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TR Expression and Function in Human Bone Marrow Stromal and Osteoblast-Like Cells

Department of Endocrinology, St. Bartholomew’s and the Royal London School of Medicine and Dentistry (A.S., M.P.P., J.P.M., J.M.B.), West Smithfield, London, United Kingdom EC1A 7BE; and Department of Hematology, LRF Center for Adult Leukemia (J.L.L.), and ICSM Molecular Endocrinology Group, Division of Medicine and Medical Research Council Clinical Sciences Center (G.R.W.), Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom W12 0NN

Address all correspondence and requests for reprints to: Dr. Ayesha Siddiqi, Molecular Endocrinology Laboratory 1.4, First Floor Dominion House, 59-60 Bartholomew Close, London, United Kingdom EC1A 7BE. E-mail: asiddiqi{at}mds.qmw.ac.uk

Abstract

Thyroid hormones influence both bone formation and bone resorption. In vitro studies demonstrate direct effects of thyroid hormones on cells of the osteoblast lineage. Transcriptional regulation by thyroid hormones is mediated by ligand-dependent transcription factors called TRs. The three main T₃-binding TR isoforms are TR1, TRß1, and TRß2. TRs have been identified in cells of the osteoblast lineage, but it is still not known whether TR isoform expression differs in primary cultures of human osteoblasts.

We used immunocytochemistry, Western blotting, nuclear binding assays, and transient transfection studies to examine the expression of functional TR isoforms in primary cultures of osteoblasts (hOb) derived from explants of trabecular bone, in human bone marrow stromal cells (hBMS), which are believed to be the source of osteoblast progenitor cells, and for comparison in the transformed human osteosarcoma cell lines MG63 and SaOs-2. TR1, TRß1, and TRß2 proteins were expressed in all cells, although expression was greatest in MG63 > hBMS > SaOs-2 > hOb. Differences between isoforms were also apparent, with TR1> TRß1 > TRß2 in all cell types. Incubation with [¹²⁵I]T₃ confirmed reversible T₃ binding to cell nuclei. Specific binding was greatest in MG63 > hBMS > SaOs-2 > hOb. Finally, endogenous TR activity was determined in transfections using a thyroid hormone response element derived from the rat GH gene linked to the luciferase reporter gene. In MG63 and hBMS cells T₃ treatment increased luciferase activity 5.5 ± 0.7-fold (P < 0.05), confirming the presence of endogenous receptors. In SaOs-2 and hOb cells, T₃ treatment had no effect on thyroid hormone response element-thymidine kinase-luciferase expression, suggesting that in these cells TR expression was too low to be detected.

These results indicate that three main TR isoforms are expressed in cells of the human osteoblast lineage, but that expression and endogenous TR activity are predominantly present in hBMS cells. Whether there are distinct mechanisms of thyroid hormone action mediated by TR1, TRß1, and TRß2 in hOb and hBMS cells remains to be shown.

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