Studies on the Mechanism Responsible for Thyrotropin Induced Expression of Microsomal/Peroxidase Antigen in FRTL-5 Cells*

Abstract

The expression of the microsomal (M) antigen on the surface and in the cytoplasm of a strain of rat thyroid cells (FRTL-5) is under the regulation of TSH. In the present report the mechanism by which TSH induces such expression was investigated with the use of human microsomal antibody-positive serum and an indirect immunofluorescence technique. Studies were also performed to ascertain whether the M antigen of FRTL-5 cells could be identified with thyroid peroxidase (TPO), as suggested by recent data obtained in human thyroid tissue. Preabsorption experiments showed that, like solubilized human thyroid microsomes, purified human TPO completely abolished the binding of microsomal antibody to FRTL-5 cells. No inhibition was obtained by preabsorption with control human tissues (placenta, liver, and spleen) or human thyroglobulin, indicating that the antigen recognized by microsomal antibody in FRTL-5 cells was TPO. After 72 h of TSH withdrawal from the culture medium the M/TPO antigen disappeared from the surface and the cytoplasm of FRTL-5 cells. Readdition of TSH (250 microU/ml) to the culture medium of cells lacking the M/TPO antigen elicited its reappearance within 24-48 h. This effect of TSH was prevented by 10 microM cycloheximide or 0.5-5 micrograms/ml actinomycin D. Two well known stimulators of the adenylate cyclase-cAMP system, cholera toxin and forskolin, mimicked TSH in inducing the reappearance of the M/TPO antigen. A similar effect was observed with use of the phosphodiesterase inhibitor isobutylmethylxanthine. Reappearance of M/TPO antigen was also produced by the cAMP analog 8-bromo-cAMP. The tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate, which stimulates thyroid cell growth through a cAMP-independent pathway, was ineffective in inducing the M/TPO antigen in FRTL-5 cells. The present data indicate that 1) thyroid peroxidase accounts for most, if not all, of the microsomal antigen of FRTL-5 cells; and 2) TSH modulates the expression of the M/TPO antigen in FRTL-5 cells by a mechanism that involves cAMP production and requires mRNA formation and subsequent protein synthesis.

In Vivo Effect of Thyrotropin on Intracellular Translocation of Thyroid Peroxidase in Rat Thyroid Cells by an Indirect Immunofluorescence Method.

“…The elimination of TSH from the culture medium of FRTL-5 resulted in the disappearance of TPO antigens from both the surface and cytoplasm of the cells within 72 h [8]. These results exhibit a striking contrast to the present experimental results which show that TPO stainability was not diminished by suppression of TSH secretion after T4 administration even for 20 days, although its location in the cytoplasm was affected.…”

“…The translocation was found to occur as early as 6 h after TSH injection. According to previous papers, the TSH-induced increase in TPO mRNA [18], TPO protein [8] and TPO activity [ 10,11] showed a lag time of more than 8-10 h in cultured pig thyroid cells. Thus, the TSH effect mentioned above seems to indicate the stimulation of translocation of preexisting TPO in the cytoplasm soon after TSH injection, although other possibilities, such as a change in the immunochemical stainability of the enzyme, cannot be completely excluded.…”

“…In addition to these intracellular organelles, some investigators demonstrated the presence of the enzyme on the external surface of microvilli [1][2][3], but the enzyme activity in such an area in normal rat [4], normal porcine [5] and normal human [6] thyroids seemed to be comparatively low. On the other hand, it is generally agreed that the peroxidase activity in microvilli and apical vesicles is stimulated by endogenous and exogenous TSH [2,4,7,8] and that these enzymes participate in the stimulated thyroid hormone formation in hyperthyroids.Recently, it has been reported that the biosynthesis of TPO mRNA in FRTL cells [9] and TPO activity in primary cultured porcine thyroid cells [10,11] are stimulated by incubation with TSH. Thus, increased synthesis of TPO and subsequent transport of the enzyme to the apical region are considered to be one of the TSH-dependent functions of thyroids.…”

“…In addition to these intracellular organelles, some investigators demonstrated the presence of the enzyme on the external surface of microvilli [1][2][3], but the enzyme activity in such an area in normal rat [4], normal porcine [5] and normal human [6] thyroids seemed to be comparatively low. On the other hand, it is generally agreed that the peroxidase activity in microvilli and apical vesicles is stimulated by endogenous and exogenous TSH [2,4,7,8] and that these enzymes participate in the stimulated thyroid hormone formation in hyperthyroids.…”

Cell Type-dependent Differences in Thyroid Peroxidase Cell Surface Expression

“…PC Cl3 cells are a rat thyrocyte line similar in origin to FRTL5 cells that contain endogenous rTPO (20,44) and have served as a suitable expression system for hTPO (45). When probed by Western blotting, there was no detectable hTPO cross-reactivity in untransfected PC Cl3 cells (Fig.…”