This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager NURSA Molecule Pages Link Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Malo, M. S. Articles by Hodin, R. A. Search for Related Content PubMed PubMed Citation Articles by Malo, M. S. Articles by Hodin, R. A.

Thyroid Hormone Positively Regulates the Enterocyte Differentiation Marker Intestinal Alkaline Phosphatase Gene via an Atypical Response Element

Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Richard A. Hodin, M.D., Department of Surgery, Massachusetts General Hospital, Gray-Bigelow 504, 55 Fruit Street, Boston, Massachusetts 02114. E-mail: rhodin{at}partners.org.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Thyroid hormone (T₃) is a critical regulator of intestinal epithelial development and homeostasis, but its mechanism of action within the gut is not well understood. We have examined the molecular mechanisms underlying the T₃ activation of the enterocyte differentiation marker intestinal alkaline phosphatase (IAP) gene. RT-PCR and Western blotting showed that thyroid hormone receptors TR1 and TRß1 were expressed in human colorectal adenocarcinoma Caco-2 cells. Northern blotting detected expression of two IAP transcripts, which were increased approximately 3-fold in response to T₃. Transient transfection studies with luciferase reporter plasmids carrying various internal and 5' deletion mutations of the IAP promoter localized a putative thyroid hormone response element (TRE) to a region approximately 620 nucleotides upstream (–620) of the ATG start codon. EMSAs using TR1-retinoid X receptor (RXR) on sequential 5' and 3' single nucleotide deletions defined the TRE between –632 and –612 (5'-TTGAACTCAgccTGAGGTTAC-3'). Compared with the consensus TRE, the IAP-TRE is novel in that it contains an everted repeat of two nonamers (not hexamers) separated by three nucleotides. Neither TR1 nor RXR binds to the IAP-TRE; however, TRß1 binds to this TRE with minimal affinity. In the presence of TR and RXR, only the TR-RXR heterodimer binds to the IAP-TRE. Mutagenesis of either nonamer abolishes the biological activity of IAP promoter. We have thus identified a novel response element that appears to mediate the T₃-induced activation of the enterocyte differentiation marker, intestinal alkaline phosphatase.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
THE HORMONE T₃ PLAYS a pivotal role in mammalian intestinal epithelial development and homeostasis, affecting the fundamental processes of growth and differentiation. Endogenous T₃ levels increase just before weaning, a period marked by a dramatic growth spurt within the gut, as well as a shift in the expression of a number of intestinal gene products, e.g. sucrase, maltase, and intestinal alkaline phosphatase (IAP) activities increase, whereas lactase and acid ß-galactosidase decrease (1). Hypothyroid animals display marked crypt-villus hypoplasia, and abnormally retain the suckling pattern of expression of the brush-border enzymes, e.g. high lactase and low IAP levels (2, 3, 4, 5, 6, 7). In addition to its effect on the developing gut, T₃ exerts dramatic effects in the adult intestine, including mucosal hyperplasia and changes in enterocyte phenotype, e.g. increase in IAP and decrease in lactase expression (2, 3, 4, 5, 6, 7, 8). T₃ mediates its effects through its nuclear receptors [thyroid hormone receptors (TRs), see below], and TR knockout mice have been examined to determine the effects of T₃ on gut development and differentiation. Although TRß knockout mice appear to have no obvious intestinal abnormalities, TR knockout mice display marked hypoplasia in both the crypts and villi, and also have decreased levels of various digestive enzymes (lactase, sucrase, aminopeptidase) and gut transcription factors (Cdx-1 and Cdx-2) (9, 10, 11, 12, 13, 14). Relatively little is known about the molecular mechanisms that govern T₃ action within the gut. As such, the present work was designed to elucidate the molecular mechanisms underlying the T₃-mediated regulation of one specific target gene, the enterocyte differentiation marker IAP gene.

IAP is a membrane-bound glycoprotein that hydrolyzes a wide variety of monophosphate esters at an alkaline pH optimum (15, 16, 17). IAP is exclusively expressed in villus enterocytes and hence serves as an excellent marker of crypt-villus differentiation (18). IAP comprises a major component of the surfactant-like particles that are seen after fat feeding (19). Although the IAP protein is thought to be involved in the process of fat digestion and absorption, a precise physiological role for IAP has not yet been determined. Recent work by Narisawa et al. (20) on IAP knockout mice has shown that IAP limits fat absorption during high-fat feeding. Previously, using both in vitro and in vivo models, we have demonstrated that the IAP gene is a direct target gene for thyroid hormone (21, 22).

T₃ action is generally mediated through its nuclear receptors (TR) (23, 24) that belong to a super family of transcription factors, which also includes receptors for retinoic acid, vitamin D, various steroids (androgen, estrogen, glucocorticoids, etc.), and others. Different isoforms of TRs have been isolated. The TR gene on human chromosome 17 generates TR1 and TR2 transcripts, which are the splice variants of a major transcript, and produce 410 and 490 amino acid proteins, respectively (23). In addition, other truncated TR proteins appear to exist and may be important in modulating T₃ action (10). The human TRß gene on chromosome 3 produces 461 and 476 amino acid proteins TRß1 and TRß2, respectively, from two splice variants of a single transcript (23). As bona fide receptors, TR1, TRß1, and TRß2 contain both DNA- and ligand (T₃)-binding domains. The TRs interact with specific DNA sequences termed thyroid hormone response elements (TREs), which generally contain a loosely conserved hexamer (AGGTCA) repeat intervened by four nonspecific nucleotides, also known as direct repeat 4 (DR4) (24, 25, 26, 27). The typical half-sites (AGGTCA) are rarely found to be 100% conserved in actual genes. TR can also bind to other forms of these two hexameric repeats, such as palindromic hexamers (28) and everted repeats (29). TRs require heterodimerization with retinoid X receptor (RXR) for their binding to DR4 and other TREs containing hexamer repeats (24, 30, 31, 32, 33). An octamer TRE (T(A/G)AGGTCA) has been shown to bind to a TR monomer, thus eliminating the necessity of TR-RXR heterodimerization for TR-mediated activity (34, 35).

In this study, we show that T₃ induces endogenous IAP gene expression in the human colorectal adenocarcinoma Caco-2 cell line. This IAP gene trans-activation appears to be mediated through an interaction between the TR-RXR complex and a unique TRE located between –632 and –612 in the IAP promoter consisting of an everted repeat of two nonamers spaced by three nonspecific nucleotides. These results provide a molecular description of how thyroid hormone activates a specific enterocyte target gene and will have implications for understanding the myriad effects of T₃ on gut development and homeostasis.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
TR1 and TRß1 Are Expressed in Caco-2 Cells
T₃ action is mediated through its receptors (TRs), and hence we determined the level of expression of different TR isoforms in Caco-2 cells using RT-PCR followed by nested PCR (Fig. 1A). An expected PCR product of 347 bp of TR1 cDNA was obtained using primers TRa1.1184F and TRa1.1530R (see Materials and Methods). Similarly, the primers TRb1.121F and TRb1.605R (see Materials and Methods) amplified the expected 485 bp fragment of the TRß1 cDNA. No PCR product was amplified for TR2 and TRß2. These results indicate that TR1 and TRß1 are expressed in Caco-2 cells, whereas TR2 and TRß2 are probably not expressed. The two extra 0.60- and 0.65-kb faint bands that are amplified in the case of TRß1 could represent other isoforms of TR, or artifacts of PCR.

View larger version (31K): [in this window] [in a new window]   Fig. 1. Activation of the IAP Gene by Endogenous TRs in the Caco-2 Cell Line A, Expression of TR mRNAs. RNA derived from Caco-2 cells was subjected to RT-PCR followed by nested PCR using isoform-specific primers (see Materials and Methods). PCR products were electrophoresed in a 2% agarose gel and stained with ethidium bromide. PCR was repeated more than three times, and the photograph of a representative gel is shown. B, Expression of TR1 and TRß1 proteins. Western blotting was performed on the lysate (UN) or nuclear extract (NE) from untransfected Caco-2 cells as well as on the lysate from Caco-2 cells transfected (TF) with an expression plasmid overexpressing TR1 or TRß1 (see Materials and Methods). The experiments were repeated more than three times, and the photograph of a representative autoradiograph is shown. C, Effects of T3 on the expression of IAP gene. Northern blots illustrating the time course of the T3 (100 nM) effects on the expression of IAP gene in Caco-2 cells. Twenty micrograms of total RNA were loaded in each lane. The 3.0- and 2.7-kb IAP mRNA bands are marked. The ß-actin control detecting equal loading is shown in the bottom panel. Northern analyses were repeated more than three times, and similar results were obtained. The figure shows the photographs of representative autoradiographs. D, Endogenous TR-mediated activation of the IAP-luciferase reporter gene. Transient transfection assays showing the time course of the T3 effects on the activation of the IAP-luciferase reporter gene in Caco-2 cells. The 2.5-kb 5' flanking region of the IAP gene proximal to the ATG start codon (between –49 and –2574) was cloned into the pGL3-Basic promoter-detection vector to construct the IAP-luciferase reporter plasmid pIAP-2574/-49 (see Materials and Methods). Cells were cotransfected with pIAP-2574/-49 and the control plasmid pRL-CMV, and treated with T3 (100 nM) for the specified period of time. Cells were then lysed, relative luciferase activity determined, and fold-activation (T3+/T3–) calculated. The results were obtained from six independent experiments, and the values are expressed as mean ± SD (P < 0.05).

T₃ Activates Endogenous IAP Gene Expression
Northern analysis shows that T₃ induces the expression of two IAP transcripts of 2.7 and 3.0 kb in Caco-2 cells (Fig. 1C). Interestingly, Henthorn et al. (36) identified only one IAP transcript in Caco-2 cells using S1 mapping. The T₃ effects are seen as early as 6 h, and maximal levels are seen at 24 h. Densitometric quantitation of bands of Northern autoradigraphs showed that each IAP transcript was increased approximately 3-fold after 24 h treatment with T₃ (100 nM). These in vitro results are consistent with our previous data from the intact rat intestine (18, 21) that also showed T₃-induced activation of the IAP gene. Hence, these in vitro and in vivo results clearly establish the IAP gene as a T₃-responsive target gene.

