help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2006-1088
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/7/3468    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, K. K.
Right arrow Articles by Kim, K. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, K. K.
Right arrow Articles by Kim, K. E.
Endocrinology Vol. 148, No. 7 3468-3476
Copyright © 2007 by The Endocrine Society

Activation of the Thyroid-Stimulating Hormone ß-Subunit Gene by LIM Homeodomain Transcription Factor Lhx2

Kee K. Kim, Seok B. Song, Kwang I. Kang, Myungchull Rhee and Kyoon Eon Kim

Departments of Biochemistry (K.K.K., S.B.S., K.E.K.) and Biology (M.R.) and Institute of Biotechnology (K.I.K.), Chungnam National University, Daejeon 305-764, Korea

Address all correspondence and requests for reprints to: Kyoon Eon Kim, Ph.D., Department of Biochemistry, Chungnam National University, Daejeon 305-764, Korea. E-mail: kyoonkim{at}cnu.ac.kr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although there is evidence that the LIM homeodomain transcription factor, Lhx2, can stimulate transcription of the glycoprotein hormone {alpha}-subunit gene, the role of Lhx2 in regulating TSH ß-subunit has not been established. In the present studies, the ability of Lhx2 to regulate transcription of the TSH ß-subunit gene was examined. In the thyrotrope-derived T{alpha}T1 cell line, Lhx2 expression was found to be induced by treatment with either TRH or cAMP, consistent with the possibility that Lhx2 may play a role in mediating the ability of this signaling pathway to stimulate TSH gene expression. Transient, forced overexpression of Lhx2 stimulated activity of a TSH ß-subunit reporter gene. Deletion studies provided evidence that the –177 to –79 region of the TSH ß-subunit promoter was necessary for stimulation of reporter gene activity by Lhx2. A gel mobility shift assay provided the evidence that Lhx2 can bind to this region of DNA. DNase I footprinting studies demonstrated that two distinct regions of the TSHß promoter, –118 to –108 and –86 to –68, are protected by Lhx2 from nuclease digestion. These regions contain repeats of the sequence, 5'-(G/T)CAAT(T/A)-3'. Mutation of this sequence, especially in the –86 to –68 region, substantially decreased Lhx2 responsiveness of the TSH ß-subunit reporter gene. In addition, a DNA fragment containing the –177 to –79 region of the TSHß promoter was found to confer Lhx2 responsiveness to a minimal promoter. These results provide multiple lines of evidence consistent with a role for Lhx2 in modulating expression of the TSH ß-subunit gene.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TSH IS SECRETED FROM thyrotropes of the anterior pituitary and regulates secretion of thyroid hormone. Because TSH is a key regulator of thyroid function (1, 2, 3), it would be useful to define the mechanisms that support the expression of TSH in the thyrotropes. TSH is a member of the glycoprotein hormone family, which are heterodimers containing an {alpha}- and ß-subunit. The {alpha}-subunit is common to all members of the glycoprotein hormone family including LH, FSH, TSH, and chorionic gonadotropin, and the ß-subunits are unique and thus confer the specific immunogenic and hormonal functions (3, 4, 5). The glycoprotein hormone subunits are encoded by distinct genes. Therefore, the mechanism supporting transcription of the TSH ß-subunit gene is essential to the synthesis of functional TSH.

Physiological modulation of TSH subunit gene expression involves both negative and positive regulation. TSH subunit genes are negatively regulated at the transcriptional level by T3 and T4 (6, 7). The hypothalamic hormone, TRH, acts positively to enhance TSH gene expression (6, 8). TRH binds to the TRH receptor and increases intracellular cAMP, which probably plays a role in mediating transcriptional regulation (9, 10). There is evidence that the pituitary-specific transcription factor, pituitary transcription factor (Pit)-1, plays a role in mediating TRH and cAMP responses of the TSH ß-subunit gene (11, 12). Indeed it has been shown that elevated cAMP leads to phosphorylation of Pit1. Whereas it is likely that Pit1 represents part of the mechanism mediating transcriptional regulation of the TSHß gene by cAMP, this does not rule out a role for other transcription factors. GATA2 is a candidate that may play a role in regulating expression of the TSH ß-subunit gene (13, 14, 15). Transfection studies have demonstrated that Pit1 can cooperate with GATA2 to activate the transcription of the TSH ß-subunit gene (14, 15). The 5' regulatory region of the TSH ß-subunit gene contains a number of Pit1 and GATA2 binding sites, which may mediate functional synergism of these factors. In addition to GATA2, Lhx3 has also been reported to functionally interact with Pit1 (16). Transfection results demonstrated that Lhx3 potentiates the expression of TSH ß-subunit gene in the presence of Pit1 (17).

The finding that Lhx3 can enhance TSHß promoter activity raises the possibility that other members of the LIM-homeodomain transcription factor family may play a role in regulating TSHß gene expression. Mouse genetic experiments have demonstrated that several different LIM-homeodomain factors are involved in pituitary organogenesis and specification of anterior pituitary cell lineages (18, 19). There is evidence that in gonadotropes, Lhx2 may play a role in both basal and GnRH-stimulated expression of the glycoprotein hormone {alpha}-subunit gene (20). Lhx2 is present in thyrotrope-derived {alpha}TSH cells and gonadotrope-derived {alpha}T3–1 cells but not in somatolactotrope-derived GH3 cells (20). In the present studies, a possible role for Lhx2 in transcription of the TSHß gene was been examined.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids and reagents
The initial TSHß-luciferase reporter construct contains –1099 to +13 bp of the rat TSH ß-subunit promoter linked to the luciferase gene in the plasmid pLuc-link2 (21). A series of 5'deletion constructs (–795, –237, –177, and –79) were prepared using KpnI, DraI, BstNI, and TaqI restriction enzyme sites, which are uniquely present in the TSH ß-subunit promoter region. In vitro site-directed mutagenesis was carried out with the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). For each mutated segment, 32mer oligos were prepared with two or three mismatched bases in the center. Multimers of –177/–79 region of TSH ß-subunit promoter were subcloned upstream of the E1b minimal promoter, which was linked to the luciferase coding sequences (21). The pcDNA3 vector (Invitrogen, San Diego, CA) was used to construct mammalian expression vectors for Pit1, GATA2, Lhx3, Lhx2, mutated Lhx2 (mt-Lhx2), and antisense Lhx2 (as-Lhx2). As a transfection reagent, a 1 mM (45 µg/liter) stock solution of branched 25 kDa polyethylenimine (Aldrich, Milwaukee, WI) was prepared (22). The molarity of the solution was calculated on the basis of the molecular weight of the monomeric unit. Solutions were sterilized by filtration through 0.2-µm membranes (Millipore, Bedford, MA). The 8-(4-chlorophenylthio)-cAMP, TRH (pGlu-Glu-Pro-NH2), and other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).

