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Glucocorticoids inhibit vascular endothelial growth factor expression in growth plate chondrocytes
Molecular and Cellular Endocrinology 197 (2002) 35 /44 www.elsevier.com/locate/mce
Glucocorticoids inhibit vascular endothelial growth factor expression in growth plate chondrocytes Joost A. Koedam *, Jeske J. Smink, Sylvia C. van Buul-Offers Department of Pediatric Endocrinology, University Medical Center Utrecht, Room KE3-139.2, P.O. Box 85090, AB-3508 Utrecht, The Netherlands
Abstract Vascular endothelial growth factor (VEGF) plays an essential role in angiogenesis in the growth plate and ultimately in regulating endochondral ossification. Since longitudinal bone growth is often disturbed in children who are treated with glucocorticoids, we investigated the effects of dexamethasone on VEGF expression by epiphyseal chondrocytes. Cells were cultured from tibial growth plates of neonatal piglets. Using Northern blotting and RT-PCR techniques, the chondrocyte-specific markers aggrecan, collagen II and CD-RAP were detected. Also the glucocorticoid receptor (GR) was expressed. VEGF protein secreted from these cells was examined by ELISA and Western immunoblotting. The VEGF121 and VEGF165 isoforms were detected in the supernatant. As determined by RT-PCR, all three major mRNA splice variants were produced, including the species encoding VEGF189. Dexamethasone (100 nM) inhibited both protein and mRNA expression by approximately 45%. Hydrocortisone (cortisol) and prednisolone also inhibited VEGF secretion, but they were less active than dexamethasone. The inhibitory actions of dexamethasone were almost completely blocked by the GR antagonist Org34116, indicating that the GR mediates these actions. Degradation of the VEGF mRNA was not accelerated by dexamethasone. Therefore, a transcriptional mechanism seems likely. Downregulation of this important growth factor could lead to disruption of the normal invasion of blood vessels in the growth plate, which could contribute to disturbed endochondral ossification and growth. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vascular endothelial growth factor; Growth plate; Chondrocyte; Glucocorticoid; Dexamethasone; Porcine
1. Introduction Longitudinal bone growth depends on the proliferation, differentiation and subsequent ossification of growth plate cartilage. The rates of chondrocyte proliferation and differentiation to the hypertrophic phenotype are determined by a complex interplay between many signaling molecules. Also the ensuing process of endochondral ossification is tightly regulated, and involves the interrelated processes of extracellular matrix degradation, apoptosis of terminal hypertrophic chondrocytes and angiogenesis: the invasion of the growth plate by new blood vessels from the metaphysis. The molecular mechanisms involved in chondrocyte differentiation and endochondral bone formation have been reviewed by Stevens and Williams (1999). Signals
* Corresponding author. Tel.: /31-30-250-4331; fax: /31-30-2505378 E-mail address: [email protected] (J.A. Koedam).
that direct the polarity of the growth plate, angiogenesis and bone formation originate in the growth plate itself, as was elegantly demonstrated by excising, inverting and reimplanting rabbit growth plates (Abad et al., 1999). While the polarity of the cartilage was maintained, the epiphyseal bone, which became adjacent to the hypertrophic zone of the growth plate, became the site of blood vessel and bone cell invasion and longitudinal bone growth. Angiogenesis can be induced by basic fibroblast growth factor (Baron et al., 1994), transferrin (Carlevaro et al., 1997) and other factors (Alini et al., 1996), but vascular endothelial growth factor (VEGF) is the quintessential angiogenic factor in this respect, as was demonstrated by treating growing mice with a soluble antagonist (Gerber et al., 1999). This treatment resulted in suppression of blood vessel invasion, accompanied by impaired resorption of terminal chondrocytes and formation of the primary spongiosa (trabeculae of the metaphyseal bone). VEGF is expressed by many tissues and cell types, including the growth plate cartilage
0303-7207/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 3 - 7 2 0 7 ( 0 2 ) 0 0 2 7 6 - 9
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(Gerber et al., 1999; Horner et al., 1999; Carlevaro et al., 2000; Garcia-Ramirez et al., 2000). Its expression can be induced by many factors, including hypoxia (Shweiki et al., 1992), estrogen (Cullinan-Bove and Koos, 1993), osteogenic protein-1/BMP-7 (Yeh and Lee, 1999), PDGF (Nauck et al., 1998) and IGF-I (Goad et al., 1996; Warren et al., 1996; Bermont et al., 2000; Miele et al., 2000; Garcia-Ramirez et al., 2000). Given its pivotal role in maintaining the normal structure of the growth plate (Gerber et al., 1999), VEGF could be a potential target for agents, which disturb longitudinal growth. Glucocorticoid hormones are well known to induce growth retardation (Blodgett et al., 1956; Allen, 1996; Rooman et al., 1999) as well as osteoporosis (Manelli and Giustina, 2000). Direct local actions of glucocorticoids on the growth plate have been demonstrated in vivo (Baron et al., 1992), which likely occur through interaction with the glucocorticoid receptor (GR) (Silvestrini et al., 1999). Although many of the inhibitory actions of pharmacological doses of glucocorticoids on skeletal growth appear to be mediated by interference with the GH/insulin-like growth factor-I axis (Jux et al., 1998), their effects on angiogenesis and in particular VEGF expression in the growth plate are worth investigating. Preliminary data from our laboratory indicate that prednisolone treatment of juvenile piglets leads to a severely disturbed invasion of blood vessels to the growth plate.1 Previously, we have used cultured rabbit costal chondrocytes to study the effects of dexamethasone on the expression of IGF binding proteins (Koedam et al., 2000). In this study, we set out to characterize the effects of pharmacological doses of dexamethasone and other glucocorticoids on VEGF expression by cultured porcine growth plate chondrocytes.
2. Materials and methods 2.1. Materials Recombinant human IGF-I was kindly provided by Eli Lilly & Co., Indianapolis, IN. Cortisol (hydrocortisone, Solu-Cortef) was from Pharmacia & Upjohn B.V. (Woerden, The Netherlands). Prednisolone (Di-Adreson-F) was from N.V. Organon (Oss, The Netherlands). The GR antagonist Org34116 was kindly provided by Dr. M. de Gooyer (N.V. Organon, Oss, The Netherlands). Dexamethasone (as dexamethasone disodium 1 Koedam, J.A., Smink, J.J., van Tilburg, C.M., Buchholz, I.M., Sakkers, R.J.B., de Meer, K., van Buul-Offers, S.C., 2001. Effects of glucocorticoid treatment on angiogenesis and apoptosis in the growth plate. In: Proceedings of the First International Conference on the Growth Plate, San Antonio, TX, June 15 /19, 2001 (Abstract 188).
