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Thyrotropin Stimulation of Cultured Thyroid Cells Increases Steady State Levels of the Messenger Ribonucleic Acid for Thyroid Peroxidase

Departments of Medicine, University of California-San Francisco San Francisco, California 94143
Veterans' Administration Medical Center San Francisco, California 94121

Address requests for reprints to: Basil Rapoport, Veterans' Administration Medical Center, Metabolism (111F), 4150 Clement Street, San Francisco, California 94121.

Abstract

We have examined the effect of TSH on thyroid peroxidase (TPO) mRNA levels in dog thyroid cell primary cultures. Freshly dispersed dog thyroid cells were cultured for up to 5 days in the absence or presence of 5 mU/ml bovine TSH. At the outset of culture, and at daily intervals thereafter, total cytoplasmic RNA was extracted and applied to Nytran paper using a slot-blot apparatus. A nick-translated cDNA fragment of the porcine TPO gene was used to probe these filters. Autoradiographs were quantified by densitometry. Nonspecific binding was negligible as determined using a pUC18 probe. During the first 2 days of culture, TPO mRNA levels declined irrespective of whether or not TSH was present in the medium. TSH did not affect this decline. Between 3 and 5 days of culture, TPO mRNA levels in control (no TSH) cells increased to 3 times the initial level (expressed relative to cellular DNA). However, during the same period TSH stimulated TPO mRNA levels 8-fold above the initial level. To confirm that the signal with the cDNA probe was actually that of dog TPO mRNA, cellular RNA (day 4 of culture) was subjected to Northern blot analysis using the same cDNA probe. Specific bands of 2.9 kilobases were detected corresponding to the known size of TPO mRNA in pig thyroid tissue. The signal of this 2.9 kilobase species was enhanced by TSH. In conclusion, the data indicate that chronic TSH stimulation raises steady state levels of TPO mRNA and provide an explanation, at least in part, for the mechanism by which TSH enhances TPO bioactivity in thyroid tissue.

FOOTNOTES

These studies were supported by NIH Grants AM-36182 and AM-19289, and the Medical Research Service of the Veterans' Administration.

Received for publication May 27, 1987. Accepted for publication October 2, 1987.

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