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General and Comparative Endocrinology 108, 80–86 (1997) Article No. GC976950

Cortisol Inhibits Glycosaminoglycan Synthesis in Cultured Rainbow Trout Cartilage Yasuaki Takagi*,† and Bjo¨rn Thrandur Bjo¨rnsson† *Otsuchi Marine Research Center, Ocean Research Institute, University of Tokyo, Akahama, Otsuchi, Iwate 028-11, Japan; and †Fish Endocrinology Laboratory, Department of Zoophysiology, Go¨teborg University, Medicinaregatan 18, S-413 90 Go¨teborg, Sweden Accepted June 6, 1997

In vitro actions of corticosteroids (cortisol, 11-deoxycortisol, cortisone, and corticosterone) as well as interaction between cortisol and triiodothyronine (T3) or recombinant human insulin-like growth factor-I (rhIGF-I) on cartilage glycosaminoglycan (GAG) synthesis in rainbow trout (Oncorhynchus mykiss) were examined. Uptake of [35S]sulfate by isolated branchial cartilage was measured as a marker for GAG synthesis. In vitro exposure of cartilage to cortisol at concentrations of 10, 100, and 1000 nM for 6 days dose-dependently inhibited sulfate uptake, while exposure to 0.1 and 1 nM cortisol had no effect. Corticosterone and 11-deoxycortisol at concentrations of 10 and 100 nM inhibited sulfate uptake slightly but not dose-dependently. Cortisone (1, 10, and 100 nM) had no effect. When cortisol (1, 10, and 100 nM) and T3 (0.075 and 0.75 nM) were simultaneously added to the culture, the T3-induced sulfate uptake was dose-dependently reduced by the presence of 10 and 100 nM cortisol. When cortisol (1, 10, and 100 nM) and rhIGF-I (0.1 and 1 nM) were added together, the sulfate uptake induced by 0.1 nM rhIGF-I was only slightly inhibited by 100 nM cortisol, but 1 nM rhIGF-I completely masked the inhibitory effect of cortisol. These data suggest that GAG synthesis in the rainbow trout cartilage is controlled by multiple interactions among stimulative hormones, such as T3 and IGF-I, and inhibitory hormones, such as cortisol. r 1997 Academic Press Establishment of an organ-culture technique for teleost cartilage (Ash, 1977; Duan and Inui, 1990; Gray

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and Kelley, 1991; McCormick et al., 1992) has enabled the in vitro assessment of cartilage matrix formation by measuring the uptake of [35S]sulfate as a marker for glycosaminoglycan (GAG) synthesis. Using this technique, in vitro stimulation of matrix formation by insulin-like growth factor-I (IGF-I) (Duan and Hirano, 1990; Gray and Kelly, 1991; McCormick et al., 1992; Cheng and Chen, 1995; Takagi and Bjo¨rnsson, 1996), insulin (Duan et al., 1992; McCormick et al., 1992; Cheng and Chen, 1995), and triiodothyronine (T3) (Takagi and Bjo¨rnsson, 1996) has been demonstrated. Furthermore, Cheng and Chen (1995) reported the synergistic effects of growth hormone (GH) with IGF-I on cartilage matrix synthesis, whereas Takagi and Bjo¨rnsson (1996) showed the additive stimulation of matrix synthesis by T3 and IGF-I, both indicating a complex hormonal control of cartilage growth in teleosts. In addition to IGF-I, insulin and T3, glucocorticoids are known to be involved in the cartilage growth regulation in mammals and birds. Excess glucocorticoids, occurring naturally as in Cushing’s syndrome or as a result of therapy, inhibit cartilage growth and hence cause somatic growth retardation in children (Preece, 1976; Peck et al., 1984). Chondrocytes of chick (Lee et al., 1978), rabbit (Blondelon et al., 1980), mouse (Maor and Silbermann, 1982), and calf (Kan et al., 1983) are reported to have glucocorticoid receptors, suggesting that glucocorticoids are the physiological modulator of chondrocyte metabolism. In vitro actions of 0016-6480/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