T₃ Activates the IAP-Luciferase Reporter Gene Expression
We constructed an IAP-luciferase reporter plasmid (pIAP-2574/-49) carrying the 2.5 kb 5' flanking region of the human IAP gene proximal to its ATG start codon (see Materials and Methods). To corroborate the Northern data on expression of the IAP gene (Fig. 1C), we determined the temporal effect of T₃-induced activation of the IAP-luciferase reporter gene in Caco-2 cells. We transiently transfected Caco-2 cells with pIAP-2574/-49, and determined luciferase activities after different time periods. After 24-h treatment of the cells with T₃ (100 nM), the IAP promoter in pIAP-2574/-49 plasmid showed about 3-fold activation by the endogenous TRs (Fig. 1D). Minimal effects of T₃ were seen as early as 6 h. These results of transfection studies are consistent with that observed by Northern analyses.

The TRE Is Located Approximately at –620 of the 5' Regulatory Region of the IAP Gene
We constructed various derivatives of pIAP-2574/-49 carrying sequential 5' deletions of the IAP promoter (see Materials and Methods). Luciferase activities in Caco-2 cells were determined by transiently transfecting the cells with these pIAP-luciferase reporter plasmids in the presence of T₃ (Fig. 2A). T₃ induces the IAP gene about 3-fold in the larger IAP reporter plasmids, but there is a major decrease in activity in the case of the pIAP-616/-49, in which the level of activity was equal to that seen in the case of the vector pGL3-Basic (carrying no insert). Shorter plasmids also showed only basal level of T₃ induction, thereby localizing the TRE in between –750 and –616, a 135-bp region (Fig. 2A). We then constructed several internal deletion mutants of the pIAP-2574/-49, focusing on this 135 bp region (Fig. 2B). Comparison of the activations of pIAP-2574m1870 and pIAP-2574m1920 indicates that the TRE is localized in the sequence between –661 and –618. Comparison of the sequence between –661 and –618 with the established consensus TRE sequence shows that the sequence around –620, between –631 and –613 (5'-TGAACTCAgccTGAGGTTA-3'), is the likely TRE. Compared with the consensus octamer TRE sequence (T(A/G)AGGTCA) (34, 35), this putative TRE sequence appears to consist of an everted repeat of two octamer half-sites that have approximately 88% homology with the consensus TRE sequence. Most of the TREs identified until now consist of a direct repeat of two hexamers and/or octamers separated by four nonspecific bases (DR4-TRE) (24, 25, 26, 27). Based on the model of DR4-TRE, the putative IAP-TRE could also be assigned as a direct repeat of two octamer half-sites that have approximately 80% homology with the consensus octamer TRE sequence. However, this sequence has only three nucleotides (instead of the expected four) separating its octamer half-sites, indicating that this may be an atypical TRE. We constructed an internal deletion mutant pIAP-2574TRE, specifically deleting the putative TRE from the plasmid pIAP-2574/-49 (Fig. 2B). This deletion of the sequence between –631 and –613 completely eliminated T₃-induced activation of the IAP-luciferase gene, indicating that this is, indeed, the only biologically active TRE in the human IAP gene within its 2.5-kb proximal promoter region (Fig. 2B).

View larger version (50K): [in this window] [in a new window]   Fig. 2. Endogenous TR-Mediated Regulation of the IAP-Luciferase Reporter Gene in Plasmids Carrying Different 5' and Internal Deletions of the IAP Promoter Luciferase reporter plasmids carrying various IAP promoter regions were used to transfect Caco-2 cells (see Materials and Methods). The control plasmid pRL-CMV was used to cotransfect the cells, which were then treated ± T3 (100 nM). The relative luciferase activity was determined after 24 h, and fold-activation (T3+/T3–) calculated. The results were obtained from six independent experiments, and the values are expressed as mean ± SD (P < 0.05). A, T3 regulation of the IAP-luciferase reporter gene in plasmids carrying different 5' deletions of the IAP promoter. Various 5' deletion mutants (pIAPs) of the pIAP-2574/-49 plasmid were constructed by deleting specific restriction fragments of the IAP promoter (see Materials and Methods). B, T3 regulation of the IAP-luciferase reporter gene in plasmids carrying internal deletions of the IAP promoter. Various pIAP plasmids carrying internal deletions of the IAP promoter were constructed by deleting specific restriction fragments as well as by in vitro site-directed mutagenesis of the pIAP-2574/-49 plasmid (see Materials and Methods). Sc, SacI; Sp, SphI; Xh, XhoI; Pv, PvuII; Pf, PflMI; Be, BstEII; Bu, Bsu36 I; Sm, SmaI; Ml, MluI; Bx, BstXI; Bt, BtrI; Ps, PstI.

View larger version (44K): [in this window] [in a new window]   Fig. 3. Binding of TR1 and RXR to the Putative IAP-TRE Analyzed by EMSA The double-stranded oligonucleotide TRE1G (Table 1) carrying the putative IAP-TRE was 5' end-labeled with 33P, and used as the probe in EMSA. TR1, TRß1, and RXR proteins were synthesized in vitro; and nuclear extract from Caco-2 cells was obtained using established protocols (see Materials and Methods). Related oligonucleotide sequences are shown in Table 1. Protein-DNA complexes were incubated at room temperature for 20 min. Fifty-fold molar excess of the unlabeled (cold) competitor double-stranded oligonucleotide was used in the related binding reaction. The protein-DNA complexes were separated by nondenaturing 5% PAGE at 4 C. EMSA for each experiment was repeated more than three times, and similar results were obtained. The figure shows the photographs of representative autoradiographs. A, Binding of TR1 and RXR to the IAP-TRE. B, Binding of TRß1 and RXR to the IAP-TRE. C, Binding of nuclear proteins of Caco-2 cells to the putative IAP-TRE. G, TRE1G; DR4, DR4 TRE; mTR, mutant IAP-TRE (mTRE, Table 1); 1, anti-TR1 antibody; ß1s, anti-TRß1 antibody (Santa Cruz); ß1u, anti-TRß1 antibody (Upstate); TnT lysate, lysate for transcription and translation in vitro.

Nuclear Extract from Caco-2 Cells Binds to the IAP-TRE
Nuclear extract from Caco-2 cells was incubated with the radiolabeled TRE1G probe, and EMSA was performed. The results are shown in Fig. 3C. Two shifted bands are observed, which proves that components of nuclear extract are capable of binding to the TRE1G oligonucleotide containing the putative TRE (lane 2). The major band was supershifted with the anti-TR1 antibody (lane 6) confirming that Caco-2 cells express endogenous TR1, which is capable of binding to the IAP-TRE and could be recognized by the anti-TR1 antibody. However, the major shifted bands could not be supershifted with anti-TRß1 antibodies (lanes 7 and 8), even though Caco-2 cells express TRß1 as evidenced by RT-PCR (Fig. 1A) and Western blotting (Fig. 1B). The minor shifted band could not be supershifted with the anti-TR1 or anti-TRß1 antibodies suggesting the nonspecific nature of this band. These data concur with the EMSA results obtained by using the in vitro-synthesized TR1 and RXR proteins (Fig. 3A) and suggest that TR1 is the major TR isoform in Caco-2 cells.

T₃ Inhibits the Binding of Anti-TR1 Antibody to the TRE-TR1-RXR Complex
Using EMSA, we determined the effect of T₃ (100 nM) on the binding of the TR1-RXR protein complex to the IAP-TRE. We also determined the effect of T₃ (100 nM) on the binding of the TR antibodies to the protein-DNA complex. The results (Fig. 4A) suggest that T₃ has no effect on the binding of the TR1-RXR protein complex to the IAP-TRE (lanes 2 and 10). In contrast, comparison between lane 6 and lane 14 shows that T₃ inhibits the binding of anti-TR1 antibody to the TRE-TR1-RXR complex. We also determined the effect of T₃ on the binding of the TR1-RXR protein complex to the IAP-TRE at different concentrations of the cold competitor (Fig. 4B). The result shows that the TR1-RXR protein complex has similar binding affinity in the presence or absence of T₃. Taken together, these results suggest that T₃ does not affect the binding affinity of the TR for the IAP-TRE, but likely causes a structural change that alters the ability of the anti-TR1 antibody to bind to TR1.

View larger version (38K): [in this window] [in a new window]   Fig. 4. Effect of T3 on the Binding of TR1-RXR Protein Complex to the IAP-TRE The double-stranded oligonucleotide TRE1G (Table 1) carrying the IAP-TRE was 5' end-labeled with 33P, and used as the probe in EMSA. TR1 and RXR proteins were synthesized in vitro (see Materials and Methods). Related oligonucleotide sequences are shown in Table 1. Fifty-fold molar excess of the unlabeled (cold) competitor double-stranded oligonucleotide was used in the related binding reaction. The protein-DNA complexes were separated by nondenaturing 5% polyacrylamide gel electrophoresis at 4 C. EMSA for each experiment was repeated more than three times, and similar results were obtained. The figure shows the photographs of representative autoradiographs. A, Effect of T3 on the binding of TR1-RXR protein complex to the IAP-TRE. The TRE1G probe was incubated with TR1 and RXR in the presence of T3 (100 nM) at room temperature for 20 min. B, Effect of T3 on the binding of TR1-RXR protein complex with different concentrations of the IAP-TRE. The probe TRE1G was incubated with different amounts (fold-excess) of unlabeled (cold) competitor TRE1G in the presence of T3 (100 nM) and TR1-RXR protein complex at room temperature for 20 min. G, TRE1G; DR4, DR4 TRE; mTR, mutant IAP-TRE (mTRE, Table 1); 1, anti-TR1 antibody; ß1s, anti-TRß1 antibody (Santa Cruz); ß1u, anti-TRß1 antibody (Upstate).

