Since hench et al.(1) first reported the use of cortisone for treatment of patients with rheumatoid arthritis, glucocorticoids (GCs) have been widely used for anti-inflammatory and immunosuppressive therapy. However, GCs have major effects on several organ systems, including the cardiovascular, endocrine, gastrointestinal, ophthalmic, and musculoskeletal systems, and these often lead to major side effects.(2) Among the most dramatic side effects of in vivo long-term GC excess is the development of GC-induced osteoporosis.(3, 4) Several lines of evidence indicate that the major mechanisms whereby GCs induce osteoporosis are decreased bone matrix deposition by osteoblasts, stimulation of osteoclastic bone resorption, and decreased intestinal calcium resorption with resulting mild secondary hyperparathyroidism (for reviews see Refs. 3, 5–8). However, the diverse and complex effects of GCs on bone metabolism are still incompletely understood, mainly due to differences in experimental outcomes in different systems and culture conditions, which could be a result of species differences, age of the experimental animals used, maturation stage of the cells studied, heterogeneity of osteoblast populations, differences between transformed cells and normal cells, or length and dose of GC treatment used. In this brief review, we will focus on the effects of GCs on bone formation. With regard to this aspect of GC effects, it has been reported in a variety of in vitro systems that GCs induce cells of the osteoblast lineage to differentiate into mature cells expressing the osteoblastic phenotype, e.g., parathyroid hormone–stimulated cAMP synthesis, osteopontin, bone sialoprotein, alkaline phosphatase (AP), and mineralized bone nodule formation.(9-22) However, in several in vitro systems, GCs have also been shown to decrease type I collagen synthesis and mRNA levels,(23-25) osteocalcin production and mRNA levels,(24, 26, 27) and the expression of insulin-like growth factor I (IGF-I)(28-30) and selected IGF-binding proteins (IGFBPs) which enhance IGF activity (e.g., IGFBP-3 and IGFBP-5).(31, 32) The reason for the observed differences is not clear, but it is known that the GC receptor can mediate not only transcriptional activation but also repression of transcriptional expression by interfering with the binding or activity of other transcription factors(33) or by interacting directly with negative glucocorticoid response elements (nGREs).(34) For example, the GC-induced inhibition of 1,25-dihydroxyvitamin D3–mediated osteocalcin transcription is likely to occur by binding of the GC receptor to a nGRE overlapping the TATA box in the proximal promoter region of osteocalcin gene.(35) In addition, mRNA levels can also be controlled by GCs through selective changes in mRNA stability.(36, 37) Indeed, Delany et al.(25) have shown that GC-mediated down-regulation of α1(I) collagen mRNA occurs by a decrease in the stability of α1(I) collagen mRNA in bone cell populations derived from fetal rat calvariae. Although a number of such mechanisms for negative-transcriptional or post-transcriptional gene regulation by GCs have been described, it is still unclear in many systems how positive and negative regulation interact to result in the observed effects (for review see Ref. 38). Furthermore, the observed differences may well be related to the fact that the effects of GCs are often dependent on the timing of GC treatment and the stage of differentiation of the osteoblast-like cells used. For example, when cultures obtained from chick embryo calvariae were continuously exposed to dexamethasone (Dex) from the onset of culture, Dex increased AP activity, but when Dex was added late in the culture period after bone had formed, AP activity decreased.(11) In addition, Dietrich et al.(39) have shown that in fetal rat calvariae, 30–100 nM cortisol stimulated collagen synthesis after 24 h, while inhibitory effects of 1 μM cortisol were seen at 48–96 h. In vivo data uniformly indicate that GCs stimulate bone resorption and decrease bone formation, resulting in bone loss.(40-42) It is important to note, however, that the inhibitory effects reported in vivo are generally seen only at pharmacological doses of the GCs used. With regard to the naturally circulating forms of GCs, some species secrete predominantly cortisol (man, guinea pig, and rabbit) and others corticosterone (mouse and rat).