Estrogens are well known for their capacity to promote bone maturation and at high doses to induce growth plate closure and thereby stop further growth. High-dose estrogen treatment has therefore been used to limit growth in extremely tall girls. However, recent data suggest that this treatment may have severe side effects, including increased risk of cancer and reduced fertility. We hypothesized that estrogenic effects in bone are mediated via ERα signaling. Twelve-week-old ovariectomized female C57BL/6 mice were subcutaneously injected for 4 weeks with E2 or selective ERα (PPT) or ERβ (DPN) agonists. After killing, tibia and femur lengths were measured, and growth plate morphology was analyzed. E2- and PPT-treated mice had shorter tibiae and femur bones when compared to vehicle-treated controls, whereas animals treated with DPN had similar bone lengths compared to controls. Growth plate height and hypertrophic zone height were reduced in animals treated with E2 or PPT but not in those treated with DPN, supporting that the effect was mediated via ERα. Moreover, PCNA staining revealed suppressed proliferation of chondrocytes in the tibia growth plate in PPT- or E2-treated mice compared to controls. Our data show that estrogenic effects on bone growth and growth plate maturation are mainly mediated via ERα. Our findings may have direct implications for the development of new and more selective treatment modalities of extreme tall stature using selective estrogen receptor modulators that may have low side effects than high-dose E2 treatment.
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Maryam Iravani, Marie Lagerquist, Claes Ohlsson, and Lars Sävendahl
Lovisa Lundholm, Milica Putnik, Michio Otsuki, Sandra Andersson, Claes Ohlsson, Jan-Åke Gustafsson, and Karin Dahlman-Wright
Obesity has become a major health problem in many parts of the world. Estrogens are known to reduce adipose tissue mass in both humans and animals but the molecular mechanisms are not well characterized. We used gene expression profiling to study long-term effects of estrogen on gene expression in mouse white adipose tissue and hypothalamus. Overall, the effects of estrogen on hypothalamic gene expression were much smaller than the corresponding effects on white adipose tissue gene expression. We characterize in detail estrogenic regulation of glutathione peroxidase 3 (GPX3). Our studies suggest that GPX3 is a direct estrogen receptor α target gene in white adipose tissue. Since obesity is correlated with oxidative stress, and GPX3 has been demonstrated to be lower in obesity and higher after weight loss, we hypothesize that GPX3 is one important mediator of effects of estrogen in relation to fat mass. Additional genes that were affected by estrogen in adipose tissue include cell death-inducing DNA fragmentation factor, α-subunit-like effector A (CIDEA), a gene shown to be related to body fat in mice. We conclude that estrogen has large effects on gene expression in white adipose tissue and hypothesize that GPX3 and CIDEA could be important mediators of the effects of estrogen on fat mass.
Louise Grahnemo, Caroline Jochems, Annica Andersson, Cecilia Engdahl, Claes Ohlsson, Ulrika Islander, and Hans Carlsten
Treatment with anti-inflammatory glucocorticoids is associated with osteoporosis. Many of the treated patients are postmenopausal women, who even without treatment have an increased risk of osteoporosis. Lymphocytes have been shown to play a role in postmenopausal and arthritis-induced osteoporosis, and they are targeted by glucocorticoids. The aim of this study was to investigate the mechanisms behind effects of glucocorticoids on bone during health and menopause, focusing on lymphocytes. Female C57BL/6 or SCID mice were therefore sham-operated or ovariectomized and 2 weeks later treatment with dexamethasone (dex), the nonsteroidal anti-inflammatory drug carprofen, or vehicle was started and continued for 2.5 weeks. At the termination of experiments, femurs were phenotyped using peripheral quantitative computed tomography and high-resolution micro-computed tomography, and markers of bone turnover were analyzed in serum. T and B lymphocyte populations in bone marrow and spleen were analyzed by flow cytometry. Dex-treated C57BL/6 mice had increased trabecular bone mineral density, but lower cortical content and thickness compared with vehicle-treated mice. The dex-treated mice also had lower levels of bone turnover markers and markedly decreased numbers of spleen T and B lymphocytes. In contrast, these effects could not be repeated when mice were treated with the nonsteroidal anti-inflammatory drug carprofen. In addition, dex did not increase trabecular bone in ovariectomized SCID mice lacking functional T and B lymphocytes. In contrast to most literature, the results from this study indicate that treatment with dex increased trabecular bone density, which may indicate that this effect is associated with corticosteroid-induced alterations of the lymphocyte populations.