Substitution Mutations in Either Half-Site Abolish the Binding of the TR1-RXR Complex to the IAP-TRE

View larger version (26K): [in this window] [in a new window]   Fig. 5. Binding of TR1 and RXR to the IAP-TREs Carrying Substitution Mutations The double-stranded oligonucleotide TRE1G (Table 1) carrying the putative IAP-TRE was 5' end-labeled with 33P, and used as the probe in EMSA. TR1 and RXR proteins were synthesized in vitro. Sequences of the IAP-TREs carrying substituted nucleotides are shown in Table 1. Protein-DNA complexes were incubated at room temperature for 20 min. Fifty-fold molar excess of the unlabeled (cold) competitor double-stranded oligonucleotide was used in the related binding reaction. The protein-DNA complexes were separated by nondenaturing 5% PAGE at 4 C. EMSA for each experiment was repeated more than three times, and similar results were obtained. The figure shows the photograph of a representative autoradiograph. Legend: M1- M10, TRE1sm1-TRE1sm10 (Table 1); mTR, mutant IAP-TRE (mTRE, Table 1); 1, anti-TR1 antibody; ßs, anti-TRß1 antibody (Santa Cruz); ßu, anti-TRß1 antibody (Upstate).

An Individual Half-Site of the IAP-TRE Does Not Bind to the TR1-RXR Complex

View this table: [in this window] [in a new window]   Table 2. Binding of the TR1-RXR Complex to Oligonucleotides Carrying 5' and 3' Deletions of the Putative IAP-TRE

View larger version (44K): [in this window] [in a new window]   Fig. 6. Binding of TR1 and RXR to the IAP-TREs Carrying Sequential Deletions and Nucleotide-Specific Mutations The double-stranded oligonucleotides (Table 2) carrying the putative wild-type IAP-TRE or its mutant derivatives were 5' end-labeled with 33P, and used as probes in EMSA. TR1 and RXR proteins were synthesized in vitro. Protein-DNA complexes were incubated at room temperature for 20 min. Except where indicated, 50-fold molar excess of the unlabeled (cold) competitor double-stranded oligonucleotide was used in the related binding reaction. The protein-DNA complexes were separated by nondenaturing 5% PAGE at 4 C. EMSA for each experiment was repeated more than three times, and similar results were obtained. The figure shows the photographs of representative autoradiographs. A, Binding of TR1 and RXR to the IAP-TREs carrying sequential deletions. Sequences of the IAP-TREs carrying sequential 5' and 3' deletions are shown in Table 2. The double-stranded oligonucleotide TRE1G (Table 2) carrying the putative wild-type IAP-TRE was used as the probe. B, Binding of TR1 and RXR to the IAP-TREs carrying substitution mutations of the extra nucleotide of the nonamers. Sequences of the IAP-TREs carrying substitution mutations of the extra nucleotides of the nonamers are shown in Table 3. The double-stranded oligonucleotide TRE1G (Table 2) carrying the putative wild-type IAP-TRE was used as the probe. C, Binding of TR1 and RXR to the IAP-TREs carrying nucleotide-specific mutations. Sequences of the IAP-TREs carrying nucleotide-specific mutations are shown in Table 3. The double-stranded oligonucleotides (Table 3) carrying the wild-type or a mutant IAP-TRE were used as probes. D, Binding of TR1 and RXR to the putative IAP-TRE in the presence of an incremental molar excess of a cold competitor. The double-stranded oligonucleotide TRE1G (Table 2) carrying the putative wild-type IAP-TRE was used as the probe. B-Y, TRE1B-TRE1Y (Table 2); G, TRE1G (Table 1); DR4, DR4 TRE (Table 1); mTR, mutant IAP-TRE (mTRE, Table 1); E-Yt, TRE1E-TRE1Yt (Table 3); U1, TREMU1 (Table 3); D9, TREMD9 (Table 3).

The Extra Nucleotides in the Nonamer Half-Sites Are Specific
We investigated the specificity of the two extra nucleotides, one at each end of the core IAP-TRE. We performed EMSA (Fig. 6B) on oligonucleotides carrying all four different types of nucleotides (A, C, G, T) in the extra positions (Table 3). For the 5' end extra nucleotide, we found that the TR1-RXR complex strongly binds to a TRE carrying T or G (Fig. 6B, lanes 9 and 12, respectively; Table 3), whereas the protein complex does not bind to a TRE containing A or C (lanes 10 and 11, respectively). Similarly, for the 3' end extra nucleotide, we found that the TR1-RXR complex strongly binds to a TRE carrying C (lane 14), whereas the protein complex does not bind to a TRE carrying T (lane 17), and a TRE containing A or G shows weak binding (lanes 15 and 16, respectively). The observed specificity of the extra nucleotides strongly suggests the nonameric nature of the half-sites of the IAP-TRE in these truncated forms of oligonucleotides. We then decided to elucidate the specificity of these two nucleotides in the context of the larger oligo TRE1G using specific point mutations at these extra positions (Table 3). We synthesized two mutant derivatives of TRE1G (TREMU1 and TREMD9, Table 3), and used them in EMSA for determining their binding property to TR-RXR complex (Fig. 6C). The results indicate that TR1-RXR protein complex probably binds to these two mutants with less efficiency. However, to determine accurate binding affinity of the TR1-RXR protein complex to these mutant oligos, we compared their competing capability with that of wild-type TRE (TRE1G) for TR1-RXR using incremental doses of cold oligos (Fig. 6D, Table 3). Comparison of intensities of respective bands of wild-type and mutant TREs shows that mutations of the two extra nucleotides reduce the binding affinity of TR1-RXR to the IAP-TRE flanked by four to six nucleotides at both ends. These results define the end nucleotide of the core IAP-TRE and strongly suggest the nonameric nature of this TRE. Similar results were obtained from the binding of the TRß1-RXR heterodimer to the wild-type and these mutant TREs (data not shown).

View this table: [in this window] [in a new window]   Table 3. Binding of the TR1-RXR Complex to Oligonucleotides Carrying Substitution Mutations of the Extra Nucleotides in the Nonamer Half-Sites of the IAP-TRE

Substitution Mutations of Internal Nucleotides of a Nonamer Half-Site Reduce the Binding of TR1-RXR to the IAP-TRE

View larger version (32K): [in this window] [in a new window]   Fig. 7. Binding of TR1 and RXR to the IAP-TREs Carrying Nucleotide-Specific Mutations The double-stranded oligonucleotides carrying the putative wild-type IAP-TRE or its mutant derivatives (Table 3) were 5' end-labeled with 33P, and used as probes in EMSA. TR1 and RXR proteins were synthesized in vitro. Protein-DNA complexes were incubated at room temperature for 20 min. Except where indicated, 50-fold molar excess of the unlabeled (cold) competitor double-stranded oligonucleotide was used in the related binding reaction. The protein-DNA complexes were separated by nondenaturing 5% PAGE at 4 C. EMSA for each experiment was repeated more than three times, and similar results were obtained. The figure shows the photographs of representative autoradiographs. A, Binding of TR1 and RXR to the IAP-TREs carrying nucleotide-specific mutations in the nonamers. Sequences of the IAP-TREs carrying nucleotide-specific mutations are shown in Table 3. The double-stranded oligonucleotides (Table 3) carrying the wild-type or a mutant IAP-TRE were used as probes. B, Binding of TR1 and RXR to the putative IAP-TRE in the presence of an incremental molar excess of a cold competitor carrying nucleotide-specific mutations in the nonamers. The double-stranded oligonucleotide TRE1G (Table 3) carrying the putative wild-type IAP-TRE was used as the probe. C, Binding of TR1 and RXR to the mutant IAP-TRE carrying nucleotide-specific mutations in the spacer region between two nonamers. Sequences of the IAP-TREs carrying nucleotide-specific mutations are shown in Table 3. The double-stranded oligonucleotides (Table 3) carrying the wild-type or a mutant IAP-TRE were used as probes. D, Binding of TR1 and RXR to the putative IAP-TRE in the presence of an incremental molar excess of a cold competitor carrying nucleotide-specific mutations in the spacer region between two nonamers. The double-stranded oligonucleotide TRE1G (Table 3) carrying the putative wild-type IAP-TRE was used as the probe. G, TRE1G (Table 1); mTR, mutant IAP-TRE (mTRE, Table 1); U23–D78, TREMU23–TREMD78 (Table 3); SR123, TREMSR123 (Table 3).

Mutations of the Spacer Region between the Two Nonamer Half-Sites Do Not Affect the Binding of TR1-RXR to the IAP-TRE

Substitution Mutations in Either Nonamer Greatly Reduces Biological Activity of the IAP-TRE
To assess the biological importance of each nonamer we performed site-directed mutagenesis to construct pIAP-luciferase reporter plasmids carrying substitution mutations for each nonamer (Table 4). Caco-2 cells were transfected with the mutant plasmids, and luciferase activity was determined in the presence of T₃ (100 nM). The results are shown in Fig. 8A and tabulated in Table 4. All mutations rendered the IAP-TRE biologically inactive.

View larger version (16K): [in this window] [in a new window]   Fig. 8. Endogenous TR-Mediated Regulation of the IAPLuciferase Reporter Gene in Plasmids Carrying Nucleotide-Specific Mutations in the IAP-TRE IAP-luciferase reporter plasmids carrying nucleotidespecific mutations in the IAP-TRE were constructed by in vitro site-directed mutagenesis, and the corresponding mutations are shown in Table 4. A mutant plasmid was used to cotransfect Caco-2 cells along with the control plasmid pRL-CMV, and the cells were then treated ±T3 (100 nM) for 24 h. The relative luciferase activity was determined, and fold-activation (T3+/T3–) calculated (see Materials and Methods). The results were obtained from six independent experiments, and the values are expressed as mean ± SD (P < 0.05). A, Regulation of mutant IAP-TREs by endogenous TRs. B, Ligand-independent regulation of mutant IAP-TREs by endogenous TRs.