Cell culture and transient transfection
The T{alpha}T1 cell line which was derived from thyrotrope cells of mouse anterior pituitary by directed oncogenesis in transgenic mice, was a generous gift from Dr. Pamela Mellon (University of California, San Diego, San Diego, CA). It was maintained in DMEM supplemented with 10% fetal bovine serum on Matrigel-coated plates (BD Biosciences, Bedford, MA) as described (23). The {alpha}TSH cell line was grown in monolayer culture in DMEM containing 10% heat-inactivated calf serum. GH3 cells were grown in monolayer culture in DMEM containing 2.5% heat-inactivated fetal bovine serum and 15% heat-inactivated horse serum. {alpha}T3–1 cells were grown in monolayer culture in DMEM containing 10% heat-inactivated fetal bovine serum. All cells were grown at 37 C in 5% CO2 incubator in accordance with the routine cell culture procedures. Cultured cells were transfected by the polyethylenimine technique as previously described (24). Luciferase activity was assayed using an LB 953 Autolumat (EG&G Berthold, Nashua, NH) as previously described (25, 26) and was normalized based on the expression of RSV-ß-galactosidase. ß-Galactosidase activity was assayed colorimetrically (27).

Electrophoretic gel mobility shift assay
T{alpha}T1 cells at approximately 80% confluence were used to prepare nuclei essentially as described (28). Expression and purification of recombinant Lhx2 were followed as described (20). DNA fragment spanning the –177/–45 bp of TSH ß-subunit promoter was end labeled with {alpha}32P-dCTP using DNA polymerase I. For the EMSA, a total volume of 20 µl reaction mixture contained 20,000 cpm DNA probe, 1 µg poly(dI-dC), and various amounts of nuclear extracts or Lhx2 homeodomain protein in 40 mM HEPES-KOH (pH 7.8), 10% glycerol, 1 mM MgCl2, 0.1 mM dithiothreitol. The reaction mixtures were incubated for 30 min at room temperature, electrophoresed through a nondenaturing polyacrylamide gel in 0.5x Tris-borate EDTA at 4 C, and then analyzed by autoradiography. For supershift assays, antiserum to Lhx2 was added to the reaction mixture before electrophoresis. Antiserum to Lhx2 was a generous gift from Dr. Mark S. Roberson (Cornell University, Ithaca, NY).

DNaseI footprinting assay
DNA-protein binding reactions were performed as described above for mobility shift assays and then subjected to DNase I digestion. At the completion of the 30-min binding reaction, 0.075 U of RQ1 DNaseI (Promega, Madison, WI) in 100 mM Tris-HCl (pH 7.5), 35 mM MgCl2 were added and incubated for an additional 5 min. The reaction was terminated by the addition of 3 µl of 0.5 M EDTA, extracted with phenol, and then ethanol precipitated. The precipitated products were electrophoresed through an 8 M urea, denaturing gel and analyzed by autoradiography.

Quantitative real-time RT-PCR
A two-step real-time PCR was carried out to analyze the expression of candidate genes. Total RNA was reverse transcribed into cDNA using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. The reaction was primed by 12–15 oligo (dT) primer. The oligonucleotide primers used in the quantitative real-time RT-PCR were as follows: TSHß forward primer (5'-TCTGTGCTGGGTATTGTATGAC-3') and TSHß reverse primer (5'-GCGGCTTGGTGCAGTAGTTG-3') were designed to amplify a 249-bp fragment; Pit1 forward primer (5'-ATTAAGTTAGGATACACCCAGACA-3') and Pit1 reverse primer (5'-CTTCCTTTCGTTTGCTCCCACCTT-3') were designed to amplify a 213-bp fragment; GATA2 forward primer (5'-ACTATGGCAGCAGTCTCTTCCATC-3') and GATA2 reverse primer (5'-AAGGTGGTGGTTGTCGTCTGAC-3') were designed to amplify a 301-bp fragment; Lhx3 forward primer (5'-CTACCTCATGGAAGACAGCCGG-3') and Lhx3 reverse primer (5'-CCTCCTGGATGCTGTCCTTGTC-3') were designed to amplify a 350-bp fragment; Lhx2 forward primer (5'-CCGTCCATCAGCAGTGACCGGGCA-3') and Lhx2 reverse primer (5'-GTTAAAGTGTGCTGGGTATTCGCC-3') were designed to amplify a 442-bp fragment. ß-Actin message as an internal control was amplified with ß-actin forward primer (5'-ATCGTGGGCCGCCCTAGGCA-3') and ß-actin reverse primer (5'-TGGCCTTACCCTTCAGAGGGG-3'). Real-time PCR was performed using the Rotorgene real-time analysis system (Rotor-Gene 2000 Robocycler; Corbett Research, Sydney, Australia) according to the manufacturer’s protocol with SYBR-Green as a fluorescent dye. All reactions were performed under identical conditions, using 35 cycles of amplification with denaturation at 95 C for 30 sec, annealing at 58 C for 30 sec, and elongation at 72 C for 30 sec. The specificity of products generated by each set of primers was examined using gel electrophoresis and further confirmed by a melting curve analysis. The relative expression level was computed with respect to the mRNA expression level of the internal standard ß-actin mRNA using the following formula, where Ct is the threshold cycle value (29, 30):

Relative mRNA expression = 1/2(Ct of gene of interest – Ct of ß-actin).

