Thyroid hormones (TH; T₄ and T₃) promote normal growth and development and also play a key role in regulation of many metabolic processes. TH exert their effects predominantly at a transcriptional level through binding to thyroid receptors (TR) in the cell nucleus (Yen, 2001). There is evidence (Larsen et al. 1981; Henneman et al. 2001) to suggest that TH transported into cells are targeted preferentially if not directly to the nucleus, rather than entering a cytoplasmic pool. Zhu et al. (1998) demonstrated that TR in the cytosol may translocate to the nucleus after TH stimulation, providing a mechanism for both selective delivery of TH to the nucleus and regulation of TR function by T₃-induced nuclear entry. We hypothesised that such a translocation mechanism might involve trafficking of TR along cytoskeletal elements and have therefore investigated the effect of disrupting the cytoskeleton on nuclear appearance of T₃ from the extracellular medium in human cell lines.

Initial experiments were performed using monolayer cultures of SHSY5Y human neuroblastoma cells. Cells were pre-incubated with cytoskeleton-disrupting drugs cytochalasin D (2 mM) or colchicine (10 mM) for 20 min. Cells were then incubated with 1 nM ¹²⁵I-T₃ for 30 min (initial-rate uptake conditions) followed by rapid processing into nuclear and cytosolic fractions using a Nuclei EZ-Prep kit (Sigma). Total exposure of cells to drug was therefore 50 min. Cellular uptake of T₃ (0.28 ± 0.04 pmol (mg protein)^-1 (30 min)^-1; mean ± S.D.) was unaffected by drugs and the ratio nuclear ¹²⁵I-T₃/cytoplasmic ¹²⁵I-T₃ remained at 0.045 ± 0.006 under all experimental conditions (n > 3 separate experiments). Similar results were obtained with BeWo human choriocarcinoma cells. We also investigated the effects of 20 min pre-incubation (50 min total exposure) with vesicular trafficking inhibitors on nuclear delivery of ¹²⁵I-T₃. Neither the endocytosis inhibitor monodansylcadaverine (50 mM) nor the vesicle recycling inhibitor chloroquine (100 mM) significantly reduced nuclear/cytoplasmic ¹²⁵I-T₃ ratio in either SHSY5Y or BeWo cells, although chloroquine treatment did significantly reduce total cellular ¹²⁵I-T₃ uptake to 50 % of control value in both cell types.

These results do not support the idea that TH delivery to cell nuclei involves trafficking along an intact cytoskeleton. The effect of chloroquine on cellular T₃ uptake may reflect inhibition of TH transporter recycling and/or recruitment at the plasma membrane.