(43) GC receptors of cortisol-secreting species (man, guinea pig, and rabbit) bind cortisol with a much higher affinity than corticosterone, whereas GC receptors of corticosterone-secreting species (rat, mouse) bind corticosterone with a much higher affinity than cortisol.(44) The data of Table 1 show relative GC binding affinities and biological potencies of various GCs in rat and man published in separate papers by several authors. In both human and animal studies, various GCs have been used, e.g., corticosterone, cortisol, cortisone, prednisone, prednisolone, or dexamethasone. Important factors involved in relative GC activities among various GC compounds are the binding affinity with the GC receptor and the biological potency.(2) Reported GC biopotencies are usually based on animal experiments, using effects on hypothalamo-pituitary-adrenal (HPA) axis suppression, glycogen deposition, anti-inflammatory effects, or eosinopenic effects as end points. In most tissues, available data suggest that all GCs act through interaction with the GC receptor(45, 46) and that the binding affinity to the receptor and biopotency closely parallel one another, although most synthetic GCs have a somewhat greater response than would be expected based on their affinity to the GC receptor.(47) However, in cells of the osteoblast lineage, no data are available concerning the relationship between binding parameters and biopotencies of GCs or the relative activities of different GCs. The mean total daily cortisol production in man is 12.8 mg (range, 6.7–20 mg), resulting in a circulating concentration of 7 × 10−7 M at peak levels (in the morning) and of 1 × 10−7 M at the lowest point of the circadian cycle.(48, 49) This amount is approximately equivalent to the contents of one 5 mg tablet of prednisolone, administration of which results in a peak level of prednisolone of 10.5 μg/dl (equivalent to 1.2 × 10−6 M cortisol).(50) Therefore, any in vivo administration in excess of this dosage will cause circulating GC levels in blood well in excess of physiological levels, likely resulting in side effects and feedback inhibition of the HPA axis.(51) Indeed, the results reported by LaRochelle et al.(52) indicated that oral administration of prednisone of <5 mg/day (equivalent to 20 mg of cortisol) did not have side effects, while 5 mg/day or more induced HPA axis suppression. In addition, Olbricht and Benker(5) reported that after treatment with low doses of prednisone (<10 mg/day), bone loss appears to be minimal or undetectable compared with controls. GC treatments with higher doses of prednisone (>10 mg/day) reduced bone mass,(53) which was most pronounced during the first months of treatment,(5) regardless of the patient's age, gender, or race.(54, 55) There is no agreement on the effects of daily dose, cumulative dose, and duration of GC treatment on the rate of bone loss.(5, 56) With regard to pharmacokinetic characteristics of GCs, in healthy men plasma concentrations after oral administration of tablets containing 5, 20, and 40 mg of prednisolone reached a maximum of 10.5 μg/dl (equivalent to 1.2 × 10−6 M cortisol), 46.2 μg/dl (equivalent to 5.1 × 10−6 M cortisol), and 66.0 μg/dl (equivalent to 7.3 × 10−6 M cortisol), respectively, at 1–2 h after oral administration, and then gradually decreased to control levels after 24 h.(50, 57) When 30 mg of prednisolone was given orally to healthy children in a single morning dose, the plasma concentration of prednisolone reached a maximum of 38–47 μg/dl (equivalent to 4.2–5.2 × 10−6 M cortisol) at 1–1.5 h after administration and declined slowly with a mean t1/2 of 2.5 h,(58) suggesting that there is no difference in pharmacokinetics of prednisolone after a single oral dose of prednisone between children and adults.(59) These data show that administration of pharmacological doses of GCs in vivo results in supraphysiological plasma levels of GCs. While a great deal of information has been obtained concerning the effects of GCs on osteoblast differentiation in vitro, relatively few data are available with regard to optimal concentrations of GCs for either stimulatory or inhibitory effects. However, available in vitro data suggest that the effectiveness of GCs is dependent on GC concentrations, i.e., low concentrations of GCs stimulate osteoblast differentiation and increase bone formation while GCs at high concentrations are less effective or inhibitory.