Chromatin immunoprecipitation (ChIP) assay
ChIP assays was performed using the reagents from Upstate (Charlottesville, VA) according to the manufacturer’s protocol with the following modifications. About 5 x 106 T{alpha}T1 cells grown in 60-mm dishes were treated with 1% formaldehyde for 10 min at room temperature and followed by quenching with 0.125 M glycine. After washing with PBS, cells were lysed with 0.4 ml of sodium dodecyl sulfate (SDS) lysis buffer. The lysate was sonicated 15 times for 10 sec each using a 150 W Sonifier Cell Disrupter 185 (Branson, Danbury, CT) at 10% power setting. This sonication method consistently yielded 200- to 2000-bp fragments with the majority of the fragments being approximately 500 bp long. For each immunoprecipitation, 0.1 ml of the sonicated cell lysate was diluted with 0.1 ml of SDS lysis buffer. The 0.2 ml diluted lysate was then further diluted 10-fold in ChIP dilution buffer. The lysate was immunoprecipitated with 2 µl of anti-Lhx2 rabbit antibody or anti-cAMP response element-binding protein (CREB) antibody. Immune complex was collected with protein-A agarose (Sigma). Immunoprecipitated DNA and input DNA were subjected to PCR using a primer set that amplifies –180 to –31 region of the TSHß gene that includes putative Lhx2 binding sites (forward, 5'-TCCAGGGAGAGGATCTAGTGAACC-3'; reverse, 5'-CCTTCGGGATAATTCCCCCCTGAT-3'). As a negative control, PCR was also performed using a primer set that amplifies the –1089 to –879 region of the TSHß gene that is not expected to be bound by Lhx2 (forward, 5'-AGAATGCACCATGAGGACTTTAAAAGTG-3'; reverse, 5'-CATTAAATGTGTAAATGACTGGGGAAGAC-3'. In addition, PCR was performed using primer set (forward, 5'-GGATGTTTTGTGTAAGGGTCAATAATATTA-3'; reverse, 5'-TTGATCATATCACATTGCAACCCTCAGATC-3'), which amplifies the –280 to –141 region of the mouse {alpha}GSU gene that contains cAMP response element (CRE).

Western blot analysis
Nuclear extracts of {alpha}TSH cells that has been transfected with Lhx2 or antisense-Lhx2 expression vector were obtained using NE-PER nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL) according to the manufacturer’s instructions. Nuclear extracts were resolved on a 10% SDS-polyacrylamide gel and then transferred to polyvinylidene difluoride membrane for immunoblotting. The membranes were incubated with 1:5000 diluted anti-Lhx2 rabbit antibody. Anti-CREB antibody and anti-PCNA (proliferating cell nuclear antigen) antibody were purchased from Santa Cruz biotechnology (Santa Cruz, CA). The secondary antibody was an antirabbit/horseradish peroxidase (Amersham, Aylesbury, UK) at 1:15,000 dilutions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The mRNA for Lhx2 is induced by TRH and cAMP in the mouse thyrotrope cell line, T{alpha}T1
To explore possible mechanisms modulating expression of the TSH ß-subunit gene, quantitative real-time PCR was used to determine the expression level of the mRNAs for Pit1, GATA2, Lhx3, and Lhx2. Mouse thyrotrope T{alpha}T1 cells were cultured in the presence or absence of TRH or cAMP and mRNA levels were determined. TSH ß-subunit mRNA was increased by TRH treatment in a dose-dependent manner (Fig. 1AGo). The mRNA for Pit1 and GATA2 were slightly increased, and Lhx3 mRNA levels were not changed over the dose range tested. Lhx2 mRNA was substantially increased by the same conditions. Similarly, substantial induction of Lhx2 mRNA was observed after cAMP treatment (Fig. 1BGo). Transfection experiments demonstrated that a transfected TSH ß-subunit promoter/luciferase reporter gene was activated by cAMP treatment in a time-dependent manner, whereas thymidine kinase (TK) promoter or minimal promoter containing only E1b TATA box did not respond to cAMP treatment (Fig. 1CGo). Interestingly, cAMP-induced activation of TSH ß-subunit construct was correlated with increased levels of Lhx2 protein, as assessed by Western blot (Fig. 1DGo). These results demonstrate that Lhx2 expression is increased by inducers of TSH ß-subunit gene expression and suggest that regulation of Lhx2 levels may play a role in mediating regulation of the TSHß expression.


Figure 1
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 1. TRH and cAMP treatments can induce Lhx2 gene expression. A, T{alpha}T1 cells were incubated for 12 h in the absence or presence of various concentrations of TRH (10–100 nM). Total RNA was isolated and expression of TSH ß-subunit, Pit1, GATA2, Lhx3, and Lhx2 mRNA were measured by a quantitative real-time PCR. Each value was normalized to that of ß-actin. B, T{alpha}T1 cells were treated with the 0.3 mM cAMP for 6 h. Total RNA was extracted and 5 µg of each sample were subjected to the quantitative real-time PCR analysis. C, T{alpha}T1 cells were transiently transfected with 2 µg of TSH ß-subunit promoter (–795 to +13) fused to the firefly luciferase gene. After 24 h of transfection, cells were treated with 0.3 mM cAMP for the indicated time. At least three individual experiments, each in triplicate, were performed and values expressed as fold induction are the mean ± SEM. The minimal promoter that contains only E1b TATA-box or TK promoter was fused to a luciferase gene, which served as a negative control. D, T{alpha}T1 cells were treated with the cAMP as in C and Western blot analysis of Lhx2 protein was performed with the nuclear extracts obtained at each time point. PCNA was employed as a loading control.

 
Lhx2 plays a role in activating the TSH ß-subunit promoter in the mouse thyrotrope cell line, T{alpha}T1
Previous studies have shown that Pit1, GATA2, and Lhx3 can modulate expression of the TSH ß-subunit gene (13, 14, 15). To compare the activity of Lhx2 with that of Pit1, GATA2, or Lhx3, T{alpha}T1 cells were transiently transfected with a TSH ß-subunit/luciferase reporter gene and expression vectors for the relevant transcription factors. It was observed that Pit1, GATA2, and Lhx3 each modestly activated the TSH ß-subunit reporter gene and Lhx2 more robustly activated the reporter gene in this context (Fig. 2Go). To determine whether endogenous Lhx2 participates in activation of the TSHß gene, an expression vector for as-Lhx2 was prepared and compared with an expression vector for Lhx2 (Fig. 3Go). Forced expression of Lhx2-induced endogenous TSH ß-subunit mRNA levels in a dose-dependent manner (Fig. 3AGo). In contrast, antisense Lhx2 substantially reduced TSHß mRNA levels (Fig. 3AGo). Western blot analysis with anti-Lhx2 antibody confirmed that as-Lhx2 construct reduced Lhx2 expression as expected (Fig. 3AGo). Activation of TSH ß-subunit reporter gene by transfection of an Lhx2 expression vector was reduced in a dose-dependent manner by antisense Lhx2 expression (Fig. 3BGo). Antisense Lhx2 did not affect the expression of TK-luciferase construct (data not shown). Moreover, cAMP- and TRH-induced TSH ß-subunit mRNA was substantially reduced by antisense Lhx2 expression (Fig. 3CGo). Likewise, increased TSH ß-subunit promoter activity by cAMP or TRH treatment was also reduced by antisense Lhx2 expression in transfection studies (data no shown). These results support a role for Lhx2 in activation of TSH ß-subunit gene.