The IAP-TRE Is a Novel TRE Consisting of an Everted Repeat of Two Nonamers
We compared the IAP-TRE with the various positively regulated TREs that have been previously described for other genes (37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51) (Table 5). It is evident that TRs recognize DNA sequences with great diversity; however, all of the positively regulated TREs are composed of a repeat of a loosely conserved hexamer (AGGTCA) sequence. Katz and Koenig (35) have identified that the dinucleotide TG or TA at the 5' end of the above-mentioned hexamer constitutes a part of a TRE. Approximately 50% of the identified TREs contain an octamer half site (Table 5). Accordingly, to define the space between two half-sites of a TRE we counted the space between the downstream octamer/hexamer and upstream hexamer/octamer (Table 5). In Fig. 9A, we summarized the results of in vitro binding of the IAP-TRE to the TR-RXR complex (EMSA) and biological (in vivo) effects of mutations of the IAP-TRE in T₃-induced activation of the IAP gene in Caco-2 cells (Fig. 9A). The data show that nucleotides at or close to the ends of each nonamer are required for biological activity as well as for in vitro binding of the TR-RXR complex. Thus, the data clearly establish that the IAP-TRE consists of two nonamers separated by three nonspecific nucleotides. Comparison of the nucleotide sequence of a consensus octamer TRE [T(G/A)AGGTCA] with the corresponding nucleotides of a nonamer in either strand of the IAP-TRE shows that the upstream nonamer in the antisense strand and the downstream nonamer in the sense strand each has two bases mismatched. Similarly, the downstream nonamer in the antisense strand and the upstream nonamer in the sense strand each has one base mismatched. Hence, to obtain maximum homology of the IAP-TRE with the consensus octamer TRE, we assigned the IAP-TRE as an everted repeat of two nonamers (Fig. 9A). Until now, no TRE has been identified containing nonamer half-sites. The IAP-TRE does not bind to TR1 alone; however, it binds to TRß1 alone making it also an atypical TRE. Further, in the presence of RXR, each TR binds to the TRE only as a heterodimer of TR-RXR. In Fig. 9B, we present the sequence of the proximal 800 bases of the IAP promoter, showing the TRE and other transcription factor binding sites.

View larger version (46K): [in this window] [in a new window]   Fig. 9. Nucleotide Sequence Analyses of the Human IAP-TRE and the 5' Flanking Region of the IAP Gene Showing the TRE and Other cis Elements A, Composition of the IAP-TRE. The nonamer half-sites of the IAP-TRE are shown in capital letters, and the nucleotides differing from their respective nucleotides of the consensus octamer (bottom panel) are underlined. The nucleotides involved in binding to TR-RXR protein complex in vitro (EMSA) are shown in bold capital letters (see Table 3 and Figs. 6 and 7). The nucleotides required for wild-type biological (in vivo) activity of the TRE are shown in bold and larger-font capital letters (see Table 4 and Fig. 8). The arrows (not broken) indicate the nonamers showing maximum homology to the consensus octamer. The broken arrows indicate the nonamers with less homology to the consensus octamer. Note: Considering the homology of the IAP-TRE half-sites with that of consensus TRE, it is most probable that the IAP-TRE is an everted repeat of two nonamers separated by three nonspecific nucleotides. B, Sequence of the human IAP gene 5' flanking region showing the IAP-TRE and other cis elements. The sequence (antisense) is numbered relative to the ATG start codon (marked +1). The IAP-TRE is underlined, and the nonamer half-sites are marked bold. The putative TATA-box (pTATA) is marked bold. The Sp1/GKLF binding sites and the relevant restriction sites are marked and underlined. Transcription start-sites, major (>>>) and minor (>), are marked. The initiation codon ATG is marked bold.

View larger version (33K): [in this window] [in a new window]   Fig. 10. Biological Activities of the Wild-Type and Mutant IAP-TREs in Caco-2 Cells Overexpressing an Individual Isoform of TR Cells were transfected with the pGL3-Basic vector, pIAP-2574/-49, or pIAP-2574TRE. Cells were also cotransfected with the control plasmid pRL-CMV along with a plasmid overexpressing an individual isoform of TR (TR1, TRß1, or TRß2). The transfectants were treated ± T3 (100 nM) for 24 h. Cells were then lysed, relative luciferase activity determined, and fold activation (T3+/T3–) calculated. The results were obtained from six independent experiments, and the values are expressed as mean ± SD (P < 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

Alkaline phosphatases are membrane-bound glycoproteins that hydrolyze a wide variety of monophosphate esters at high pH optima and are widely distributed in different tissues (36). Depending on their biochemical nature and tissue distribution, the alkaline phosphatases are classified as placental, placental-like, intestinal (fetal and adult), and liver, bone, kidney (L/B/K) alkaline phosphatases. The human intestinal alkaline phosphatase (IAP) gene maps to chromosome 2q34–37 and produces a 528-amino acid polypeptide. The IAP protein has been identified as a content of the surfactant-like particles, the unilamellar secreted membrane associated with the process of lipid absorption and isolated from the apical surface of enterocytes (19). The IAP protein has also been identified as a Hela cell tumor-associated antigen (53). A recent report by Narisawa et al. (20) on IAP knockout mice demonstrated that IAP limits fat absorption during high-fat feeding. The IAP gene is overexpressed in hyperthyroid intestine, especially in the duodenum, thus indicating the T₃-mediated activation of this gene (22). Hypothyroidism is associated with villus atrophy and a decrease in the expression of the 3.0-kb IAP mRNA species, further supporting the involvement of T₃ in the positive regulation of the IAP gene (54).

The molecular mechanisms by which T₃ exerts its effect on the gut are not well understood. There have been few in vitro studies on T₃ action in intestinal cells, one exception being a study by Giannella et al. (55), showing that T₃ induces Na,K-ATPase gene expression in the Caco-2 colon cancer cell line. However, Na,K-ATPase is a ubiquitous trans-membrane protein mostly expressed in kidney, intestine, skeletal muscle, and cardiac muscle. In addition to IAP, lactase, and Na,K-ATPase, it will be of interest to identify the full range of T₃-responsive genes in the gut, similar to what has been done in other tissues such as the pituitary (56). Wu et al. (38) have tabulated the T₃-responsive genes that have been studied at the molecular level identifying their respective TREs; however, the status of expression of these genes in the gut is not known.

Various forms of TR and TRß isoforms are expressed in the gut; however, using RT-PCR we found expression of only the TR1 and TRß1 receptors in the colorectal adenocarcinoma Caco-2 cell line (Fig. 1A). We observed two 0.60 and 0.65 kb extra bands in the case of TRß1, which were produced by nested PCR, indicating the possible existence of other TRß isoforms in these cells. Western blotting on whole-cell lysates, as well as nuclear extract, confirmed the production of TR1 and TRß1 receptors by Caco-2 cells, which corroborates with the RT-PCR data. Based upon the Northern blot analyses, the basal level of TRs in Caco-2 cells was capable of approximately 3-fold activation of the endogenous IAP gene after T₃ treatment (Fig. 1C). Using S1 mapping, Henthorn et al. (15) have shown only one IAP transcript in the Caco-2 cells; however, we observed two transcripts in the Northern blots. This apparent inconsistency could be due to subtype variations among the Caco-2 cells or the relatively lower sensitivity of S1 mapping. Similar to the activation of endogenous IAP mRNA expression, transient transfections in Caco-2 cells with IAP-luciferase reporter constructs also showed approximately 3-fold activation of the IAP gene by T₃ (Figs. 1D and 2A).

Previously identified naturally occurring TREs generally consist of two hexamer half-sites arranged as either DR4, palindrome, inverted palindrome, or everted palindrome (24, 25, 26, 27, 28, 29, 30, 31, 32, 33). The TREs with hexamer half-sites bind only to TR-RXR heterodimer. An octamer TRE [T(A/G)AGGTCA] has been shown to bind to a TR monomer, thus eliminating the necessity of TR-RXR heterodimerization for TR-mediated activity (34, 35). Most previous work on these octamer TREs has been performed in vitro with synthesized oligonucleotides elucidating the precise sequences, spacing, and orientation (34, 35). Recently, Toyoda et al. (40) have described the TRE1 from the human type 1 deiodinase gene (hdio1) as a naturally occurring, biologically functional TRE consisting of two relatively independent octamer sequences (DR10) that do not require the RXR family of proteins for function.

Using transient transfection assays with IAP-luciferase reporter constructs carrying various 5' and internal deletion mutants of the IAP promoter (Fig. 2), we were able to convincingly localize the TRE to a 135-bp region in the proximal IAP promoter around –620. We compared the sequence of this 135-bp region with consensus DR4 and octamer TREs (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35). We identified a putative IAP-TRE (5'-TGAACTCAgccTGAGGTTA-3') consisting of two octamer half-sites separated by three nucleotides instead of the expected four nucleotides. With respect to the spacing of three nucleotides between octamer half-sites, the IAP-TRE resembles the rat prestin TRE (Table 5). The TRE for rat uncoupling protein 1 consists of a downstream hexamer and upstream octamer separated by three nucleotides (Table 5). In addition, the pTRE for human thyroid hormone receptor ß1 consists of a direct repeat of two hexamers separated by three nucleotides, and the dTRE consists of an everted repeat of a downstream octamer and an upstream hexamer (Table 5). By internal deletion mutation of the identified TRE, we showed that this TRE was, indeed, the only biologically active TRE in the 2.5-kb proximal promoter region of the IAP gene. However, our result does not exclude the possibility of another TRE outside the region of the IAP promoter examined in this work. Most in vivo work on T₃ regulation of the IAP gene has been performed in rodents. However, by sequence comparison, we could not identify a close homology (>70%) of the identified human IAP-TRE within the 2.5-kb proximal promoter region of the mouse IAP gene. It will be interesting to investigate the localization and structure of the mouse IAP-TRE and compare the underlying mechanisms with those found for the human IAP gene.

