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Effects of Glucocorticoids on Cartilage Growth and Response to IGF-I in the Tilapia (Oreochromis mossambicus)
General and Comparative Endocrinology 121, 289 –294 (2001) doi:10.1006/gcen.2000.7599, available online at http://www.idealibrary.com on
Effects of Glucocorticoids on Cartilage Growth and Response to IGF-I in the Tilapia (Oreochromis mossambicus)
Jason P. Datuin, Kevin P. Ng, Tyrone B. Hayes, and Howard A. Bern Department of Integrative Biology, Group in Endocrinology and Cancer Research Laboratory, University of California, Berkeley, California 94720-3140 Accepted November 27, 2000
INTRODUCTION
To study the effects of glucocorticoids and IGF-I on the modulation of growth in the tilapia Oreochromis mossambicus, we employed an epiceratobranchial cartilage radioisotope incorporation assay, wherein radiolabeled sulfate and thymidine uptakes are measured in vitro to indicate proteoglycan synthesis and cell proliferation, respectively. Cartilage explants were cultured with cortisol or dexamethasone with or without recombinant bovine insulin-like growth factor-I. Cortisol directly inhibited sulfate uptake at 100 and 1000 ng/mL concentrations in a concentration-dependent manner but inhibited thymidine uptake significantly only at the 1000 ng/mL concentration. Dexamethasone inhibited sulfate and thymidine uptake at concentrations similar to the effective concentrations of cortisol. Cortisol did not inhibit IGF-I stimulation of sulfate uptake at any of the concentrations tested. Furthermore, cortisol did not inhibit thymidine uptake when IGF-I was present in the medium. Cortisol appears to act directly on cartilage and not by interacting with the IGF-I system. However, the physiologically significant role of cortisol is mainly an inhibitory one on cartilage metabolism. The data generally indicate an inhibitory role for glucocorticoids on cartilage growth but an inability to counter the stimulation of sulfate uptake by IGF-I.
Cartilage growth is affected by several hormones, most importantly growth hormone (GH) and insulinlike growth factor-I (IGF-I). An organ culture technique has been established for teleosts to estimate cartilage proteoglycan synthesis and DNA synthesis by measuring [ 35S]sulfate and [ 3H]thymidine incorporation, respectively (Duan and Inui, 1990; Gray and Kelley, 1991; McCormick et al., 1992). In vivo administration of GH increases cartilage matrix proteoglycan synthesis in the Japanese eel, Anguilla japonica (Duan and Inui, 1990), and in the goby, Gillichthys mirabilis (Gray and Kelley, 1991), whereas GH treatments did not affect growth in vitro. Matrix proteoglycan synthesis increased when cartilages were cultured with GH and liver slices (Komourdjian and Idler, 1970). These findings suggested that cartilage synthesis was mediated by a growth factor produced by the liver. This hypothesis was supported by later studies that found increases in matrix proteoglycan synthesis in the cartilage after IGF-I treatment (Duan and Inui, 1990; Gray and Kelley, 1991; McCormick et al., 1992; Marchant and Moroz, 1993; Tsai et al., 1994; Cheng and Chen, 1995; Takagi and Bjørnsson, 1996, 1997; Gelsleichter and Musick, 1999).
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Triiodothyronine (T 3) or thyroxine (T 4) increased sulfate incorporation in cartilages in vitro (Barrington and Rawdon, 1967; Takagi and Bjørnsson, 1996). In addition, T 3 increased GH mRNA transcription and GH secretion (Moav and McKeown, 1992; Luo and McKeown, 1991). Other investigators reported that thyroid hormones did not affect cartilage matrix synthesis (Komourdjian and Idler, 1970; McCormick et al., 1992; Gelsleichter and Musick, 1999). Insulin stimulated cartilage matrix synthesis (Duan et al., 1992; McCormick et al., 1992; Cheng and Chen, 1995). GH and IGF-I synergistically stimulated growth of cartilage cultures in the carp, Cyprinus carpio (Cheng and Chen, 1995). Triiodothyronine and IGF-I simultaneously added to the culture additively stimulated cartilage growth in rainbow trout, Oncorhynchus mykiss (Takagi and Bjørnsson, 1996). Another important hormonal regulator of growth is the glucocorticoid cortisol (Wendelaar Bonga, 1997). An inhibition of growth after in vivo administration of cortisol occurs in teleosts (Davis et al., 1985; Barton et al., 1987). In vitro addition of cortisol at concentrations of 10 nM or more inhibits proteoglycan synthesis in O. mykiss cartilage (Takagi and Bjørnsson, 1997). Corticosterone inhibits proteoglycan synthesis in an elasmobranch, the clearnose skate, Raja eleganteria (Gelsleichter and Musick, 1999). Not much is known about the effects of glucocorticoids together with growth factors on cartilage synthesis in teleosts. Cortisol (10 nM or more) inhibits proteoglycan synthesis stimulated by T 3. However, cortisol at 1, 10, or 100 nM does not inhibit proteoglycan synthesis stimulated by IGF-I at physiological concentrations (1 nM), but when IGF-I levels are reduced (0.1 nM) and cortisol levels are high (100 nM), inhibition does occur (Takagi and Bjørnsson, 1997). The effect of cortisol together with growth factors needs to be studied in other teleost species to understand more fully the regulation of growth by cortisol. Other parameters may be physiologically important in regulating cartilage growth. Insulin-like growth factor-binding proteins (IGFBPs) play an important role in regulating the bioactivity of IGF-I and thus growth by affecting the ratio of free to bound IGF-I, clearance rate, and targeting of IGF-I to specific cells. IGFBPs are present in teleosts, including tilapia (Kelley et al., 1992; Park et al., 2000), as in mammals (Siharath and Bern, 1993), but it is not known whether they are affected by
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Datuin et al.