Figure 2
View larger version (16K):
[in this window]
[in a new window]

  		FIG. 2. A TSH ß-subunit reporter gene can be stimulated by expression of Lhx2. A reporter gene containing the TSH ß-subunit promoter (–795 to +13) upstream of the firefly luciferase gene was cotransfected into T{alpha}T1 cells with expression vectors for Pit1, GATA2, Lhx3, or Lhx2. E1b TATA-box promoter and TK promoter were used as a negative control. Each set of transfections was performed in triplicate and values expressed as fold induction are mean ± SEM of three independent experiments.

 

Figure 3
View larger version (23K):
[in this window]
[in a new window]

  		FIG. 3. An expression vector for as-Lhx2 can reduce the ability of Lhx2 to activate TSHß gene expression. A, T{alpha}T1 cells were grown for 24 h and then transfected with increasing amounts of expression vector for Lhx2 (0.1, 0.5, 1 µg) or as-Lhx2 (0.1, 0.5, 1 µg). After 24 h, total RNA was isolated and expression levels of TSH ß-subunit mRNA were measured by a quantitative real-time PCR, which were then normalized to that of ß-actin. Lower panels represent Western blot data performed with anti-Lhx2 or PCNA antibody. B, T{alpha}T1 cells were cotransfected with 2 µg of TSH ß-subunit promoter (–795 to +13) linked to firefly luciferase, together with 0.5 µg of Lhx2 in pcDNA3 and increasing amount of as-Lhx2 expression construct (0.1, 1, and 3 µg). Cells were lysed and luciferase activities measured at 24 h after transfection. C, T{alpha}T1 cells were grown for 24 h and then treated with 0.3 mM cAMP for 6 h or 100 nM TRH for 12 h in the absence or presence of expression vector for as-Lhx2 (1 µg). Total RNA was isolated and expression levels of TSH ß-subunit mRNA were measured by a quantitative real-time PCR, which were then normalized to that of ß-actin.

 
The proximal region of TSH ß-subunit promoter is necessary for the activation by Lhx2
To define the region of the Lhx2 5' flanking sequence necessary for the stimulation by Lhx2, a series of 5'-deletion constructs (–795/+13-Luc, –237/+13-Luc, –177/+13-Luc, and –79/+13-Luc) were prepared and transfected into T{alpha}T1, {alpha}TSH, and GH3 cells with an Lhx2 expression vector. In all cell lines, Lhx2 activated the TSH ß-subunit promoter in a dose-dependent manner (data not shown). As expected, activation of TSH ß-subunit promoter by the Lhx2 expression vector was greater in GH3 cells than in {alpha}TSH and T{alpha}T1 cells (Fig. 4Go), consistent with the very low amount of endogenous Lhx2 in GH3 cells (20). In all cell lines tested, a proximal region spanning from –177 to –79 of the TSH ß-subunit promoter was found to be essential for stimulation by Lhx2. An Lhx2 mt-Lhx2 in which an alanine was substituted for a cysteine in two zinc finger motifs of the LIM domain had little or no activity, consistent with the known importance of the LIM domain.


Figure 4
View larger version (15K):
[in this window]
[in a new window]

  		FIG. 4. The –177 to –79 region of the TSH ß-subunit promoter is important for the activation by Lhx2 in T{alpha}T1, {alpha}TSH, {alpha}T3–1, and GH3 cells. T{alpha}T1 (A), {alpha}TSH (B), {alpha}T3–1 (C), and GH3 (D) cells were transiently transfected with 2 µg of 5' serial deletion constructs of TSH ß-subunit promoter fused to the firefly luciferase, together with Lhx2 or mt-Lhx2 in pcDNA3 (0.5 µg). Each set of transfections was performed in triplicate, and results are expressed as the fold induction relative to the pcDNA3 control vector ± SEM.

 
The –177 to –79 region of the TSH ß-subunit gene confers Lhx2 responsiveness to a minimal promoter
As shown above, deletion analysis demonstrated that the region between –177 and –79 is necessary for Lhx2-mediated TSH ß-subunit activation. To determine whether this region is sufficient to confer Lhx2-mediated regulation, multiple copies of the sequences between –177 and –79 were subcloned upstream of the E1b minimal promoter fused to the luciferase reporter gene. All of these constructs that contained the –177 to –79 region of the TSHß gene were found to be responsive to transfection of an Lhx2 expression vector both in {alpha}TSH and GH3 cells (Fig. 5Go). Interestingly, as the number of copies of the –177 to –79 region increased, basal expression increased and the fold-activation obtained with the Lhx2 expression vector was reduced. In particular, the increase in basal expression resulting from the multimerization of the –177 to –79 region is much more prominent in {alpha}TSH cells than GH3 cells. Consequently, promoter response to the forced overexpression of Lhx2 is almost entirely lost in {alpha}TSH cells, whereas a very significant Lhx2-dependent activation is maintained in GH3 cells. Such difference in fold activation could well be explained by the difference in the amounts of endogenous Lhx2 between the two cell lines. In fact, Roberson et al. (20) demonstrated that Lhx2 mRNA is abundant in {alpha}TSH cells but is extremely low in GH3 cells. Multiple copies of this region may permit greater responsiveness to the abundant endogenous Lhx2 in {alpha}TSH cells. In any case, these studies provide evidence that the –177 to –79 region of the TSHß gene is sufficient to confer Lhx2 responsiveness to a heterologous promoter.