Toyoda et al. (40) have shown that the hDIO1-TRE, presumably consisting of two independent DR10 octamers (Table 5), preferentially binds to two monomers (double occupancy) of TR1 in the absence of T₃, whereas in the presence of T₃ (100 nM), TR double occupancy is impaired, and binding of a single monomer (single occupancy) is also observed. The authors (40) have also shown that RXR as well as the TR-RXR heterodimer does not bind to the hDIO1-TRE. In contrast to the binding of TR to hDIO1-TRE, our EMSA data (Fig. 3A) show that TR1 cannot independently bind to the IAP-TRE, and only the TR1-RXR complex can bind to the TRE efficiently. However, our EMSA data show that TRß1 alone can bind to the IAP-TRE although with less affinity (Fig. 3B), and such binding is abolished in the presence of RXR, when only the TRß1-RXR heterodimer binds to the IAP-TRE. We also observed that TRß1 alone binds to the downstream half-site of the IAP-TRE (data not shown), and it will be interesting to further investigate such binding of TRß1 that might reveal the nucleotide composition of any likely isoform-specific TRE. Like the hDIO-TRE, the IAP-TRE also does not bind to RXR. In contrast to the effect of T₃ on the hDIO1-TRE, our results (Fig. 4) show that T₃ has no effect on binding of the TR1-RXR complex to the IAP-TRE. However, we found that T₃ inhibits the binding of the anti-TR1 antibody to TRE-TR1-RXR complex. We presume that binding of T₃ to TR1 changes the conformation of TR1 thus making it inaccessible to the antibody.

Using EMSA, we compared the binding of in vitro-synthesized TR1-RXR and TRß1-RXR complexes to the IAP-TRE (Fig. 3, A and B) with the binding of nuclear extract from Caco-2 cells to the IAP-TRE (Fig. 3C). The TR1-RXR complex, as well as the nuclear extract, is capable of binding to the IAP-TRE and can be supershifted with anti-TR1 antibody (Fig. 3C). The polyclonal anti-TRß1 antibody from Upstate Biotechnology (Lake Placid, NY) was able to supershift TRE-TRß1-RXR complex, whereas monoclonal anti-TRß1 antibody from Santa Cruz Biotechnology (Santa Cruz, CA) was not able to bind to the TRE-TRß1-RXR complex (Fig. 3B). Although Caco-2 cells express TRß1 (Fig. 1, A and B) we found that anti-TRß1 antibodies do not generate any supershifted band (Fig. 3C). We believe that the amount of TRß1 expressed in Caco-2 cells was probably not sufficient to bind to the TRE in competition with TR1. We also found that T₃ has similar effects on binding of in vitro-synthesized protein complex as well as on binding of nuclear proteins to the IAP-TRE (data not shown).

EMSA results (Fig. 5) on the IAP-TRE containing substitution mutations demonstrated that each half-site of the IAP-TRE is at least an octamer or it could be larger than an octamer. Efficient binding of TR1 alone to an octamer (TAAGGTCA) has been described (34); however, neither octamer in the IAP-TRE can independently bind to the TR1-RXR complex (Fig. 6), thus proving the necessity of both the half-sites for efficient binding to this TRE. Based on the sequence of the antisense strand, the upstream octamer of the IAP-TRE (TGAACTCA) varies by three bases, whereas the downstream octamer (TGAGGTTA) varies by two bases from the above octamer (TAAGGTCA) sequence (Fig. 9A). Similarly, based on the sequence of the sense strand, the octamers of the IAP-TRE (TAACCTCA and TGAGTTCA) each varies by two bases from the above-mentioned octamer (TAAGGTCA) sequence (Fig. 9A). It appears that binding to a single octamer TRE probably requires very stringent homology to TAAGGTCA.

Using EMSA on sequential deletion mutations of a large TRE, we show that the half site of the IAP-TRE is a nonamer (Fig. 6A). We studied the specificity of the extra nucleotides within the nonamers of the IAP-TRE, and we indeed found the specific role of these nucleotides for the binding of the TR1-RXR complex to the IAP-TRE (Fig. 6, B–D). The specificity of the extra nucleotides strongly indicates that the IAP-TRE is a repeat of nonamers. It will be interesting to investigate the biological role of these extra nucleotides in regard to the T₃ activation of the IAP gene. To further determine the nonameric nature of half-sites of the IAP-TRE, we also performed EMSA on the mutant IAP-TREs carrying internal mutations. These results also showed that nucleotides at or close to the ends of each nonamer are also involved in the binding of the TRE to the TR-RXR complex (Fig. 7, A and B). However, it remains possible that the extra nucleotides outside each hexameric half-site (AGGTCA) could be parts of the flanking sequences that are involved in the modulation of activity of the response element (57, 58). It will be interesting to perform methylation interference studies (34), which could identify the nucleotides that contact with the TR-RXR protein complex.

The use of EMSA on oligonucleotides carrying sequential deletions of single nucleotides enabled us to determine the upstream and downstream boundaries of the IAP-TRE. Until now, EMSA has not been used to determine the 5' and 3' boundaries of other identified TREs, although this is probably a valid method for determining the boundaries of any cis element. We predict that boundaries of other TREs could be elucidated using this protocol, and subsequent analysis might generate a broader consensus among the TREs.

The in vitro site-directed mutagenesis of the IAP-TRE shows that the dinucleotide TG at the 5' end of the either octamer in the antisense strand is required to confer the wild-type TRE activity in Caco-2 cells (Fig. 8A and Table 4). These mutations could also be assigned as the 3' end dinucleotide (CA) of the octamers in the sense strand. Hence the dinucleotide TG at the 5' end of either octamer in the antisense strand must be a part of the TRE. Our EMSA data show that nucleotides at or close to the ends of each nonamer are required for in vitro binding of the TR-RXR complex (Figs. 6 and 7). Thus, the possibility that the IAP-TRE is composed of a repeat of two hexamers separated by seven or five nonspecific nucleotides was considered very unlikely. The EMSA results, supported by the mutagenesis data, strongly suggest that the IAP-TRE most likely consists of a repeat of two nonamers separated by three nucleotides. Based on maximum homology of the IAP-TRE with the consensus octamer TRE, we assigned the IAP-TRE as an everted repeat of two nonamers (Fig. 9A). However, it will require crystallographic data on TRE-TR-RXR complex to conclusively define whether the IAP-TRE is an everted repeat or direct repeat of nonamers, or otherwise. Until now, no TRE with nonamer half-sites has been reported. However, we believe that many of the identified TREs, especially the octamer TREs, could be in fact nonameric TREs if the boundaries of the TREs were determined. We observed that TR1-RXR complex can efficiently bind to the nonamers of the IAP-TRE separated by extensively variable spacer regions (data not shown). Such observation probably indicates a generalized principle of mechanism of TR-RXR binding to an everted repeat that allows extensive variability of the length of the spacer region. However, to establish such a principle extensive work will be required to study in vitro and in vivo receptor functions on different direct and everted repeats.

Analyses of TREs (Table 5) indicate that TRs exert their effects by interaction with TREs of different forms and structures. Our results (Table 4), as well as other results (34, 35), indicate that the dinucleotide TG or TA at the 5' end of the downstream hexamer half-site of a TRE is indeed a part of a TRE. Thus, we think that the rule of DR4 does not apply to a TRE containing TG or TA at the 5' end of the downstream hexamer half-site. To define the space between two half-sites of a TRE we counted the space between the downstream octamer/hexamer and upstream hexamer/octamer (Table 5). We predict that the existence of a TG or TA at the 5' end of a hexamer half-site allows the TR1-RXR complex to bind to relatively diverse sequences compared with the consensus DR4 TRE sequence.

To determine the existence of another TRE as well as to ascertain whether the identified TRE was an isoform-specific TRE, we examined the effects of exogenous TRs on a mutant of the identified IAP-TRE. Knockout mouse data suggest that TR1 is the major TR involved in intestinal development and homeostasis (9, 10, 11, 12, 13, 14). However, the wild-type IAP reporter gene was highly activated in response to T₃ when the cells were cotransfected with a plasmid expressing a bona fide TR (TR1, TRß1, or TRß2) that contains both DNA and ligand (T₃) binding domains (Fig. 10). No exogenous TR was able to activate the IAP reporter construct carrying the deletion of the IAP-TRE. Thus, it was confirmed that there was no other TRE within the 2.5-kb proximal promoter region of the IAP gene, and also that the identified TRE was not an isoformspecific TRE. The varying levels of IAP activation by TR1 (10-fold), TRß1 (16-fold), and TRß2 (9-fold) are of uncertain significance but may reflect different affinities of the TRs for the IAP-TRE or it may also reflect different levels of expression of TR isoforms in the transfected cells.

Ligand-independent repression of positively controlled T₃-responsive genes has been described (59, 60, 61, 62). In the transient transfections, we also observed ligand-independent repression of the IAP gene by approximately 75% with cotransfected TR expression plasmids (data not shown). Ligand-independent repression was abolished in the IAP-TRE mutants (Fig. 8B). It is worth noting that Caco-2 cells express only small amount of endogenous TRs. As such, to precisely define the TR-mediated repression of the IAP promoter will require studies in a cell line that does not express any endogenous TR. The unliganded TR acts as a repressor as a result of its association with a complex of proteins, including corepressors [e.g.nuclear receptor coactivator protein (N-CoP)/silencing mediator for retinoid and thyroid hormone receptors (SMRT)], mSinA, ski proto-oncoprotein, and histone deacetylase. Upon binding to the ligand, the repressor complex is replaced by the other proteins, including coactivators such as cAMP response element binding protein-binding protein/p300, chromatin assembly factor (CAF), and other proteins associated with histone hyperacetylation resulting in gene activation (59, 60, 61, 62). It is possible that TRs in the Caco-2 cells recognize the atypical IAP-TRE by their interaction with other tissue-specific factors. We have previously shown that the gut-enriched Kruppel-like factor (KLF) 4 activates the IAP gene through a guanine-cytosine (GC)-rich cis element (Fig. 9B), and cotransfection studies showed that KLF4 and TR/T₃ synergistically activate the IAP gene (63). It will be interesting to investigate the role of other possible corepressor and coactivator factors in Caco-2 cells that associate with TRs as well as to characterize the various TREs in other gut-specific genes determining whether any tissue-specific pattern of TREs exists.

