Tetsuya Tagami

A point mutation of the T3 receptor β1 gene in a kindred of generalized resistance to thyroid hormone

Mutations of the thyroid hormone receptor (TR) beta 1 gene have recently been detected in several unrelated families with generalized resistance to thyroid hormone (GRTH). We now report a novel point mutation in the TR beta 1 gene in a case of a Korean-Japanese kindred. The intracellular localization and the amount of TR proteins were considered to be normal by the immunocytochemical study of cultured skin fibroblasts from the patients using anti-T3 receptor antibody. The cDNA of the T3-binding domain of the TR beta 1 gene, synthesized from the total RNA of the patients fibroblasts, was amplified by the polymerase chain reaction, and was sequenced. A point mutation, A to G, in one allele at 1612 resulting in an amino acid substitution from lysine 438 to glutamic acid was detected. The same mutation was identified in one allele in each of the affected members. In vitro translation products of the mutant TR beta 1 gene showed decreased T3-binding activity. These data suggest that a TR mutation is predominantly responsible for GRTH, irrespective of ethnic background.

Comparison of the functional properties of three different truncated thyroid hormone receptors identified in subjects with resistance to thyroid hormone.

The tau4 domain in the extreme carboxyl (C) terminal region of thyroid hormone receptor (TR) is important to transactivation. We identified three truncated TRbeta1s with 11 (F451X), 13 (E449X) and 16 (C446X) amino acid deletions within this domain in subjects with resistance to thyroid hormone (RTH). F451X and C446X were found in a 6-year-old Japanese girl and a 31-year-old American male, respectively, who had both severe mental retardation. E449X was identified in a 16-year-old Japanese boy with no remarkable clinical symptoms except for goiter. Transient expression study revealed that all three mutants had negligible T3 binding and transcriptional activities. Each mutant TRbeta1 exhibited not only very strong dominant negative activity against wild TRbeta1, but also marked silencing activity. Interestingly, the dominant negative activity and silencing activity were significantly stronger in F451X than in E449X and C446X (P < 0.05). Gel-shift experiments revealed no apparent differences in homodimer formations of wild-type or mutant TRbeta1 proteins and in heterodimer formations with retinoid X receptor (RXR). These observations indicate that the tau4 domain affects diverse TR functions, and that the region between 11 and 13 C-terminal amino acids influences ligand-independent TR functions, including dominant negative and silencing activities. The central nervous system involvement is not necessarily determined by the dominant negative potency of the mutant TRbeta1 and other environmental or genetic factors may influence the RTH manifestations.

Functional properties of a mutant T3 receptor β (R338W) identified in a subject with pituitary resistance to thyroid hormone

Previously, we identified a point mutation of the T3 receptor (TR) beta gene (R338W) in a patient with pituitary resistance to thyroid hormone (PRTH). The mutation existed in one of two hot spot areas in TRbeta gene where clusters of mutations have been found in subjects with generalized resistance to thyroid hormone (GRTH). Interestingly, R338W induces the phenotypical features responsible for PRTH. In the present study, we examined the functional properties of R338W in comparison with those of a GRTH-mutant, K443E. The levels of thyroid hormones and inappropriately elevated TSH (SITSH) were similar between subjects with K443E and R338W. Transcriptional activities and dominant negatives potencies were measured by CAT assay in CV1 cells transfected with each mutant TRbeta1 or along with wild-type TR. When a reporter gene containing T3-responsive elements (TRE), TRE-pal2, DR4 or myosin heavy chain alpha subunit, was used, transcriptional activation induced by R338W was higher than that by K443E. At 50 nM T3, K443E decreased the transcriptional activity of wild-type TRbeta1 on TRE-pal2 by 31.5%, while R338W reduced by 13.6% (n = 15, P < 0.05). Co-expression of retinoid X receptor (RXR) alpha increased transcriptional activity of R338W and K443E, but not of wild-type TRbeta1. Dominant negative activity on TRE-TSHalpha subunit of R338W was milder than that of K443E. When T3-binding activities of mutant TRbeta1s expressed in the cells were assayed under the same cell conditions for CAT assay, both mutant TRbeta1 showed remarkably reduced activity with no difference between the two. Gel mobility shift assay using TRE-DR4 showed poor homodimer formation of R338W. Heterodimerization with RXRalpha was similar between R338W, K443E and wild-type TRbeta1. The result of the present study suggested that R338W had relatively mild transcriptional and dominant negative activities on several TREs including TRE-TSHalpha subunit. We also showed poor homodimerization of R338W, which might be related to its weak dominant negative potency.