Figure 5
View larger version (22K):
[in this window]
[in a new window]

  		FIG. 5. The –177 to –79 region of the TSH ß-subunit promoter confer Lhx2 responsiveness to a minimal promoter. {alpha}TSH (A) and GH3 (B) cells were transiently transfected with either 2 µg of a reporter construct containing zero, one, two, or three copies of Lhx2 response element upstream of the E1b TATA-box linked to a luciferase gene together with 0.5 µg of Lhx2 in pcDNA3. Each set of transfections was performed in triplicate and values expressed as relative light unit are mean ± SEM of three independent experiments.

 
Lhx2 binds to the –177 to –45 region of the TSH ß-subunit promoter both in vitro and in vivo
To explore a physical interaction of Lhx2 with the TSH ß-subunit promoter, a DNA fragment corresponding to the –177 to –45 region of the TSH ß-subunit promoter was used in an EMSA (Fig. 6Go). Nuclear extracts of {alpha}TSH cells produced two slowly migrating complexes (Fig. 6AGo, lane 2). Addition of an excess of the unlabeled –179 to –45 DNA fragment greatly reduced these complexes (Fig. 6AGo, lanes 3–5). The two complexes were supershifted by addition of Lhx2 antiserum and also appeared to be stabilized as there was an obvious increase in the intensity of the supershifted complex (Fig. 6AGo, lane 6). These results provide evidence that Lhx2 can physically interact with the –177 to –45 region of the TSH ß-subunit promoter. The EMSA was also performed with purified recombinant His-tagged Lhx2147–427 protein. This region of the protein contains the DNA binding, homeodomain. The recombinant DNA binding fragment of Lhx2 produced slowly migrating complexes (Fig. 6BGo, lane 2). Specificity of the binding reaction was again confirmed by the reduced intensity of the shifted bands with the addition of an excess of unlabeled competitor DNA (Fig. 6BGo, lane 3). Additionally, anti-His antibody could partially supershift the complexes (Fig. 6BGo, lane 4).


Figure 6
View larger version (43K):
[in this window]
[in a new window]

  		FIG. 6. Lhx2 can bind to the –177 to –45 region of the TSH ß-subunit promoter in vitro. An EMSA was performed with the –177 to –45 DNA fragment from the TSH ß-subunit promoter. A, A radiolabeled DNA probe containing the –177 to –45 region was incubated with nuclear extracts from {alpha}TSH cells. Lane 1, Probe only; lane 2, with nuclear extracts; lane 3–5, with 50-, 100-, and 200-fold molar excess of unlabeled –177 to –45 DNA; lane 6, with 1 µl Lhx2 antiserum; lane 7, with 1 µl Lhx2 antiserum and no nuclear extracts added. B, A radiolabeled DNA probe containing –177 to –45 region of the TSH ß-subunit promoter was incubated with recombinant Lhx2 homeodomain protein. Lane 1, Probe only; lane 2, with recombinant Lhx2 homeodomain protein; lane 3, 200-fold molar excess of unlabeled –177 to –45 DNA; lane 4, with 1 µl His-antibody; lane 5, with 1 µl His-antibody and no nuclear extracts added. Complexes were resolved by a nondenaturing PAGE.

 
To identify Lhx2 binding sites at the nucleotide level, the same DNA fragment was subjected to the DNaseI footprinting assay with the recombinant Lhx2147–427 protein. Whereas some apparent hypersensitive sites were observed, two major footprint regions (–118 to –108 and –84 to –68) were visible in the sense strand with just one footprint (–86 to –68) in the antisense strand (Fig. 7Go). Interestingly, each footprint shares 5'-(G/T)CAAT(T/A)-3' sequences, which was repeated twice in the –86 to –68 region.


Figure 7
View larger version (104K):
[in this window]
[in a new window]

  		FIG. 7. The Lhx2 homeodomain binds specifically to the –118/–108 and –86/68 region of the TSH ß-subunit promoter in vitro. Recombinant Lhx2 homeodomain was expressed in Escherichia coli as a fusion protein with an amino-terminal polyhistidine extension. The recombinant protein was purified using nickel chelate agarose chromatography. After incubation of radiolabeled DNA with the Lhx2 homeodomain, the reaction was partially digested with DNaseI and then analyzed by denaturing PAGE and autoradiography. Regions protected by Lhx2 are indicated by the box on the right side of each panel. The DNA fragment used in the binding reaction was also subjected to the Maxam and Gilbert chemical modification and cleavage reactions to use as a size standard.

 
In vivo recruitment of Lhx2 to the TSH ß-subunit promoter was examined by a ChIP assay. T{alpha}T1 cells were treated with formaldehyde to cross-link nuclear proteins to their in vivo binding sites in DNA. After sonication and immunoprecipitation with rabbit anti-Lhx2 or anti-CREB antibody, the precipitated Lhx2-DNA complexes were analyzed by PCR using primers specific for putative Lhx2 binding sites (–180 to –31). Anti-Lhx2-treated chromatin but not nonimmune control rabbit IgG-precipitated chromatin gave a readily detectable TSH ß-specific band after PCR amplification, indicating the presence of specific Lhx2-TSH ß promoter complex in vivo (Fig. 8Go). The specificity of precipitation by the Lhx2 antibody was confirmed by the absence of PCR product using primers designed to amplify other region of TSH ß-promoter (–1089 to –879). In addition, putative Lhx2 binding sites were not amplified in chromatin treated with anti-CREB antibody, whereas CRE sequences in the mouse {alpha}GSU gene were readily amplified in the same chromatin. These results provide support for the conclusion that Lhx2 indeed binds to the TSH ß-subunit promoter in vivo.


Figure 8
View larger version (35K):
[in this window]
[in a new window]

  		FIG. 8. Lhx2 binds to the TSH ß-subunit promoter in vivo. A ChIP assay was performed with T{alpha}T1 cells. T{alpha}T1 cells were grown in the absence (upper) or presence (lower) of 0.5 mM cAMP. Anti-Lhx2 or anti-CREB antibody was used to precipitate chromatin from T{alpha}T1 cells. Preimmune serum was used in parallel as a negative control. Precipitated DNA fragments were amplified in PCRs using primers representing the –180- to –31-bp region and –1089- to –879-bp region of the mouse TSH ß-subunit promoter and the –280- to –141-bp region (including CRE) of the {alpha}GSU gene. The products were separated by agarose gel electrophoresis. Mouse genomic DNA (Input) was served as a positive control for the PCR.

