Nuclear Binding of [125I]Triiodothyronine in Dispersed Cultured Skin Fibroblasts from Patients with Resistance to Thyroid Hormone*

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Abstract
To reexamine the possibility of a defect in nuclear receptors for T3 in patients with resistance to thyroid hormone, we have developed a method for measuring the nuclear uptake of [125I]T3 by intact dispersed fibroblasts grown from human skin. Fibroblasts were grown in serial passage, dispersed with mild trypsin digestion, and incubated at 37 C in serum-free medium with increasing concentrations of [125I]T3 in the presence or absence of unlabeled T3. After 60 min, cells were collected, and nuclei were isolated and counted for radioactivity. With fibrobiasts from eight normal subjects (four males and four females; age, neonate to 53 yr), the nuclear binding of [125I]T3 was saturable and demonstrated a single class of high affinity receptors, with a mean (±SE) binding capacity (Ro) of 3800 ± 500 nuclear sites/cell and a mean (±SE) apparent dissociation constant (Kd) of 1.0 ± 0.1 × 1O−10 M. Analysis of [125I]T3 nuclear binding in normal fibroblasts performed between passages 3–15 showed no significant variation in these parameters. Competition of nuclear [125I]T3 binding by thyroid hormone analogs showed affinity characteristics similar to those seen with nuclear T3 receptors in other tissues, such as rat liver. When fibroblasts from four patients from four different kindreds with thyroid hormone resistance [three males (age, 1 6/12 to 17 yr) and one female (age, 11.5 years)] were analyzed at multiple passages (3–12), no significant differences in the mean (±SE) characteristics of nuclear [125I]T3 binding were detectable (Ro = 5380 ± 650 sites/cell; Kd = 1.28 ± 0.17 × 10−10 M). However, the values for both nuclear T3 binding parameters from one patient's fibroblasts were above the normal range. We conclude that fibroblasts from three patients from three separate families with the syndrome of resistance to thyroid hormone have normal affinity and capacity for nuclear binding of [I25I]T3. The fourth patient's cells had a slight decrease in affinity (i.e. increased Kd) and a slightly elevated nuclear binding capacity for T3, but it is unclear whether these characteristics in nuclear T3 binding parameters are related to this patient's resistance to thyroid hormone. Assuming that these fibroblasts manifest resistance to thyroid hormone, these data suggest that the target organ defect in most cases of thyroid hormone resistance is not in the number or affinity of nuclear T3 receptors or in T3 cellular or nuclear transport mechanisms; the defect(s) may reside in nonnuclear T3 receptors, in postreceptor processes, or in qualitative properties of nuclear T3 receptors not demonstrated in conventional binding assays. Alternatively, it is possible that tissue resistance due to a nuclear receptor abnormality is present in these patients, but is not shared by cultured fibroblasts.