In conclusion, we have identified a novel TRE that is involved in T₃-mediated positive regulation of the enterocyte differentiation marker IAP gene. Further studies of T₃ regulation of this gene in association with other gut-specific transcription factors will undoubtedly shed light on the molecular mechanisms governing the fundamental process of gut development, homeostasis, and differentiation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Materials
T₃ was purchased from Sigma (St. Louis, MO). DNA restriction enzymes and DNA modifying enzymes were purchased from New England Biolabs (Beverly, MA). Superscript II reverse transcriptase and Trizol reagent for RNA preparation were obtained from Invitrogen Life Technologies (Carlsbad, CA). Taq DNA polymerase and TNT T7 Quick Coupled Transcription/Translation System were obtained from Promega (Madison, WI). Polydeoxyinosinic-deoxycytidylic acid was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). SuperFect transfection reagent, the kit for DNA extraction from agarose gel, and also the kit for large scale DNA preparation were obtained from QIAGEN (Valencia, CA). Radionucleotides were purchased from PerkinElmer Life Sciences (Boston, MA), and the oligonucleotides were synthesized by Biosource International (Camarillo, CA) and Sigma Genosys (The Woodlands, TX). The monoclonal anti-TR1 antibody was purchased from Covance (Richmond, CA). The monoclonal anti-TRß1 antibody was obtained from the Santa Cruz Biotechnology (Santa Cruz, CA), and the polyclonal anti-TRß1 antibody was purchased from the Upstate Biotechnology Inc (Charlottesville, VA).

Plasmids
The pGL3-Basic mammalian promoter-detection vector and the pRL-CMV control plasmid expressing Renilla luciferase were obtained from Promega. The human RXR, TR1, TRß1, and TRß2 expression plasmids were the derivatives of the pSG5 vector (Stratagene, Cedar Creek, TX), and were the kind gifts of Anthony Hollenberg (Beth Israel Deaconess Medical Center, Boston, MA).

The 2.5-kb SacI-BamHI fragment from the plasmid IAP_2.4CAT (64), carrying the human IAP promoter region (–2574 to –49, relative to translation initiation codon ATG), was subcloned into the pGL3-Basic vector digested with SacI and BglII, thus constructing the plasmid pIAP-2574/-49. Various 5' deletion mutants were constructed by digesting the plasmid pIAP-2574/-49 with SacI and another appropriate restriction enzyme followed by generation of blunt-ended fragments, agarose gel purification of the desired band and recircularization. The internal deletion plasmid pIAP-2574-753/-617 was generated by deleting the 135-bp PflMI-BstEII restriction fragment from pIAP-2574/-49. The other internal deletion mutants were constructed by replacing the above 135 bp PflMI-BstEII restriction fragment of pIAP-2574/-49 with oligonucleotides representing specific sequences of this 135-bp region. The plasmid pIAP-2574TRE was generated by deleting –631 to –613 bases by PCR-mediated mutagenesis. The novel joint(s) in each plasmid was determined by DNA sequencing (65, 66).

Cell Culture
The Caco-2 human colorectal adenocarcinoma cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA) and was maintained in DMEM (Invitrogen Life Technologies) supplemented with 10% fetal bovine serum (FBS, Sigma, St. Louis, MO), 2 mM L-glutamine, and 100 U/ml each of penicillin and streptomycin (Invitrogen Life Technologies). DMEM containing FBS stripped of thyroid hormone (T₃) was prepared by treating FBS with ion-exchange resin and charcoal (67). The cells were grown at 37 C in the presence of 5% CO₂ and were split by trypsinization when they reached about 80–90% confluence.

RNA Preparation
Total RNA was prepared using Trizol reagent from Invitrogen Life Technologies following the manufacturer’s instructions. Where indicated, cells were treated with T₃ (100 nM) for the specified period of time before RNA extraction.

RT-PCR
First-strand cDNA was synthesized using RNA from Caco-2 cells with poly-deoxythymidine primer and Superscript II reverse transcriptase after the instructions of the manufacturer. PCR was then performed with gene specific primers using Taq DNA polymerase from Promega. The list of primers used in RT-PCR is given below:

1. TRa1.1153F: 5'-ccg cac ttc tgg ccc aag ctg ctg atg-3' 27 (bases)

2. TRa1.1184F: 5'-gtg act gac ctc cgc atg atc ggg gcc tg-3' 29 (bases)

3. TRa1.1530R: 5'-cac act gtc ctt tcc ata gca agt tc-3' 26 (bases)

4. TRa2.1620R: 5'-cca gct ttc agg cac ctc ctg ctc ttg-3' 27 (bases)

5. TRb1.121F: 5'-cgg ctc cag ggc act ggt aat ttg gc-3' 26 (bases)

6. TRb2.151F: 5'-ggc agc aat tgc tac atg cag tcc ac-3' 26 (bases)

7. TRb1.605R: 5'-cag agc tcg tcc ttg tct aag taa ctg c-3' 28 (bases)

8. TRb1.610R: 5'-gtc taa gta act ggg gat gta ccc t-3' 25 (bases)

Western Blotting
Western blotting was performed according the protocols described in Nomura et al. (60). Proteins were extracted from untransfected Caco-2 cells as well as from Caco-2 cells transiently transfected with an expression plasmid overexpressing TR1 or TRß1. Approximately 15 µg of plasmid DNA were used to transfect 50% confluent Caco-2 cells in a 10-cm culture dish. Forty-eight hours after transfection, cells were lysed in the lysis buffer [50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100] that also contained a complete range of protease inhibitors (Roche Applies Science, Indianapolis, IN); and whole-cell lysates were prepared. Nuclear extract from Caco-2 cells was prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents kit from Pierce (Rockford, IL) according to instructions from the manufacturer (see below). The respective lysates and nuclear extract were analyzed by Western blotting using anti-TR1 or anti-TRß1 antibodies. The lysate or nuclear extract was mixed in Laemmli denaturing loading buffer [15 mM Tris-HCl (pH 6.8), 0.5% sodium dodecyl sulfate (SDS), 2.5% glycerol, 0.5% betamercaptoethanol, and 0.001% bromophenol blue], boiled for 10 min, and then electrophoresed through a Ready Gel Tris-HCl Gel (10% polyacrylamide resolving gel, 4% polyacrylamide stacking gel; Bio-Rad, Hercules, CA) in Tris-glycine-SDS running buffer (Boston Bioproducts, Ashland, MA). Proteins were electrotransferred onto nitrocellulose membranes, and blocked with 5% (wt/vol) nonfat milk (Bio-Rad) for 1 h at room temperature. The blot was then incubated overnight at 4 C in the presence of the respective antibody (1:1000 dilution). The horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution; Bio-Rad) was added to the blot and incubated for 1 h at room temperature. The protein bands were identified by developing the blot with Immun-Star horseradish peroxidase chemiluminescent kit from Bio-Rad.

Northern Hybridization
Northern hybridization was performed following the protocol described in Hodin et al. (18). Twenty micrograms of total RNA were mixed with glyoxal loading dye and electrophoresed through an agarose gel prepared in Northern Max Gly Gel-Prep/Running Buffer (Ambion, Austin, TX). Capillary transfer was performed to transfer RNA onto a nylon membrane (Amersham Pharmacia Biotech) and this RNA was cross-linked to the nylon membrane using the Stratalinker 1880 UV hybridization oven (Stratagene, Cedar Creek, TX) according to the manufacturer’s instructions. About 50 ng of ³²P-labeled DNA probe was prepared using Ready-To-Go DNA Labeling Beads from Amersham Pharmacia Biotech, typically achieving a specific activity of about 5 x 10⁸ cpm/µg DNA. The IAP probe was a 1.9-kb PstI fragment derived from human IAP cDNA (15) and was obtained from ATCC. The actin probe was a 1.0-kb PstI fragment derived from the mouse ß-actin cDNA (68). Hybridization was performed in Rapid-hyb buffer (Amersham Pharmacia Biotech, Buckinghamshire, UK) for overnight at 65 C. The membrane was then washed twice with low stringency wash buffer containing 2x saline sodium citrate (SSC) and 0.1% SDS at 65 C for 30 min each followed by high stringency wash with 0.1x SSC/0.1% SDS (Boston Bioproducts) at 65 C for 30 min. The membrane was then subjected to autoradiography.

DNA Sequencing
DNA sequencing of each plasmid was determined by the Sequencing Core Facility at the Department of Molecular Biology, Massachusetts General Hospital (Boston, MA), using dye-labeled dideoxy nucleotide chain-terminators.

Transient Transfection and Luciferase Reporter Assays
Transient transfection and luciferase reporter assays were performed according to the protocols described in Siddique et al. (63). Caco-2 cells were plated at a density of 300,000 cells per well of a six-well plate in DMEM containing FBS stripped of T₃. Cells were grown overnight and transient transfections were performed using SuperFect reagent from QIAGEN. The test plasmid DNA (1.5 µg/well) was cotransfected with control plasmid pRL-CMV DNA (0.5 µg/well), and the total amount of DNA was kept the same for each transfection by adding nonspecific plasmid TF12 DNA. The control Renilla luciferase activity (pRL-CMV) was used to determine transfection efficiency as well as to normalize the firefly luciferase activity data. After transfection, the cells were grown for a further 24 h in DMEM containing FBS stripped of T₃. The cells were then maintained in stripped media and treated ± T₃ (100 nM) for another 24 h, except the experiments requiring T₃ treatment for a specific period of time. Firefly and Renilla luciferase assays were then performed on cell lysates using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions.

In Vitro Protein Synthesis
TNT T7 Quick Coupled Transcription/Translation System (Promega) was used for in vitro synthesis of human TR1, TRß1, and RXR from the derivatives of pSG5 (Stratagene) carrying the relevant coding sequences under the control of the T7 promoter.

Preparation of Nuclear Extract
Nuclear extract was prepared from 2 x 10⁶ Caco-2 cells using NE-PER Nuclear and Cytoplasmic Extraction Reagents kit from Pierce (Rockford, IL) according to instructions from the manufacturer. Halt Protease Inhibitor Cocktail (Pierce) was added to the extract, and stored at –80 C.