glucocorticoids. In this study, the direct effect of glucocorticoids on cartilage growth in the tilapia, Oreochromis mossambicus, is examined, along with their interaction with IGF-I.
MATERIALS AND METHODS Animals Juvenile tilapia, O. mossambicus, were provided by Pacific Aquafarms (Nilands, CA). All fish had been exposed to 17␣-methyltestosterone as fry. They were housed in 560-liter flow-through freshwater tanks in a temperature-controlled (28°) room under a simulated natural photoperiod (12L:12D). Fish were fed Rangen Floating Catfish Chow (Buhl, ID) twice daily ad libitum. Fish were fasted 1 week before study.
Organ Culture Cartilages were dissected and cultured as described by Duan and Inui (1990) for the Japanese eel, A. japonica, with modifications to optimize conditions for tilapia. Cartilage explants from the first two pairs of gill arches from each fish were placed in a defined culture medium for preincubation (Lee, 1996). A group of cartilages was freeze-killed at ⫺80° and later used for determination of nonspecific isotope uptake. Cartilages were preincubated for 2 h in a defined culture medium and then transferred to 24-well microplates, each well containing 0.5 mL culture medium along with 200 units/ml penicillin and streptomycin and 6 Ci/mL each of [ 35S]sulfate (NEN Life Science Products, Boston, MA) and [ 3H]thymidine (Amersham, Piscataway, NJ). The following hormones were added: cortisol (F; Sigma), a synthetic glucocorticoid, dexamethasone (DEX; Sigma), recombinant bovine insulin-like growth factor-1 (rbIGF-I; Monsanto, St. Louis, MO), or a combination of one of the glucocorticoids with IGF-I. Radioactivity was measured as disintegrations per minute (dpm) by a scintillation counter (Beckman LS6500) programmed to measure simultaneously [ 35S]sulfate and [ 3H]thymidine. Specific uptake was determined by subtracting nonspecific uptake from total uptake. Nonspecific uptake values were less that 1% of the total values. The dpm
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of each sample was normalized to its own dry weight to determine dpm/g cartilage. For each test concentration, seven or eight fish were represented by seven or eight cartilages, one cartilage from each fish.
Hormones Cortisol was first dissolved in ethanol and then diluted with Ringer to appropriate concentrations (1, 10, 100, or 1000 ng/ml). Final culture medium contained less than 0.01% ethanol. DEX and F were prepared on the day of culture. DEX was diluted with tilapia Ringer to one of the following concentrations: 0.1, 1, 10, 100, or 1000 ng/ml. Recombinant bovine IGF-I was added alone at 100 ng/mL or in combination with F or DEX. Ethanol was added to all control cultures in appropriate amounts.
Statistical Analyses Statistical comparisons were analyzed by the Kruskal–Wallis test followed by the Mann–Whitney U test. Significance was accepted as P ⱕ 0.05. Statistics were performed using the Statview 4.5 software package.
RESULTS Addition of Cortisol Alone and in Combination with IGF-I Cortisol inhibited sulfate uptake at 100 and 1000 ng/mL (P ⬍ 0.05). Cortisol inhibited thymidine uptake only at 1000 ng/mL (P ⬍ 0.05). The concentration of 100 ng/mL of IGF-I significantly stimulated sulfate uptake over controls (P ⬍ 0.05). IGF-I had no effect on thymidine uptake. Cortisol did not inhibit the IGF-I stimulation of sulfate or thymidine uptake at any concentration tested (Fig. 1).
FIG. 1. Effects of cortisol (F) at 0, 1, 10, 100, and 1000 ng/mL and F at 0, 1, 10, and 100 ng/mL combined with IGF-I at 100 ng/mL on sulfate and thymidine uptake by cartilage in vitro. Sulfate uptake was inhibited by F at 100 and 1000 ng/mL (P ⬍ 0.05; significance indicated by *). Thymidine uptake was inhibited by F at 1000 ng/mL (P ⬍ 0.05; significance indicated by *). IGF-I-exposed cartilage showed higher sulfate uptake than controls (P ⬍ 0.05; significance indicated by †); thymidine uptake was not affected. Values are means ⫾SE (n ⫽ 8). This figure is representative of three replicate experiments yielding similar results.