phosphate) was from Merck Sharpe & Dohme (Haarlem, The Netherlands). Platelet-derived growth factor (PDGF)-AB, pronase (‘‘Protease’’, P-6911), collagenase (C-9891), actinomycin D, BSA, salmon sperm DNA and Alcian Blue 8GX were from Sigma Chemical Co. (St. Louis, MO). Rabbit polyclonal anti-VEGF antibody and blocking peptide were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Dulbecco’s modified Eagle’s medium (DMEM), L-glutamine, penicillin, streptomycin, fetal calf serum (FCS), and 100 bp DNA marker ladder were obtained from Life Technologies Ltd. (Paisley, UK). Peroxidase-conjugated donkey anti-rabbit antibody, Oligo(dT)12 18 primer, RediPrime Random Primer labeling mixture, [a-32P]dCTP (10 mCi/ml), nylon membrane (Hybond-N/, 0.2 mm) and Hyperfilm ECL film were obtained from Amersham Pharmacia Biotech (Little Chalfont, UK). Centricon-10 ultrafiltration membranes and PVDF membranes (Immobilon-P) were from Millipore Corp. (Bedford, MA). Restriction enzymes, AmpliTaq DNA polymerase, modifying enzymes and TriPure isolation reagent were purchased from Roche Molecular Biochemicals (Mannheim, Germany). M-MLV Reverse Transcriptase was from Promega Corp. (Madison, WI). PCR primers were prepared by Isogen Bioscience B.V. (Maarssen, The Netherlands). The TOPO TA Cloning kit was from Invitrogen Corp., Carlsbad, CA. 2.2. Cell culture The culture conditions of the growth plate chondrocytes were identical to those we used earlier for rabbit costal chondrocytes (Koedam et al., 2000). In short, cartilage growth plates were dissected from the distal and proximal tibiae of neonatal piglets. The piglets were used and sacrificed for unrelated experiments by the Neonatology Department of the University Medical Center Utrecht. These experiments did not interfere with the normal physiology of the growth plate cartilage. The cells were released from the extracellular matrix by subsequent digestion with protease (1 mg/ml in serum-free culture medium, 30 min, 37 8C) and collagenase (0.5 mg/ml in culture medium containing 10% FCS, 6 h at 37 8C). The cells released from the tissue were filtered through a 70 mm nylon cell strainer, centrifuged, counted in a Bu¨rker counting chamber and seeded in 75 cm2 tissue culture flasks. The cells were grown in Dulbecco’s modified Eagle’s medium containing 1 g/l glucose, 4 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 10% FCS at 37 8C in a 5% CO2 and humid atmosphere. Generally, second passage cells were used for experiments, but identical results were obtained with first and third passage cells. When cells were treated with growth factors and hormones followed by VEGF ELISA or Western blotting, they were seeded (40,000 or 80,000 cells per well) on 24-well
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plates. For isolation of RNA from cells, 1/106 cells were seeded in 25 cm2 flasks. Conditioned medium was prepared by incubating the cells in medium containing 10% FCS, which had been stripped of steroids by adsorption with dextran-coated charcoal. In order to analyze the secreted VEGF protein by electrophoresis and Western immunoblotting, serumfree medium supplemented with 10 nM IGF-I and 0.005% BSA was used instead. In a few cases, also the amount of VEGF secreted under these conditions was measured by ELISA, and no differences were found between both milieus for collecting conditioned medium, neither in the amount of VEGF secreted, nor in the inhibitory effect of dexamethasone. Experiments in which RNA was to be isolated from the cells were always performed in the presence of 10% stripped FCS. 2.3. Analysis of VEGF protein Chondrocyte-conditioned medium (1 ml) was collected after 48 h, centrifuged for 10 min at 3000 /g (4 8C) and stored at /80 8C. VEGF concentrations were measured by ELISA (R&D Systems, Inc., Minneapolis, MN) according to the manufacturer’s instructions. The adherent cells were washed three times with PBS and lysed in 100 ml 2% SDS, 125 mM Tris /HCl, pH 6.8, followed by boiling for 5 min. Ten-microliter was used to measure the protein content (using the BCA reagent, Pierce, Rockford, IL). VEGF levels were normalized to the protein content of the cells. For Western immunoblotting, the conditioned media were concentrated 10-fold on Centricon-10 ultrafiltration membranes and loaded on a 12% gel for reducing SDS-PAGE. VEGF was visualized by electrotransfer to PVDF membranes, followed by incubation with rabbit polyclonal antibody (1/200 dilution) and peroxidaseconjugated donkey anti-rabbit antibody (1/4000 dilution). The bands were visualized using the SuperSignal WestPico Chemiluminescent Substrate of Pierce (Rockford, IL) and Hyperfilm ECL film. Molecular weights were calculated using BioRad (Hercules, CA) broadrange markers as standards. 2.4. RNA isolation and Northern blot analysis Cells in confluent 25 cm2 flasks were washed with PBS and lysed in TriPure reagent. RNA was isolated according to the manufacturer’s instructions. Ten-microgram of RNA was separated on a denaturing (7% formaldehyde) 1% agarose gel and blotted onto a nylon membrane in 10/ SSC using a vacuum blotter (Appligene-Oncor, Illkirch, France) followed by crosslinking with UV radiation. The filter was prehybridized with 50 mg/ml denatured salmon sperm DNA in hybridization mix (0.1 g/ml dextran sulfate, 5 / Denhardt’s, 3/ SSC, 0.1% SDS) and hybridized with the indicated DNA
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probes for 16 h at 55 8C (65 8C for GAPDH) in hybridization mix. Probes (see below) were labeled with [a-32P]dCTP using the random hexamer RediPrime kit. The blots were washed up to a stringency of 0.5 / SSC, 0.1% SDS at 55 8C (0.1 / SSC, 0.1% SDS at 65 8C for GAPDH, collagen II). Quantification of the bands on the blots was performed by phosphorimaging using a BioRad GS-363 Molecular Imager System followed by analysis with BioRad Molecular Analyst software (version 1.5). Values were corrected for RNA loading by dividing by the GAPDH signal in the same lane. In addition, the filters were exposed to Fuji Super RX (Du¨sseldorf, Germany) film. The cDNA probes for collagen types I and II, aggrecan and GAPDH were described before (Koedam et al., 2000). A probe for porcine CD-RAP (cartilagederived retinoic acid-sensitive protein) (Dietz and Sandell, 1996) was prepared by RT-PCR from total RNA isolated from the cultured chondrocytes as described below. The PCR product was ligated into the pCRIITOPO TA Cloning vector. The cDNA probe for human VEGF was a kind gift of Dr. M.F. Gebbink and Dr. E.E. Voest (University Medical Center Utrecht). The 520 bp insert encompassing the sequence encoding VEGF121 (Weindel et al., 1992) was excised using Nco I/Bgl II double digestion.
2.5. RT-PCR and Southern blotting One microgram of total cellular RNA was reverse transcribed using 1 mg of oligo(dT) as primer and 200 U of Reverse Transcriptase for 90 min at 42 8C in a total volume of 25 ml. Two-microliter of cDNA was amplified by PCR using the following primers: CD-RAP, forward 5?-ATGCCCAAGCTGGCTGAC-3? and reverse 5?ATGCTACTGGGGAAATAGC-3? (240 bp product); Bcl-2, forward 5?-GTCGAATCAGCTATTTACTGC3? and reverse 5?-GGCTTAAGGTACTGGATGATA3? (565 bp product); GAPDH, forward 5?-CTCAAGATTGTCAGCAATGC-3? and reverse 5?-TTGCCCACAGCCTTGGCA-3? (226 bp product); GR, forward 5?-GTGAGTACCTCTGGAGGACA-3? and reverse 5?CTTTGCCCATTTCACTGC-3? (761 bp product). The VEGF primers (forward 5?-AGTGTGTGCCCACTGAGGAG, corresponding to nt 278 /297 in exon 3 and reverse 5?-ACCGCCTCGGCTTGTCACAT, corresponding to nt 627/648 in exon 8) were selected, because they are identical in human and porcine VEGF, and have identical melting temperatures. In each case, the PCR consisted of an initial denaturation step for 5 min at 94 8C, followed by 30 /35 cycles (1 min at 94 8C, 1 min at 58 8C, 1 min at 72 8C) and a final extension step (10 min at 72 8C) in a total volume of 50 ml containing 10 pmol of primers. PCR products were analyzed on a 1% agarose gel.
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Fig. 1. Characterization of cultured primary growth plate chondrocytes. Total RNA was isolated from confluent second passage monolayer cultures and analyzed for the expression of chondrocyte-specific markers. (A) Autoradiogram of Northern blots. (B) Ethidium bromide staining of RT-PCR product. Col II, collagen II; aggr, aggrecan; CD-RAP, cartilage-derived retinoic acid-sensitive protein; M, 100 bp marker.
For Southern blotting of VEGF, 4 ml from the PCR reaction was loaded on a 1% agarose gel. Southern blotting and hybridization with 32P-labeled VEGF cDNA probe was essentially carried out as described above for Northern blotting. 2.6. Statistics Data from VEGF ELISA were normalized for total protein content and data from Northern blots were normalized for GAPDH content. Results are expressed as mean9/S.E.M. Statistical differences between two groups were determined by two-tailed Student’s t-test. Statistical differences between multiple treatments were determined by one-way ANOVA using InStat version 3.00 (GraphPad Software, Inc., San Diego, CA). The Tukey /Kramer post hoc test was used to determine significance between treatment groups.
