Study of TTF-1 gene expression in dog thyrocytes in primary culture

TTF-1 is a homeodomain-containing transcription factor mainly expressed in the thyroid where it controls the tissue-specific expression of the thyroglobulin, thyroperoxidase and TSH receptor genes. It is therefore potentially implicated in the hormonal control exerted by thyrotropin via the second messenger cyclic AMP on the transcription of these genes in thyrocytes. In order to investigate whether there exists a relationship between the stimulation of the cAMP pathway and TTF-1 gene expression in these cells, we have compared the amounts of TTF-1 protein, its state of phosphorylation and its subcellular distribution in control and cAMP-stimulated dog thyrocytes in primary culture. Dog TTF-1 was expressed in bacteria as a fusion protein and antibodies were raised against the dog TTF-1 moiety. Stimulation of the thyrocytes by cyclic AMP agonist only marginally increased TTF-1 gene expression as shown for the mRNA by RNase protection assay and for the protein by immunoblotting and immunoprecipitation of extracts from 35S-methionine labelled cells. The phosphorylation state of TTF-1 was investigated by immunoprecipitation of extracts from 32P-labelled thyrocytes. Phosphorylation level appeared to be essentially unaffected by forskolin treatment of the cells. We also looked for differences in the use of phosphorylation sites by partial proteolytic digestion of immunoprecipitated 32P-labelled TTF-1 with Glu-C and Asp-N endoproteases. Comparison of radioactivity distribution amongst the generated fragments did not reveal any difference in the pattern of TTF-1 phosphorylation in control and forskolin conditions. Lastly, in situ detection of TTF-1 by immunofluorescence demonstrated that the protein was localized in the nucleus of the cells, irrespective of the culture conditions. No major change in TTF-1 gene expression upon stimulation of the thyrocyte with a cAMP agonist could thus be detected in this study. The absence of an obvious modification of the TTF-1 protein itself in response to cAMP stimulation may indicate that other transcription factor(s) or co-factor(s) are involved in the control exerted by cAMP on the expression of thyroid-specific genes.