THYROID-SPECIFIC EXPRESSION AND CYCLIC ADENOSINE-3',5'-MONOPHOSPHATE AUTOREGULATION OF THE THYROTROPIN RECEPTOR GENE INVOLVES THYROID TRANSCRIPTION FACTOR-I

The chimeric chloramphenicol acetyltransferase (CAT) construct, pTRCAT5'-199, containing the TSH receptor (TSHR) minimal promoter, -199 to -39 base pairs (bp), exhibits the thyroid specificity and TSH/cAMP autoregulation evident in TSHR gene expression. The present report shows that a cis-acting element between -189 and -175 bp, which binds thyroid transcription factor-1 (TTF-1), is involved in both activities. The 22 bp between -199 and -178 contains a positive element important for expression of the TSHR minimal promoter in rat FRTL-5 thyroid cells. DNAase I footprinting shows that extracts from functioning FRTL-5, but not nonfunctioning FRT thyroid or Buffalo rat liver (BRL) cells, protect a region between -189 and -175 bp. The protection is duplicated by TTF-1, and the protected element has only a two-base mismatch from the consensus TTF-1 element identified in the thyroglobulin (TG) and thyroid peroxidase minimal promoters. Gel mobility shift analyses reveal that FRTL-5 thyroid cell nuclear extracts form a specific protein/DNA complex with this region, which is prevented by the TTF-1 binding element from the TG promoter; FRT and BRL cell nuclear extracts do not have TTF-1 and do not form this complex. A role for the TSHR/TTF-1 binding element in thyroid-specific expression of the TSHR gene is evidenced as follows. Overexpression of TTF-1 in 887 or BRL cells, which have no TTF-1, increased the activity of pTRCAT5'-199, but not pTRCAT5' 177, which has no TTF-1 binding element. A nonsense mutation of the TTF1 binding element eliminated TTF-1-induced activation of TSHR promoter activity in 887 or BRL cells and reduced TSHR promoter activity in FRTL-5 thyroid cells. In contrast, mutation of this element to the TTF-1 consensus sequence of the TG or thyroid peroxidase promoter had no significant influence on TSHR promoter activity. The activity of the TSHR/ TTF-1 binding element requires a functioning cAMP response element (CRE). Thus, TTF-1 activity is lost when the CRE site is mutated to a nonfunctional, nonpalindromic sequence; it is, in contrast, maximized when CRE activity is maximized by its mutation to a consensus AP1 element. TTF-1 phosphorylation is important for binding and activity. Thus, binding of TTF-1 to the TSHR/TTF-1 element is phosphatase-sensitive and is increased by treating nuclear extracts with the catalytic subunit of protein kinase A. Overexpression of the catalytic subunit of PKA enhances TTF-1-increased activity of the TSHR minimal promoter. TSH/cAMP regulation of TTF-1 is involved in TSH/cAMP positive and negative autoregulation of the TSHR gene. Thus, within the first 2 h after TSH is given to FRTL-5 cells maintained without TSH for 7 days, TSH/cAMP increases the formation of the specific TTF-1/TSHR complex in association with increased TSHR gene expression. After 2 h, there is a decrease in TTF-1/TSHR complex formation which is coincident with TSH-induced down-regulation of TTF-1 and TSHR mRNA levels, as a function of both time and TSH concentration. The TSH-induced decrease in TTF-1 mRNA levels is duplicated by forskolin and inhibited by cycloheximide, as is the case for the ability of TSH to decrease TSHR mRNA levels. We suggest, therefore, that TSHR gene expression reflects, in part, a dynamic balance between the ability of TSH/cAMP to phosphorylate TTF-1 (positive autoregulation) and to decrease TTF-1 mRNA levels (negative autoregulation). Using oligo C from the TG promoter, we show that the TSH/cAMP biphasic regulation of TSHR gene expression is not duplicated, because TSH induces an increase in TG oligo C complex formation with another protein in the nuclear extracts, most likely Pax-8. Biphasic regulation of TSHR gene expression thus appears to reflect the specificity of the TTF-1/TSHR interaction.