lage-specific gene CD-RAP (0.8 kb) (Fig. 1A). CD-RAP could be amplified from cDNA obtained from these cells (Fig. 1B), further illustrating the phenotype. Collagen I mRNA, specific for fibroblasts, osteoblasts and de-differentiated chondrocytes, was expressed at very low levels, whereas in NIH 3T3 mouse fibroblasts a strong signal was observed (not shown). Staining of sulfated proteoglycans in the extracellular matrix with Alcian blue also showed that the chondrocyte cultures were not contaminated with fibroblasts or osteoblasts and that the chondrocytes were not de-differentiated (not shown). The GR was detected by RT-PCR, yielding the expected 761 bp product (Fig. 3, middle panel). Treatment of the cells with dexamethasone did not affect the expression level of the GR (not shown, Smink et al., 2002a). 3.2. Secretion of VEGF protein from chondrocytes
3. Results 3.1. Characterization of growth plate chondrocytes The chondrocyte phenotype of the cultured porcine epiphyseal cells was maintained, as shown in Fig. 1. Typical chondrocytic markers were expressed, as assessed by Northern blotting of total RNA isolated from the cells. Specifically, transcripts were detected for collagen II (5.4 kb), aggrecan (8.0 kb) and the carti-
When cells were cultured in the presence of serum, VEGF was secreted into the supernatant. Accumulated over a 48 h period, 400/1600 pg/ml (1.3 /4.6 pg/mg protein) was measured with an ELISA assay. In the absence of serum, VEGF production was stimulated by PDGF (threefold at 5 nM) and by IGF-I (1.89/0.2-fold at 10 nM, n/5 independent experiment, P B/0.02). In the presence of 10 nM IGF-I, 600 /1300 pg/ml (2.3 /6.1 pg/mg) VEGF accumulated over a 48 h period. Dexamethasone caused a dose-dependent decrease of se-
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Fig. 2. Inhibitory effect of glucocorticoids on VEGF secretion. Porcine epiphyseal chondrocytes were cultured in 24-well plates and conditioned medium was collected 48 h after the addition of hormone and/or GR antagonist Org34116 (Org). Dex, dexamethasone; Cort, cortisol; Pred, prednisolone. (A) Org34116 at 1 mM and 10 mM was added to the cells 1 h before the addition of dexamethasone. VEGF was measured by ELISA assay. Values are the mean9/S.E.M. of four experiments, each performed in duplicate and normalized for protein content in each well and for the control wells in each experiment. Two experiments were carried out in 10% serum, and two experiments were carried out in serum-free medium supplemented with 10 nM IGF-I and 0.005% BSA. The results from these two conditions were identical and were pooled. *P B/0.01, compared with controls without dexamethasone; #P B/0.01, compared with corresponding dexamethasone concentration in the absence of Org34116. (B) Values (ELISA assay) are from one experiment (carried out in serum-free medium supplemented with 10 nM IGF-I), which was repeated with identical results. (C) Western blot of VEGF secreted from chondrocytes. Conditioned medium (serum-free, supplemented with 10 nM IGF-I) was collected, concentrated and electrophoressed under reducing conditions. VEGF protein was detected using a rabbit polyclonal antibody. 20, 23, mass of proteins in kDa.
creted VEGF protein (Fig. 2A). The GR antagonist Org34116 (Karst et al., 1997) largely reversed the inhibitory action of dexamethasone, indicating the involvement of functional GRs in the action of dexamethasone. When compared with the inhibitory effect of dexamethasone, cortisol and prednisolone were less potent, but at 1 mM were also able to cause 80% inhibition of VEGF secretion (Fig. 2B). On Western immunoblot, two species of secreted VEGF were detected at 20 and 23 kDa (Fig. 2C). When the antibody was first preabsorbed with VEGF blocking peptide, these bands were not detected (not shown). Based on their relative weights and the fact that they were released from the cells, these forms most likely represent the VEGF121 and VEGF165 isoforms. VEGF121 lacks both heparin-binding regions, making it highly diffusible, while VEGF165 still retains some cellbinding properties. The intensities of both isoforms decreased in parallel in response to dexamethasone treatment, while Org34116 blocked the action of dexamethasone. Thus, the results obtained from the im-
munoblot completely correspond to the ELISA measurements. Similarly, cortisol and prednisolone inhibited the secretion of both VEGF isoforms (not shown). 3.3. RT-PCR and Southern blotting RT-PCR was performed on RNA isolated from epiphyseal chondrocytes to characterize the splice variants of VEGF mRNA (Fig. 3). Oligonucleotide primers were chosen to correspond with sequences in exons 3 and 8, allowing to distinguish between forms which contain both exons 6 and 7 (VEGF189), or lacking exon 6 (VEGF165) or lacking both exons 6 and 7 (VEGF121) (Tischer et al., 1991). The major amplicon observed migrated just above the 300 bp marker, corresponding well with the predicted size of 297 bp for the VEGF165 species. Likewise, minor bands representing the VEGF121 (165 bp) and VEGF189 (369 bp) isoforms were detected. Also several larger products were weakly visible. None of these bands corresponded to the
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Fig. 3. RT-PCR and Southern blot of VEGF isoforms. Total RNA from chondrocytes was reverse-transcribed to cDNA, followed by PCR using primer pairs corresponding with exons 3 and 8. Samples were run in duplicate, and either stained with ethidium bromide or transferred to a PVDF membrane followed by hybridization with a 32P-labeled VEGF cDNA probe. In addition to PCR for VEGF (V), PCR was performed for Bcl-2 (B), GAPDH (G) and the GR. M, 100 bp marker.
VEGF206 isoform (Houck et al., 1991), which would contain an extra insertion of 51 bp between exons 6 and 7 and would thus be expected to yield a PCR product of 420 bp. On the corresponding Southern blot, all the PCR-amplified products hybridized with the VEGF probe. As negative controls, three unrelated PCR products (from Bcl-2, GAPDH and the GR) were transferred to the filter, but they failed to hybridize with the VEGF probe. In conclusion, porcine epiphyseal chondrocytes produce all major splice variants of VEGF, with VEGF165 being the most abundant.
halflife was just under 2 h, and was not altered in the presence of dexamethasone, indicating that the downregulation of VEGF by dexamethasone is not caused by the destabilization of its mRNA.
3.4. Dexamethasone effect on VEGF mRNA Northern blots of total RNA extracted from chondrocytes were hybridized with a VEGF cDNA probe. A major transcript of approximately 4.8 kb was observed, together with minor transcripts of approximately 1.3 and 5.3 kb (Fig. 4). Expression of all transcripts was inhibited by dexamethasone. At 100 nM dexamethasone, the abundance of the major transcript was decreased to 57.59/5.2% (n/4 independent experiments, P B/0.01). A time-course experiment, in which RNA was isolated from cells at different times after addition of dexamethasone, indicated that the maximum inhibition of VEGF mRNA expression was reached after 1 h (data not shown). As for the protein expression, the inhibitory effect of dexamethasone was blocked by the GR antagonist Org34116. To test whether dexamethasone decreased the abundance of VEGF mRNA by accelerating its degradation, the transcription inhibitor actinomycin D was used. Actinomycin D was added to the cells 1 h after the addition of dexamethasone, and the stability of the VEGF mRNA was determined. As shown in Fig. 5, the
Fig. 4. Northern blot of VEGF mRNA. Chondrocytes were treated with 100 nM dexamethasone (Dex) or 10 mM Org34116 (Org) or the combination of both for 24 h. Total RNA was isolated and hybridized with 32P-labeled VEGF cDNA probe as described in Section 2. Rehybridization with a GAPDH probe was performed to check for equal loading of the lanes. A representative experiment is shown, which was repeated with identical results. Sizes of transcripts are indicated in kb.
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Fig. 5. Stability of VEGF mRNA. Chondrocytes were cultured in 25 cm2 flasks in the presence of 10% serum and 100 nM dexamethasone was added to one-half of the flasks. After 1 h, actinomycin D (5 mg/ml) was added to all the flasks. RNA was isolated at 1, 2, 3 and 4 h after the addition of actinomycin D. VEGF mRNA was assayed by Northern blot as described for Fig. 4. The lower panel shows the quantification of the major VEGF transcript, corrected for GAPDH content. The graph shows the average result of two experiments.
