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Studies on the mechanism responsible for thyrotropin-induced expression of microsomal/peroxidase antigen in FRTL-5
L Chiovato1, P Vitti, A Lombardi
1Cattedra di Endocrinologia e Medicina Costituzionale, University of Pisa, Italy.
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.
Insights
Thyroid-stimulating hormone (TSH) regulates microsomal antigen expression in rat thyroid cells, identifying it as thyroid peroxidase (TPO). TSH
Area of Science:
- Endocrinology
- Molecular Biology
- Cell Biology
Background:
- Microsomal (M) antigen expression in FRTL-5 rat thyroid cells is regulated by TSH.
- Previous studies suggested a link between M antigen and thyroid peroxidase (TPO) in human thyroid tissue.
Purpose of the Study:
- To investigate the mechanism by which TSH induces M antigen expression in FRTL-5 cells.
- To determine if the M antigen in FRTL-5 cells is identical to TPO.
Main Methods:
- Indirect immunofluorescence technique using human microsomal antibody-positive serum.
- Preabsorption experiments with purified human TPO, solubilized human thyroid microsomes, and control tissues.
- TSH withdrawal and readdition experiments, with and without cycloheximide, actinomycin D, cholera toxin, forskolin, isobutylmethylxanthine, 8-bromo-cAMP, and 12-O-tetradecanoyl-phorbol 13-acetate.
Main Results:
- Preabsorption experiments confirmed that the M antigen recognized by microsomal antibody in FRTL-5 cells is TPO.
- TSH withdrawal led to the disappearance of M/TPO antigen, while readdition restored its expression.
- TSH-induced M/TPO antigen reappearance was dependent on mRNA and protein synthesis and mimicked by cAMP pathway activators, but not by a cAMP-independent tumor promoter.
Conclusions:
- Thyroid peroxidase accounts for the majority of the microsomal antigen in FRTL-5 cells.
- TSH modulates M/TPO antigen expression via a cAMP-dependent pathway requiring mRNA and protein synthesis.