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Cortisol and Trout Cartilage Growth

glucocorticoids on cartilage matrix synthesis have been extensively studied in experimental animals and appear to differ depending on the glucocorticoid concentrations and/or the presence of growth factors. Pharmacological concentrations of glucocorticoids (100 µM or higher), without somatomedin stimulus, inhibit cartilage matrix synthesis in organ-cultured embryonic chick pelvic cartilage (Kilgore et al., 1979) and calf costochondral growth plate cartilage (Hill, 1981). More physiological concentrations of glucocorticoids, without any somatomedin stimulus, are inhibitory in rat costal cartilage (Tessler and Salmon, 1975), but have no effect in calf costochondral growth plate cartilage (Hill, 1981). On the other hand, glucocorticoids under the stimulus of plasma or growth factors such as IGF-I or fibroblast growth factor have been reported to increase matrix synthesis in chick femur (Calcagno et al., 1970), rabbit costal chondrocytes (Kato and Gaspodarowicz, 1985; Takano et al., 1985), and rabbit cranofacial chondrocytes (Takigawa et al., 1988). In teleosts, cortisol has been reported to have an inhibitory action on somatic growth in vivo (Davis et al., 1985; Barton et al., 1987), but little information is available on its effects in vitro. Thus, the mechanisms underlying the growth-inhibiting actions of cortisol in teleosts is still unclear. The aim of this study was to investigate the in vitro actions of corticosteroids on cartilage matrix synthesis in teleost fish. Uptake of [35S]sulfate by isolated branchial cartilage of rainbow trout was measured as a marker for GAG synthesis. Effects of corticosteroids (cortisol, cortisone, corticosterone, and 11-deoxycortisol) as well as interaction between cortisol and T3 or recombinant human IGF-I (rhIGF-I) on sulfate uptake were examined using a chemically defined culture medium.

trout pellets (EWOS or Nosan). The fish were fasted for 7 days before the experiments, which were conducted during May–July.

Organ Culture The organ culture of epibranchial and ceratobranchial cartilage was conducted largely according to Takagi and Bjo¨rnsson (1996). Briefly, the fish were anesthetized in 0.05% 2-phenoxyethanol or in 0.01% tricaine methanesulfonate buffered to pH 7.0 with NaHCO3 and killed by decapitation. Epibranchial and ceratobranchial cartilage from first three pairs of gill arches were separated and washed in ice-cold culture medium (BGJb medium, Fitton Jackson modification; Sigma Chemical Co. Ltd.) containing 100 U penicillin-G potassium salt/ml and 0.1 mg streptomycin sulfate/ml (both from Sigma). The cartilage was transferred into 24-well microplates containing 0.5 ml medium per well and incubated at 15–18° under an atmosphere of 5% CO2–95% air for 6 days. The medium was changed after 3 days and [35S]Na2SO4 (37 kBq/ml; Amersham Int. plc) was included during the last 3 days of the culture. After incubation, the cartilage was rinsed in a 0.9% NaCl solution, washed in a 0.5 M Na2SO4 solution for at least 3 h, and rerinsed in a 0.9% NaCl solution. Wet weight was measured, and the tissue was dissolved in 200 µl tissue solubilizer (Soluen, Packard). Scintillation fluid (5 ml; Hionic Fluor, Packard, or Optiphase HiSafe3, Wallac) was added and the radioactivity was measured by a liquid scintillation counter (Packard Tri-Carb 1500 or Wallac 1409). After 6 days of culture, cartilage was fully viable, as no histological signs of necrosis or cell deformity were observed by light microscopy (Takagi and Bjo¨rnsson, 1996).

Hormones

MATERIALS AND METHODS Fish Yearling rainbow trout (Oncorhynchus mykiss) weighing 70–130 g were obtained from a commercial dealer in Kamaishi, Japan or at Antens Laxodling AB, Sweden. Fish were maintained in running fresh water at about 12° and fed daily ad libitum with commercial

Cortisol, cortisone, corticosterone, and 11-deoxycortisol (all from Sigma) were dissolved first in ethanol at a concentration of 10 mM, diluted 100 times by the culture medium, and stored at 220°. T3 (Sigma) was dissolved in 0.9% NaCl solution containing 0.001 M NaOH at a concentration of 75 mM and stored at 220°. rhIGF-I (Genzyme) was dissolved in 0.01 M CH3COOH solution containing 0.5% BSA (Sigma) at a concentration of 1 mM and kept at 280°.

