Tomoaki Mitsuhashi

DNA binding and interaction with the nuclear receptor corepressor of thyroid hormone receptor are required for ligand-independent stimulation of the mouse preprothyrotropin-releasing hormone gene.

A negative thyroid hormone response element (TRE) in the mouse preprothyrotropin-releasing hormone (TRH) gene was previously mapped within the proximal promoter element between -83 and +53 that contained a TRE half-site motif at -57 (-57TGACCT-51). In transfection experiments, the promoter activity is stimulated by unliganded thyroid hormone receptor (TR) and T3 reverses the basal promoter stimulation. In this study, we determined whether the direct binding of TR to the TRE half-site in the mouse TRH gene is required for the ligand-independent stimulation using a transient transfection assay into CV-1 cells and electrophoretic mobility shift assays (EMSA). In addition, the role of a corepressor protein for the ligand-independent stimulation was examined using a putative splicing variant of the nuclear receptor corepressor (N-CoRI). Point mutations introduced into the TRE half-site at -57 eliminated the binding of TR and the stimulatory effect of unliganded TR. Two mutant TRs lacking DNA-binding activity and two CoR box mutant TRs showed no stimulation in the wild-type TRH promoter. The cotransfected N-CoRI potentiated the ligand-independent stimulation by the wild-type TR, but did not compensate for the impaired function of the CoR box mutant TR. In EMSA, TR strongly bound as homodimers and weakly as heterodimers with retinoid X receptor (RXR) to the element containing the TRE half-site at -57. Binding of TR to the TRE half-site was essential to form homo- and heterodimers, and the RXR binding site appeared to be located downstream of the TRE half-site. In vitro translated N-CoRI preferentially bound TR homodimers over TR/RXR heterodimers. These results collectively suggest that the DNA-bound TR/corepressor complex might be directly involved in the ligand-independent stimulation of the mouse TRH gene promoter.

Developmental expression of mRNAs from a rat C-erbA genomic locus.

The thyroid hormone receptor alpha gene is alternatively spliced to give the alpha receptor and a variant which does not bind thyroid hormones. An additional alpha-like receptor (Rev-erbA alpha) is transcribed on the opposite strand and overlaps the unique variant sequence. The alpha receptor in rat brain reaches peak levels 5-15 days after birth. The variant remains high from 2 days before birth to old age. The Rev-erbA alpha mRNA increases beginning 5 days after birth, maximizing in adulthood. Thyroid hormone decreases and hypothyroidism increases the level of these 3 messages. We conclude that the levels of these messages are similarly controlled by thyroid hormone but have different ontogenetic controls. No functional results can be seen in vivo from potential anti sense pairing of Rev-erbA alpha with the variant.

In vivo activation of the constitutive androstane receptor β (CARβ) by treatment with dehydroepiandrosterone (DHEA) or DHEA sulfate (DHEA-S)

We investigated whether dehydroepiandrosterone (DHEA) or DHEA‐sulfate (S) affected the activities of nuclear receptors, with special reference to constitutive androstane receptor β (CARβ). Administration of DHEA or DHEA‐S enhanced the DNA binding of hepatic nuclear extracts to responsive elements for the retinoic acid receptor, the retinoic acid receptor β 2 and the peroxisome proliferator activated receptor. The bound complexes were shown to be the CARβ‐RXR heterodimer by antibody‐supershift assays. The expression of a target gene of CARβ, Cyp2b10, was increased in liver by DHEA or DHEA‐S treatment, suggesting that DHEA or DHEA‐S actually activated CARβ in vivo. It was suggested that the metabolic conversion of DHEA, DHEA‐S to CARβ ligands could occur in vivo and the metabolites could regulate the expression of CARβ target gene expression. Our results provide new insights into the in vivo relationship between DHEA/DHEA‐S and CARβ activation.

Changes in Thyrotropin Binding Inhibiting Immunoglobulins (TBII) in sera of patients with Graves’ disease at the time of relapse or exacerbation

Thyrotropin Binding Inhibiting Immunoglobulins (TBII) were measured in sera of 240 patients with Graves’ disease who were followed 0–25 yr as a cross-sectioned study (21 untreated, 189 under therapy and 30 T3-suppressible and drug-discontinued patients) by using solubilized porcine thyroid TSH receptor. Assays were performed by using 50 μl of serum. All untreated 21 patients showed positive TBII. Frequency of positive patients decreased yearly with treatment although 36% of patients remained positive after 6 yr of therapy. After that time TBII were positive in 61 % of follow-up patients and in 16 positive patients who have been treated for more than 10 yr, drug therapy could not be stopped because of recurrence. TBII were positive in 6 of 30 T3-suppressible patients. As a longitudinal study changes in TBII were studied in 10 patients at the time of relapse or exacerbation. TBII increased in parallel with increases in thyroid hormone concentrations in 3 of 10 patients. Six of the others showed earlier or later TBII increases than those in thyroid hormones. One patient did not show any change in TBII, albeit thyroid hormone concentrations were found to be increased. Our observations suggest that abnormal IgGs detected as TBII in sera of patients with Graves’ disease by the present method do not explain the occurrence of hyperthyroidism.