(10, 39, 60-62) We have shown previously that Dex increases the number of colonies of differentiated osteoblasts producing bone (bone nodule formation) present in populations of cells derived from both adult rat vertebrae and fetal rat calvariae (e.g., Refs. 12, 21, 22, 63). In these studies, we have shown that in bone cell populations derived from fetal rat calvariae or adult female and male rat vertebrae the maximally stimulatory effect of Dex was seen at 1 × 10−8 M, and that the effects decreased at higher concentrations of Dex (1 × 10−7 M to 1 × 10−6 M). Similar dose-related effects of corticosterone on osteoblast proliferation and differentiation were found, i.e., a maximal stimulatory effect of corticosterone on bone nodule formation at 3 × 10−7 M, and decreased effects at higher concentrations (3 × 10−6 to 10−5 M) (Ishida and Heersche submitted for publication; Bellows et al. submitted for publication). Importantly, the maximally effective concentrations of GCs to stimulate osteoblast proliferation and differentiation are within the range of physiological circulating levels of GCs (1 × 10−7 M to 1 × 10−6 M of corticosterone in rats).(64-67) A possible explanation for the dose-dependent effects of GCs may be that high concentrations of GCs cause a decrease in osteoblast proliferation. Scutt et al.(68) reported that 10−9−10−5 M Dex treatment resulted in a dose-dependent inhibition of osteoblast precursor cell proliferation in rat bone marrow cultures (maximum inhibition at 10−5 M Dex). Alternatively, high concentrations of GCs may down-regulate GC receptors, e.g., a 4-day incubation with 10−9−10−6 M Dex resulted in a dose-dependent decrease in the number of nuclear GC receptors (maximally 95% inhibition at 10−6 M Dex) in human cervical carcinoma HeLa S3 cells.(69) Similar results were obtained in mouse pituitary tumor AtT-20 cells(47) and human lymphocytes.(70) Thus, it is possible that the agonist-induced decrease in the receptor number could attenuate the osteoblast response to GCs. Further investigation of the mechanism underlying such dose-dependent effects of GCs on osteoblasts will be an important focus for future studies. In human osteoblast-like cell populations, maximally stimulatory effects of GCs on osteoblast differentiation are seen at 10−8−10−7 M Dex, e.g., secretion of latent transforming growth factor-beta (TGF-β), gene expression of betaglycan (TGF-β type III receptor), alkaline phosphatase mRNA expression, and formation of a mineralized extracellular matrix.(71-74) Again, these maximally effective concentrations to stimulate osteoblast differentiation are close to or within the range of physiological circulating GC plasma levels (1 × 10−7 M to 7 × 10−7 M of cortisol in humans).(48, 75-77) Taken together, these human and animal data suggest that in vivo the circulating GC plasma levels both in humans and animals are similar to the concentrations that have maximally stimulatory effects on osteoblast differentiation in vitro. The data presented here support the view that physiological concentrations of GCs in vivo are equivalent to doses that have maximally stimulatory effects on osteoblast differentiation in vitro and result in sustaining optimal osteoblast differentiation. That in vivo no additional stimulatory but only inhibitory effects on bone formation have been observed by any dose of administered GCs is in agreement with the in vitro observations that increasing the concentration beyond the optimal concentration decreases osteoblast differentiation. Since long-term administration of GCs causes supraphysiological plasma GC levels, this likely results in decreased osteoblast differentiation compared with that at physiological plasma GC levels. This consideration explains GC-induced bone loss in vivo, emphasizes an important role for GCs in the normal regulation of osteoblast differentiation, and resolves the apparent discrepancy between the in vitro and in vivo effects of GCs on bone formation and osteoblast differentiation. We thank Dr. H. Morishita for helpful discussions on glucocorticoid pharmacokinetics.
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Ishida et al. (1998) studied this question.
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