Again the standard concentration of 100 ng/ml IGF-I significantly stimulated sulfate uptake (P ⬍ 0.05) and had no effect on thymidine uptake compared to controls. As with cortisol, DEX did not inhibit the stimulation of sulfate uptake by IGF-I at any concentration tested (Fig. 2).
Addition of DEX Alone and in Combination with IGF-I
DISCUSSION
DEX inhibited sulfate uptake at all concentrations (P ⬍ 0.05). DEX inhibited thymidine uptake at 100 and 1000 ng/mL (P ⬍ 0.05).
Takagi and Bjørnsson (1997) provided the first evidence that cortisol significantly inhibited proteoglycan synthesis and consequently growth of cartilage of a
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FIG. 2. Effects of dexamethasone (DEX) at 0, 0.1, 1, 10, 100, and 1000 ng/mL and DEX at 0, 0.1, 1, 10, 100, and 1000 ng/mL combined with IGF-I at 100 ng/mL on sulfate and thymidine uptake by cartilage in vitro. Sulfate uptake was inhibited by DEX at 0.1, 1, 10, 100, and 1000 ng/mL (P ⬍ 0.05; significance indicated by *). Thymidine uptake was inhibited by DEX at 100 and 1000 ng/mL (P ⬍ 0.05; significance indicated by *). IGF-I-exposed cartilage showed higher sulfate uptake than controls (P ⬍ 0.05; significance indicated by †); thymidine uptake was not affected. Values are means ⫾SE (n ⫽ 8). This figure is representative of three replicate experiments yielding similar results.
teleost fish, the rainbow trout, O. mykiss. The present study differed from their study in the species used, the duration of culture, and the consideration of another parameter: thymidine uptake. Cortisol significantly inhibited sulfate uptake at both 100 and 1000 ng/mL and thymidine uptake at 1000 ng/mL. Foo and Lam (1993) showed that in unstressed tilapia, O. mossambicus, serum cortisol concentration was less than 10 ng/mL. However, in tilapia stressed by netting, serum cortisol concentration rose to around 65 ng/mL, and netting followed by confinement caused serum cortisol concentration to increase to around 120 ng/mL. These data show that
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Datuin et al.
the experimental concentrations of cortisol used by us were within the physiological range to be expected for O. mossambicus; the inhibition is dose-dependent. Our data agree with those of Takagi and Bjørnsson (1997) for O. mykiss cartilage. The concentration of 100 ng/mL of rbIGF-I stimulated sulfate uptake, as expected, but not thymidine uptake. Juvenile O. mossambicus have a high growth rate, and cartilage cells may be maximally mitotically active. As thymidine uptake is a measure of cell division, added IGF-I may not be able to stimulate cartilage further. Takagi and Bjørnsson (1996), using a similar assay system, reported that IGF-I had no effect on thymidine uptake by O. mykiss cartilage. The authors suggested that a nutrient-rich culture medium may induce chondrocyte proliferation and thus thymidine uptake, masking any stimulatory effect of additional hormones to the culture. Cortisol did not inhibit IGF-I stimulation of sulfate uptake at any of the concentrations tested. Furthermore, cortisol did not inhibit thymidine uptake with IGF-I present in the medium. Cortisol appears to act directly on cartilage and not by interacting with the IGF-I system. The physiologically significant role of cortisol would mainly seem to be inhibitory to cartilage metabolism and growth. Cortisol may not act directly on cartilage in vivo. Mammalian data suggest that autocrine/paracrine factors are the most important factors in control of cartilage growth (Froger-Gaillard et al., 1989). Cortisol could act indirectly on cartilage growth by affecting the production and action of local IGF-I and/or its binding proteins. Nishioka et al. (1985) found that incubating O. mossambicus pituitary with cortisol resulted in increased GH secretion, but Takagi and Bjørnsson (1997) and the current study found that cortisol in vitro inhibited growth of cartilage. The effect of cortisol on liver production of IGF-I and IGFBPS is unknown, however. Whereas cortisol may inhibit cartilage growth, IGF-I (100 ng/mL) was able to mask any effect of cortisol. This result implies that in the presence of high levels of circulating IGF-I, cortisol may have no effect on cartilage growth. The impact of cortisol on the liver has not been adequately studied. In addition to direct effects on liver production and secretion of IGF-I and the IGFBPs, cortisol may down-regulate liver GH recep-
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tors and thus decrease IGF-I production by the liver. This view is supported by mammalian data. Beauloye et al. (1999) has shown that high concentrations of DEX (1 M) inhibit GH induction of IGF-I mRNA, and DEX directly inhibits the expression of GHR mRNA. These effects may be important for euryhaline teleosts during periods of seawater adaptation, as high cortisol levels would result in higher GH levels, both hormones being favorable to seawater adaptation. Moreover, as cortisol would have an overall growth-inhibiting effect, energy might thus be available for seawater adaptation. Dexamethasone is used ubiquitously in mammalian systems as a synthetic glucocorticoid binding to the glucocorticoid receptor with high affinity. Dexamethasone is inhibitory to sulfate and thymidine uptake at concentrations similar to the effective concentrations of cortisol used in the present study. Takagi and Bjørnsson (1997) cultured cartilage for 6 days before recording a cortisol effect. Two differences are apparent between their culture system and that used in the current study that may account for the difference in time required for response. First, the O. mykiss used by Takagi and Bjørnsson were much larger and presumably older than the subadult O. mossambicus used in the current system. As the growth rates for both species decrease appreciably with age, the O. mossambicus were likely growing faster, leading to a faster response time in our system. Second, the preferred temperature of the two species differs greatly, represented by different incubation temperatures. We incubated cultures at 28°, whereas Takagi and Bjørnsson incubated at 15–18°. This temperature difference would lead to different metabolic rates and slower responsiveness by O. mykiss tissues. However, in both studies, cortisol essentially proved to be inhibitory to fish cartilage growth.
