The aim of the present study was to examine whether triiodo-L-thyronine (T 3 ) or L-thyroxine (T 4 ) rapidly activated the mitogen-activated protein kinase (MAPK) intracellular signalling cascade in osteoblast-like cells and investigate whether this activation was initiated at the integrin a V b 3 cell surface receptor. Using PCR and western blotting, the expression of integrin a V b 3 mRNA and protein was demonstrated in the human osteoblast-like cell lines MG-63 and SaOS-2. The treatment of MG-63 cells with T 3 (10 nM) or T 4 (100 nM) for 10 min stimulated extracellular signal-regulated kinase activity (ERK, a component of the MAPK pathway) as determined by fluorescent immunocytochemistry and an immunocomplex activity assay (T 3 by 10 . 7-fold, P!0 . 01 and T 4 by 10 . 4-fold, P!0 . 01 compared with control). T 3 (10 nM) and T 4 (100 nM) also significantly stimulated thymidine incorporation into MG-63 cells by 2 . 3G0 . 7-fold (P!0 . 01) and 2 . 1G0 . 1-fold (P!0 . 05) respectively. To establish whether transient ERK activation via the integrin a V b 3 cell surface receptor mediated these effects, MG-63 cells were pretreated for 30 min with the specific MAPK kinase inhibitor, U0126 (1 mM), or an antiintegrin a V b 3 -blocking antibody. Both pretreatments significantly inhibited T 3 -and T 4 -stimulated ERK activation and abolished T 3 -stimulated thymidine incorporation (P!0 . 01).T 4 -stimulated incorporation was significantly inhibited from 2 . 1-to 1 . 3-fold above control (P!0 . 05). Thus, our results suggest that T 3 and T 4 rapidly stimulate ERK activation in MG-63 cells via integrin a V b 3 and that one functional effect of this ERK activation is increased DNA synthesis.
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 T(3)-binding TR isoforms are TRalpha1, TRbeta1, and TRbeta2. 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. TRalpha1, TRbeta1, and TRbeta2 proteins were expressed in all cells, although expression was greatest in MG63 > hBMS > SaOs-2 > hOb. Differences between isoforms were also apparent, with TRalpha1> TRbeta1 > TRbeta2 in all cell types. Incubation with [(125)I]T(3) confirmed reversible T(3) 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 T(3) treatment increased luciferase activity 5.5 +/- 0.7-fold (P < 0.05), confirming the presence of endogenous receptors. In SaOs-2 and hOb cells, T(3) 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 TRalpha1, TRbeta1, and TRbeta2 in hOb and hBMS cells remains to be shown.
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