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MC Erlandsson, CA Jonsson, MK Lindberg, C Ohlsson, and H Carlsten

Raloxifene is a selective estrogen receptor modulator approved for the prevention of osteoporosis in postmenopausal women. It is selective by having estrogen-agonistic effects on bone, vessels and blood lipids while it is antagonistic on mammary and uterine tissue. Our aim was to study the agonistic and antagonistic properties of the raloxifene analogue LY117018 (LY) on uterus, bone, B lymphopoiesis and B cell function. Oophorectomized and sham-operated animals were treated with s.c. injections of equipotent anti-osteoporotic doses of 17beta-estradiol (E2) (0.1 mg/kg) or LY (3 mg/kg) or vehicle as controls. Effects on bone mineral density (BMD) were studied using peripheral quantitative computed tomography, uterine weight was examined, B lymphopoiesis was examined using flow cytometry and B cell function in bone marrow and spleen was studied by the use of an ELISPOT assay. E2 and LY had similar effects on BMD and bone marrow B lymphopoiesis, while LY had a clear antagonistic effect on endogenous estrogen in uterine tissue and no stimulating effect on the frequency of Ig-producing B cells in sham-operated animals. Our results are discussed in the context of estrogen receptor biology, relations between the immune system and bone metabolism and also with respect to the estrogen-mediated effects on rheumatic diseases.

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JM Kindblom, O Nilsson, T Hurme, C Ohlsson, and L Savendahl

Indian Hedgehog (Ihh) has been reported to control the rate of cartilage differentiation during skeletal morphogenesis in rodents through a negative feedback loop involving parathyroid hormone related protein (PTHrP). The role of Ihh and PTHrP in the regulation of human epiphyseal chondrocytes is unknown. The aim of the current study was to examine the expression and localization of Ihh and PTHrP in the human growth plate at various pubertal stages. Growth plate biopsies were obtained from patients subjected to epiphyseal surgery and the expression of Ihh and PTHrP was detected by immunohistochemistry. We show that Ihh and PTHrP are expressed mainly in early hypertrophic chondrocytes in the human growth plate. The levels of expression of Ihh and PTHrP are higher in early stages of puberty than later. Our results suggest that Ihh and PTHrP are present in the human growth plate and that Ihh and PTHrP may be involved in the regulation of pubertal growth in humans.

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C. Ohlsson, A. Nilsson, O. Isaksson, J. Bentham, and A. Lindahl

ABSTRACT

The effects of tri-iodothyronine (T3) and insulin-like growth factor-I (IGF-I) on [3H]thymidine incorporation, alkaline phosphatase (ALP) activity and IGF-I receptor mRNA levels were studied in rat epiphyseal chondrocytes cultured in monolayer.

Chondrocytes from enzymatically digested rat tibia epiphyseal growth plates were seeded in monolayer culture and precultured for 7–14 days in Ham's F-12 medium supplemented with 10% (v/v) newborn calf serum and 1% (v/v) of a serum substitute. After preculture the medium was changed to Ham's F-12 medium containing 1% (v/v) serum from hypophysectomized rats, and the effects of T3 and/or IGF-I on DNA synthesis ([3H]thymidine incorporation), ALP activity (a late marker of differentiated epiphyseal chondrocytes) and IGF-I receptor mRNA levels were studied.

ALP activity was increased by T3 in a dose-dependent manner with a maximal response at 10 μg T3/1 (678 ±86% compared with control culture). The increase in ALP activity was accompanied by a concomitant decrease in [3H]thymidine incorporation (52 ±14% compared with control culture). Human GH (hGH; 50 μg/l) and IGF-I (25 μg/l) had no stimulatory effect on ALP activity. However IGF-I (10 μg/l) exerted an inhibition on the T3 (10 μg/l)-induced increase in ALP activity (64 ± 9% compared with T3-treated culture). T3 (3 μg/l) inhibited the increase in [3H]thymidine incorporation caused by 25 μg IGF-I/l(51 ± 13% compared with IGF-I-treated culture). Furthermore, IGF-I receptor mRNA levels were increased by 10 μg T3/l (137 ±4·2% compared with control culture) while no effect of hGH (50 μg/l) or IGF-I (25 μg/l) was demonstrated.