ACKNOWLEDGMENTS We acknowledge Dr. Richard S. Nishioka for his advice during this study. We thank Sascha Emami, Isabel Hsu, Kevin Ko, Karen Lee, Richard Park, and Nguyen Tan for their help and technical assistance. We thank Colin Bornia of Pacific Aquafarms for generously providing animals and advice. Support has been generously provided by NOAA, National Sea Grant College Program, U.S.
Department of Commerce, under Grant No. NA 89AA-D-SG 138R, Project R/F-145 to H.A.B., a traineeship from 1997–1999 under Project No. R/A-94 to J.P.D., and a traineeship from 1996 –1998 under Project No. R/A-94 to K.P.N. from the California Sea Grant College Program. The views expressed herein are those of the authors and do not necessarily reflect the views of the NOAA or any of its subagencies.
REFERENCES Barrington, E. J. W., and Rawdon, B. B. (1967). Influence of thyroxine upon the uptake of 35S-labelled sulfate into the branchial arch skeleton of the rainbow trout (Salmo gairdnerii). Gen. Comp. Endocrinol. 9, 116 –128. Barton, B. A., Schreck, C. B., and Barton, L. D. (1987). Effect of chronic cortisol administration and daily acute stress on growth, physiological conditions and stress responses in juvenile rainbow trout. Diseases Aquat. Org. 2, 173–185. Beauloye, V., Ketelslegers, J. M., Moreau, B., and Thissen, J. P. (1999). Dexamethasone inhibits both growth hormone (GH)-induction of insulin-like growth factor-I (IGF-I) mRNA and GH receptor (GHR) mRNA levels in rat primary cultured hepatocytes. Growth Horm. IGF Res. 9, 205–211. Cheng, C. M., and Chen, T. T. (1995). Synergism of GH and IGF-1 in stimulation of sulphate uptake by teleostean branchial cartilage in vitro. J. Endocrinol. 147, 67–73. Davis, K. B., Torrance, P., Parker, N. C., and Suttle, M. A. (1985). Growth, body, composition and hepatic tyrosine aminotransferase activity in cortisol-fed channel catfish, Ictalurus punctatus. J. Fish Biol. 29, 177–184. Duan, C. (1997). The insulin-like growth factor system and its biological actions in fish. Am. Zool. 37, 491–503. Duan, C. (1998). Nutritional and developmental regulation of insulin-like growth factors in fish. J. Nutr. 128, 306S–314S. Duan, C., and Inui, Y. (1990). Effects of recombinant eel growth hormone on the uptake of [ 35S]sulfate by ceratobranchial cartilages of the Japanese eel, Anguilla japonica. Gen. Comp. Endocrinol. 79, 320 –325. Duan, C., Noso, T., Moriyama, S., Kawauchi, H., and Hirano, T. (1992). Eel insulin: Isolation, characterization and stimulatory actions on [ 35S]sulfate and [ 3H]thymidine uptake in the branchial cartilage of the eel in vitro. J. Endocrinol. 133, 221–230. Foo, J. T. W., and Lam, T. J. (1993). Serum cortisol response to handling stress and the effect of cortisol implantation on testosterone level in the tilapia, Oreochromis mossambicus. Aquaculture 115, 145–158. Froger-Gaillard, B., Hossenlopp, P., Adolphe, M., and Binoux, M. (1989). Production of insulin-like growth factors and their binding proteins by rabbit articular chondrocytes: Relationships with cell multiplication. Endocrinology 124, 2365–2372. Gelsleichter, J., and Musick, J. (1999). Effects of insulin-like growth factor-I, corticosterone, and 3,3⬘,5-tri-iodo-l-thyronine on glycosaminoglycan synthesis in vertebral cartilage of the clearnose skate, Raja eleganteria. J. Exp. Zool. 284, 549 –556.