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

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Hormones to be tested were included from the beginning of the culture. Stock solutions of the hormones were thawed and diluted by the solvents to the appropriate concentrations and 5 µl of the diluted stock solutions was added to 0.5 ml culture medium to obtain final culture concentrations. The solvent alone was added to the control cultures. A maximum of 12 pieces of epibranchial and ceratobranchial cartilage were removed from the first three pairs of gill arches from each fish. When different hormones and doses were tested, care was taken to include a cartilage piece from each individual fish in all groups, in order to minimize the influence of individual variation in sulfate uptake.

Statistical Analyses To determine if sulfate uptake differed significantly among treatments, a Friedman test was used. To determine the difference between control and one of the hormone-treated groups, Wilcoxon signed rank test was applied. Significance was set at P , 0.05. The results are presented as means 6 SEM.

RESULTS

Takagi and Bjo¨rnsson

FIG. 2. Dose–response of in vitro exposure to cortisone, corticosterone, and 11-deoxycortisol on the [35S]sulfate uptake by rainbow trout cartilage. The values are means 6 SEM (n 5 6). *P , 0.05 compared with the culture without hormones (control).

100, and 1000 nM, cortisol dose-dependently inhibited sulfate uptake. The sulfate uptake in cartilage treated with 1000 nM cortisol was about 30% of that in control cartilage. Corticosterone and 11-deoxycortisol at 10 and 100 nM slightly reduced sulfate uptake, but their effects were not dose-dependent (Fig. 2). Cortisone at 1, 10, and 100 nM showed no effect (Fig. 2).

Effects of Corticosteroids The effects of cortisol on sulfate uptake are shown in Fig. 1. Cortisol at 0.1 and 1 nM had no effect, but at 10,

FIG. 1. Dose–response of cortisol exposure in vitro on the [35S]sulfate uptake by rainbow trout cartilage. The values are means 6 SEM (n 5 6). *P , 0.05 compared with the culture without cortisol.


Interaction between Cortisol and T3 or rhIGF-I Both T3 (0.075 and 0.75 nM) and rhIGF-I (0.1 and 1 nM) stimulated sulfate uptake dose-dependently (Figs. 3 and 4). However, the stimulative potency of IGF-I was much greater than that of T3. IGF-I at 1.0 nM increased the uptake more than 10-fold over the controls, whereas 0.75 nM T3 increased the uptake only 2- to 3-fold. When cortisol (1, 10, and 100 nM) and T3 (0.075 and 0.75 nM) were simultaneously added to the culture, the T3-induced sulfate uptake was significantly reduced by the presence of 10 and 100 nM cortisol (Fig. 3). Cortisol at 100 nM reduced the sulfate uptake by 50% when T3 was not present, whereas under the stimulus of 0.75 nM T3, cortisol at 100 nM reduced the uptake by 40%. When cortisol (1, 10, and 100 nM) and rhIGF-I (0.1 and 1 nM) were added together, the sulfate uptake induced by 0.1 nM rhIGF-I was only slightly inhibited by 100 nM cortisol, but 1


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nM rhIGF-I completely masked the inhibitory effect of cortisol (Fig. 4).

DISCUSSION The present study provides the first evidence that cortisol, the major corticosteroid circulating in teleost fish plasma, significantly inhibits the glycosaminoglycan (GAG) synthesis and thus growth of teleost cartilage. However, the inhibitory action of cortisol is masked by 1 nM levels of IGF-I. When trout cartilage was cultured in the medium supplemented with cortisol alone, cortisol at a concentration of 10 nM or higher clearly inhibited GAG synthesis. The circulating levels of cortisol in unstressed salmonids are reported to be in the range of 4 to 40 ng/ml (11–110 nM ), reaching peak levels during salmonid parr–smolt transformation (Specker and Schreck, 1982; Barton et al., 1985; Young et al., 1989). The reported dissociation constant (Kd ) values for cortisol receptors in salmonid fish gill (Chakraborti et al., 1987) and liver (Chakraborti and Weisbart, 1987; Pottinger, 1990) are at the order of 1029 to 1028 M. Although no reports on the specific binding sites for glucocorticoids and their characterization in teleost

