Endocrine Abstracts

T3 and fibroblast growth factors (FGFs) are critical regulators of chondrocyte proliferation during bone formation and we identified recently that T3 modulates skeletal FGF receptor-1 (FGFR1) activity. FGFRs signal via several routes including mitogen-activated protein kinase (MAPK), STAT and phospholipase C gamma pathways. Mechanisms determining which pathways are activated by FGFRs are poorly understood but include alternative splicing of exons encoding variable domains within the FGFR extracellular region. We investigated T3 effects on FGFR mRNA splicing and FGF2-stimulated MAPK activity in differentiating chondrogenic ATDC5 cells. RT-PCR of the region spanning immunoglobulin domain-1 and the acid box (Ig1/AB) of the extracellular region can detect four mRNA isoforms for each FGFR and analysis of immunoglobulin domain-3 and the trans-membrane (Ig3/TM) region can detect two additional variants. Two FGFR1, four FGFR2 and one FGFR3 Ig1/AB isoforms were identified and their expression increased as ATDC5 cell differentiation progressed. A single Ig3/TM isoform was identified for each FGFR and expression of only the FGFR3 mRNA increased as cells differentiated. T3 (100nM, 6h, culture days 6, 12, 21 and 28) did not alter expression of any FGFR1 isoform or the FGFR2 Ig1/AB mRNAs, but stimulated expression of the FGFR2 Ig3/TM variant and each of the FGFR3 Ig1/AB and Ig3/TM mRNA isoforms. In experiments to examine FGF responsiveness, cells were cultured in the absence or presence of T3 (100nM, 6 days) and then treated with FGF2 (5ng/ml, 0-30min). FGF2-stimulated activation of MAPK was increased 2.5-fold in T3-treated cells. In contrast, when cells were cultured in the absence of hormone and then treated with T3 for 6 hours, FGF2-stimulated activation of MAPK was decreased 1.75-fold, demonstrating that effects of T3 on FGF signalling depend on the duration of T3 treatment. These data indicate that control of FGF signalling in bone involves gene-specific T3-regulated expression of FGFRs.