A cDNA clone encoding a human cytosolic thyroid hormone-binding protein (p58) has been isolated. The human sequence was found to be homologous to that of rat pyruvate kinase (EC 2.7.1.40) subtype M2. p58 is a monomer that has --5% the enzymatic activity of the tetrameric pyruvate kinase M2. The tetrameric M2 does not bind 3,3',5-triiodo-L-thyronine (T3). Binding of p58 to T3 and its analogs resulted in the inhibition of its pyruvate kinase activity. The apparent K; values of T3, L-thyroxine, and D-T3 are 30 aM, 100 nM, and 2 mM, respectively. L-Thyronine and 3,3',5'-triiodo-L-thyronine had no effect. This order of activity correlates with the thermogenic effects reported for T3 and its analogs. Conversion of p58 to the tetramer is reversible and is under the control of fructose 1,6-bisphosphate. The conversion is inhibited by T3 in a dose-dependent manner. Since pyruvate kinase is a key enzyme in regulating cellular ADP, ATP, and pyruvate, our findings suggest that p58 may be involved in mediating some of the cellular metabolic effects induced by thyroid hormones.The thyroid hormone 3,3',5-triiodo-L-thyronine (T3) plays an essential role in maintaining fetal and neonatal development, regulating amino acid and electrolyte transport into the cell, modulating carbohydrate, protein, and lipid metabolism, and increasing the rate of oxidative phosphorylation. No single mechanism has been found to account for all the diverse actions of-thyroid hormones.Some of the thyroid hormone effects are initiated through the interaction of T3 with nuclear receptors (see reviews in refs. 1 and 2). However, some ofthe effects of T3, such as the enhancement of enzymatic activity (3,4) and cellular amino acid accumulation and ATP synthesis (5) that occur in the absence of protein synthesis, have to be accounted for by alternative mechanism(s). Therefore, extranuclear T3 binding sites that might mediate such activities have been sought. Cheng and coworkers (6) purified a cytosolic thyroid hormone-binding protein (p58) to homogeneity. The purified protein retains its T3-binding activity and specificity (6), and two monoclonal antibodies against p58 have been developed (7). p58 has an apparent molecular weight of 60,000 by gel filtration and 58,000 by SDS/PAGE. To study its cellular function and the roles it plays in T3 action, we isolated and sequenced the cDNA that encodes p58A We found that p58 is a monomer of pyruvate kinase (ATP:pyruvate 02-phosphotransferase, EC 2.7.1.40) subtype M2 and that its conversion to the tetrameric pyruvate kinase is regulated by fructose 1,6-bisphosphate (Fru-1,6-P2).

The expression of surface markers associated with activation and characterization was compared among T cells in thyroid glands and peripheral blood of 10 patients with Graves' hyperthyroidism receiving chronic antithyroid drug therapy, in peripheral blood of 15 patients with untreated hyperthyroid Graves' disease, and in peripheral blood of 21 normal subjects using two-color flow cytometry. In the chronically treated Graves' disease patients, the percentage of activated T cells (HLA-DR+ T cells) among total T cells was significantly higher in thyroid tissue than in peripheral blood, and the increase in percent activated T cells was also significant among both helper/inducer T cell (CD4+ cell) and suppressor/cytotoxic T cell (CD8+ cell) subsets. The percentage of activated T cells in peripheral blood was not significantly different between chronically treated hyperthyroid Graves' patients and normal subjects, whereas the percentage of activated T cells in the peripheral blood from untreated hyperthyroid Graves' disease patients was significantly higher than that in normal subjects or chronically treated hyperthyroid Graves' patients. The percentages of CD4+ cells and CD8+ cells among total T cells were not different between thyroid tissues and peripheral blood in patients with chronically treated hyperthyroid Graves' disease. When CD4+ were further divided into helper T cells (CD4+2H4- cells) and suppressor-inducer T cells (CD4+2H4+ cells) using two-color flow cytometry, the percentage of helper T cells among CD4+ cells was significantly higher in thyroid tissue than in peripheral blood, resulting in an increased ratio of CD4+2H4- cells to CD4+2H4+ cells. The percentage of CD4+2H4+ cells in peripheral blood, however, was not significantly different among untreated and chronically treated Graves' disease patients and normal subjects. From the findings of abnormalities in intrathyroidal T cell subsets, we suggest that the decrease in the function of suppressor T cells within the thyroids of Graves' disease patients may be due to a decrease in CD4+2H4+ cells within thyroid tissue.

Class II major histocompatibility complex (MHC) antigens have been demonstrated on the surface of thyroid epithelial cells (thyrocytes) from patients with autoimmune thyroid disease. The present study was designed to investigate how the expression of class II MHC antigens is involved in autoimmune processes in Graves' disease by studying cellular interactions among thyrocytes, lymphocytes within thyroid glands (TG), and peripheral blood (PB) lymphocytes. Thyrocytes were prepared by collagenase digestion, and T or non-T cells were separated by E-rosette formation. Thyrocytes were cocultured in the presence or absence of interferon-gamma, and the expression of HLA-DR antigens on cultured thyrocytes was examined by an indirect immunofluorescence method using monoclonal anti-HLA-DR antibody and monoclonal anti-HLA-DQ antibody. The cellular interactions were assessed as the proliferative response of T cells to autologous stimulators, such as thyrocytes or lymphocytes. Expression of HLA-DR antigens on thyrocytes after culture for 18 h in the absence of interferon-gamma was found in two thirds of the patients with Graves' disease studied (n = 18). Interferon-gamma induced and maintained the expression of HLA-DR antigens on thyrocytes. The percentages of HLA-DR+T cells were significantly higher among TG-T cells than among PB-T cells [32.6 +/- 12.4% (+/- SD) vs. 12.2 +/-5.0%; n = 18; P less than 0.01]. Thyrocytes from Graves' patients induced proliferation of both autologous PB-T cells and TG-T cells, and TG-T cells stimulated proliferation of autologous PB-T cells. In conclusion, interferon-gamma induces HLA-DR antigen expression on thyrocytes from patients with Graves' disease, and these cells induce proliferation of autologous T cells, which may, in turn, act on thyrocytes to perpetuate the process.