4. Discussion In this study, we have examined the effects of glucocorticoids, in particular dexamethasone, on the expression of VEGF. We cultured primary chondrocytes from the epiphyseal growth plates of neonatal piglets. This model system was used earlier in the study of dexamethasone effects on the expression of IGF-binding proteins (Smink et al., 2002a). In many ways, the phenotype of these cells resembles that of the rabbit costal chondrocytes, which we described earlier to study the expression of IGFBPs (Koedam et al., 2000). Although no attempts were made to separate the various layers of the growth plate, it is assumed that the in vitro phenotype most closely resembles that of the proliferative zone. We found no evidence of de-differentiation during our experiments, as demonstrated by a lack of decreased collagen type II expression or increased collagen type I expression during passaging of the cells. We demonstrate here that these chondrocytes synthesize and secrete VEGF, an important angiogenic factor,
and that its production is regulated by IGF-I and PDGF. VEGF protein has been detected earlier by immunohistochemistry in growth plate cartilage of human neonates (Horner et al., 1999), chicken and mouse embryos (Carlevaro et al., 2000) and human fetuses (Garcia-Ramirez et al., 2000). Similarly, VEGF mRNA expression was found by in situ hybridization in juvenile (Gerber et al., 1999) and embryonic (Colnot and Helms, 2001) mouse growth plates. In most of these cases, VEGF was exclusively found in the hypertrophic zone; only Garcia-Ramirez et al. (2000) also detected VEGF protein in the resting and proliferative zones. In agreement with our result, they also found VEGF secreted by cultured proliferative chondrocytes. The sizes of the VEGF transcripts cannot be precisely linked to the various splice variants. Since the 297 bp PCR product representing the VEGF165 species is the most abundant species, it is tempting to speculate that the major 4.8 kb transcript represents this variant. Bermont et al. (2000) came to the same conclusion for a 4.5 kb transcript. A 4.4 or 4.5 kb transcript has been
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reported for many cell types, e.g. human fetal vascular smooth muscle cells (Tischer et al., 1991), endometrial adenocarcinoma cells (Bermont et al., 2000) and human breast cancer cells (Ruohola et al., 1999). In all these cell types, also a 3.7 kb transcript was found, which was usually the predominant species. In addition, this 3.7 kb form was found in, e.g. human fetal epiphyseal chondrocytes (Garcia-Ramirez et al., 2000) and mouse osteoblastic cells (Saadeh et al., 2000). We did not, however, detect this species, a discrepancy for which we have no explanation. The minor 5.3 kb band was also reported by others, including Tischer et al. (1991), Bermont et al. (2000). The 1.3 kb VEGF transcript has not been described before, although it is weakly visible on a Northern blot in the paper by GarciaRamirez et al. (2000). Downregulation of VEGF expression by dexamethasone has previously been demonstrated in rat glioma cells (Heiss et al., 1996; Machein et al., 1999), human vascular smooth muscle cells (Nauck et al., 1998), mouse and rat pituitary folliculostellate cells (Gloddek et al., 1999) and porcine brain endothelial cells (Fischer et al., 2001). Many mechanisms exist by which glucocorticoids can repress gene function (Webster and Cidlowski, 1999). Since the GR is present in our cells, and the specific antagonist Org34116 blocked the inhibitory action of dexamethasone on VEGF expression, the dexamethasone /GR complex is most likely the mediator of VEGF repression. The 5?-promoter region of the VEGF gene does not contain a glucocorticoid-responsive element (GRE), but it does contain four AP-1 binding sites (Tischer et al., 1991). The transcription factor AP-1 is activated by growth factors via mitogenactivated protein (MAP) kinases. Since the induction of VEGF expression by IGF-I, at least in NIH3T3 fibroblasts, involves the MAP kinase signaling pathway (Miele et al., 2000), it is likely that in our experiments (in which IGF-I was present) AP-1 activity is involved. It is conceivable, therefore, that a mechanism operates by which the ligand-occupied GR prevents the AP-1 transactivator complex from stimulating the VEGF gene (Schu¨le et al., 1990). In this case, GR binding to DNA and GR-dependent gene transcription are not required. It is not known which factors regulate VEGF expression in the growth plate. IGF-I is an obvious candidate, however, since others and we have detected expression of this growth factor in the growth plate (Smink et al., 2002b; Reinecke et al., 1999). IGF-I production in cultured rat epiphyseal chondrocytes, especially when stimulated by GH, is inhibited by 100 nM dexamethasone (Jux et al., 1998). Similarly, dexamethasone blocks the IGF-I-stimulated rise in type 1 IGF receptor molecules (Jux et al., 1998). In addition to a direct effect on the AP-1 transactivator complex as described above, glucocorticoids could therefore also regulate
VEGF expression indirectly through interactions with the somatotropic axis. Another well-established inducer of VEGF expression is hypoxia (Shweiki et al., 1992), mediated by the transcriptional activator hypoxia-inducible factor 1 (HIF-1) (Semenza, 2001). Because the cartilage is not vascularized, it has been widely assumed that chondrocytes, especially in the hypertrophic zone, are hypoxic. Using a hypoxia-sensing drug and immunohistochemistry, Shapiro et al. (1997) found that hypertrophic cartilage, at least in the chicken, is actually not oxygendeficient. In contrast, Schipani et al. (2001), using a similar technique, recently reported that hypoxia does occur in the growth plates of developing mice. VEGF expression in the growth plate appears to be regulated through both HIF-1-dependent and -independent mechanisms (Schipani et al., 2001). The possible role of glucocorticoids in regulating these mechanisms needs to be addressed, especially since hypoxia-induced VEGF expression seems to be much less susceptible to dexamethasone inhibition than VEGF expression induced by serum and growth factors (Heiss et al., 1996; Machein et al., 1999). We also explored another possibility by which dexamethasone could regulate VEGF expression, namely by destabilizing its mRNA. We found no evidence for such a post-transcriptional regulation. When transcription was interrupted by actinomycin D, the halflife of the VEGF mRNA was unaffected by the presence of dexamethasone. Glucocorticoid-regulated mRNA turnover is thought to involve AUUUA sequences (AU response elements or AREs) in the 3?-untranslated region of mRNAs (Malter, 1989; Peppel et al., 1991), and these sequences are present in the VEGF transcript (Claffey et al., 1998). The regulation of the stability of VEGF mRNA is very complex, however, and involves sequences in the 3?-untranslated region as well as the 5?untranslated region and the coding region (Dibbens et al., 1999). This complexity is also illustrated by the different results, which have been reported with respect to the ability of IGF-I to stabilize VEGF mRNA. While IGF-I increased VEGF mRNA halflife in colon carcinoma cells (Warren et al., 1996) and endometrial adenocarcinoma cells (Bermont et al., 2000), it had no such effect in osteoblastic cells (Goad et al., 1996) and fibroblasts (Miele et al., 2000). VEGF is of utmost importance for the vascular invasion of the growth plate cartilage and the resulting processes of apoptosis and bone formation (Gerber et al., 1999). It is therefore highly likely that the negative effect of glucocorticoids on VEGF expression by epiphyseal chondrocytes contributes to the mechanism by which these steroid hormones cause growth retardation and/or osteoporosis.
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Acknowledgements We thank Ms. C.M.P.C.D. Peeters and Dr. F. Groenendaal for making the tibia of neonatal piglets available to us.
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acid and peptide in rat tibial growth plate and articular chondrocytes in vivo. Endocrinology 141, 2847 /2853. Rooman, R., Koster, J.G., Bloemen, R., Gresnigt, R., van BuulOffers, S.C., 1999. The effect of dexamethasone on body and organ growth of normal and IGF-II transgenic mice. J. Endocrinol. 163, 543 /552. Ruohola, J.K., Valve, E.M., Karkkainen, M.J., Joukov, V., Alitalo, K., Ha¨rko¨nen, P.L., 1999. Vascular endothelial growth factors are differentially regulated by steroid hormones and antiestrogens in breast cancer cells. Mol. Cell. Endocrinol. 149, 29 /40. Saadeh, P.B., Mehrara, B.J., Steinbrech, D.S., Spector, J.A., Greenwald, J.A., Chin, G.S., Ueno, H., Gittes, G.K., Longaker, M.T., 2000. Mechanism of fibroblast growth factor-2 modulation of vascular endothelial growth factor expression by osteoblastic cells. Endocrinology 141, 2075 /2083. Schipani, E., Ryan, H.E., Didrckson, S., Kobayashi, T., Knight, M., Johnson, R.S., 2001. Hypoxia in cartilage: HIF-1a is essential for chondrocyte growth arrest and survival. Genes Dev. 15, 2865 / 2876. Schu¨le, R., Rangarajan, P., Kliewer, S., Ransone, L.J., Bolado, J., Yang, N., Verma, I.M., Evans, R.M., 1990. Functional antagonism between oncoprotein c-Jun and glucocorticoid receptor. Cell 62, 1217 /1226. Semenza, G.L., 2001. Hypoxia-inducible factor 1: oxygen homeostasis and disease pathophysiology. Trends Mol. Med. 7, 345 / 350. Shapiro, I.M., Mansfield, K.D., Evans, S.M., Lord, E.M., Koch, C.J., 1997. Chondrocytes in the endochondral growth plate cartilage are not hypoxic. Am. J. Physiol. 272, C1134 /C1143. Shweiki, D., Itin, A., Soffer, D., Keshet, E., 1992. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 359, 843 /845.