Åsa Tivesten, Anna Barlind, Kenneth Caidahl, Natalia Klintland, Antonio Cittadini, Claes Ohlsson, and Jörgen Isgaard
Growth hormone (GH) deficiency is associated with abnormal vascular reactivity and development of atherosclerosis. GH treatment in GH deficient states restores systemic vascular resistance, arterial compliance, endothelium-dependent and endothelium-independent vasodilation, and may reverse markers of early atherosclerosis. However, very little is known about the molecular mechanisms underlying these effects. In the present study, male Sprague Dawley rats were hypophysectomized and treated for two weeks with GH (recombinant human GH, 2 mg/kg/day) or saline as s.c. injections twice daily. GH decreased aortic systolic blood pressure compared with saline-treated animals, while the diastolic blood pressure was not significantly changed. GH treatment increased cardiac output as determined by Doppler-echocardiography and the calculated systemic vascular resistance was markedly reduced. In order to identify GH-regulated genes of importance for vascular function, aortic mRNA levels were analyzed by the microarray technique and correlated to the systolic blood pressure levels. Using this approach, we identified 18 GH-regulated genes with possible impact on vascular tone and atherogenesis. In particular, mRNA levels of the inwardly rectifying potassium channel Kir6.1 and the sulfonylurea receptor 2B, which together form the vascular smooth muscle ATP-sensitive potassium channel, were both up-regulated by GH treatment and highly correlated to systolic blood pressure. Our findings establish a major role for GH in the regulation of vascular physiology and gene expression. Increased expression of the ATP-sensitive potassium channel, recently shown to be crucial in the regulation of vascular tone, constitutes a possible mechanism by which GH governs vascular tone.
Thomas Funck-Brentano, Karin H Nilsson, Robert Brommage, Petra Henning, Ulf H Lerner, Antti Koskela, Juha Tuukkanen, Martine Cohen-Solal, Sofia Movérare-Skrtic, and Claes Ohlsson
WNT signaling is involved in the tumorigenesis of various cancers and regulates bone homeostasis. Palmitoleoylation of WNTs by Porcupine is required for WNT activity. Porcupine inhibitors are under development for cancer therapy. As the possible side effects of Porcupine inhibitors on bone health are unknown, we determined their effects on bone mass and strength. Twelve-week-old C57BL/6N female mice were treated by the Porcupine inhibitors LGK974 (low dose = 3 mg/kg/day; high dose = 6 mg/kg/day) or Wnt-C59 (10 mg/kg/day) or vehicle for 3 weeks. Bone parameters were assessed by serum biomarkers, dual-energy X-ray absorptiometry, µCT and histomorphometry. Bone strength was measured by the 3-point bending test. The Porcupine inhibitors were well tolerated demonstrated by normal body weight. Both doses of LGK974 and Wnt-C59 reduced total body bone mineral density compared with vehicle treatment (P < 0.001). Cortical thickness of the femur shaft (P < 0.001) and trabecular bone volume fraction in the vertebral body (P < 0.001) were reduced by treatment with LGK974 or Wnt-C59. Porcupine inhibition reduced bone strength in the tibia (P < 0.05). The cortical bone loss was the result of impaired periosteal bone formation and increased endocortical bone resorption and the trabecular bone loss was caused by reduced trabecular bone formation and increased bone resorption. Porcupine inhibitors exert deleterious effects on bone mass and strength caused by a combination of reduced bone formation and increased bone resorption. We suggest that cancer targeted therapies using Porcupine inhibitors may increase the risk of fractures.
