishes. Statistics were by ANOVA. Data are from one representative experiment. *P , 0.05 IGF-I versus IGF-I 1 1a,25(OH)2D3; **P , 0.05 1a,25(OH)2D3 versus IGF-I 1 1a,25(OH)2D3; #P , 0.05 versus solvent control.

cells as an important component of the action of 1a,25(OH)2D3. Whether recruitment of IGFIR-positive cells is a reflection of cell differentiation cannot be decided from the above data, because the primary cultures used are inherently heterogeneous. Alternatively, the additive effects of IGF-I and 1a,25(OH)2D3 could be explained by distinct responsive subpopulations, which might be recruited by either hormone alone. Stimulation of AP activity by both 1a,25(OH)2D3 and IGF-I would argue for chondrocyte maturation. The lack of modulation of IGFIR on the mRNA and on protein level as well as the absence of proliferative effects of IGF-I during coincubation with high-dose (1028 M) 1a,25(OH)2D3 is surprising. The mechanisms involved remain unexplained, but this finding is in line with our previous observation of a biphasic effect of 1a,25(OH)2D3 on proliferation [10, 15, 31]. The in vitro antiproliferative effect of high concentrations of 1a,25(OH)2D3 (1028 M) has previously been documented by our laboratory [10, 38] and others [39 – 44].

The stimulatory effect of 1a,25(OH)2D3 on DNA synthesis was specific, because this was not seen with the stereoisomer 1b,25(OH)2D3. Figure 4 shows that the effects of IGF-I and 1a,25(OH)2D3 were additive. This indicates that the two agonists operate, at least in part, through separate effectors or pathways. Some effects of 1a,25(OH)2D3 are mediated by nongenomic mechanisms [45– 48]. In single cell studies we found a slow, long lasting increase in free ionized intracellular calcium within two minutes after exposure to 1a,25(OH)2D3 [49]. Furthermore, we demonstrated in previous studies that stimulation of growth by 1a,25(OH)2D3 was dependent on transmembrane calcium flux through L-type voltage gated channels [31] arguing for rapid actions of 1a,25(OH)2D3. However, the effect of 1a,25(OH)2D3 on increased [125I]-aIR3 binding was obliterated by actinomycin D, suggesting that nuclear mechanisms are involved despite the fact that only minor changes in IGFIR mRNA were detected. Within the precision of our methods, the increase in IGFIR mRNA tended to be smaller than the increase in [125I]-aIR3 binding. We cannot decide whether this discrepancy is due to stabilization of IGFIR mRNA by 1a,25(OH)2D3 or to the long half life of IGFIR mRNA (Rodrı´guez, unpublished observation). The signal transduction sequences involved in the interaction between somatotropic and calciotropic hormones on growth cartilage chondrocytes have not yet been elucidated. To the best of our knowledge, no calcium- or 1a,25(OH)2D3-sensitive response sequences have been documented in the IGFIR gene. Furthermore, IGF-I failed to affect binding of the VDR to its cognate response element in osteosarcoma cells [50]. We visualized and quantitated the IGF-I receptor in rat (and rabbit) cells by binding with a mAb directed against the human IGFIR, that is, mAb aIR3. Binding was specific since hepatocytes of two-month-old rats that practically do not express IGFIR failed to stain; furthermore, staining was obliterated by an excess of cold IGF-I, while excess of IGF-II did not affect immune staining. When evaluating aIR3 binding by chondrocytes, we were concerned about the confounding effect of potential IGF-I synthesis by chondrocytes. Under the basal conditions of the experiment, little IGF-I (0.16 ng/ml) was found in the supernatant. At this concentration, no inhibition of aIR3 binding was noted in control experiments (data not given). The additive effect of IGF-I and 1a,25(OH)2D3 on chondrocyte proliferation and AP activity resembles previous observations

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1159

Fig. 6. Modulation of [3H]-1a,25(OH)2D3binding by IGF-I and IGF-II. Symbols are: (Œ) IGF-I; (ƒ) IGF-II; (F) control. Subconfluent chondrocytes, synchronized by preculture in serum-free medium for 24 hours (and by implication in the absence of 1a,25(OH)2D3) were cultured in F-12/DMEM 1/1 medium, 1.2 mmol nominal calcium concentration with 0.2% BSA and 0.1% FCS. IGF-I (60 ng/ml final concentration) or IGF-II (60 ng/ml) were added for the entire 48-hour culture period. Specific binding of [3H]-1a,25(OH)2D3 was determined in KTED whole cell extracts as described in the Methods section. IGF-I, but not IGF-II increased maximal specific binding of [3H]1a,25(OH)2D3 without changes in KD. One representative experiment is shown. Nmax solvent control, 2424 bound molecules/cell; IGF-I, 12,000; IGF-II, 3320. Kd solvent control, 5.7 3 10210 M; IGF-I, 5.7; IGF-II, 5.8.