Silvestrini, G., Mocetti, P., Ballanti, P., DiGrezia, R., Bonucci, E., 1999. Cytochemical demonstration of the glucocorticoid receptor in skeletal cells of the rat. Endocr. Res. 25, 117 /128. Smink, J.J., Koedam, J.A., Koster, J.G., van Buul-Offers, S.C., 2002a. Dexamethasone-induced growth inhibition of porcine growth plate chondrocytes is accompanied by changes in levels of IGF axis components. J. Endocrinol. 174, 343 /352. Smink, J.J., Koster, J.G., Gresnigt, M.G., Rooman, R., Koedam, J.A., van Buul-Offers, S.C., 2002b. IGF and IGF-binding protein expression in the growth plate of normal, dexamethasone-treated and human IGF-II transgenic mice. J. Endocrinology (in press). Stevens, D.A., Williams, G.R., 1999. Hormone regulation of chondrocyte differentiation and endochondral bone formation. Mol. Cell. Endocrinol. 151, 195 /204. Tischer, E., Mitchell, R., Hartman, T., Silva, M., Gospodarowicz, D., Fiddes, J.C., Abraham, J.A., 1991. The human gene for vascular endothelial growth factor: multiple protein forms are encoded through alternative exon splicing. J. Biol. Chem. 266, 11947 / 11954. Warren, R.S., Yuan, H., Matli, M.R., Ferrara, N., Donner, D.B., 1996. Induction of vascular endothelial growth factor by insulinlike growth factor I in colorectal carcinoma. J. Biol. Chem. 271, 29483 /29488. Webster, J.C., Cidlowski, J.A., 1999. Mechanism of glucocorticoidreceptor-mediated repression of gene expression. Trends Endocrinol. Metab. 10, 396 /402. Weindel, K., Marme´, D., Weich, H.A., 1992. AIDS-associated Kaposi’s sarcoma cells in culture express vascular endothelial growth factor. Biochem. Biophys. Res. Commun. 183, 1167 /1174. Yeh, L.-C.C., Lee, J.C., 1999. Osteogenic protein-1 increases gene expression of vascular endothelial growth factor in primary cultures of fetal rat calvaria cells. Mol. Cell. Endocrinol. 153, 113 /124.
Glucocorticoids inhibit vascular endothelial growth factor expression in growth plate chondrocytes Joost A. Koedam *, Jeske J. Smink, Sylvia C. van Buul-Offers Department of Pediatric Endocrinology, University Medical Center Utrecht, Room KE3-139.2, P.O. Box 85090, AB-3508 Utrecht, The Netherlands
Abstract Vascular endothelial growth factor (VEGF) plays an essential role in angiogenesis in the growth plate and ultimately in regulating endochondral ossification. Since longitudinal bone growth is often disturbed in children who are treated with glucocorticoids, we investigated the effects of dexamethasone on VEGF expression by epiphyseal chondrocytes. Cells were cultured from tibial growth plates of neonatal piglets. Using Northern blotting and RT-PCR techniques, the chondrocyte-specific markers aggrecan, collagen II and CD-RAP were detected. Also the glucocorticoid receptor (GR) was expressed. VEGF protein secreted from these cells was examined by ELISA and Western immunoblotting. The VEGF121 and VEGF165 isoforms were detected in the supernatant. As determined by RT-PCR, all three major mRNA splice variants were produced, including the species encoding VEGF189. Dexamethasone (100 nM) inhibited both protein and mRNA expression by approximately 45%. Hydrocortisone (cortisol) and prednisolone also inhibited VEGF secretion, but they were less active than dexamethasone. The inhibitory actions of dexamethasone were almost completely blocked by the GR antagonist Org34116, indicating that the GR mediates these actions. Degradation of the VEGF mRNA was not accelerated by dexamethasone. Therefore, a transcriptional mechanism seems likely. Downregulation of this important growth factor could lead to disruption of the normal invasion of blood vessels in the growth plate, which could contribute to disturbed endochondral ossification and growth. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vascular endothelial growth factor; Growth plate; Chondrocyte; Glucocorticoid; Dexamethasone; Porcine
1. Introduction Longitudinal bone growth depends on the proliferation, differentiation and subsequent ossification of growth plate cartilage. The rates of chondrocyte proliferation and differentiation to the hypertrophic phenotype are determined by a complex interplay between many signaling molecules. Also the ensuing process of endochondral ossification is tightly regulated, and involves the interrelated processes of extracellular matrix degradation, apoptosis of terminal hypertrophic chondrocytes and angiogenesis: the invasion of the growth plate by new blood vessels from the metaphysis. The molecular mechanisms involved in chondrocyte differentiation and endochondral bone formation have been reviewed by Stevens and Williams (1999). Signals
* Corresponding author. Tel.: /31-30-250-4331; fax: /31-30-2505378 E-mail address: [email protected] (J.A. Koedam).
that direct the polarity of the growth plate, angiogenesis and bone formation originate in the growth plate itself, as was elegantly demonstrated by excising, inverting and reimplanting rabbit growth plates (Abad et al., 1999). While the polarity of the cartilage was maintained, the epiphyseal bone, which became adjacent to the hypertrophic zone of the growth plate, became the site of blood vessel and bone cell invasion and longitudinal bone growth. Angiogenesis can be induced by basic fibroblast growth factor (Baron et al., 1994), transferrin (Carlevaro et al., 1997) and other factors (Alini et al., 1996), but vascular endothelial growth factor (VEGF) is the quintessential angiogenic factor in this respect, as was demonstrated by treating growing mice with a soluble antagonist (Gerber et al., 1999). This treatment resulted in suppression of blood vessel invasion, accompanied by impaired resorption of terminal chondrocytes and formation of the primary spongiosa (trabeculae of the metaphyseal bone). VEGF is expressed by many tissues and cell types, including the growth plate cartilage
0303-7207/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 3 - 7 2 0 7 ( 0 2 ) 0 0 2 7 6 - 9
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(Gerber et al., 1999; Horner et al., 1999; Carlevaro et al., 2000; Garcia-Ramirez et al., 2000). Its expression can be induced by many factors, including hypoxia (Shweiki et al., 1992), estrogen (Cullinan-Bove and Koos, 1993), osteogenic protein-1/BMP-7 (Yeh and Lee, 1999), PDGF (Nauck et al., 1998) and IGF-I (Goad et al., 1996; Warren et al., 1996; Bermont et al., 2000; Miele et al., 2000; Garcia-Ramirez et al., 2000). Given its pivotal role in maintaining the normal structure of the growth plate (Gerber et al., 1999), VEGF could be a potential target for agents, which disturb longitudinal growth. Glucocorticoid hormones are well known to induce growth retardation (Blodgett et al., 1956; Allen, 1996; Rooman et al., 1999) as well as osteoporosis (Manelli and Giustina, 2000). Direct local actions of glucocorticoids on the growth plate have been demonstrated in vivo (Baron et al., 1992), which likely occur through interaction with the glucocorticoid receptor (GR) (Silvestrini et al., 1999). Although many of the inhibitory actions of pharmacological doses of glucocorticoids on skeletal growth appear to be mediated by interference with the GH/insulin-like growth factor-I axis (Jux et al., 1998), their effects on angiogenesis and in particular VEGF expression in the growth plate are worth investigating. Preliminary data from our laboratory indicate that prednisolone treatment of juvenile piglets leads to a severely disturbed invasion of blood vessels to the growth plate.1 Previously, we have used cultured rabbit costal chondrocytes to study the effects of dexamethasone on the expression of IGF binding proteins (Koedam et al., 2000). In this study, we set out to characterize the effects of pharmacological doses of dexamethasone and other glucocorticoids on VEGF expression by cultured porcine growth plate chondrocytes.