Niklas Andersson, Ulrika Islander, Emil Egecioglu, Elin Löf, Charlotte Swanson, Sofia Movérare-Skrtic, Klara Sjögren, Marie K Lindberg, Hans Carlsten, and Claes Ohlsson
It is generally believed that estrogens exert their bone sparing effects directly on the cells within the bone compartment. The aim of the present study was to investigate if central mechanisms might be involved in the bone sparing effect of estrogens. The dose–response of central (i.c.v) 17β-estradiol (E2) administration was compared with that of peripheral (s.c.) administration in ovariectomized (ovx) mice. The dose–response curves for central and peripheral E2 administration did not differ for any of the studied estrogen-responsive tissues, indicating that these effects were mainly peripheral. In addition, ovx mice were treated with E2 and/or the peripheral estrogen receptor antagonist ICI 182,780. ICI 182,780 attenuated most of the estrogenic response regarding uterus weight, retroperitoneal fat weight, cortical BMC and trabecular bone mineral content (P<0.05). These findings support the notion that the primary target tissue that mediates the effect of E2 on bone is peripheral and not central.
Johan Svensson, Åsa Tivesten, Klara Sjögren, Olle Isaksson, Göran Bergström, Subburaman Mohan, Johan Mölne, Jörgen Isgaard, and Claes Ohlsson
The GH/-IGF-I axis is important for kidney size and function and may also be involved in the development of renal failure. In this study, the role of liver-derived endocrine IGF-I for kidney size and function was investigated in mice with adult liver-specific IGF-I inactivation (LI-IGF-I−/− mice). These mice have an 80–85% reduction of serum IGF-I level and compensatory increased GH secretion. Seven-month-old as well as 24-month-old LI-IGF-I−/− mice had decreased kidney weight. Glomerular filtration rate, assessed using creatinine clearance as well as creatinine clearance corrected for body weight, was unchanged. The 24-h urine excretion of sodium and potassium was increased in the LI-IGF-I−/− mice. In the 24-month-old mice, there was no between-group difference in kidney morphology. Microarray and real-time PCR (RT-PCR) analyses showed a high renal expression of IGF-II in the control mice, whereas in the LI-IGF-I−/− mice, there was a tissue-specific decrease in the renal IGF-II mRNA levels (−79%, P < 0.001 vs controls using RT-PCR). In conclusion, deficiency of circulating liver-derived IGF-I in mice results, despite an increase in GH secretion, in a global symmetrical decrease in kidney size, increased urinary sodium and potassium excretion, and a clear down regulation of renal IGF-II expression. However, the LI-IGF-I−/− mice did not develop kidney failure or nephrosclerosis. One may speculate that liver-derived endocrine IGF-I induces renal IGF-II expression, resulting in symmetrical renal growth.
Joyce Emons, Andrei S Chagin, Torun Malmlöf, Magnus Lekman, Åsa Tivesten, Claes Ohlsson, Jan M Wit, Marcel Karperien, and Lars Sävendahl
Longitudinal bone growth is regulated in the growth plate. At the end of puberty, growth velocity diminishes and eventually ceases with the fusion of the growth plate through mechanisms that are not yet completely understood. Vascular endothelial growth factor (VEGF) has an important role in angiogenesis, but also in chondrocyte differentiation, chondrocyte survival, and the final stages of endochondral ossification. Estrogens have been shown to up-regulate VEGF expression in the uterus and bone of rats. In this study, we investigated the relation between estrogens and VEGF production in growth plate chondrocytes both in vivo and in vitro. The expression of VEGF protein was down-regulated upon ovariectomy and was restored upon estradiol (E2) supplementation in rat growth plates. In cultured rat chondrocyte cell line RCJ3.1C5.18, E2 dose dependently stimulated 121 and 189 kDa isoforms of VEGF, but not the 164 kDa isoform. Finally, VEGF expression was observed at both protein and mRNA levels in human growth plate specimens. The protein level increased during pubertal development, supporting a link between estrogens and local VEGF production in the growth plate. We conclude that estrogens regulate VEGF expression in the epiphyseal growth plate, although the precise role of VEGF in estrogen-mediated growth plate fusion remains to be clarified.
