The paired-domain transcription factor Pax8 binds to the upstream enhancer of the rat sodium/iodide symporter gene and participates in both thyroid-specific and cyclic-AMP-dependent transcription

Abstract

The gene encoding the Na/I symporter (NIS) is expressed at high levels only in thyroid follicular cells, where its expression is regulated by the thyroid-stimulating hormone via the second messenger, cyclic AMP (cAMP). In this study, we demonstrate the presence of an enhancer that is located between nucleotides -2264 and -2495 in the 5'-flanking region of the NIS gene and that recapitulates the most relevant aspects of NIS regulation. When fused to either its own or a heterologous promoter, the NIS upstream enhancer, which we call NUE, stimulates transcription in a thyroid-specific and cAMP-dependent manner. The activity of NUE depends on the four most relevant sites, identified by mutational analysis. The thyroid-specific transcription factor Pax8 binds at two of these sites. Mutations that interfere with Pax8 binding also decrease transcriptional activity of the NUE. Furthermore, expression of Pax8 in nonthyroid cells results in transcriptional activation of NUE, strongly suggesting that the paired-domain protein Pax8 plays an important role in NUE activity. The NUE responds to cAMP in both protein kinase A-dependent and -independent manners, indicating that this enhancer could represent a novel type of cAMP responsive element. Such a cAMP response requires Pax8 but also depends on the integrity of a cAMP responsive element (CRE)-like sequence, thus suggesting a functional interaction between Pax8 and factors binding at the CRE-like site.

FIG. 1

DNA sequence of the 5′-flanking region and part of the coding region of the rat NIS gene. The first nucleotide of the translation start codon is designated +1. The enhancer fragment described in detail in this study is shown in bold letters, and the TATA box and major transcription start site are indicated.

FIG. 2

Cell-type-specific promoter activity of rNIS-LUC chimeric plasmids. Schematic representations of various chimeric LUC plasmids containing different fragments from rNIS 5′-flanking region in front of its own promoter (A) or of the TK promoter (B). The activity of each construct is expressed relative to that of pGL3-basic. The results presented are the means for at least three separate experiments, with each experiment done in duplicate. In all transfections, a CAT expression vector was introduced to normalize for transfection efficiency.

FIG. 3

Hormonal regulation of the promoter activity of rNIS-TKLUC chimeric plasmids in FRTL-5 cells. Schematic representations of rNIS-TKLUC chimeric plasmids and their relative LUC activities in FRTL-5 cells cultured in 44 medium containing four hormones with (+) or without TSH or forskolin (forsk.). After transfection, cells were cultured for 72 h in starvation medium containing 0.2% calf serum and then incubated for an additional 72 h in medium containing 5% calf serum and the indicated additives. The activity of each construct is expressed relative to that of pGL3-basic. The results presented are the means for at least three separate experiments, with each experiment done in duplicate. In all transfections, a CAT expression vector was introduced to normalize for transfection efficiency.

FIG. 4

The NUE does not respond to forskolin in nonthyroid cells. (A) CRE-CAT and pNISTKLUC3 were transiently transfected, with or without an expression vector for cPKA in Rat-1 cells. Cells were plated 48 h prior to transfection. After transfection, cells were cultured in Dulbecco modified Eagle medium containing 5% calf serum in the absence or presence of 10 μM forskolin for 48 h. (B) Normalized CAT (for CRE-CAT) and LUC (for pNISTKLUC3) activity. Results are expressed as fold activation of the value obtained in the absence (−) of both cPKA and forskolin (Forsk.). The means of three independent experiments in duplicate are shown.

FIG. 5

The NUE responds in thyroid cells to PKA-independent and -dependent pathways. (A) Experimental strategy. The entire experiment was performed under conditions that keep FRTL-5 thyroid cells in a state refractory to cAMP induction (6H) of CREB-dependent promoters. (B) Normalized CAT (for CRE-CAT) and LUC (for pNISTKLUC3) activity. Results are expressed as fold activation of the value obtained in the absence (−) of both cPKA and forskolin (Forsk.). The means of three independent experiments in duplicate are shown. (C) Activation of CRE-CAT and pNISTKLUC3 in response to different amounts of cPKA expression vector in the refractory phase of FRTL-5 cells. Means and standard deviations of relative activity of CRE-CAT (squares) and pNISLUC3 (circles) were plotted.