FIG. 3. In vitro effects of T3, cortisol, and their combination on the [35S]sulfate uptake by rainbow trout cartilage. The values are means 6 SEM (n 5 6). *P , 0.05 compared with the cultures without cortisol (open bars).

FIG. 4. In vitro effects of rhIGF-I, cortisol, and their combination on the [35S]sulfate uptake by rainbow trout cartilage. The values are means 6 SEM. Numbers of cartilage used were 11, 12, and 6 when 1, 10, and 100 nM cortisol was tested, respectively. *P , 0.05 compared with the cultures without cortisol (open bars).

chondrocytes are available at present, these data suggest that the lowest effective concentration of cortisol observed in this study is within the physiological levels and thus the present data are probably of physiological relevance. The present results are in good agreement with data by Tessler and Salmon (1975), who reported that the lowest effective doses of dexamethasone and cortisol for inhibiting GAG synthesis in cultured rat costal cartilage were 1028 and 1027 M, respectively. In contrast, in calf costochondral growth plate, cortisol at 1023 M was needed to significantly inhibit GAG synthesis, with lower concentrations having no effect (Hill, 1981). Thus, effective concentrations of cortisol for inhibition of GAG synthesis may differ among species, cartilage types, and/or culture conditions. In mammals, it is well known that chondrocyte metabolism is controlled in an autocrine/paracrine manner by locally produced growth factors, such as IGF-I and basic fibroblast growth factor (FrogerGaillard et al., 1989; Hill et al., 1992). Thus, in vitro inhibition of GAG synthesis by cortisol does not imply that this is a direct effect, as it is possible that cortisol indirectly inhibits GAG synthesis through its effects on the production and action of such local factors. In fact, direct effects of cortisol on GAG synthesis, without any


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confounding effects of such locally produced growth factors, have yet to be demonstrated clearly. As no studies on local production of growth factor(s) in teleost chondrocytes are presently available, careful examination of the mechanisms of inhibitory effects of cortisol on GAG synthesis is needed in future. Although cortisol is the major corticosteroid circulating in teleost plasma, generally low levels of cortisone, corticosterone, and 11-deoxycortisol are also circulating (cf. Gorbman et al., 1983). These corticosteroids, however, appear to have only minor inhibitory effects on cartilage growth in the rainbow trout, since inhibitory effects of corticosterone and 11-deoxycortisol on GAG synthesis were weak and dose-independent, while cortisone showed no effect. Similarly, cortisol was the most effective in increasing hypoosmoregulatory ability in tilapia larvae in vivo, while 11-deoxycortisol was only slightly effective, and corticosterone had no effect (Hwang and Wu, 1993). Receptor-binding data support these findings of the present study, as the binding affinities of 11-deoxycortisol, corticosterone, and/or cortisone for glucocorticoid receptors are much lower than that of cortisol in eel intestinal cells (DiBattista et al., 1983), eel gill (Sandor et al., 1984), brook trout liver (Chakraborti and Weisbart, 1987), rainbow trout liver (Pottinger, 1990), and in chinook salmon brain (Knoebl et al., 1996). In contrast, 11-deoxycortisol was equipotent with cortisol in increasing Na1,K1ATPase activity in coho salmon gill in vitro, while cortisone was less potent (McCormick and Bern, 1989). Supporting the high potency of 11-deoxycortisol in the gills of salmonids, Chakraborti et al. (1987) found higher affinity of 11-deoxycortisol than cortisol to glucocorticoid receptors in brook trout gills. Furthermore, in the mature plaice, substantial levels of 11deoxycortisol produced by the ovary are found in plasma (Canario and Scott, 1990; Scott and Canario, 1990). These data suggest that 11-deoxycortisol may also have important corticosteroid functions depending on teleost species and tissues. In mammals and birds, glucocorticoids are reported to interact with growth factors such as IGF-I and stimulate cartilage matrix synthesis. In organ-cultured calf growth plate cartilage, cortisol at a concentration of 1026 M appeared to increase the proteoglycan synthesis in the presence of a somatomedin stimulus of normal rat plasma, although similar concentrations of