2. Materials and methods 2.1. Materials Recombinant human IGF-I was kindly provided by Eli Lilly & Co., Indianapolis, IN. Cortisol (hydrocortisone, Solu-Cortef) was from Pharmacia & Upjohn B.V. (Woerden, The Netherlands). Prednisolone (Di-Adreson-F) was from N.V. Organon (Oss, The Netherlands). The GR antagonist Org34116 was kindly provided by Dr. M. de Gooyer (N.V. Organon, Oss, The Netherlands). Dexamethasone (as dexamethasone disodium 1 Koedam, J.A., Smink, J.J., van Tilburg, C.M., Buchholz, I.M., Sakkers, R.J.B., de Meer, K., van Buul-Offers, S.C., 2001. Effects of glucocorticoid treatment on angiogenesis and apoptosis in the growth plate. In: Proceedings of the First International Conference on the Growth Plate, San Antonio, TX, June 15 /19, 2001 (Abstract 188).
phosphate) was from Merck Sharpe & Dohme (Haarlem, The Netherlands). Platelet-derived growth factor (PDGF)-AB, pronase (‘‘Protease’’, P-6911), collagenase (C-9891), actinomycin D, BSA, salmon sperm DNA and Alcian Blue 8GX were from Sigma Chemical Co. (St. Louis, MO). Rabbit polyclonal anti-VEGF antibody and blocking peptide were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Dulbecco’s modified Eagle’s medium (DMEM), L-glutamine, penicillin, streptomycin, fetal calf serum (FCS), and 100 bp DNA marker ladder were obtained from Life Technologies Ltd. (Paisley, UK). Peroxidase-conjugated donkey anti-rabbit antibody, Oligo(dT)12 18 primer, RediPrime Random Primer labeling mixture, [a-32P]dCTP (10 mCi/ml), nylon membrane (Hybond-N/, 0.2 mm) and Hyperfilm ECL film were obtained from Amersham Pharmacia Biotech (Little Chalfont, UK). Centricon-10 ultrafiltration membranes and PVDF membranes (Immobilon-P) were from Millipore Corp. (Bedford, MA). Restriction enzymes, AmpliTaq DNA polymerase, modifying enzymes and TriPure isolation reagent were purchased from Roche Molecular Biochemicals (Mannheim, Germany). M-MLV Reverse Transcriptase was from Promega Corp. (Madison, WI). PCR primers were prepared by Isogen Bioscience B.V. (Maarssen, The Netherlands). The TOPO TA Cloning kit was from Invitrogen Corp., Carlsbad, CA. 2.2. Cell culture The culture conditions of the growth plate chondrocytes were identical to those we used earlier for rabbit costal chondrocytes (Koedam et al., 2000). In short, cartilage growth plates were dissected from the distal and proximal tibiae of neonatal piglets. The piglets were used and sacrificed for unrelated experiments by the Neonatology Department of the University Medical Center Utrecht. These experiments did not interfere with the normal physiology of the growth plate cartilage. The cells were released from the extracellular matrix by subsequent digestion with protease (1 mg/ml in serum-free culture medium, 30 min, 37 8C) and collagenase (0.5 mg/ml in culture medium containing 10% FCS, 6 h at 37 8C). The cells released from the tissue were filtered through a 70 mm nylon cell strainer, centrifuged, counted in a Bu¨rker counting chamber and seeded in 75 cm2 tissue culture flasks. The cells were grown in Dulbecco’s modified Eagle’s medium containing 1 g/l glucose, 4 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 10% FCS at 37 8C in a 5% CO2 and humid atmosphere. Generally, second passage cells were used for experiments, but identical results were obtained with first and third passage cells. When cells were treated with growth factors and hormones followed by VEGF ELISA or Western blotting, they were seeded (40,000 or 80,000 cells per well) on 24-well
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plates. For isolation of RNA from cells, 1/106 cells were seeded in 25 cm2 flasks. Conditioned medium was prepared by incubating the cells in medium containing 10% FCS, which had been stripped of steroids by adsorption with dextran-coated charcoal. In order to analyze the secreted VEGF protein by electrophoresis and Western immunoblotting, serumfree medium supplemented with 10 nM IGF-I and 0.005% BSA was used instead. In a few cases, also the amount of VEGF secreted under these conditions was measured by ELISA, and no differences were found between both milieus for collecting conditioned medium, neither in the amount of VEGF secreted, nor in the inhibitory effect of dexamethasone. Experiments in which RNA was to be isolated from the cells were always performed in the presence of 10% stripped FCS. 2.3. Analysis of VEGF protein Chondrocyte-conditioned medium (1 ml) was collected after 48 h, centrifuged for 10 min at 3000 /g (4 8C) and stored at /80 8C. VEGF concentrations were measured by ELISA (R&D Systems, Inc., Minneapolis, MN) according to the manufacturer’s instructions. The adherent cells were washed three times with PBS and lysed in 100 ml 2% SDS, 125 mM Tris /HCl, pH 6.8, followed by boiling for 5 min. Ten-microliter was used to measure the protein content (using the BCA reagent, Pierce, Rockford, IL). VEGF levels were normalized to the protein content of the cells. For Western immunoblotting, the conditioned media were concentrated 10-fold on Centricon-10 ultrafiltration membranes and loaded on a 12% gel for reducing SDS-PAGE. VEGF was visualized by electrotransfer to PVDF membranes, followed by incubation with rabbit polyclonal antibody (1/200 dilution) and peroxidaseconjugated donkey anti-rabbit antibody (1/4000 dilution). The bands were visualized using the SuperSignal WestPico Chemiluminescent Substrate of Pierce (Rockford, IL) and Hyperfilm ECL film. Molecular weights were calculated using BioRad (Hercules, CA) broadrange markers as standards. 2.4. RNA isolation and Northern blot analysis Cells in confluent 25 cm2 flasks were washed with PBS and lysed in TriPure reagent. RNA was isolated according to the manufacturer’s instructions. Ten-microgram of RNA was separated on a denaturing (7% formaldehyde) 1% agarose gel and blotted onto a nylon membrane in 10/ SSC using a vacuum blotter (Appligene-Oncor, Illkirch, France) followed by crosslinking with UV radiation. The filter was prehybridized with 50 mg/ml denatured salmon sperm DNA in hybridization mix (0.1 g/ml dextran sulfate, 5 / Denhardt’s, 3/ SSC, 0.1% SDS) and hybridized with the indicated DNA
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probes for 16 h at 55 8C (65 8C for GAPDH) in hybridization mix. Probes (see below) were labeled with [a-32P]dCTP using the random hexamer RediPrime kit. The blots were washed up to a stringency of 0.5 / SSC, 0.1% SDS at 55 8C (0.1 / SSC, 0.1% SDS at 65 8C for GAPDH, collagen II). Quantification of the bands on the blots was performed by phosphorimaging using a BioRad GS-363 Molecular Imager System followed by analysis with BioRad Molecular Analyst software (version 1.5). Values were corrected for RNA loading by dividing by the GAPDH signal in the same lane. In addition, the filters were exposed to Fuji Super RX (Du¨sseldorf, Germany) film. The cDNA probes for collagen types I and II, aggrecan and GAPDH were described before (Koedam et al., 2000). A probe for porcine CD-RAP (cartilagederived retinoic acid-sensitive protein) (Dietz and Sandell, 1996) was prepared by RT-PCR from total RNA isolated from the cultured chondrocytes as described below. The PCR product was ligated into the pCRIITOPO TA Cloning vector. The cDNA probe for human VEGF was a kind gift of Dr. M.F. Gebbink and Dr. E.E. Voest (University Medical Center Utrecht). The 520 bp insert encompassing the sequence encoding VEGF121 (Weindel et al., 1992) was excised using Nco I/Bgl II double digestion.
2.5. RT-PCR and Southern blotting One microgram of total cellular RNA was reverse transcribed using 1 mg of oligo(dT) as primer and 200 U of Reverse Transcriptase for 90 min at 42 8C in a total volume of 25 ml. Two-microliter of cDNA was amplified by PCR using the following primers: CD-RAP, forward 5?-ATGCCCAAGCTGGCTGAC-3? and reverse 5?ATGCTACTGGGGAAATAGC-3? (240 bp product); Bcl-2, forward 5?-GTCGAATCAGCTATTTACTGC3? and reverse 5?-GGCTTAAGGTACTGGATGATA3? (565 bp product); GAPDH, forward 5?-CTCAAGATTGTCAGCAATGC-3? and reverse 5?-TTGCCCACAGCCTTGGCA-3? (226 bp product); GR, forward 5?-GTGAGTACCTCTGGAGGACA-3? and reverse 5?CTTTGCCCATTTCACTGC-3? (761 bp product). The VEGF primers (forward 5?-AGTGTGTGCCCACTGAGGAG, corresponding to nt 278 /297 in exon 3 and reverse 5?-ACCGCCTCGGCTTGTCACAT, corresponding to nt 627/648 in exon 8) were selected, because they are identical in human and porcine VEGF, and have identical melting temperatures. In each case, the PCR consisted of an initial denaturation step for 5 min at 94 8C, followed by 30 /35 cycles (1 min at 94 8C, 1 min at 58 8C, 1 min at 72 8C) and a final extension step (10 min at 72 8C) in a total volume of 50 ml containing 10 pmol of primers. PCR products were analyzed on a 1% agarose gel.