FIG. 6

NUE mutants and relative transcriptional activity in FRTL-5 cells. The sequence of wild-type NUE is shown at the top. Mutants are numbered from 2-1 to 16-2, and only the nucleotides that are different from those of the wild-type sequence are indicated for each mutant. Transcriptional activity of each mutant is expressed as a percentage of that of pNISTKLUC3. rNISA, -B, -C, and -D are the regions that showed significantly lower LUC activity upon mutagenesis.

FIG. 7

Binding of known thyroid-specific transcription factors to NUE. (A) DNase I footprinting obtained on the rNIS enhancer fragment in the absence of added proteins (−) or in the presence of either Pax8 PD or TTF1 HD. A sequence ladder of the rNIS fragment (G, A, T, and C) was used as size marker. The regions protected by PD (PA and PB) and HD (TA and TB) are depicted as lines. (B) Summary of the regions on rNIS upstream enhancer protected from DNase I digestion. Protected sequences obtained with PD (PA and PB) or HD (TA and TB) are indicated.

FIG. 8

Pax8 stimulates rNIS enhancer activity in HeLa cells. (A) pNISTKLUC3 (3 μg) and CMV-CAT (1 μg) were transfected in HeLa cells with the expression vectors of Pax8 (0.5 μg of CMV-Pax8), cPKA (1 μg of C-PKA), and TTF1 (0.5 μg of CMV-TTF1). Activity of pNISTKLUC3 without Pax8, cPKA, and TTF1 was set at 1, and mean relative activities and standard deviations of cotransfected pNISTKLUC3 with the various expression vectors from three separate experiments are shown. pBluescriptII was used to adjust the total amount of DNA transfected. HeLa cells were incubated 48 h after transfection and harvested, and LUC activity and CAT amount were measured. (B) Comparison of the effects of cPKA and/or Pax8 on pNISTKLUC3 and mutants of the CRE-like sequence in NUE. Three micrograms of mutants 6-2 and 7-1, derived from pNISTKLUC3, and 1 μg of CMV-CAT were transfected in HeLa cells with or without 1 μg of C-PKA and/or 0.5 μg of CMV-Pax8. Activity of pNISTKLUC3 without Pax8 and cPKA was set at 1, and the mean relative activities and standard deviations of cotransfected test plasmids with the various expression vectors from three separate experiments are shown. pBluescriptII was used to adjust the total amount of DNA transfected. HeLa cells were incubated for 48 h after transfection and harvested, and LUC activity and CAT amount were measured.

FIG. 9

Binding of Pax8 protein on rNISA and rNISC. (A) Band shift assays were performed with ³²P-labeled oligonucleotides corresponding to the Pax8 binding sites PA and PB. Each probe was incubated without extracts (−) or with extracts from FRTL-5 cells, mock-transfected HeLa cells (HeLa), or HeLa cells transfected with a Pax8 expression vector (HeLa-Pax8) in the presence (+) or absence (−) of either anti-Pax8 (P) or anti-TTF1 (T) antibody. Where indicated, a Pax8 synthetic peptide used to generate the antibody was added. The DNA-Pax8 complexes (Pax8), DNA-ubiquitous factor complex (asterisk), and supershift by anti-Pax8 antibody (Supershift) are indicated by arrows. (B) Competition experiments, using either PA or PB oligonucleotide. The complex formed by FRTL-5 nuclear protein with each oligonucleotide was challenged with 100-fold excess of itself (self) (cold) or cold oligonucleotides carrying either the 5-1m or 9-1m mutation. (C) Alignments between Pax8 consensus binding sequence (Pax8cons) and probes used in this study. Nucleotides in PA and PB matching the consensus sequence are indicated by vertical lines. Identical nucleotides between the PA and PB binding sites are indicated by white letters on a black background, and nucleotides changed by mutations 5-1m and 9-1m are underlined.