STUDY OF TTF-1 GENE-EXPRESSION IN DOG THYROCYTES IN PRIMARY CULTURE

TTF-1 is a homeodomain-containing transcription factor mainly expresse d in the thyroid where it controls the tissue-specific expression of t he thyrogobulin, thyroperoxidase and TSH receptor genes. It is therefo re potentially implicated in the hormonal control exerted by thyrotrop in via the second messenger cyclic AMP on the transcription of these g enes in thyrocytes. In order to investigate whether there exists a rel ationship between the stimulation of the cAMP pathway and TTF-1 gene e xpression in these cells, we have compared the amounts of TTF-1 protei n, its state of phosphorylation and its subcellular distribution in co ntrol and cAMP-stimulated dog thyrocytes in primary culture. Dog TTF-1 was expressed in bacteria as a fusion protein and antibodies were rai sed against the dog TTF-1 moiety. Stimulation of the thyrocytes by cyc lic AMP agonist only marginally increased TTF-1 gene expression as sho wn for the mRNA by RNase protection assay and for the protein by immun oblotting and immunoprecipitation of extracts from S-35-methionine lab elled cells. The phosphorylation state of TTF-1 was investigated by im munoprecipitation of extracts from P-32-labelled thyrocytes. Phosphory lation level appeared to be essentially unaffected by forskolin treatm ent of the cells. We also looked for differences in the use of phospho rylation sites by partial proteolytic digestion of immunoprecipitated P-32-labelled TTF-1 with Glu-C and Asp-N endoproteases. Comparison of radioactivity distribution amongst the generated fragments did not rev eal any difference in the pattern of TTF-1 phosphorylation in control and forskolin conditions. Lastly, in situ detection of TTF-1 by immuno fluorescence demonstrated that the protein was localized in the nucleu s of the cells, irrespective of the culture conditions. No major chang e in TTF-1 gene expression upon stimulation of the thyrocyte with a cA MP agonist could thus be detected in this study. The absence of an obv ious modification of the TTF-1 protein itself in response to cAMP stim ulation may indicate that other transcription factor(s) or co-factor(s ) are involved in the control exerted by cAMP on the expression of thy roid-specific genes.