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294 Gray, E. S., and Kelley, K. M. (1991). Growth regulation in the gobiid teleost, Gillichthys mirabilis: Roles of growth hormone, hepatic growth hormone receptors and insulin-like growth factor-1. J. Endocrinol. 131, 57– 66. Itagane, Y., Yanada, H., Fujita, K., and Isshiki, G. (1991). Interactions between steroid hormones and insulin-like growth factor-I in rabbit chondrocytes. Endocrinology 128, 1419 –1424. Kelley, K. M., Siharath, K., and Bern, H. A. (1992). Identification of insulin-like growth factor-binding proteins in the circulation of four teleost fish species. J. Exp. Zool. 263, 220 –224. Komourdjian, M. P., and Idler, D. R. (1970). Hepatic mediation of hormonal and nutritional factors influencing the in vitro sulfur uptake by rainbow trout bone. Gen. Comp. Endocrinol. 36, 33–39. Lee, K. L. (1996). “The Effects of Salinity on the Growth of Tilapia, Oreochromis mossambicus.” M.A. thesis, Group in Endocrinology, University of California at Berkeley. Luo, D., and McKeown, B. A. (1991). The effect of thyroid hormone and glucocorticoids on carp growth hormone-releasing factor (GRF)-induced growth hormone (GH) release in rainbow trout, Oncorhynchus mykiss. Comp. Biochem. Physiol. A-Comp. Physiol. 99, 621– 626. Marchant, T. A., and Moroz, B. M. (1993). Hormonal influences on in vitro [ 35S]sulfate by gill arches from the goldfish (Carassius auratus L.). Fish Physiol. Biochem. 11, 393–399. McCormick, S. D., Tsai, P. I., Kelly, K. M., Nishioka, R. S., and Bern, H. A. (1992). Hormonal control of sulfate uptake by branchial cartilage of coho salmon: Role of IGF-1. J. Exp. Zool. 262, 166 –171. Moav, B., and McKeown, B. A. (1992). Thyroid hormone increases transcription of growth hormone mRNA in rainbow trout pituitary. Horm. Met. Res. 24, 10 –14.
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Nishioka, R. S., Park, R., Shepherd, B. S., Grau, E. G., and Bern, H. A. (1997). Effects of homologous pituitary hormone treatment on serum IGFBPs in hypophysectomized tilapia, Oreochromis mossambicus. XIII International Congress of Comparative Endocrinology, November 16 –21, 1997, P.56 (abstract P 1–38), Yokohama, Japan. Nishioka, R. S., Grau, E. G., and Bern, H. A. (1985). In vitro release of growth hormone from the pituitary gland of tilapia, Oreochromis mossambicus. Gen. Comp. Endocrinol. 60, 90 –94. Park, R., Shepherd, B. S., Nishioka, R. S., Grau, E. G., and Bern, H. A. (2000). Effects of homologous pituitary hormone treatment on serum insulin-like growth factor-binding proteins (IGFBPs) in hypophysectomized tilapia, Oreochromis mossambicus, with special reference to a novel 20 kDA IGFBP. Gen. Comp. Endocrinol. 117, 404 – 412. Siharath, K., and Bern, H. A. (1993). The physiology of insulin-like growth factor (IGF) and its binding proteins in teleost fishes. Proc. Zool. Soc. Calcutta Haldane Commem. Vol. pp. 113–124. Takagi, Y., and Bjørnsson, B. T. (1996). Regulation of cartilage glycosaminoglycan synthesis in the rainbow trout, Oncorhynchus mykiss, by 3,3⬘,5-tri-iodo-l-thyronine and IGF-1. J. Endocrinol. 149, 357–365. Takagi, Y., and Bjørnsson, B. T. (1997). Cortisol inhibits glycosaminoglycan synthesis in cultured rainbow trout cartilage. Gen. Comp. Endocrinol. 108, 80 – 86. Tsai, P. I., Madsen, S. S., McCormick, S. D., and Bern, H. A. (1994). Endocrine control of cartilage in coho salmon: GH influence in vivo on the response to IGF-1 in vitro. Zool. Sci. 11, 299 –303. Wendelaar Bonga, S. E. (1997). The stress response in fish. Physiol. Rev. 77, 591– 625.
Effects of Glucocorticoids on Cartilage Growth and Response to IGF-I in the Tilapia (Oreochromis mossambicus)
Jason P. Datuin, Kevin P. Ng, Tyrone B. Hayes, and Howard A. Bern Department of Integrative Biology, Group in Endocrinology and Cancer Research Laboratory, University of California, Berkeley, California 94720-3140 Accepted November 27, 2000
INTRODUCTION
To study the effects of glucocorticoids and IGF-I on the modulation of growth in the tilapia Oreochromis mossambicus, we employed an epiceratobranchial cartilage radioisotope incorporation assay, wherein radiolabeled sulfate and thymidine uptakes are measured in vitro to indicate proteoglycan synthesis and cell proliferation, respectively. Cartilage explants were cultured with cortisol or dexamethasone with or without recombinant bovine insulin-like growth factor-I. Cortisol directly inhibited sulfate uptake at 100 and 1000 ng/mL concentrations in a concentration-dependent manner but inhibited thymidine uptake significantly only at the 1000 ng/mL concentration. Dexamethasone inhibited sulfate and thymidine uptake at concentrations similar to the effective concentrations of cortisol. Cortisol did not inhibit IGF-I stimulation of sulfate uptake at any of the concentrations tested. Furthermore, cortisol did not inhibit thymidine uptake when IGF-I was present in the medium. Cortisol appears to act directly on cartilage and not by interacting with the IGF-I system. However, the physiologically significant role of cortisol is mainly an inhibitory one on cartilage metabolism. The data generally indicate an inhibitory role for glucocorticoids on cartilage growth but an inability to counter the stimulation of sulfate uptake by IGF-I.