Both T3 and IGF-I were shown to interact with epiphyseal chondrocytes and both substances seemed to affect cell proliferation and maturation and therefore longitudinal bone growth. Furthermore, the results indicated that IGF-I is important for proliferation of the cells while T3 initiates the terminal differentiation of epiphyseal chondrocytes.

Journal of Endocrinology (1992) 135, 115–123

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C Nilsson, D Swolin-Eide, C Ohlsson, E Eriksson, HP Ho, P Bjorntorp, and A Holmang

Leptin is involved in regulating food intake, energy balance and bone formation. Increasing evidence suggests that leptin is also involved in fetal growth and development. The aim of this study was to determine if increased maternal leptin is followed by changes in body composition, skeletal growth or hormonal regulation in the adult rat offspring. Pregnant rats were given injections of either human recombinant leptin (3.5 mg/kg, i.p.) or vehicle on days 8, 10 and 12 of gestation. Both genders of leptin-exposed offspring showed significantly reduced adipose tIssue weight at adult age. Skeletal growth and cortical bone dimensions were significantly reduced. Circulating testosterone levels were significantly increased in female leptin-exposed offspring, and male leptin-exposed offspring had significant testicular enlargement. No significant effects were seen on circulating leptin levels or hypothalamic protein levels of the leptin receptor. The results demonstrate that maternally administered leptin is involved in fetal growth and development, leading to lean offspring with reduced skeletal growth.

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D Swolin-Eide, J Dahlgren, C Nilsson, K Albertsson Wikland, A Holmang, and C Ohlsson

Events occurring early in life or prenatally are able to play important roles in the pathogenesis of diseases in adult life. Different sorts of stress or hormonal influences, during particular periods of pregnancy, may result in persisting or transient changes in physiology. Glucocorticoids are used for the treatment of a variety of diseases, to promote organ maturation and to prevent preterm delivery. Glucocorticoids are also known to affect skeletal growth and adult bone metabolism. The aim of the present study was to investigate whether exposure to dexamethasone (Dex) during fetal life has any effect on skeletal growth and/or bone mineral density in adult rat offspring. Pregnant rats were given injections of either Dex (100 micro g/kg) or vehicle on days 9, 11 and 13 of gestation. Dex-exposed male but not female rat offspring showed transient increases in crown-rump length and tibia and femur lengths at 3-6 weeks of age. In contrast, the cortical bone dimensions were altered in 12-week-old female but not male Dex-exposed offspring. The areal bone mineral densities of the long bones and the spine, as determined by dual X-ray absorptiometry, and trabecular as well as cortical volumetric bone mineral density, as measured using peripheral quantitative computerized tomography, were unchanged in both male and female Dex-exposed offspring. In conclusion, prenatal Dex exposure affects skeletal growth in a gender-specific manner, while the mineralization of bones is unaffected in both male and female offspring.

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M C Slootweg, J P Salles, C Ohlsson, C P de Vries, M J E Engelbregt, and J C Netelenbos

Abstract

Growth hormone (GH) exerts direct differentiative and proliferative effects on osteoblasts. We studied 125I-labeled human (h) GH binding to primary mouse osteoblasts derived from collagenase-treated 18-day fetal mouse calvaria. Scatchard analysis of the data revealed a single class of high affinity GH receptors (apparent K a= 5·74 × 109 m −1) with 2200 sites per cell. Affinity cross-linking and SDS-PAGE electrophoresis showed two bands with apparent molecular masses of 120 and 70 kDa. Mouse osteoblasts express GH receptor mRNA with gene transcripts of 4·2 and 1·2 kb, at levels which reach approximately 1/6 of those in mouse liver and 1/3 of those in mouse muscle. Two populations of undifferentiated and diffentiated osteoblasts, obtained by sequential collagenase digestion of mouse calvaria, were used to study the relationship between osteoblastic phenotype and GH receptor expression. Although the affinity of the receptors in undifferentiated and differentiated cells was the same, the capacity was significantly higher (1·45 ± 1·0% vs 2·39 ± 0·9%, P=0·03) in differentiated cells. This stresses the specific importance of the osteoblast as a target cell for GH. The differentiating potential of the vitamin A derivative retinoic acid was subsequently used experimentally to induce differentiation in the cells. Retinoic acid increased 125I-hGH binding to preosteoblasts (153%, P=0·02). Together, these data demonstrate the presence of a high affinity GH receptor in mouse osteoblasts which is related to differentiation.