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Fig. 1. Characterization of cultured primary growth plate chondrocytes. Total RNA was isolated from confluent second passage monolayer cultures and analyzed for the expression of chondrocyte-specific markers. (A) Autoradiogram of Northern blots. (B) Ethidium bromide staining of RT-PCR product. Col II, collagen II; aggr, aggrecan; CD-RAP, cartilage-derived retinoic acid-sensitive protein; M, 100 bp marker.
For Southern blotting of VEGF, 4 ml from the PCR reaction was loaded on a 1% agarose gel. Southern blotting and hybridization with 32P-labeled VEGF cDNA probe was essentially carried out as described above for Northern blotting. 2.6. Statistics Data from VEGF ELISA were normalized for total protein content and data from Northern blots were normalized for GAPDH content. Results are expressed as mean9/S.E.M. Statistical differences between two groups were determined by two-tailed Student’s t-test. Statistical differences between multiple treatments were determined by one-way ANOVA using InStat version 3.00 (GraphPad Software, Inc., San Diego, CA). The Tukey /Kramer post hoc test was used to determine significance between treatment groups.
lage-specific gene CD-RAP (0.8 kb) (Fig. 1A). CD-RAP could be amplified from cDNA obtained from these cells (Fig. 1B), further illustrating the phenotype. Collagen I mRNA, specific for fibroblasts, osteoblasts and de-differentiated chondrocytes, was expressed at very low levels, whereas in NIH 3T3 mouse fibroblasts a strong signal was observed (not shown). Staining of sulfated proteoglycans in the extracellular matrix with Alcian blue also showed that the chondrocyte cultures were not contaminated with fibroblasts or osteoblasts and that the chondrocytes were not de-differentiated (not shown). The GR was detected by RT-PCR, yielding the expected 761 bp product (Fig. 3, middle panel). Treatment of the cells with dexamethasone did not affect the expression level of the GR (not shown, Smink et al., 2002a). 3.2. Secretion of VEGF protein from chondrocytes
3. Results 3.1. Characterization of growth plate chondrocytes The chondrocyte phenotype of the cultured porcine epiphyseal cells was maintained, as shown in Fig. 1. Typical chondrocytic markers were expressed, as assessed by Northern blotting of total RNA isolated from the cells. Specifically, transcripts were detected for collagen II (5.4 kb), aggrecan (8.0 kb) and the carti-
When cells were cultured in the presence of serum, VEGF was secreted into the supernatant. Accumulated over a 48 h period, 400/1600 pg/ml (1.3 /4.6 pg/mg protein) was measured with an ELISA assay. In the absence of serum, VEGF production was stimulated by PDGF (threefold at 5 nM) and by IGF-I (1.89/0.2-fold at 10 nM, n/5 independent experiment, P B/0.02). In the presence of 10 nM IGF-I, 600 /1300 pg/ml (2.3 /6.1 pg/mg) VEGF accumulated over a 48 h period. Dexamethasone caused a dose-dependent decrease of se-
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Fig. 2. Inhibitory effect of glucocorticoids on VEGF secretion. Porcine epiphyseal chondrocytes were cultured in 24-well plates and conditioned medium was collected 48 h after the addition of hormone and/or GR antagonist Org34116 (Org). Dex, dexamethasone; Cort, cortisol; Pred, prednisolone. (A) Org34116 at 1 mM and 10 mM was added to the cells 1 h before the addition of dexamethasone. VEGF was measured by ELISA assay. Values are the mean9/S.E.M. of four experiments, each performed in duplicate and normalized for protein content in each well and for the control wells in each experiment. Two experiments were carried out in 10% serum, and two experiments were carried out in serum-free medium supplemented with 10 nM IGF-I and 0.005% BSA. The results from these two conditions were identical and were pooled. *P B/0.01, compared with controls without dexamethasone; #P B/0.01, compared with corresponding dexamethasone concentration in the absence of Org34116. (B) Values (ELISA assay) are from one experiment (carried out in serum-free medium supplemented with 10 nM IGF-I), which was repeated with identical results. (C) Western blot of VEGF secreted from chondrocytes. Conditioned medium (serum-free, supplemented with 10 nM IGF-I) was collected, concentrated and electrophoressed under reducing conditions. VEGF protein was detected using a rabbit polyclonal antibody. 20, 23, mass of proteins in kDa.
creted VEGF protein (Fig. 2A). The GR antagonist Org34116 (Karst et al., 1997) largely reversed the inhibitory action of dexamethasone, indicating the involvement of functional GRs in the action of dexamethasone. When compared with the inhibitory effect of dexamethasone, cortisol and prednisolone were less potent, but at 1 mM were also able to cause 80% inhibition of VEGF secretion (Fig. 2B). On Western immunoblot, two species of secreted VEGF were detected at 20 and 23 kDa (Fig. 2C). When the antibody was first preabsorbed with VEGF blocking peptide, these bands were not detected (not shown). Based on their relative weights and the fact that they were released from the cells, these forms most likely represent the VEGF121 and VEGF165 isoforms. VEGF121 lacks both heparin-binding regions, making it highly diffusible, while VEGF165 still retains some cellbinding properties. The intensities of both isoforms decreased in parallel in response to dexamethasone treatment, while Org34116 blocked the action of dexamethasone. Thus, the results obtained from the im-
munoblot completely correspond to the ELISA measurements. Similarly, cortisol and prednisolone inhibited the secretion of both VEGF isoforms (not shown). 3.3. RT-PCR and Southern blotting RT-PCR was performed on RNA isolated from epiphyseal chondrocytes to characterize the splice variants of VEGF mRNA (Fig. 3). Oligonucleotide primers were chosen to correspond with sequences in exons 3 and 8, allowing to distinguish between forms which contain both exons 6 and 7 (VEGF189), or lacking exon 6 (VEGF165) or lacking both exons 6 and 7 (VEGF121) (Tischer et al., 1991). The major amplicon observed migrated just above the 300 bp marker, corresponding well with the predicted size of 297 bp for the VEGF165 species. Likewise, minor bands representing the VEGF121 (165 bp) and VEGF189 (369 bp) isoforms were detected. Also several larger products were weakly visible. None of these bands corresponded to the
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Fig. 3. RT-PCR and Southern blot of VEGF isoforms. Total RNA from chondrocytes was reverse-transcribed to cDNA, followed by PCR using primer pairs corresponding with exons 3 and 8. Samples were run in duplicate, and either stained with ethidium bromide or transferred to a PVDF membrane followed by hybridization with a 32P-labeled VEGF cDNA probe. In addition to PCR for VEGF (V), PCR was performed for Bcl-2 (B), GAPDH (G) and the GR. M, 100 bp marker.
VEGF206 isoform (Houck et al., 1991), which would contain an extra insertion of 51 bp between exons 6 and 7 and would thus be expected to yield a PCR product of 420 bp. On the corresponding Southern blot, all the PCR-amplified products hybridized with the VEGF probe. As negative controls, three unrelated PCR products (from Bcl-2, GAPDH and the GR) were transferred to the filter, but they failed to hybridize with the VEGF probe. In conclusion, porcine epiphyseal chondrocytes produce all major splice variants of VEGF, with VEGF165 being the most abundant.
halflife was just under 2 h, and was not altered in the presence of dexamethasone, indicating that the downregulation of VEGF by dexamethasone is not caused by the destabilization of its mRNA.
3.4. Dexamethasone effect on VEGF mRNA Northern blots of total RNA extracted from chondrocytes were hybridized with a VEGF cDNA probe. A major transcript of approximately 4.8 kb was observed, together with minor transcripts of approximately 1.3 and 5.3 kb (Fig. 4). Expression of all transcripts was inhibited by dexamethasone. At 100 nM dexamethasone, the abundance of the major transcript was decreased to 57.59/5.2% (n/4 independent experiments, P B/0.01). A time-course experiment, in which RNA was isolated from cells at different times after addition of dexamethasone, indicated that the maximum inhibition of VEGF mRNA expression was reached after 1 h (data not shown). As for the protein expression, the inhibitory effect of dexamethasone was blocked by the GR antagonist Org34116. To test whether dexamethasone decreased the abundance of VEGF mRNA by accelerating its degradation, the transcription inhibitor actinomycin D was used. Actinomycin D was added to the cells 1 h after the addition of dexamethasone, and the stability of the VEGF mRNA was determined. As shown in Fig. 5, the
Fig. 4. Northern blot of VEGF mRNA. Chondrocytes were treated with 100 nM dexamethasone (Dex) or 10 mM Org34116 (Org) or the combination of both for 24 h. Total RNA was isolated and hybridized with 32P-labeled VEGF cDNA probe as described in Section 2. Rehybridization with a GAPDH probe was performed to check for equal loading of the lanes. A representative experiment is shown, which was repeated with identical results. Sizes of transcripts are indicated in kb.