Cartilage growth is affected by several hormones, most importantly growth hormone (GH) and insulinlike growth factor-I (IGF-I). An organ culture technique has been established for teleosts to estimate cartilage proteoglycan synthesis and DNA synthesis by measuring [ 35S]sulfate and [ 3H]thymidine incorporation, respectively (Duan and Inui, 1990; Gray and Kelley, 1991; McCormick et al., 1992). In vivo administration of GH increases cartilage matrix proteoglycan synthesis in the Japanese eel, Anguilla japonica (Duan and Inui, 1990), and in the goby, Gillichthys mirabilis (Gray and Kelley, 1991), whereas GH treatments did not affect growth in vitro. Matrix proteoglycan synthesis increased when cartilages were cultured with GH and liver slices (Komourdjian and Idler, 1970). These findings suggested that cartilage synthesis was mediated by a growth factor produced by the liver. This hypothesis was supported by later studies that found increases in matrix proteoglycan synthesis in the cartilage after IGF-I treatment (Duan and Inui, 1990; Gray and Kelley, 1991; McCormick et al., 1992; Marchant and Moroz, 1993; Tsai et al., 1994; Cheng and Chen, 1995; Takagi and Bjørnsson, 1996, 1997; Gelsleichter and Musick, 1999).
© 2001 Academic Press
0016-6480/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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Triiodothyronine (T 3) or thyroxine (T 4) increased sulfate incorporation in cartilages in vitro (Barrington and Rawdon, 1967; Takagi and Bjørnsson, 1996). In addition, T 3 increased GH mRNA transcription and GH secretion (Moav and McKeown, 1992; Luo and McKeown, 1991). Other investigators reported that thyroid hormones did not affect cartilage matrix synthesis (Komourdjian and Idler, 1970; McCormick et al., 1992; Gelsleichter and Musick, 1999). Insulin stimulated cartilage matrix synthesis (Duan et al., 1992; McCormick et al., 1992; Cheng and Chen, 1995). GH and IGF-I synergistically stimulated growth of cartilage cultures in the carp, Cyprinus carpio (Cheng and Chen, 1995). Triiodothyronine and IGF-I simultaneously added to the culture additively stimulated cartilage growth in rainbow trout, Oncorhynchus mykiss (Takagi and Bjørnsson, 1996). Another important hormonal regulator of growth is the glucocorticoid cortisol (Wendelaar Bonga, 1997). An inhibition of growth after in vivo administration of cortisol occurs in teleosts (Davis et al., 1985; Barton et al., 1987). In vitro addition of cortisol at concentrations of 10 nM or more inhibits proteoglycan synthesis in O. mykiss cartilage (Takagi and Bjørnsson, 1997). Corticosterone inhibits proteoglycan synthesis in an elasmobranch, the clearnose skate, Raja eleganteria (Gelsleichter and Musick, 1999). Not much is known about the effects of glucocorticoids together with growth factors on cartilage synthesis in teleosts. Cortisol (10 nM or more) inhibits proteoglycan synthesis stimulated by T 3. However, cortisol at 1, 10, or 100 nM does not inhibit proteoglycan synthesis stimulated by IGF-I at physiological concentrations (1 nM), but when IGF-I levels are reduced (0.1 nM) and cortisol levels are high (100 nM), inhibition does occur (Takagi and Bjørnsson, 1997). The effect of cortisol together with growth factors needs to be studied in other teleost species to understand more fully the regulation of growth by cortisol. Other parameters may be physiologically important in regulating cartilage growth. Insulin-like growth factor-binding proteins (IGFBPs) play an important role in regulating the bioactivity of IGF-I and thus growth by affecting the ratio of free to bound IGF-I, clearance rate, and targeting of IGF-I to specific cells. IGFBPs are present in teleosts, including tilapia (Kelley et al., 1992; Park et al., 2000), as in mammals (Siharath and Bern, 1993), but it is not known whether they are affected by
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Datuin et al.
glucocorticoids. In this study, the direct effect of glucocorticoids on cartilage growth in the tilapia, Oreochromis mossambicus, is examined, along with their interaction with IGF-I.
MATERIALS AND METHODS Animals Juvenile tilapia, O. mossambicus, were provided by Pacific Aquafarms (Nilands, CA). All fish had been exposed to 17␣-methyltestosterone as fry. They were housed in 560-liter flow-through freshwater tanks in a temperature-controlled (28°) room under a simulated natural photoperiod (12L:12D). Fish were fed Rangen Floating Catfish Chow (Buhl, ID) twice daily ad libitum. Fish were fasted 1 week before study.