Journal of Endocrinology (1996) 150, 465–472

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JM Kindblom, S Gothe, D Forrest, J Tornell, B Vennstrom, and C Ohlsson

Thyroid hormone receptor alpha 1, beta 1 and beta 2-deficient mice (TR alpha 1-/-beta-/- mice) demonstrate growth retardation and defective ossification in the epiphyses associated with an inhibition of the GH/IGF-I axis. There are differences between TR alpha 1-/-beta-/- mice (receptor deficient) and the hypothyroid animal model (ligand deficient). Such differences include possible repressive actions exerted by unliganded receptors in the ligand-deficient (hypothyroid) model but not in the receptor-deficient model. In the present study we have investigated whether or not GH substitution rescues the skeletal phenotype of TR alpha 1-/-beta-/- mice. TR alpha 1-/-beta-/- and wild-type (WT) mice were treated with GH from day 18 until 10 weeks of age. GH substitution of mutant mice resulted in a significant and sustained stimulatory effect on the body weight that was not seen in WT mice. GH-treated mutant mice but not GH-treated WT mice demonstrated increased length and periosteal circumference of the femur. However, GH substitution did not reverse the defective ossification seen in TR alpha 1-/-beta-/- mice. TR alpha 1-/-beta-/- mice displayed increased width of the proximal tibial growth plate, which was caused by increased width of the proliferative but not the hypertrophic layer. GH substitution did not restore the disturbed morphology of the growth plate in TR alpha 1-/-beta-/- mice. In summary, GH substitution reverses the growth phenotype but not the defective ossification in TR alpha 1-/-beta-/- mice. Our data suggest that TRs are of importance both for the regulation of the GH/IGF-I axis and for direct effects on cartilage.

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MK Lindberg, SL Alatalo, JM Halleen, S Mohan, JA Gustafsson, and C Ohlsson

There are two known estrogen receptors, estrogen receptor-alpha (ER alpha) and estrogen receptor-beta (ER beta), which may mediate the actions of estrogen. The aim of the present study was to compare fat content, skeletal growth and adult bone metabolism in female mice lacking ER alpha (ERKO), ER beta (BERKO) or both ERs (DERKO). We demonstrate that endogenous estrogens decrease the fat content in female mice via ER alpha and not ER beta. Interestingly, the longitudinal bone growth was decreased in ERKO, increased in BERKO, but was intermediate in DERKO females, demonstrating that ER alpha and ER beta exert opposing effects in the regulation of longitudinal bone growth. The effects on longitudinal bone growth were correlated with similar effects on serum levels of IGF-I. A complex regulation of the trabecular bone mineral density (BMD), probably caused by a disturbed feedback regulation of estrogen and testosterone, was observed in female ER-inactivated mice. Nevertheless, a partial functional redundancy for ER alpha and ER beta in the maintenance of the trabecular BMD was observed in the female mice at 60 days of age. Thus, ER alpha and ER beta may have separate effects (regulation of fat), opposing effects (longitudinal bone growth) or partial redundant effects (trabecular BMD at 60 days of age), depending on which parameter is studied.