 
Mutation of Lhx2 binding sites diminishes Lhx2 responsiveness of the TSH ß-subunit promoter
Based on the result of the DNaseI footprinting assay, site-directed mutagenesis was performed on the two footprinted regions as shown in Fig. 9AGo. Basically, each mutation was designed to destroy the 5'-(G/T)CAAT(T/A)-3' sequences present repeatedly in the footprinted regions. Interestingly, the response to an Lhx2 expression was significantly reduced by Mt2 mutation in the –86 to –68 region but not by Mt1 mutation in the –118 to –108 region (Fig. 9BGo). Double mutation of both the regions exhibited a similar response to Lhx2 as that obtained with –86 to –68 mutation alone. Because Mt2 mutation is located within a Pit1 binding site incidentally, it was necessary to confirm that this mutation does not influence the Pit1 binding. To this end, effect of Mt2 mutation on basal promoter activity was examined in three different cell lines in which endogenous Lhx2 and Pit1 levels are quite different: abundant Lhx2 but no Pit1 proteins in {alpha}TSH cells (31), a normal concentration of Lhx2 and active Pit1 proteins in T{alpha}T1 cells, and abundant Pit1 but extremely low Lhx2 proteins in GH3 cells (20). Basal activity of Mt2-TSH ß-subunit promoter, when compared with that of Wt-TSH ß-subunit promoter, was very low in {alpha}TSH cells, lower in T{alpha}T1 cells and marginally lower in GH3 cells (Fig. 9CGo). This result indicates that Mt2 mutation affects Lhx2 binding alone because {alpha}TSH cells contain Lhx2 essentially, T{alpha}T1 cells contain both Pit1 and Lhx2, and GH3 cells contain Pit1 essentially. To further support the notion that Mt2 mutation does not affect the binding of Pit1 protein in the context of TSH ß-subunit promoter, a cotransfection experiment was carried out, in which Wt-TSHß or Mt2-TSHß/luciferase reporter construct was transiently cotransfected with the Pit1, GATA2, or Lhx2 expression vector into the T{alpha}T1 cells (Fig. 9DGo). Significant decrease of fold induction was observed in the Mt2-TSHß construct whenever Lhx2 was overexpressed, whereas almost the same fold induction was obvious in the Wt-TSHß and Mt2-TSHß constructs only when Lhx2 expression vector was not included. Taken together, these results strongly suggest that the Mt2 mutation affects TSH ß-subunit promoter transactivation by Lhx2 essentially and has no significant effect on transactivation by either Pit1 or GATA2.


Figure 9
View larger version (36K):
[in this window]
[in a new window]

  		FIG. 9. Disruption of the repeated sequences in –86 to –68 but not in –118 to –108 region significantly reduces the Lhx2 responsiveness of the TSH ß-subunit promoter in {alpha}TSH, T{alpha}T1, and GH3 cells. A, Mutations were placed in the –118 to –68 region as indicated. These mutations were incorporated within the context of a DNA fragment representing –795 to +13 of the TSH ß-subunit promoter, which was linked to the luciferase gene. B, Two micrograms of the wild-type or each mutant TSH ß-subunit promoter construct were transfected into {alpha}TSH cells together with 1 µg of the Lhx2 in pcDNA3. Each set of transfections was performed in triplicate, and values expressed as fold induction over basal activities by Lhx2 are mean ± SEM of three independent experiments. C, Two micrograms of the wild-type or Mt2-TSH ß-subunit promoter construct was transfected into {alpha}TSH, T{alpha}T1, and GH3. Each set of transfections was performed in triplicate, and values expressed as percent of basal promoter activity over wild type are mean ± SEM of three independent experiments. D, Two micrograms of the wild-type or Mt2-TSH ß-subunit promoter construct was transfected into T{alpha}T1cells together with Pit1, GATA2, or Lhx2 in pcDNA3 as indicated. Each set of transfections was performed in triplicate, and values expressed as fold induction over basal activities are mean ± SEM of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present studies provide evidence that Lhx2 interacts with the TSH ß-subunit promoter and activates it. Lhx2 was initially identified in immature murine B cell lines (32). Although Lhx2 is expressed in lymphoid cells, particularly in pre-B cell stage, it was also detected in the embryonic and adult brain. Mice with homozygous disruption of the Lhx2 gene die during embryonic development probably due to defects in erythrocyte development and with defects in eye development and forebrain development (33). At this time, a possible role of Lhx2 in pituitary development has not been established. In contrast, Lhx3 and Lhx4 clearly play a role in normal pituitary development (17, 34, 35). Some evidence for a possible role of Lhx2 in regulating glycoprotein hormone gene expression was provided with the observation that Lhx2 can activate the {alpha}-subunit promoter (20). The present studies suggest that Lhx2 can play a role as an activator of the TSH ß-subunit promoter. Under the conditions tested, Lhx2 was found to be much more potent than Lhx3 in activating the TSH ß-subunit promoter. Bach et al. (17) previously reported that Lhx3 alone did not stimulate the TSH ß-subunit promoter. Based on footprint and southwestern data, Haugen et al. (36) suggested that the nuclear protein that is necessary for basal activity of the TSH ß-subunit promoter is probably not Lhx3. In the present work, transfection studies have shown that Lhx2 can activate the TSHß promoter. In particular, expression of Lhx2 in somatotrope-derived GH3 cells that have low levels of endogenous Lhx2 resulted in much higher activation of the TSH ß-subunit promoter rather than in thyrotrope-derived cells.

We have determined that the –177 to –79 region of the TSHß 5' flanking region is necessary and sufficient for Lhx2 responsiveness. DNA binding, footprinting, and ChIP studies suggest Lhx2 interacts with this region in vitro and in vivo. Roberson et al. (20) previously demonstrated that 5'-TACTTAGCTAATTA-3' sequence in mouse {alpha}-subunit gene represent the Lhx2-binding site, designating it as PGBE. It is interesting that repeated sequences in two Lhx2 footprints of the TSH ß-subunit promoter look similar to the downstream half of the PGBE palindrome, suggesting that Lhx2 recognizes a similar DNA element in both the {alpha}- and TSH ß-subunit promoter. It is noteworthy that two footprints in the TSH ß-subunit promoter partially overlap the previously identified Pit1 binding sites (17). Recently MED220 was reported to associate with Pit1 and GATA2 on TSH ß-subunit promoter, functioning as transcriptional coactivator in thyrotropes (37). Because Lhx2 binding sequences are very close to or partially overlapped with the Pit1 and GATA2 binding region in TSH ß-subunit promoter, Lhx2 may either associate with MED220 also or compete with Pit1 and GATA2 for the association with MED220. Further study will be necessary to examine such association between various transcription factors on the TSH ß-subunit promoter.