indicating that the action of IGF-I is also amplified by other growth factors in vivo [51]. In osteoblast cell lines synergism of IGF-I and 1a,25(OH)2D3 on alkaline phosphatase activity and collagen synthesis was reported [29]. Similar observations on alkaline phosphatase activity and Gla protein secretion were made in cultivated human bone cells [30]. In contrast, supplementation of 1a,25(OH)2D3 normalized growth in vitamin D deficient rats without any change in serum IGF-I concentrations, although circulating IGF-I levels may not necessarily reflect local IGF-I concentrations in the growth plate (paracrine secretion) [52]. Furthermore, effects of IGF-binding proteins modulating IGF-I action [53–55] were not excluded. Chondrocytes in primary culture express VDR as shown previously [10, 18]. Incubation of the cultures with IGF-I increased the binding of radiolabeled 1a,25(OH)2D3. This effect was stereospecific. It was not the result of increased chondrocyte proliferation induced by IGF-I, because IGF-II stimulated DNA synthesis to a similar extent as IGF-I, but did not affect VDR expression. These observations are in line with studies reporting up-regulation of VDR by IGF-I in 3T3 fibroblasts and in human breast cancer cells [56]. The intriguing selectivity for IGF-I versus IGF-II in VDR up-regulation and in additive action with 1a,25(OH)2D3 on proliferation cannot be explained by our results. However, the affinity of IGF-I for the IGFIR normally is higher than that of IGF-II as demonstrated in articular chondrocytes [57] and bone cells [reviewed in 58]. In our study we were not able to mimic all of the effects of IGF-I by high concentrations of IGF-II as demonstrated in osteosarcoma cells [59]. This suggests a selective action of IGF-I via IGFIR. We could demonstrate IGFIR expression not only in cultured cells, but also ex vivo in rat tibial growth plates (results not shown). These findings are in contrast to results obtained by Schalch et al, who described action of IGF-I via the type 2 IGF/mannose-6-phosphate receptor on costal chondrocytes [60]. These differences might be due to cell source, cell culture condition and maturation state of cells used. Our study confirms that the increase in VDR precedes the rise in DNA synthesis [31]. As shown previously [19], short-term incubation of chondrocytes with 1a,25(OH)2D3 at low concentrations causes homologous up-regulation of specific binding of

1a,25(OH)2D3. Significant regulation of VDR protein stability was described in osteoblasts [61]. Although the effect of IGF-I on VDR protein expression measured by radioligand binding was dependent on transcription, as shown by inhibition by actinomycin D, we could not demonstrate an increase of VDR mRNA upon incubation with IGF-I. This suggests that VDR protein upregulation occurred primarily independent of VDR mRNA accumulation. Therefore, an expression of a costimulatory subunit such as retinol X receptor [reviewed in 62] and/or translational modification of the VDR transcript might participate in the significant up-regulation of VDR protein observed. We considered several possibilities to explain stimulation of 1a,25(OH)2D3 binding by IGF-I. Cell morphology assessed by phase contrast microscopy showed no major alteration in the cell geometry. We further indirectly assessed whether IGF-I might have altered the amount of plasma membrane available for aIR3 binding: no change in cell protein content (as a rough index of cell hypertrophy) and no morphological evidence of cell hypotrophy were found. Furthermore, analysis of the supernatant for detached cells was consistently negative. We measured DNA synthesis, an index of chondrocyte proliferation after stimulation with IGF-I and 1a,25(OH)2D3, respectively. These measurements were carried out in cells cultured in 0.2% BSA to avoid the confounding influence of IGF and vitamin D metabolites, respectively, which are present in FCS. Under these conditions, however, cells did not replicate in a time frame of two days and started to deteriorate after 72 hours. While IGF-I was effective in stimulating cell proliferation, it additionally increased activity of alkaline phosphatase as a marker of chondrocyte differentiation, in contrast to what had previously been observed in chicken chondrocytes [34]. Further evidence of induction of cell differentiation by IGF-I was provided by the observation of stimulated colony formation in long-term cultures incubated with IGF-I. In the clinical setting of growth retardation due to chronic renal failure in children, disturbances of the vitamin D system and the somatotropic axis are simultaneously present [11]. If the additive effects of IGF-I and 1a,25(OH)2D3 can be confirmed in vivo, restoration of both 1a,25(OH)2D3 and GH/IGF status may be required to fully restore abnormal growth in these children. Our

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in vitro experiments also suggest mutual inhibition of the interaction when high concentrations of 1a,25(OH)2D3 are achieved. There is no documentation for this possibility, but it should be considered in the light of our recent animal experiments, in which daily low dose 1a,25(OH)2D3 increased the GH effect on uremic growth retardation, while high dose 1a,25(OH)2D3 (pulse therapy) did not [63]. ACKNOWLEDGMENTS This study was supported by the Deutsche Forschungsgemeinschaft (Kl 630/5-1). We appreciate the generous help of J. Reichrath, M.D., University of Homburg, in setting up the technique for immunocytology and immunohistology. P. Fernandez received a scientific grant from MEC (Spain), and J. Rodriguez from FICYT, supported by the 2nd Regional Plan of Investigation of the Principado of Asturias (Spain). Reprint requests to Gu ¨nter Klaus, M.D., Department of Pediatrics, University of Marburg, Deutschhausstr. 12, 35033 Marburg, Germany. E-mail: [email protected]

APPENDIX Abbreviations used in this article are: AB-1, polyclonal IGF-I antibody; AP, alkaline phosphatase activity; FCS, fetal calf serum; GH, growth hormone; IGF, insulin-like growth factor; IGFIR, IGF-type 1 receptors; LGT, low gel temperature; mAb, monoclonal antibody; RT-PCR, reverse transcriptase-polymerase chain reaction; SL, standard low gel temperature; VDR, vitamin D receptor.

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