Activation of the thyroid peroxidase gene in human thyroid cells: effect of thyrotrophin, forskolin and phorbol ester

Abstract

The enzyme thyroid peroxidase (TPO) plays a central role in thyroid hormone synthesis and is the target for the autoimmune attack in lymphocytic thyroiditis. We have examined the activation of the TPO gene in cultured human thyrocytes using slot-blot hybridization with a synthetic 40 mer oligonucleotide probe derived from the nucleotide sequence of the human TPO gene. The oligonucleotide probe was shown by Northern blotting to hybridize specifically to an approximately 3 kb RNA species from thyroid tissue of patients with Graves' disease, but not to RNA preparations from human or bovine retinal tissue, providing compelling evidence for the specificity of the probe for TPO mRNA. Addition of TSH (10 mU/ml) to primary thyroid cultures for 4 h led to increased TPO mRNA levels which were maximal after 48 h and significantly higher than basal even after 7 days of co-culture. Activation of TPO mRNA by TSH showed dose dependency over a wide range (0.01-100 mU/ml), with a maximal effect at 10 mU TSH/ml in cells cultured for a period of 72 h. Comparison of TPO mRNA levels with the accumulation of thyroglobulin mRNA levels following stimulation by TSH indicated that the induction of the gene encoding thyroglobulin precedes transcription of the TPO gene. The adenylate cyclase activator forskolin (1-100 microM) mimicked TSH in increasing TPO mRNA levels whilst, in contrast, the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA; 0.01-1 microM) led to levels of TPO mRNA that were lower than basal. Thus TSH induces a specific dose-dependent activation of TPO mRNA which is mimicked by agents which increase cyclic AMP. In contrast, TPA-induced activation of protein kinase C inhibits this response.

Thyroid Hormone Synthesis

“…188 Expression of the human TPO gene is, in part, regulated by a thyroidspecific enhancer located 5.5 kb upstream of the transcriptional start site. [192][193][194] In immunoblot analyses, TPO appears as a doublet of 110 and 105 kDa, a phenomenon that is not explained by the translation of TPO2. 190,191 As noted, follicular TG, acting in a counterregulatory fashion to the stimulatory effect of TSH, downregulates the expression of TTF1, TTF2, and PAX8, a mechanism that leads to a decrease in NIS, TPO, TG, and TSH receptor expression.…”