EMSA
EMSA was performed according to the protocol described in Giralt et al. (37). The complementary oligonucleotides were annealed and radiolabeled by the kinasing reaction with T₄ polynucleotide kinase in the presence of [-³³P]ATP. The radiolabeled probe was purified twice by passing it through Micro Bio-Spin 6 chromatography columns (Bio-Rad), followed by determination of the specific activity, which usually measured about 10⁸ cpm/µg DNA. Approximately 10 ng of radiolabeled probe were incubated at room temperature for 20 min with 1 µl of synthesized TR and/or RXR each in 10 µl of binding buffer containing 20 mM HEPES (pH 7.7), 50 mM NaCl, 1 mM dithiothreitol, 0.1 mM EDTA, 5 µM nonspecific oligo, 10% glycerol, and 2 µg of polydeoxyinosinic-deoxycytidylic acid. The samples were electrophoresed in a 5% polyacrylamide gel in a cold room (4 C), followed by drying of the gel and autoradiography.

In Vitro Mutagenesis
PCR mutagenesis was performed to introduce a mutation(s) in the putative TRE for functional analysis as described in Wilber and Xu (69). PCR primers were synthesized with a specific mutation followed by PCR amplification and ligation of the PCR product into an appropriate plasmid. Each mutant was sequenced to verify the mutation(s).

	FOOTNOTES

 
This work was supported by National Institutes of Health Research Grants DK47186 and DK50623 (to R.A.H.)

Abbreviations: DR, Direct repeat; FBS, fetal bovine serum; hdio1, human type 1 deiodinase gene; IAP, intestinal alkaline phosphatase; KLF, Kruppel-like factor; mTRE, mutant IAP-TRE; RXR, retinoid X receptor; TR, thyroid hormone receptor; SDS, sodium dodecyl sulfate; TRE, thyroid hormone response element.

Received for publication September 12, 2003. Accepted for publication May 3, 2004.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

1. Henning SJ 1981 Postnatal development: coordination of feeding, digestion, and metabolism. Am J Physiol 241:G199–G214
2. Yeh KY, Moog M 1977 Influence of the thyroid and adrenal glands on the growth of the intestine of the suckling rat, and on the development of intestinal alkaline phosphatase and disaccharidase activities. J Exp Zool 200:337–347[CrossRef][Medline]
3. Israel EJ, Pang KY, Harmatz PR, Walker WA 1987 Structural and functional maturation of rat gastrointestinal barrier with thyroxine. Am J Physiol 252:G762–G767
4. Wall AJ, Middleton WR, Pearse AG, Booth CC 1970 Intestinal mucosal hyperplasia following induced hyperthyroidism in the rat. Virchows Arch B Cell Pathol 6:79–87
5. Leblond CP, Carriere R 1955 The effect of growth hormone and thyroxine on the mitotic rate of the intestinal mucosa of the rat. Endocrinology 56:261–266
6. Tutton PJ 1976 The influence of thyroidectomy and of triiodothyronine administration on epithelial cell proliferation in the jejunum of rat. Virchows Arch B Cell Pathol 20:139–142
7. Celano P, Jumawan J, Koldovsky O 1977 Thyroxine-evoked decrease of jejunal lactase activity in adult rats. Gastroenterology 73:425–427[Medline]
8. Watson WC, Tuckerman JF 1971 Effect of thyroid status on intestinal alkaline phosphatase levels in the rat. Endocrinology 88:1524–1527[Medline]
9. Plateroti M, Chassande O, Fraichard A, Gauthier K, Freund JN, Samarut J, Kedinger M 1999 Involvement of T₃R- and ß-receptor subtypes in mediation of T₃ functions during postnatal murine intestinal development. Gastroenterology 116:1367–1378[CrossRef][Medline]
10. Plateroti M, Gauthier K, Domon-Dell C, Freund JN, Samarut J, Chassande O 2001 Functional interference between thyroid hormone receptor (TR) and natural truncated TR isoforms in the control of intestine development. Mol Cell Biol 21:4761–4772[Abstract/Free Full Text]
11. Gauthier K, Chassande O, Plateroti M, Roux JP, Legrand C, Pain B, Rousset B, Weiss R, Trouillas J, Samarut J 1999 Different functions for the thyroid hormone receptors TR and TRß in the control of thyroid hormone production and post-natal development. EMBO J 18:623–631[CrossRef][Medline]
12. Fraichard A, Chassande O, Plateroti M, Roux JP, Trouillas J, Dehay C, Legrand C, Gauthier K, Kedinger M, Malaval L, Rousset B, Samarut J 1997 The T₃R gene encoding a thyroid hormone receptor is essential for post-natal development and thyroid hormone production. EMBO J 16:4412–4420[CrossRef][Medline]
13. Forrest D, Erway LC, Ng L, Altschuler R, Curran T 1996 Thyroid hormone receptor ß is essential for development of auditory function. Nat Genet 13:354–357[CrossRef][Medline]
14. Forrest D, Hanebuth E, Smeyne RJ, Everds N, Stewart CL, Wehner JM, Curran T 1996 Recessive resistance to thyroid hormone in mice lacking thyroid hormone receptor ß: evidence for tissue-specific modulation of receptor function. EMBO J 15:3006–3015[Medline]
15. Henthorn PS, Raducha M, Edwards YH, Weiss MJ, Slaughter C, Lafferty MA, Harris H 1987 Nucleotide and amino acid sequences of human intestinal alkaline phosphatase: close homology to placental alkaline phosphatase. Proc Natl Acad Sci USA 84:1234–1238[Abstract/Free Full Text]
16. McComb RB, Bowers Jr GN, Posen S 1979 Alkaline phosphatase. New York: Plenum Publishing Corp.
17. Goldstein DJ, Rogers C, Harris H 1982 Evolution of alkaline phosphatases in primates. Proc Natl Acad Sci USA 79:879–883[Abstract/Free Full Text]
18. Hodin RA, Chamberlain SM, Meng S 1995 Pattern of rat intestinal brush-border enzyme gene expression changes with epithelial growth state. Am J Physiol 269:C385–C391
19. Mahmood A, Shao JS, Alpers DH 2003 Rat enterocytes secrete SLPs containing alkaline phosphatase and cubilin in response to corn oil feeding. Am J Physiol Gastrointest Liver Physiol 285:G433–G441
20. Narisawa S, Huang L, Iwasaki A, Hasegawa H, Alpers DH, Millan JL 2003 Accelerated fat absorption in intestinal alkaline phosphatase knockout mice. Mol Cell Biol 23:7525–7530[Abstract/Free Full Text]
21. Hodin RA, Chamberlain SM, Upton MP 1992 Thyroid hormone differentially regulates rat intestinal brush border enzyme gene expression. Gastroenterology 103:1529–1536[Medline]
22. Hodin RA, Shei A, Morin M, Meng S 1996 Thyroid hormone and the gut: selective transcriptional activation of a villus-enterocyte marker. Surgery 120:138–143[CrossRef][Medline]
23. Lazar MA 1993 Thyroid hormone receptors: multiple forms, multiple possibilities. Endocr Rev 14:184–193[Abstract/Free Full Text]
24. Zhang J, Lazar MA 2000 The mechanism of action of thyroid hormones. Annu Rev Physiol 62:439–466[CrossRef][Medline]
25. Forman BM, Samuels HH 1990 Interactions among a subfamily of nuclear hormone receptors: the regulatory zipper model. Mol Endocrinol 4:1293–1301[Abstract/Free Full Text]
26. Umesono K, Murakami KK, Thompson CC, Evans RM 1991 Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors. Cell 65:1255–1266[CrossRef][Medline]
27. Umesono K, Evans RM 1989 Determinants of target gene specificity for steroid/thyroid hormone receptors. Cell 57:1139–1146[CrossRef][Medline]
28. Glass CK, Franco R, Weinberger C, Albert VR, Evans RM, Rosenfeld MG 1987 A c-erb-A binding site in rat growth hormone gene mediates trans-activation by thyroid hormone. Nature 329:738–741[CrossRef][Medline]
29. Ribeiro RC, Apriletti JW, Yen PM, Chin WW, Baxter JD 1994 Heterodimerization and deoxyribonucleic acid-binding properties of a retinoid X receptor-related factor. Endocrinology 135:2076–2085[Abstract]
30. Rastinejad F, Perlmann T, Evans RM, Sigler PB 1995 Structural determinants of nuclear receptor assembly on DNA direct repeats. Nature 375:203–211[CrossRef][Medline]
31. Kliewer SA, Umesono K, Mangelsdorf DJ, Evans RM 1992 Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signalling. Nature 355:446–449[CrossRef][Medline]
32. Zhang XK, Lehmann J, Hoffmann B, Dawson MI, Cameron J, Graupner G, Hermann T, Tran P, Pfahl M 1992 Homodimer formation of retinoid X receptor induced by 9-cis retinoic acid. Nature 358:587–591[CrossRef][Medline]
33. Yu VC, Delsert C, Andersen B, Holloway JM, Devary OV, Naar AM, Kim SY, Boutin JM, Glass CK, Rosenfeld MG 1991RXR ß: a coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate response elements. Cell 67:1251–1266
34. Katz RW, Koenig RJ 1993 Nonbiased identification of DNA sequences that bind thyroid hormone receptor 1 with high affinity. J Biol Chem 268:19392–19397[Abstract/Free Full Text]
35. Katz RW, Koenig RJ 1994 Specificity and mechanism of thyroid hormone induction from an octamer response element. J Biol Chem 269:18915–18920[Abstract/Free Full Text]
36. Henthorn PS, Raducha M, Kadesch T, Weiss MJ, Harris H 1988 Sequence and characterization of the human intestinal alkaline phosphatase gene. J Biol Chem 263:12011–12019[Abstract/Free Full Text]
37. Giralt M, Park EA, Gurney AL, Liu JS, Hakimi P, Hanson RW 1991 Identification of a thyroid hormone response element in the phosphoenolpyruvate carboxykinase (GTP) gene. Evidence for synergistic interaction between thyroid hormone and cAMP cis-regulatory elements. J Biol Chem 266:21991–21996[Abstract/Free Full Text]
38. Wu Y, Xu B, Koenig RJ 2001 Thyroid hormone response element sequence and the recruitment of retinoid X receptors for thyroid hormone responsiveness. J Biol Chem 276:3929–3936[Abstract/Free Full Text]
39. Martinez de Arrieta C, Morte B, Coloma A, Bernal J 1999 The human RC3 gene homolog, NRGN contains a thyroid hormone-responsive element located in the first intron. Endocrinology 140:335–343[Abstract/Free Full Text]
40. Toyoda N, Zavacki AM, Maia AL, Harney JW, Larsen PR 1995 A novel retinoid X receptor-independent thyroid hormone response element is present in the human type 1 deiodinase gene. Mol Cell Biol 15:5100–5112[Abstract]
41. Furlow JD, Kanamori A 2002 The transcription factor basic transcription element-binding protein 1 is a direct thyroid hormone response gene in the frog Xenopus laevis. Endocrinology 143:3295–3305[Abstract/Free Full Text]
42. Norman MF, Lavin TN, Baxter JD, West BL 1989 The rat growth hormone gene contains multiple thyroid response elements. J Biol Chem 264:12063–12073[Abstract/Free Full Text]
43. Jansen MS, Cook GA, Song S, Park EA 2000 Thyroid hormone regulates carnitine palmitoyltransferase I gene expression through elements in the promoter and first intron. J Biol Chem 275:34989–34997[Abstract/Free Full Text]
44. Sakurai A, Suzuki S, Katai M, Miyamoto T, Kobayashi H, Nakajima K, Ichikawa K, DeGroot LJ, Hashizume K 1995 Transcriptional regulation of human thyroid hormone receptor ß 1 gene expression: effect of human retinoid X receptor and identification of a transcriptional silencer region. Mol Cell Endocrinol 110:103–112[CrossRef][Medline]
45. Weber T, Zimmermann U, Winter H, Mack A, Kopschall I, Rohbock K, Zenner HP, Knipper M 2002 Thyroid hormone is a critical determinant for the regulation of the cochlear motor protein prestin. Proc Natl Acad Sci USA 99:2901–2906[Abstract/Free Full Text]
46. Garcia-Fernandez LF, Urade Y, Hayaishi O, Bernal J, Munoz A 1998 Identification of a thyroid hormone response element in the promoter region of the rat lipocalin-type prostaglandin D synthase (ß-trace) gene. Brain Res Mol Brain Res 55:321–330[Medline]
47. Desvergne B, Favez T 1997 The major transcription initiation site of the SV40 late promoter is a potent thyroid hormone response element. Nucleic Acids Res 25:1774–1781[Abstract/Free Full Text]
48. Torrance CJ, Usala SJ, Pessin JE, Dohm GL 1997 Characterization of a low affinity thyroid hormone receptor binding site within the rat GLUT4 gene promoter. Endocrinology 138:1215–1223[Abstract/Free Full Text]
49. Zavacki AM, Harney JW, Brent GA, Larsen PR 1996 Structural features of thyroid hormone response elements that increase susceptibility to inhibition by an RTH mutant thyroid hormone receptor. Endocrinology 137:2833–2841[Abstract]
50. Farsetti A, Desvergne B, Hallenbeck P, Robbins J, Nikodem VM 1992 Characterization of myelin basic protein thyroid hormone response element and its function in the context of native and heterologous promoter. J Biol Chem 267:15784–15788[Abstract/Free Full Text]
51. Park HY, Davidson D, Raaka BM, Samuels HH 1993 The herpes simplex virus thymidine kinase gene promoter contains a novel thyroid hormone response element. Mol Endocrinol 7:319–330[Abstract/Free Full Text]
52. Szilagyi A, Lerman S, Barr RG, Stern J, Colacone A, McMullan S 1991 Reversible lactose malabsorption and intolerance in Graves’ disease. Clin Invest Med 14:188–197[Medline]
53. Latham KM, Stanbridge EJ 1990 Identification of the HeLa tumor-associated antigen, p75/150, as intestinal alkaline phosphatase and evidence for its transcriptional regulation. Proc Natl Acad Sci USA 87:1263–1267[Abstract/Free Full Text]
54. Hodin RA, Meng S, Chamberlain SM 1994 Thyroid hormone responsiveness is developmentally regulated in the rat small intestine: a possible role for the -2 receptor variant. Endocrinology 135:564–568[Abstract]
55. Giannella RA, Orlowski J, Jump ML, Lingrel JB 1993 Na(+)-K(+)-ATPase gene expression in rat intestine and Caco-2 cells: response to thyroid hormone. Am J Physiol 265:G775–G782
56. Miller LD, Park KS, Guo QM, Alkharouf NW, Malek RL, Lee NH, Liu ET, Cheng SY 2001 Silencing of Wnt signaling and activation of multiple metabolic pathways in response to thyroid hormone-stimulated cell proliferation. Mol Cell Biol 21:6626–6639[Abstract/Free Full Text]
57. Olson DP, Koenig RJ 1997 5'-Flanking sequences in thyroid hormone response element half-sites determine the requirement of retinoid X receptor for receptor-mediated gene expression. J Biol Chem 272:9907–9914[Abstract/Free Full Text]
58. Kim HS, Crone DE, Sprung CN, Tillman JB, Force WR, Crew MD, Mote PL, Spindler SR 1992 Positive and negative thyroid hormone response elements are composed of strong and weak half-sites 10 nucleotides in length. Mol Endocrinol 6:1489–1501[Abstract/Free Full Text]
59. Heinzel T, Lavinsky RM, Mullen TM, Soderstrom M, Laherty CD, Torchia J, Yang WM, Brard G, Ngo SD, Davie JR, Seto E, Eisenman RN, Rose DW, Glass CK, Rosenfeld MG 1997 A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression. Nature 387:43–48[CrossRef][Medline]
60. Nomura T, Khan MM, Kaul SC, Dong HD, Wadhwa R, Colmenares C, Kohno I, Ishii S 1999 Ski is a component of the histone deacetylase complex required for transcriptional repression by Mad and thyroid hormone receptor. Genes Dev 13:412–423[Abstract/Free Full Text]
61. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835–839[CrossRef][Medline]
62. Rachez C, Lemon BD, Suldan Z, Bromleigh V, Gamble M, Naar AM, Erdjument-Bromage H, Tempst P, Freedman LP 1999 Ligand-dependent transcription activation by nuclear receptors requires the DRIP complex. Nature 398:824–828[CrossRef][Medline]
63. Siddique A, Malo MS, Ocuin LM, Hinnebusch BF, Abedrapo MA, Henderson JW, Zhang W, Mozumder M, Yang VW, Hodin RA 2003 Convergence of the thyroid hormone and gut-enriched Kruppel-like factor pathways in the context of enterocyte differentiation. J Gastrointest Surg 7:1053–1061[CrossRef][Medline]
64. Kim JH, Meng S, Shei A, Hodin RA 1999 A novel Sp1-related cis element involved in intestinal alkaline phosphatase gene transcription. Am J Physiol 276:G800–G807
65. Sanger F, Nicklen S, Coulson AR 1977 DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467[Abstract/Free Full Text]
66. Tabor S, Richardson CC 1987 DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci USA 84:4767–4771[Abstract/Free Full Text]
67. Samuels HH, Stanley F, Casanova J 1979 Depletion of L-3,5,3'-triiodothyronine and L-thyroxine in euthyroid calf serum for use in cell culture studies of the action of thyroid hormone. Endocrinology 105:80–85[Abstract/Free Full Text]
68. Cleveland DW, Lopata MA, MacDonald RJ, Cowan NJ, Rutter WJ, Kirschner MW 1980 Number and evolutionary conservation of - and ß-tubulin and cytoplasmic ß- and -actin genes using specific cloned cDNA probes. Cell 20:95–105[CrossRef][Medline]
69. Wilber JF, Xu AH 1998 The thyrotropin-releasing hormone gene: cloning, characterization, and transcriptional regulation in the central nervous system, heart, and testis. Thyroid 8:897–901[Medline]