Organ Culture Cartilages were dissected and cultured as described by Duan and Inui (1990) for the Japanese eel, A. japonica, with modifications to optimize conditions for tilapia. Cartilage explants from the first two pairs of gill arches from each fish were placed in a defined culture medium for preincubation (Lee, 1996). A group of cartilages was freeze-killed at ⫺80° and later used for determination of nonspecific isotope uptake. Cartilages were preincubated for 2 h in a defined culture medium and then transferred to 24-well microplates, each well containing 0.5 mL culture medium along with 200 units/ml penicillin and streptomycin and 6 Ci/mL each of [ 35S]sulfate (NEN Life Science Products, Boston, MA) and [ 3H]thymidine (Amersham, Piscataway, NJ). The following hormones were added: cortisol (F; Sigma), a synthetic glucocorticoid, dexamethasone (DEX; Sigma), recombinant bovine insulin-like growth factor-1 (rbIGF-I; Monsanto, St. Louis, MO), or a combination of one of the glucocorticoids with IGF-I. Radioactivity was measured as disintegrations per minute (dpm) by a scintillation counter (Beckman LS6500) programmed to measure simultaneously [ 35S]sulfate and [ 3H]thymidine. Specific uptake was determined by subtracting nonspecific uptake from total uptake. Nonspecific uptake values were less that 1% of the total values. The dpm
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Effects of Glucocorticoids on Cartilage Growth
of each sample was normalized to its own dry weight to determine dpm/g cartilage. For each test concentration, seven or eight fish were represented by seven or eight cartilages, one cartilage from each fish.
Hormones Cortisol was first dissolved in ethanol and then diluted with Ringer to appropriate concentrations (1, 10, 100, or 1000 ng/ml). Final culture medium contained less than 0.01% ethanol. DEX and F were prepared on the day of culture. DEX was diluted with tilapia Ringer to one of the following concentrations: 0.1, 1, 10, 100, or 1000 ng/ml. Recombinant bovine IGF-I was added alone at 100 ng/mL or in combination with F or DEX. Ethanol was added to all control cultures in appropriate amounts.
Statistical Analyses Statistical comparisons were analyzed by the Kruskal–Wallis test followed by the Mann–Whitney U test. Significance was accepted as P ⱕ 0.05. Statistics were performed using the Statview 4.5 software package.
RESULTS Addition of Cortisol Alone and in Combination with IGF-I Cortisol inhibited sulfate uptake at 100 and 1000 ng/mL (P ⬍ 0.05). Cortisol inhibited thymidine uptake only at 1000 ng/mL (P ⬍ 0.05). The concentration of 100 ng/mL of IGF-I significantly stimulated sulfate uptake over controls (P ⬍ 0.05). IGF-I had no effect on thymidine uptake. Cortisol did not inhibit the IGF-I stimulation of sulfate or thymidine uptake at any concentration tested (Fig. 1).
FIG. 1. Effects of cortisol (F) at 0, 1, 10, 100, and 1000 ng/mL and F at 0, 1, 10, and 100 ng/mL combined with IGF-I at 100 ng/mL on sulfate and thymidine uptake by cartilage in vitro. Sulfate uptake was inhibited by F at 100 and 1000 ng/mL (P ⬍ 0.05; significance indicated by *). Thymidine uptake was inhibited by F at 1000 ng/mL (P ⬍ 0.05; significance indicated by *). IGF-I-exposed cartilage showed higher sulfate uptake than controls (P ⬍ 0.05; significance indicated by †); thymidine uptake was not affected. Values are means ⫾SE (n ⫽ 8). This figure is representative of three replicate experiments yielding similar results.
Again the standard concentration of 100 ng/ml IGF-I significantly stimulated sulfate uptake (P ⬍ 0.05) and had no effect on thymidine uptake compared to controls. As with cortisol, DEX did not inhibit the stimulation of sulfate uptake by IGF-I at any concentration tested (Fig. 2).
Addition of DEX Alone and in Combination with IGF-I
DISCUSSION
DEX inhibited sulfate uptake at all concentrations (P ⬍ 0.05). DEX inhibited thymidine uptake at 100 and 1000 ng/mL (P ⬍ 0.05).
Takagi and Bjørnsson (1997) provided the first evidence that cortisol significantly inhibited proteoglycan synthesis and consequently growth of cartilage of a
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FIG. 2. Effects of dexamethasone (DEX) at 0, 0.1, 1, 10, 100, and 1000 ng/mL and DEX at 0, 0.1, 1, 10, 100, and 1000 ng/mL combined with IGF-I at 100 ng/mL on sulfate and thymidine uptake by cartilage in vitro. Sulfate uptake was inhibited by DEX at 0.1, 1, 10, 100, and 1000 ng/mL (P ⬍ 0.05; significance indicated by *). Thymidine uptake was inhibited by DEX at 100 and 1000 ng/mL (P ⬍ 0.05; significance indicated by *). IGF-I-exposed cartilage showed higher sulfate uptake than controls (P ⬍ 0.05; significance indicated by †); thymidine uptake was not affected. Values are means ⫾SE (n ⫽ 8). This figure is representative of three replicate experiments yielding similar results.