Both the mRNA and protein for Lhx2 were rapidly increased in thyrotrope cells by TRH or cAMP treatment. To our knowledge, this is the first study showing up-regulation of Lhx2 mRNA by TRH. Previous studies in other tissues have shown that expression of Lhx2 is influenced by Six3 in forehead development of zebrafish (38), and Lhx2 is regulated by phosphorylated mothers against decapentaplegic signaling in hematopoietic stem cells (39). Previous studies of TRH regulation of TSH subunit gene expression in thyrotropes provided evidence that TRH effects on the {alpha}-subunit promoter are mediated by CRE and PGBE elements, whereas effects on TSH ß-subunit promoter are mediated by Pit1 binding sites, leading to recruitment of CREB-binding protein (9). The present findings suggest that TRH/cAMP-induced increases in Lhx2 play a role in activating the TSHß promoter. Because Lhx2 can also bind to PGBE of the {alpha}-subunit promoter (20), TRH-induced increases in Lhx2 mRNA and protein may provide a mechanism to coordinate stimulation of the TSH {alpha}- and ß-subunit gene expression.


   Footnotes
 
This work was supported by a grant from the Ministry of Commerce, Industry, and Energy through the Research Center for Bio-Medicinal Resources at Pai Chai University.

Disclosure Summary: The authors have nothing to disclose.

First Published Online April 19, 2007

Abbreviations: as-Lhx2, Antisense Lhx2; ChIP, chromatin immunoprecipitation; CRE, cAMP response element; CREB, cAMP response element-binding protein; Ct, threshold cycle value; mt-Lhx2, mutated Lhx2; PCNA, proliferating cell nuclear antigen; Pit, pituitary transcription factor; SDS, sodium dodecyl sulfate; TK, thymidine kinase.

Received August 9, 2006.