Recognition of rat liver and kidney nuclear T3 receptors by an antibody against c-erb A peptide

It has been reported that c-erb A encodes nuclear T3 receptors (NT3R). Based on the sequence of c-erb A cDNA, we synthesized a polypeptide consisting of 15 amino acids, the sequence of which has high homology between c-erb A alpha 1 and beta. The antibody against this c-erb A peptide not only immunoprecipitated rat liver and kidney NT3R but also inhibited T3 binding to NT3R. In a displacement study, the inhibition of [125I]T3-binding by the antibody was parallel to that by T3 in terms of the concentration of the competitor added in the incubation mixture. Scatchard analysis revealed that the antibody decreased the value for the association constant in a dose dependent manner. The antibody did not bind T3 itself. The results show that the antibody against c-erb A peptide recognizes rat liver and kidney NT3R and that the sequence encoding this peptide, the closest carboxyl-terminal of c-erb A may be critical or at least closely related to the hormone binding.

3,5,3'-Triiodothyronine stimulates retinoic acid-induced differentiation in HL-60 cells

Abstract The human acute promyelocytic leukemia (APL) cell line HL-60 differentiates to functionally mature granulocytes by incubation with all- trans -retinoic acid (RA). Since T3 and RA are important in cell differentiation and development, and since their receptors are highly homological, we investigated the T3 effects on RA-induced HL-60 cell differentiation. Although T3 alone did not induce cell differentiation, RA-mediated differentiation was significantly enhanced in the presence of 10 −7 M T3. This effect of T3 was considered to be mediated, at least in part, by increased intracellular cAMP, since the phosphodiesterase inhibitor enhanced, and the protein kinase A antagonist partially blocked, T3 potentiation. When HL-60 cells were pretreated with RA for 20 h, T3 alone stimulated the cell differentiation. The time-course study showed that incubation with RA for 12 h was necessary for HL-60 cells to be primed to respond to T3 for differentiation. The present finding that T3 potentiates RA-induced HL-60 cell differentiation may raise the possibility that T3 supplement increases clinical remission in APL patients who are treated with RA.

Starvation-induced decrease in the maximal binding capacity for triiodothyronine of the thyroid hormone receptor is due to a decrease in the receptor protein.

Biological responses to thyroid hormones are mediated by the nuclear thyroid hormone receptor (TR). Alterations in the maximal triiodothyronine (T3)-binding capacity (Cmax) of TR measured using a ligand binding assay have been reported under some pathophysiological conditions. Northern blot analysis has indicated that TR mRNA concentrations do not necessarily correlate with Cmax levels. For example, although the decrease in Cmax in rat liver induced by prolonged fasting is well established, TR mRNA concentrations have been reported to be constant. In the present study, we examined starvation-induced changes in TR by Western blot with anti-TR(alpha 1 + beta)antiserum and by Scatchard plot analysis. Starvation of rats for 72 hours decreased Cmax in the liver to 72.5% of control levels. The 47- and 55-kd TR proteins detected in hepatic nuclear extract by Western blotting also decreased to 64% and 66% of control values, respectively. The starvation-induced changes in Cmax and TR protein levels paralleled the change in total hepatic nuclear protein concentration. These results suggest that the decrease in T3-binding activity of the TR is due to a reduction of the TR protein itself.