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N Andersson, VV Surve, D Lehto-Axtelius, C Ohlsson, R Hakanson, K Andersson, and B Ryberg

Both ovariectomy (Ovx) and gastrectomy (Gx) induce osteopaenia in rats and humans. While the effect of Ovx has been ascribed to oestrogen deficiency, the underlying mechanism behind Gx is poorly understood. Alendronate, oestrogen and parathyroid hormone (PTH) are known to prevent the osteopaenia induced by Ovx in rats. The purpose of the present study was to determine whether alendronate, oestrogen or PTH could also prevent Gx-evoked osteopaenia. Rats were Ovx-, Gx-, or were sham-operated (Sham) and were then treated with alendronate (50 micro g/kg/day), oestrogen (10 micro g/kg/day) or PTH(1-84) (75 micro g/kg/day) for eight weeks. At sacrifice, serum PTH was unaffected by surgery (Ovx, 64+/-8 pg/ml; Gx, 75+/-13 pg/ml; Sham, 58+/-11 pg/ml). The bone mineral density (BMD) of the fifth lumbar vertebra (L5) was analysed. Ovx and Gx reduced the BMD (ash weight/Volume) of the L5 by 15+/-4% and 22+/-3% respectively. Trabecular BMD and the cortical bone mineral content (BMC) of the femur were assessed using peripheral computed tomography. Both Ovx and Gx markedly reduced trabecular BMD in the metaphyseal area of the distal femur (Ovx, -37+/-7%; Gx, -49+/-7%). The cortical BMC of the femur was only slightly reduced. Alendronate prevented trabecular bone loss after both Ovx and Gx, while oestrogen and PTH prevented trabecular bone loss after Ovx but not after Gx. In conclusion, the bisphosphonate alendronate prevented both Ovx- and Gx-induced trabecular bone loss. In contrast, PTH and oestrogen prevented Ovx-induced but not Gx-induced trabecular bone loss, suggesting that the mechanism behind the trabecular bone loss in Ovx rats differs from that in Gx rats. The results support the notion that the mechanism of action for the bone-sparing effect of these drugs differs. The ability of alendronate, and probably also other bisphosphonates, to prevent Gx-evoked osteopaenia in the rat might be of potential clinical interest when dealing with post-Gx osteopaenia in humans.

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I Pettersson, G Muccioli, R Granata, R Deghenghi, E Ghigo, C Ohlsson, and J Isgaard

Recent experimental data demonstrate cardiovascular effects of the GH secretagogues (GHSs) hexarelin and ghrelin, the proposed natural ligand for the GHS receptor. Moreover, specific cardiac binding sites for GHSs have been suggested. The aim of the present study was to investigate if the natural ligand ghrelin and synthetic GHS peptide hexarelin and analogues have direct effects on the cardiomyocyte cell line, H9c2. Hexarelin stimulated thymidine incorporation in a dose-dependent manner with significant responses at 3 micro M (147+/-3% of control, P<0.01) and elicited maximal effects at concentrations around 30 micro M. This activity was seen already after 12 h of incubation with a maximal effect after 18 h (176+/-9% of control, P<0.01). Ghrelin also had a significant stimulatory effect on thymidine incorporation (129+/-2% of control at 3 micro M and 18 h, P<0.05). The stimulatory effect on thymidine incorporation of hexarelin, Tyr-Ala-hexarelin, EP80317 and ghrelin was specific and no stimulatory effect was observed with the truncated GH-releasing peptide EP51389 or the non-peptidyl GHS MK-0677. In competitive binding studies, (125)I-labeled Tyr-Ala-hexarelin was used as radioligand and competition curves showed displacement with hexarelin, Tyr-Ala-hexarelin, EP80317 and ghrelin, whereas MK-0677 and EP51389 produced very little displacement at 1 micro M concentration, adding further support for an alternative subtype binding site in the heart compared with the pituitary. In conclusion, we have demonstrated a dose-dependent and specific stimulation of cardiomyocyte thymidine incorporation by natural and synthetic GHS analogues, suggesting increased cell proliferation and binding of GHS to H9c2 cardiomyocyte cell membranes. These findings support potential peripheral effects of GHS on the cardiovascular system independent of an increased GH secretion.