teleost fish, the rainbow trout, O. mykiss. The present study differed from their study in the species used, the duration of culture, and the consideration of another parameter: thymidine uptake. Cortisol significantly inhibited sulfate uptake at both 100 and 1000 ng/mL and thymidine uptake at 1000 ng/mL. Foo and Lam (1993) showed that in unstressed tilapia, O. mossambicus, serum cortisol concentration was less than 10 ng/mL. However, in tilapia stressed by netting, serum cortisol concentration rose to around 65 ng/mL, and netting followed by confinement caused serum cortisol concentration to increase to around 120 ng/mL. These data show that
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Datuin et al.
the experimental concentrations of cortisol used by us were within the physiological range to be expected for O. mossambicus; the inhibition is dose-dependent. Our data agree with those of Takagi and Bjørnsson (1997) for O. mykiss cartilage. The concentration of 100 ng/mL of rbIGF-I stimulated sulfate uptake, as expected, but not thymidine uptake. Juvenile O. mossambicus have a high growth rate, and cartilage cells may be maximally mitotically active. As thymidine uptake is a measure of cell division, added IGF-I may not be able to stimulate cartilage further. Takagi and Bjørnsson (1996), using a similar assay system, reported that IGF-I had no effect on thymidine uptake by O. mykiss cartilage. The authors suggested that a nutrient-rich culture medium may induce chondrocyte proliferation and thus thymidine uptake, masking any stimulatory effect of additional hormones to the culture. Cortisol did not inhibit IGF-I stimulation of sulfate uptake at any of the concentrations tested. Furthermore, cortisol did not inhibit thymidine uptake with IGF-I present in the medium. Cortisol appears to act directly on cartilage and not by interacting with the IGF-I system. The physiologically significant role of cortisol would mainly seem to be inhibitory to cartilage metabolism and growth. Cortisol may not act directly on cartilage in vivo. Mammalian data suggest that autocrine/paracrine factors are the most important factors in control of cartilage growth (Froger-Gaillard et al., 1989). Cortisol could act indirectly on cartilage growth by affecting the production and action of local IGF-I and/or its binding proteins. Nishioka et al. (1985) found that incubating O. mossambicus pituitary with cortisol resulted in increased GH secretion, but Takagi and Bjørnsson (1997) and the current study found that cortisol in vitro inhibited growth of cartilage. The effect of cortisol on liver production of IGF-I and IGFBPS is unknown, however. Whereas cortisol may inhibit cartilage growth, IGF-I (100 ng/mL) was able to mask any effect of cortisol. This result implies that in the presence of high levels of circulating IGF-I, cortisol may have no effect on cartilage growth. The impact of cortisol on the liver has not been adequately studied. In addition to direct effects on liver production and secretion of IGF-I and the IGFBPs, cortisol may down-regulate liver GH recep-
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Effects of Glucocorticoids on Cartilage Growth
tors and thus decrease IGF-I production by the liver. This view is supported by mammalian data. Beauloye et al. (1999) has shown that high concentrations of DEX (1 M) inhibit GH induction of IGF-I mRNA, and DEX directly inhibits the expression of GHR mRNA. These effects may be important for euryhaline teleosts during periods of seawater adaptation, as high cortisol levels would result in higher GH levels, both hormones being favorable to seawater adaptation. Moreover, as cortisol would have an overall growth-inhibiting effect, energy might thus be available for seawater adaptation. Dexamethasone is used ubiquitously in mammalian systems as a synthetic glucocorticoid binding to the glucocorticoid receptor with high affinity. Dexamethasone is inhibitory to sulfate and thymidine uptake at concentrations similar to the effective concentrations of cortisol used in the present study. Takagi and Bjørnsson (1997) cultured cartilage for 6 days before recording a cortisol effect. Two differences are apparent between their culture system and that used in the current study that may account for the difference in time required for response. First, the O. mykiss used by Takagi and Bjørnsson were much larger and presumably older than the subadult O. mossambicus used in the current system. As the growth rates for both species decrease appreciably with age, the O. mossambicus were likely growing faster, leading to a faster response time in our system. Second, the preferred temperature of the two species differs greatly, represented by different incubation temperatures. We incubated cultures at 28°, whereas Takagi and Bjørnsson incubated at 15–18°. This temperature difference would lead to different metabolic rates and slower responsiveness by O. mykiss tissues. However, in both studies, cortisol essentially proved to be inhibitory to fish cartilage growth.
ACKNOWLEDGMENTS We acknowledge Dr. Richard S. Nishioka for his advice during this study. We thank Sascha Emami, Isabel Hsu, Kevin Ko, Karen Lee, Richard Park, and Nguyen Tan for their help and technical assistance. We thank Colin Bornia of Pacific Aquafarms for generously providing animals and advice. Support has been generously provided by NOAA, National Sea Grant College Program, U.S.
Department of Commerce, under Grant No. NA 89AA-D-SG 138R, Project R/F-145 to H.A.B., a traineeship from 1997–1999 under Project No. R/A-94 to J.P.D., and a traineeship from 1996 –1998 under Project No. R/A-94 to K.P.N. from the California Sea Grant College Program. The views expressed herein are those of the authors and do not necessarily reflect the views of the NOAA or any of its subagencies.
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