Lina Lawenius, Hannah Colldén, Karin Horkeby, Jianyao Wu, Louise Grahnemo, Liesbeth Vandenput, Claes Ohlsson, and Klara Sjögren
Studies in postmenopausal women and ovariectomized mice show that the probiotic mix Lacticaseibacillus paracasei DSM13434, Lactiplantibacillus plantarum DSM 15312 and DSM 15313 (L. Mix) can protect from bone loss caused by sex steroid deficiency. Whether probiotic bacteria can protect bone also in sex steroid-deficient males is less studied. We used the orchiectomized mouse as a model for age-dependent bone loss caused by decreasing sex hormone levels in males. We treated 10-week-old male mice with either vehicle (veh) or L. Mix for 6 weeks, starting 2 weeks before orchiectomy (orx) or sham surgery. Importantly, mice treated with L. Mix had a general increase in total body bone mineral density (BMD) and lean mass (P ≤ 0.05) compared with veh-treated mice. Detailed computer tomography analysis of dissected bones showed increased trabecular BMD of the distal metaphyseal region of the femur in L. Mix compared to veh-treated orx mice (+8.0%, P ≤ 0.05). In the vertebra, L. Mix treatment increased trabecular bone volume fraction BV/TV (+11.5%, P ≤ 0.05) compared to veh in orx mice. Also, L. Mix increased the levels of short-chain fatty acids (SCFAs) such as propionate and acetate and important intermediates in SCFA synthesis such as succinate and lactate in the cecal content of male mice. In conclusion, L. Mix treatment resulted in a general increase in BMD in adult male mice and prevented trabecular bone loss in femur and vertebra of orx mice. These bone protective effects of L. Mix were associated with increased levels of SCFAs in the cecal content of male mice.
Annica Andersson, Anna E Törnqvist, Sofia Moverare-Skrtic, Angelina I Bernardi, Helen H Farman, Pierre Chambon, Cecilia Engdahl, Marie K Lagerquist, Sara H Windahl, Hans Carlsten, Claes Ohlsson, and Ulrika Islander
Apart from the role of sex steroids in reproduction, sex steroids are also important regulators of the immune system. 17β-estradiol (E2) represses T and B cell development, but augments B cell function, possibly explaining the different nature of immune responses in men and women. Both E2 and selective estrogen receptors modulators (SERM) act via estrogen receptors (ER). Activating functions (AF)-1 and 2 of the ER bind to coregulators and thus influence target gene transcription and subsequent cellular response to ER activation. The importance of ERαAF-1 and AF-2 in the immunomodulatory effects of E2/SERM has previously not been reported. Thus, detailed studies of T and B lymphopoiesis were performed in ovariectomized E2-, lasofoxifene- or raloxifene-treated mice lacking either AF-1 or AF-2 domains of ERα, and their wild-type littermate controls. Immune cell phenotypes were analyzed with flow cytometry. All E2 and SERM-mediated inhibitory effects on thymus cellularity and thymic T cell development were clearly dependent on both ERαAFs. Interestingly, divergent roles of ERαAF-1 and ERαAF-2 in E2 and SERM-mediated modulation of bone marrow B lymphopoiesis were found. In contrast to E2, effects of lasofoxifene on early B cells did not require functional ERαAF-2, while ERαAF-1 was indispensable. Raloxifene reduced early B cells partly independent of both ERαAF-1 and ERαAF-2. Results from this study increase the understanding of the impact of ER modulation on the immune system, which can be useful in the clarification of the molecular actions of SERMs and in the development of new SERM.