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Identification of Target Cells for the Genomic Effects of Estrogens in Bone

Center for Bone Research, Departments of Internal Medicine (S.H.W., M.K.L., N.A., C.O.) and Rheumatology and Inflammation Research (C.J., A.K., C.H., H.C.), Institute of Medicine, Gothenburg University, 413 45 Gothenburg, Sweden; Department of Biosciences and Nutrition at NOVUM (J.I., J.-Å.G., K.P.), Karolinska Institute, SE-141 04 Huddinge, Sweden; and Hubrecht Laboratory (P.T.v.d.S.), Netherlands Institute for Developmental Biology, 3584 CT Utrecht, The Netherlands

Address all correspondence and requests for reprints to: Claes Ohlsson, Department of Internal Medicine, Division of Endocrinology, Gröna Stråket 8, 413 45 Gothenburg, Sweden. E-mail: claes.ohlsson{at}medic.gu.se.

Estrogen has bone protective effects, but the exact mechanism behind these effects remains unclear. The aim of the present study was to identify the primary target cells in bone for the classical genomic effects of estrogens in vivo. For this purpose we have used reporter mice with a luciferase gene under the control of three estrogen-responsive elements (EREs), enabling detection of in vivo activation of gene transcription. Three-month-old ovariectomized mice were treated with a single dose (50 µg/kg) 17ß-estradiol (E2). Luciferase activity was analyzed in several tissues and in different bone marrow-derived lymphocyte enriched/depleted preparations using MacsMouse CD19 (for B lymphocytes) or CD90 (for T lymphocytes) MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Histological characterization of cells with high luciferase content was performed using immunohistochemistry. Both cortical bone and bone marrow displayed a rapid (within 1 h) and pronounced E2-induced increase in luciferase activity. The luciferase activity in total bone marrow and in bone marrow depleted of lymphocytes was increased six to eight times more than in either B-lymphocyte or T-lymphocyte enriched cell fractions 4 h after the E2 injection, demonstrating that mature lymphocytes are not major direct targets for the genomic effect of estrogens in bone. Immunohistochemistry identified clear luciferase staining in hypertrophic growth plate chondrocytes, megakaryocytes, osteoblasts, and lining cells, whereas no staining was seen in proliferative chondrocyte. Although most of the osteocytes did not display any detectable luciferase staining, a subpopulation of osteocytes both in cortical and trabecular bone stained positive for luciferase. In conclusion, hypertrophic growth plate chondrocytes, megakaryocytes, osteoblasts, lining cells, and a subpopulation of osteocytes were identified to respond to estrogen via the classical ERE-mediated genomic pathway in bone. Furthermore, our findings indicate that possible direct estrogenic effects on the majority of osteocytes, not staining positive for luciferase, on proliferative chondrocytes and on mature lymphocytes are mediated by non-ERE actions.

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