NURSA Molecule Pages Link:

Nuclear Receptors:   TRα  |  TRβ  |  RXRα

Ligands:   Thyroid hormone

This article has been cited by other articles:

				G. Doyon, S. St-Jean, M. Darsigny, C. Asselin, and F. Boudreau Nuclear Receptor Co-repressor Is Required to Maintain Proliferation of Normal Intestinal Epithelial Cells in Culture and Down-modulates the Expression of Pigment Epithelium-derived Factor J. Biol. Chem., September 11, 2009; 284(37): 25220 - 25229. [Abstract] [Full Text] [PDF]

				N. S. Belaguli, M. Zhang, M. Rigi, M. Aftab, and D. H. Berger Cooperation between GATA4 and TGF-beta signaling regulates intestinal epithelial gene expression Am J Physiol Gastrointest Liver Physiol, June 1, 2007; 292(6): G1520 - G1533. [Abstract] [Full Text] [PDF]

				M. Plateroti, E. Kress, J. I. Mori, and J. Samarut Thyroid Hormone Receptor {alpha}1 Directly Controls Transcription of the {beta}-Catenin Gene in Intestinal Epithelial Cells Mol. Cell. Biol., April 15, 2006; 26(8): 3204 - 3214. [Abstract] [Full Text] [PDF]

				M. S. Malo, M. Mozumder, X. B. Zhang, S. Biswas, A. Chen, L.-C. Bai, J. L. Merchant, and R. A. Hodin Intestinal alkaline phosphatase gene expression is activated by ZBP-89 Am J Physiol Gastrointest Liver Physiol, April 1, 2006; 290(4): G737 - G746. [Abstract] [Full Text] [PDF]

				F. Alkhoury, M. S. Malo, M. Mozumder, G. Mostafa, and R. A. Hodin Differential regulation of intestinal alkaline phosphatase gene expression by Cdx1 and Cdx2 Am J Physiol Gastrointest Liver Physiol, August 1, 2005; 289(2): G285 - G290. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager NURSA Molecule Pages Link Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Malo, M. S. Articles by Hodin, R. A. Search for Related Content PubMed PubMed Citation Articles by Malo, M. S. Articles by Hodin, R. A.