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Fig. 5. Stability of VEGF mRNA. Chondrocytes were cultured in 25 cm2 flasks in the presence of 10% serum and 100 nM dexamethasone was added to one-half of the flasks. After 1 h, actinomycin D (5 mg/ml) was added to all the flasks. RNA was isolated at 1, 2, 3 and 4 h after the addition of actinomycin D. VEGF mRNA was assayed by Northern blot as described for Fig. 4. The lower panel shows the quantification of the major VEGF transcript, corrected for GAPDH content. The graph shows the average result of two experiments.
4. Discussion In this study, we have examined the effects of glucocorticoids, in particular dexamethasone, on the expression of VEGF. We cultured primary chondrocytes from the epiphyseal growth plates of neonatal piglets. This model system was used earlier in the study of dexamethasone effects on the expression of IGF-binding proteins (Smink et al., 2002a). In many ways, the phenotype of these cells resembles that of the rabbit costal chondrocytes, which we described earlier to study the expression of IGFBPs (Koedam et al., 2000). Although no attempts were made to separate the various layers of the growth plate, it is assumed that the in vitro phenotype most closely resembles that of the proliferative zone. We found no evidence of de-differentiation during our experiments, as demonstrated by a lack of decreased collagen type II expression or increased collagen type I expression during passaging of the cells. We demonstrate here that these chondrocytes synthesize and secrete VEGF, an important angiogenic factor,
and that its production is regulated by IGF-I and PDGF. VEGF protein has been detected earlier by immunohistochemistry in growth plate cartilage of human neonates (Horner et al., 1999), chicken and mouse embryos (Carlevaro et al., 2000) and human fetuses (Garcia-Ramirez et al., 2000). Similarly, VEGF mRNA expression was found by in situ hybridization in juvenile (Gerber et al., 1999) and embryonic (Colnot and Helms, 2001) mouse growth plates. In most of these cases, VEGF was exclusively found in the hypertrophic zone; only Garcia-Ramirez et al. (2000) also detected VEGF protein in the resting and proliferative zones. In agreement with our result, they also found VEGF secreted by cultured proliferative chondrocytes. The sizes of the VEGF transcripts cannot be precisely linked to the various splice variants. Since the 297 bp PCR product representing the VEGF165 species is the most abundant species, it is tempting to speculate that the major 4.8 kb transcript represents this variant. Bermont et al. (2000) came to the same conclusion for a 4.5 kb transcript. A 4.4 or 4.5 kb transcript has been
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reported for many cell types, e.g. human fetal vascular smooth muscle cells (Tischer et al., 1991), endometrial adenocarcinoma cells (Bermont et al., 2000) and human breast cancer cells (Ruohola et al., 1999). In all these cell types, also a 3.7 kb transcript was found, which was usually the predominant species. In addition, this 3.7 kb form was found in, e.g. human fetal epiphyseal chondrocytes (Garcia-Ramirez et al., 2000) and mouse osteoblastic cells (Saadeh et al., 2000). We did not, however, detect this species, a discrepancy for which we have no explanation. The minor 5.3 kb band was also reported by others, including Tischer et al. (1991), Bermont et al. (2000). The 1.3 kb VEGF transcript has not been described before, although it is weakly visible on a Northern blot in the paper by GarciaRamirez et al. (2000). Downregulation of VEGF expression by dexamethasone has previously been demonstrated in rat glioma cells (Heiss et al., 1996; Machein et al., 1999), human vascular smooth muscle cells (Nauck et al., 1998), mouse and rat pituitary folliculostellate cells (Gloddek et al., 1999) and porcine brain endothelial cells (Fischer et al., 2001). Many mechanisms exist by which glucocorticoids can repress gene function (Webster and Cidlowski, 1999). Since the GR is present in our cells, and the specific antagonist Org34116 blocked the inhibitory action of dexamethasone on VEGF expression, the dexamethasone /GR complex is most likely the mediator of VEGF repression. The 5?-promoter region of the VEGF gene does not contain a glucocorticoid-responsive element (GRE), but it does contain four AP-1 binding sites (Tischer et al., 1991). The transcription factor AP-1 is activated by growth factors via mitogenactivated protein (MAP) kinases. Since the induction of VEGF expression by IGF-I, at least in NIH3T3 fibroblasts, involves the MAP kinase signaling pathway (Miele et al., 2000), it is likely that in our experiments (in which IGF-I was present) AP-1 activity is involved. It is conceivable, therefore, that a mechanism operates by which the ligand-occupied GR prevents the AP-1 transactivator complex from stimulating the VEGF gene (Schu¨le et al., 1990). In this case, GR binding to DNA and GR-dependent gene transcription are not required. It is not known which factors regulate VEGF expression in the growth plate. IGF-I is an obvious candidate, however, since others and we have detected expression of this growth factor in the growth plate (Smink et al., 2002b; Reinecke et al., 1999). IGF-I production in cultured rat epiphyseal chondrocytes, especially when stimulated by GH, is inhibited by 100 nM dexamethasone (Jux et al., 1998). Similarly, dexamethasone blocks the IGF-I-stimulated rise in type 1 IGF receptor molecules (Jux et al., 1998). In addition to a direct effect on the AP-1 transactivator complex as described above, glucocorticoids could therefore also regulate
VEGF expression indirectly through interactions with the somatotropic axis. Another well-established inducer of VEGF expression is hypoxia (Shweiki et al., 1992), mediated by the transcriptional activator hypoxia-inducible factor 1 (HIF-1) (Semenza, 2001). Because the cartilage is not vascularized, it has been widely assumed that chondrocytes, especially in the hypertrophic zone, are hypoxic. Using a hypoxia-sensing drug and immunohistochemistry, Shapiro et al. (1997) found that hypertrophic cartilage, at least in the chicken, is actually not oxygendeficient. In contrast, Schipani et al. (2001), using a similar technique, recently reported that hypoxia does occur in the growth plates of developing mice. VEGF expression in the growth plate appears to be regulated through both HIF-1-dependent and -independent mechanisms (Schipani et al., 2001). The possible role of glucocorticoids in regulating these mechanisms needs to be addressed, especially since hypoxia-induced VEGF expression seems to be much less susceptible to dexamethasone inhibition than VEGF expression induced by serum and growth factors (Heiss et al., 1996; Machein et al., 1999). We also explored another possibility by which dexamethasone could regulate VEGF expression, namely by destabilizing its mRNA. We found no evidence for such a post-transcriptional regulation. When transcription was interrupted by actinomycin D, the halflife of the VEGF mRNA was unaffected by the presence of dexamethasone. Glucocorticoid-regulated mRNA turnover is thought to involve AUUUA sequences (AU response elements or AREs) in the 3?-untranslated region of mRNAs (Malter, 1989; Peppel et al., 1991), and these sequences are present in the VEGF transcript (Claffey et al., 1998). The regulation of the stability of VEGF mRNA is very complex, however, and involves sequences in the 3?-untranslated region as well as the 5?untranslated region and the coding region (Dibbens et al., 1999). This complexity is also illustrated by the different results, which have been reported with respect to the ability of IGF-I to stabilize VEGF mRNA. While IGF-I increased VEGF mRNA halflife in colon carcinoma cells (Warren et al., 1996) and endometrial adenocarcinoma cells (Bermont et al., 2000), it had no such effect in osteoblastic cells (Goad et al., 1996) and fibroblasts (Miele et al., 2000). VEGF is of utmost importance for the vascular invasion of the growth plate cartilage and the resulting processes of apoptosis and bone formation (Gerber et al., 1999). It is therefore highly likely that the negative effect of glucocorticoids on VEGF expression by epiphyseal chondrocytes contributes to the mechanism by which these steroid hormones cause growth retardation and/or osteoporosis.
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Acknowledgements We thank Ms. C.M.P.C.D. Peeters and Dr. F. Groenendaal for making the tibia of neonatal piglets available to us.
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