Accepted for publication April 11, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Vassart G, Dumont JE 1992 The thyrotropin receptor and the regulation of thyrocyte function and growth. Endocr Rev 13:596–611[CrossRef][Medline]
  2. Szkudlinski MW, Fremont V, Ronin C, Weintraub BD 2002 Thyroid-stimulating hormone and thyroid-stimulating hormone receptor structure-function relationships. Physiol Rev 82:473–502[Abstract/Free Full Text]
  3. Pierce JG, Parsons TF 1981 Glycoprotein hormones: structure and function. Annu Rev Biochem 50:465–495[CrossRef][Medline]
  4. Shupnik MA, Ridgway EC, Chin WW 1989 Molecular biology of thyrotropin. Endocr Rev 10:459–475[Medline]
  5. Magner JA 1990 Thyroid-stimulating hormone: biosynthesis, cell biology, and bioactivity. Endocr Rev 11:354–385[Medline]
  6. Carr FE, Shupnik MA, Burnside J, Chin WW 1989 Thyrotropin-releasing hormone stimulates the activity of the rat thyrotropin ß-subunit gene promoter transfected into pituitary cells. Mol Endocrinol 3:717–724[Abstract]
  7. Yusta B, Alarid ET, Gordon DF, Ridgway EC, Mellon PL 1998 The thyrotropin ß-subunit gene is repressed by thyroid hormone in a novel thyrotrope cell line, mouse T {alpha}T1 cells. Endocrinology 139:4476–4482[Abstract/Free Full Text]
  8. Carr FE, Burnside J, Chin WW 1989 Thyroid hormones regulate rat thyrotropin ß gene promoter activity expressed in GH3 cells. Mol Endocrinol 3:709–716[Abstract]
  9. Hashimoto K, Zanger K, Hollenberg AN, Cohen LE, Radovick S, Wondisford FE 2000 cAMP response element-binding protein-binding protein mediates thyrotropin-releasing hormone signaling on thyrotropin subunit genes. J Biol Chem 275:33365–33372[Abstract/Free Full Text]
  10. Sun Y, Lu X, Gershengorn MC 2003 Thyrotropin-releasing hormone receptors—similarities and differences. J Mol Endocrinol 30:87–97[Abstract]
  11. Steinfelder HJ, Hauser P, Nakayama Y, Radovick S, McClaskey JH, Taylor T, Weintraub BD, Wondisford FE 1991 Thyrotropin-releasing hormone regulation of human TSHB expression: role of a pituitary-specific transcription factor (Pit-1/GHF-1) and potential interaction with a thyroid hormone-inhibitory element. Proc Natl Acad Sci USA 88:3130–3134[Abstract/Free Full Text]
  12. Steinfelder HJ, Radovick S, Wondisford FE 1992 Hormonal regulation of the thyrotropin ß-subunit gene by phosphorylation of the pituitary-specific transcription factor Pit-1. Proc Natl Acad Sci USA 89:5942–5945[Abstract/Free Full Text]
  13. Dasen JS, O’Connell SM, Flynn SE, Treier M, Gleiberman AS, Szeto DP, Hooshmand F, Aggarwal AK, Rosenfeld MG 1999 Reciprocal interactions of Pit1 and GATA2 mediate signaling gradient-induced determination of pituitary cell types. Cell 97:587–598[CrossRef][Medline]
  14. Gordon DF, Lewis SR, Haugen BR, James RA, McDermott MT, Wood WM, Ridgway EC 1997 Pit-1 and GATA-2 interact and functionally cooperate to activate the thyrotropin ß-subunit promoter. J Biol Chem 272:24339–24347[Abstract/Free Full Text]
  15. Gordon DF, Woodmansee WW, Black JN, Dowding JM, Bendrick-Peart J, Wood WM, Ridgway EC 2002 Domains of Pit-1 required for transcriptional synergy with GATA-2 on the TSH ß gene. Mol Cell Endocrinol 196:53–66[CrossRef][Medline]
  16. Sloop KW, Parker GE, Hanna KR, Wright HA, Rhodes SJ 2001 LHX3 transcription factor mutations associated with combined pituitary hormone deficiency impair the activation of pituitary target genes. Gene 265:61–69[CrossRef][Medline]
  17. Bach I, Rhodes SJ, Pearse 2nd RV, Heinzel T, Gloss B, Scully KM, Sawchenko PE, Rosenfeld MG 1995 P-Lim, a LIM homeodomain factor, is expressed during pituitary organ and cell commitment and synergizes with Pit-1. Proc Natl Acad Sci USA 92:2720–2724[Abstract/Free Full Text]
  18. Sheng HZ, Zhadanov AB, Mosinger Jr B, Fujii T, Bertuzzi S, Grinberg A, Lee EJ, Huang SP, Mahon KA, Westphal H 1996 Specification of pituitary cell lineages by the LIM homeobox gene Lhx3. Science 272:1004–1007[Abstract]
  19. Sheng HZ, Moriyama K, Yamashita T, Li H, Potter SS, Mahon KA, Westphal H 1997 Multistep control of pituitary organogenesis. Science 278:1809–1812[Abstract/Free Full Text]
  20. Roberson MS, Schoderbek WE, Tremml G, Maurer RA 1994 Activation of the glycoprotein hormone {alpha}-subunit promoter by a LIM-homeodomain transcription factor. Mol Cell Biol 14:2985–2993[Abstract/Free Full Text]
  21. Schoderbek WE, Kim KE, Ridgway EC, Mellon PL, Maurer RA 1992 Analysis of DNA sequences required for pituitary-specific expression of the glycoprotein hormone {alpha}-subunit gene. Mol Endocrinol 6:893–903[Abstract]
  22. Boussif O, Lezoualc’h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, Behr JP 1995 A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci USA 92:7297–7301[Abstract/Free Full Text]
  23. Alarid ET, Windle JJ, Whyte DB, Mellon PL 1996 Immortalization of pituitary cells at discrete stages of development by directed oncogenesis in transgenic mice. Development 122:3319–3329[Abstract]
  24. Song SB, Rhee M, Roberson MS, Maurer RA, Kim KE 2003 Gonadotropin-releasing hormone-induced stimulation of the rat secretogranin II promoter involves activation of CREB. Mol Cell Endocrinol 199:29–36[CrossRef][Medline]
  25. Kaiser UB, Sabbagh E, Katzenellenbogen RA, Conn PM, Chin WW 1995 A mechanism for the differential regulation of gonadotropin subunit gene expression by gonadotropin-releasing hormone. Proc Natl Acad Sci USA 92:12280–12284[Abstract/Free Full Text]
  26. de Wet JR, Wood KV, DeLuca M, Helinski DR, Subramani S 1987 Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7:725–737[Abstract/Free Full Text]
  27. Edlund T, Walker MD, Barr PJ, Rutter WJ 1985 Cell-specific expression of the rat insulin gene: evidence for role of two distinct 5' flanking elements. Science 230:912–916[Abstract/Free Full Text]
  28. Zhang T, Wolfe MW, Roberson MS 2001 An early growth response protein (Egr) 1 cis-element is required for gonadotropin-releasing hormone-induced mitogen-activated protein kinase phosphatase 2 gene expression. J Biol Chem 276:45604–45613[Abstract/Free Full Text]
  29. Gibson UE, Heid CA, Williams PM 1996 A novel method for real time quantitative RT-PCR. Genome Res 6:995–1001[Abstract/Free Full Text]
  30. Gelmini S, Tricarico C, Vona G, Livi L, Melina AD, Serni S, Cellai E, Magrini S, Villari D, Carini M, Serio M, Forti G, Pazzagli M, Orlando C 2001 Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) for the measurement of prostate-specific antigen mRNA in the peripheral blood of patients with prostate carcinoma using the taqman detection system. Clin Chem Lab Med 39:385–391[CrossRef][Medline]
  31. Sarapura VD, Wood WM, Woodmansee WW, Haakinson DJ, Dowding JM, Gordon DF, Ridgway EC 2006 Pituitary tumors arising from glycoprotein hormone {alpha}-subunit-deficient mice contain transcription factors and receptors present in thyrotropes. Pituitary 9:11–18[CrossRef][Medline]
  32. Xu Y, Baldassare M, Fisher P, Rathbun G, Oltz EM, Yancopoulos GD, Jessell TM, Alt FW 1993 LH-2: a LIM/homeodomain gene expressed in developing lymphocytes and neural cells. Proc Natl Acad Sci USA 90:227–231[Abstract/Free Full Text]
  33. Porter FD, Drago J, Xu Y, Cheema SS, Wassif C, Huang SP, Lee E, Grinberg A, Massalas JS, Bodine D, Alt F, Westphal H 1997 Lhx2, a LIM homeobox gene, is required for eye, forebrain, and definitive erythrocyte development. Development 124:2935–2944[Abstract]
  34. Zhadanov AB, Copeland NG, Gilbert DJ, Jenkins NA, Westphal H 1995 Genomic structure and chromosomal localization of the mouse LIM/homeobox gene Lhx3. Genomics 27:27–32[CrossRef][Medline]
  35. Zhadanov AB, Bertuzzi S, Taira M, Dawid IB, Westphal H 1995 Expression pattern of the murine LIM class homeobox gene Lhx3 in subsets of neural and neuroendocrine tissues. Dev Dyn 202:354–364[Medline]
  36. Haugen BR, McDermott MT, Gordon DF, Rupp CL, Wood WM, Ridgway EC 1996 Determinants of thyrotrope-specific thyrotropin ß promoter activation. Cooperation of Pit-1 with another factor. J Biol Chem 271:385–389[Abstract/Free Full Text]
  37. Gordon DF, Tucker EA, Tundwal K, Hall H, Wood WM, Ridgway EC 2006 MED220/thyroid receptor-associated protein 220 functions as a transcriptional coactivator with Pit-1 and GATA-2 on the thyrotropin-ß promoter in thyrotropes. Mol Endocrinol 20:1073–1089[Abstract/Free Full Text]
  38. Ando H, Kobayashi M, Tsubokawa T, Uyemura K, Furuta T, Okamoto H 2005 Lhx2 mediates the activity of Six3 in zebrafish forebrain growth. Dev Biol 287:456–468[Medline]
  39. Utsugisawa T, Moody JL, Aspling M, Nilsson E, Carlsson L, Karlsson S 2006 A road map toward defining the role of Smad signaling in hematopoietic stem cells. Stem Cells 24:1128–1136[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/7/3468    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, K. K.
Right arrow Articles by Kim, K. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, K. K.
Right arrow Articles by Kim, K. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals