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Endocrinology Vol. 145, No. 12 5623-5628
Copyright © 2004 by The Endocrine Society

Thyrotropin-Releasing Hormone-Stimulated Thyrotropin Expression Involves Islet-Brain-1/c-Jun N-Terminal Kinase Interacting Protein-1

Hiroshi Abe, Koji Murao, Hitomi Imachi, Wen M. Cao, Xiao Yu, Kazuya Yoshida, Norman C. W. Wong, Margaret A. Shupnik, Jacques-Antoine Haefliger, Gérard Waeber and Toshihiko Ishida

First Department of Internal Medicine (H.A., K.M., H.I., W.M.C., X.Y., K.Y., T.I.), Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan; Departments of Medicine and Biochemistry and Molecular Biology (N.C.W.W.), Faculty of Medicine, University of Calgary, Health Sciences Center, Calgary, Alberta, Canada T2N 4N1; Department of Internal Medicine (M.A.S.), Division of Endocrinology and Metabolism, University of Virginia, Charlottesville, Virginia 22903; and Department of Internal Medicine B (J.-A.H., G.W.), Centre Hospitalier Universitaire Vandois-University Hospital, 1011 Lausanne, Switzerland

Address all correspondence and requests for reprints to: Koji Murao, M.D., Ph.D., First Department of Internal Medicine, Faculty of Medicine, Kagawa University, 1750-1, Miki-cho, Kita-gun, Kagawa 761-0793, Japan. E-mail: mkoji{at}kms.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Islet-brain-1 (IB1)/c-Jun N-terminal kinase interacting protein 1 (JIP-1) is a scaffold protein that is expressed at high levels in neurons and the endocrine pancreas. IB1/JIP-1 interacts with the c-Jun N-terminal kinase and mediates the specific physiological stimuli (such as cytokines). However, the potential role of the protein in the pituitary has not been evaluated. Herein, we examined expression of the gene encoding IB1/JIP-1 and its translated product in the anterior pituitary gland and a pituitary cell line, GH3. We then examined the potential role of IB1/JIP-1 in controlling TSH-ß gene expression. Exposure of GH3 cells to TRH stimulated the expression of IB1/JIP-1 protein levels, mRNA, and transcription of the promoter. The increase of IB1/JIP-1 content by transient transfection study of a vector encoding IB1/JIP-1 or by the stimulation of TRH stimulates TSH-ß promoter activity. This effect is not found in the presence of a mutated nonfunctional (IB1S59N) IB1/JIP-1 protein. Together, these facts point to a central role of the IB1/JIP-1 protein in the control of TRH-mediated TSH-ß stimulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TSH IS A MEMBER of the pituitary glycoprotein hormone family. Other members of the same family include FSH, LH, and chorionic gonadotropin. These hormones consist of two subunits, the {alpha}- and ß-subunits. The {alpha}-subunit ({alpha}-glycoprotein hormone subunit) is common to all members of this family, but the ß-subunit is unique and confers specific biological activity to each heterodimeric protein (1). The TSH subunit genes are coordinately regulated at a transcriptional level by thyroid hormone, dopamine, and TRH (1). In anterior pituitary cells, TRH binds to a membrane receptor and activates phospholipase C, leading to calcium mobilization and protein kinase C (PKC) activation (1, 2). The actions of these intracellular signaling pathways ultimately lead to an increase in transcription of the TSH-ß, {alpha}-subunit, and prolactin genes (1). Several nuclear proteins bind to TRH-responsive elements on TSH promoter in addition to Pit-1 (3). The combined actions of these factors participate in stimulating gene activity via activation of a selected intracellular signaling pathway. Many pituitary proteins are phosphorylated in response to TRH, PKC, and Ca2+, but most of these proteins have not been identified (1).

The gene encoding human islet-brain-1 (IB1; encoded by mapk8ip1 gene) has been identified and maps to chromosome 11p11.2-p12 (4). IB1 was initially isolated from a pancreatic ß-cell cDNA library (5), and the resulting protein had DNA-binding activity that transactivated the GTII cis-element of glucose transporter (GLUT) 2 (5). Subsequent studies showed that rat and human IB1 were the homolog of murine c-Jun N-terminal kinase interacting protein-1 (JIP-1), a murine cytoplasmic inhibitor of the c-Jun amino-terminal kinase (JNK)-activated pathway (6). IB1/JIP-1 is recognized as a mammalian scaffold protein that is involved in the regulation of the JNK signaling pathway (5, 6, 7, 8). The scaffold protein IB1/JIP-1 ensures the formation, compartmentalization, and specificity of this physically ordered signaling module, so that a defined pool of JNK might be recruited by specific physiological stimuli (such as cytokines) but protected from activation by irrelevant ones (9). IB1 differs from JIP-1 by the insertion of a 47-amino acid region in its carboxyl-terminal region. This insertion contains a phosphotyrosine interaction domain and a helix-loop-helix motif (5). A S59N mutation close (IB1S59N) to the JNK binding domain of IB1 was recently described and shown to be associated with a monogenic late-onset type 2 diabetes mellitus (10). The IB1 is mainly expressed in pancreatic ß-cells and neuronal cells (5); furthermore, Pellet et al. (11) show that the IB1 mRNA is found in the pituitary gland. However, expression of IB1 in the pituitary does not reveal any information regarding its potential role in this tissue. One clue to IB1 function came from the knowledge that the IB1 protein regulated GLUT2 gene expression via a cis-acting element, GTII. A DNA homology search revealed the presence of three putative GTII elements within the human TSH-ß promoter region. These findings prompted us to examine the potential role of IB1 on TSH expression in pituitary cells. Our findings demonstrate that the IB1/JIP-1 is involved in the control of TRH-mediated TSH-ß transcription.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
TRH and 8-bromo-cAMP were purchased from Sigma (St. Louis, MO).

Cell culture
GH3 cells (Japan Health Sciences Foundation, Osaka, Japan) were cultured in Ham’s F10 media (ICN Biomedicals, Inc., Aurora, OH) supplemented with 15% fetal bovine serum and 2.5% horse serum in a humidified atmosphere containing 5% CO2. Male Wistar rats were killed by decapitation, and the anterior pituitary lobes, liver, kidney, spleen, and brain were removed and the cells dispersed as previously described (12). The isolated cells were suspended in DMEM containing 5% horse serum, 2.5% fetal calf serum, and 1% nonessential amino acids, and the cells were seeded at a concentration of 1 x 105 cell/dish. These cells were maintained in culture at 37 C in humidified air with 5% CO2.

Northern blot analysis
Total RNA was extracted from the rat tissues including brain, liver, kidney, anterior pituitary, and GH3 cells. A full-length cDNA of rat IB1 was synthesized by PCR using reverse-transcribed RNA from GH3 cells and labeled with [32P]deoxycytidine triphosphate (3000 Ci/mmol) using the random priming method (Takara, Tokyo, Japan). The primer sequences for rat IB1 mRNA were as follows: sense primer, 5'-ATGATCGTGATGGTGCCGTC-3'; and antisense primer, 5'-ACTGAACCTGCAGGTGCTGA-3'. Electrophoresis and hybridization were performed as described previously (13). Blots were also probed with rat ß-actin to assess equal loading of samples (14). After autoradiography at room temperature for 24 h, hybridization signals were detected using a Bioimaging Analyzer (BAS 1000; Fuji Photo Film, Tokyo, Japan).

Western blot analysis
Cultured cells were lysed as described previously (15). The proteins extracts were resuspended under reducing conditions, and 15 µg was fractionated by size on 7.5% sodium dodecyl sulfate-polyacrylamide gel and then transferred to polyvinylidene difluoride membrane for immunoblotting. The membranes were incubated for 1 h at 4 C with 0.2% Tween 20 in PBS containing an anti-IB1 antiserum (1:250 dilution) as described previously (5). An antibody for glyceraldehyde-3-phosphate dehydrogenase (1:1000 dilution; Trevigen, Gaithersburg, MD) was used as the internal standard for cytosolic extract. Abundance of the basal transcriptional factor IID (TFIID) was detected using an antibody at a dilution of 1:1000 as the internal standard for nuclear extract (Santa Cruz Biotechnology, Santa Cruz, CA). Antibody binding was visualized using a chemiluminescence detection kit (ECL; Amersham Pharmacia Biotech, Buckinghamshire, UK).

Electrophoretic mobility shift analysis
Nuclear extracts from GH3 cells treated with various doses of TRH for 12 h were prepared as described previously (16). A synthetic DNA duplex spanning GTII (5'-CCTCTTAAGACTCTAATTACCCT-3'; Nihon Bioservice, Asagiri, Japan) was radiolabeled at the 5'-end by incubating each strand separately with [{gamma}32P]-ATP and polynucleotic kinase before annealing (5). Each binding reaction of 20 µl contained 25 mM HEPES, 50 mM KCl, 1 mM EDTA, 0.5 mM spermidine, 0.6 mM dithiothreitol, 12% glycerol, 5 µg of polydeoxyinosinic deoxycytidylic acid, 1 fmol of radiolabeled probe, and 20 µg of nuclear extract. In competition analysis, 50-fold molar excess of unlabeled competitor DNA was added to the reaction before the addition of the nuclear extracts. All reactions were incubated at room temperature for 20 min and then separated on a native 6% polyacrylamide gel. Electrophoresis was performed at 10 V/cm2 for 3 h at 4 C. The gel was then dried, and the signals were detected using a Bioimaging Analyzer (Fuji Photo Film, Tokyo, Japan).

