Endocrine Abstracts

T3 is essential for skeletal development and T3-actions in bone are mediated mainly by the nuclear receptor TRα1. We previously identified that FGFR1 is a T3-responsive gene in osteoblasts by subtraction hybridization. In mice that harbor a dominant negative mutation PV, FGFR1 mRNA expression is reduced in TRα1^PV mutants that exhibit skeletal hypothyroidism and increased in TRβ^PV/PV mice with skeletal thyrotoxicosis. In this study, primary calvarial osteoblasts from wild-type and TRβ^PV/PV littermates were cultured in the absence and presence of T3. Quantitative RT-PCR revealed FGFR1 mRNA expression was increased 25-fold in TRβ^PV/PV osteoblasts relative to wild-type cells maintained in T3, but elevated only 2.3-fold in the absence of T3. These data indicate that, although FGFR1 mRNA expression is increased in TRβ^PV/PV osteoblasts in the absence and presence of hormone, the mutant cells remain exquisitely sensitive to T3, demonstrating that T3-action in TRβ^PV/PV osteoblasts is mediated via the unaffected TRα1 protein. We next performed electrophoretic mobility shift assays to identify and characterize a TRE between positions −279/−264 of the Fgfr1 promoter that showed strong and specific binding with TR/RXR heterodimers but weaker specific binding with TR/TR homodimers. Mutational analysis of the putative binding site attenuated TR/RXR and TR/TR specific binding and confirmed the identity of the TRE, which consisted of a classical direct repeat-4 (DR4) motif. We further performed transient transfection studies using T3-treated CV-1 cells co-transfected with TRα1 or TRβ1 and a range of CAT-reporter gene constructs linked to 5′-deletion mutants of the Fgfr1 gene 5′-flanking region. Initial results indicate that truncation of the Fgfr1 5′-flanking region containing the putative TRE leads to a loss of T3-responsiveness without affecting basal promoter activity. These data indicate that the effects of T3 in the skeleton are mediated, at least in part, via TRα1-dependent transcriptional regulation of FGFR1 expression.