cortisol are ineffective and higher concentrations are inhibitory without somatomedin stimulus (Hill, 1981). Kato and Gaspodarowicz (1985) reported that cortisol and dexamethasone dose-dependently increased the proteoglycan synthesis in rabbit chondrocyte cultures at concentrations of 10210–1025 M only when culture medium was supplemented with transferrin, high density lipoprotein, fibroblast growth factor, and insulin. These data suggest that cortisol stimulates cartilage matrix synthesis under the stimulus of growth factors. Furthermore, when rabbit chondrocytes were treated with 5 3 10211–1027 M cortisol or dexamethasone before the addition of IGF-I, glucocorticoids synergistically accelerated proteoglycan synthesis mediated by IGF-I (Itagane et al., 1991), suggesting the priming effects of glucocorticoids on the action of IGF-I. In the present study, however, cortisol did not stimulate GAG synthesis under the presence of T3 or IGF-I, both of which are the significant stimulators of GAG synthesis in trout cartilage. With the presence of T3, cortisol inhibited the T3-induced GAG synthesis at concentrations of 10 nM or higher, the concentrations at which cortisol alone inhibited the GAG synthesis. On the other hand, cortisol did not show any inhibitory effects when physiological concentration (1 nM) of IGF-I was present in the culture medium. These data indicate that, in the rainbow trout, physiological function of the cortisol on cartilage metabolism is to inhibit GAG synthesis. Since cortisol is ever present in fish plasma, and frequently reaching high levels, a masking effect of IGF-I over the inhibitory action of cortisol on the cartilage GAG synthesis may be of importance in preventing undesirable growth retardation. In coho salmon, circulating plasma levels of IGF-I, measured by homologous radioimmunoassay, were 50–120 ng/ml depending on fish developmental stages (Moriyama et al., 1994). As for mammals, IGF-binding proteins (IGFBPs) exist in teleost plasma (cf. Siharath and Bern, 1994) and thus only a small part of circulating IGF-I exists as free IGF-I (Moriyama et al., 1994), which is thought to be an active form. In mammals, around 1% of circulating IGF-I is reported to be free (Takada et al., 1994). If the situation is similar in fish, the free IGF-I levels in coho salmon would be 0.5–1.2 ng/ml (0.07– 0.17 nM). In the present study, in the presence of 0.1 nM IGF-I, only the highest dose (100 nM) of cortisol was inhibitory. These data suggest that, in vivo, the


effects of cortisol on cartilage growth may only be of importance when plasma cortisol levels are high, such as when fish are stressed, and/or IGF-I levels are low. In mammals, it appears that the growth inhibiting action of glucocorticoids may be mediated, at least in part, by IGFBPs, which modulate IGF-I bioactivity. Glucocorticoids are reported to affect circulating levels and/or hepatic production of IGFBPs in vivo (Luo et al., 1990) and in vitro (Unterman et al., 1991; Villafuerte et al., 1995). Although this has yet to be investigated, it is thus possible that growth inhibiting actions of cortisol in teleosts in vivo may also mediated by IGFBPs. In summary, the present study clearly demonstrates the inhibitory action of cortisol on cartilage GAG synthesis in the rainbow trout. It is suggested that GAG synthesis is controlled by multiple interactions, both among stimulative hormones, such as T3 and IGF-I, and also inhibitory hormones, such as cortisol. IGF-I may mask the inhibitory action of cortisol on GAG synthesis during normal growth.

ACKNOWLEDGMENTS This study was supported in part by grants from the Swedish Council for Forestry and Agricultural Research and the Swedish Natural Science Research Council to B.Th.B. A Swedish Institute Fellowship to Y.T. is also acknowledged.

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