TSH stimulation of human thyroglobulin and thyroid peroxidase gene transcription is partially dependent on internalization

The TSH receptor (TSHR) is the major regulator of thyroid hormone biosynthesis in human thyrocytes by regulating the transcription of a number of genes including thyroglobulin (TG) and thyroperoxidase (TPO). Until recently, it was thought that TSHR initiated signal transduction pathways only at the cell-surface and that internalization was primarily involved in TSHR desensitization and downregulation. Studies primarily in mouse cells showed that TSHR internalization regulates gene transcription at an intracellular site also. However, this has not been shown for genes involved in thyroid hormone biosynthesis in human thyrocytes. We used human thyrocytes in primary culture. In these cells, the dose-response to TSH for gene expression is biphasic with low doses upregulating gene expression and higher doses decreasing gene expression. We used two approaches to inhibit internalization. In the first, we used inhibitors of dynamins, dynasore and dyngo-4a. Pretreatment with dynasore or dyngo-4a markedly inhibited TSH upregulation of TG and TPO mRNAs, as well as TG secretion. In the second, we used knockdown of dynamin 2, which is the most abundant dynamin in human thyrocytes. We showed that dynamin 2 knockdown inhibited TSHR internalization and decreased the TSH-stimulated levels of TG and TPO mRNAs and proteins. Lastly, we showed that the level of the activatory transcription factor phosphorylated cAMP response element binding protein (pCREB) in the cell nuclei was reduced by 68% when internalization was inhibited. We conclude that upregulation of genes involved in thyroid hormone synthesis in human thyrocytes is, in part, dependent on internalization leading to nuclear localization of an activated transcription factor(s).