Transfection of GH3 cells and luciferase reporter gene assay
The expression vectors used in our studies contained IB1 or its mutant S59N IB1 (IB1S59N; a mutation in exon 2 leading to a serine-to-asparagine substitution at codon 59, S59N has been described previously) (5, 10). The reporter plasmid containing the rat TSH-ß promoter (pTSH-LUC) or IB1 promoter (pIB1-LUC) was constructed as described previously (2, 17). Purified reporter plasmid was transfected into GH3 cells (at 60% confluence) using conventional cationic liposome transfection methods (Lipofectamine; Life Technologies, Gaithersburg, MD). One microgram of Rous sarcoma virus-ß-galactosidase was added to all transfections to monitor the efficiency of DNA uptake by the cells (16). All assays were corrected for ß-galactosidase activity, and total amount of protein per reaction was identical. Twenty-microliter aliquots were taken for the luciferase assay, which was performed according to the manufacturer’s instructions (ToyoInk, Tokyo, Japan).

Statistical analysis
Statistical comparisons were made by one-way ANOVA and Student’s t test, with P < 0.05 considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IB1/JIP-1 is expressed in transformed and primary pituitary cells
The first step in determining the potential role of IB1 in the pituitary was to measure its level of expression in the tissue. We examined the expression of IB1 message in several rat tissues and GH3 cells by Northern blot analysis. The message was detected at an expected size consistent with previous report (4). IB1 was strongly expressed in the brain and pituitary and was also expressed in GH3 cells. Little expression was seen in liver, kidney, and spleen. Furthermore, whether the IB1/JIP-1 transcript was translated into protein and the comparison between nuclear and cytosolic IB1/JIP-1 content were assessed using Western blot analysis. IB1/JIP-1 was strongly expressed in the brain, pituitary, and GH cells in both nuclear and cytosolic extracts. Little expression was seen in the liver, and no IB1/JIP-1 was detected in the kidney (Fig. 1Go, B and C). These results clearly show that IB1 mRNA and protein are present in both GH3 and rat pituitary cells.



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  		FIG. 1. Tissue distribution of IB1/JIP-1. A, Presence of IB1 mRNA in rat anterior pituitary and GH3 cells was examined by Northern blot analysis of 20 µg total RNA from these cells. The blots were hybridized with the coding region of rat IB1 [32P] cDNA (upper panel). Picture of ethidium bromide-stained 28S rRNA is shown as internal control (lower panel). Lane B, brain; lane P, anterior pituitary; lane K, kidney; lane L, liver; lane S, spleen; lane G, GH3. B and C, IB1 protein expression in the pituitary and GH3 cells. Cytosolic (B) and nuclear (C) extract from GH3 cells and rat brain, pituitary, kidney, and liver were extracted and subjected to Western blot analysis as described in Materials and Methods. Abundance of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (B) or TFIID (C) served as a control and is shown on the bottom of each lane. An independently performed identical experiment gave similar results. Lane B, brain; lane P, pituitary; lane K, kidney; lane L, liver; lane G, GH3.

 
TRH induces IB1/JIP-1 in the pituitary cells
The presence of IB1 expression does not reveal any information regarding its potential role in the pituitary. A DNA homology search revealed the potential binding sites of IB1 within the TSH-ß promoter region. This finding prompted us to determine the potential role of IB1 in pituitary TSH gene expression. Therefore, rat pituitary cells were exposed to TRH, a peptide that stimulated TSH gene expression. Results showed that TRH-treated cells had increased abundance of the IB1/JIP-1 transcript (Fig. 2AGo). Like TRH, 8-bromo-cAMP also increased IB1/JIP-1 expression in a dose-dependent manner, with maximal stimulation at a concentration of 10 nmol/liter and 1 µmol/liter, respectively (Fig. 2Go, B and C). The increase in IB1/JIP-1 appeared to be specific because Western blot analysis showed that abundance of nuclear IB1/JIP-1 protein was also increased after treatment with TRH (Fig. 2DGo), but a general factor TFIID was not increased. Because IB1/JIP-1 interacted with the GTII site of the GLUT2 promoter (5) and TRH induction of IB1/JIP-1 abundance in GH3 cells, this manipulation may also enhance IB1/JIP-1 binding to the GTII element. Therefore, we used electrophoretic mobility shift analysis to measure GTII site binding activity in nuclear extract from GH3 cells that were exposed for 12 h to varying doses of TRH (Fig. 2EGo). Results showed a dose-dependent increase in GTII site binding activity after exposure to TRH. Furthermore, the binding activity to the GTII site was abolished in the presence of 50-fold molar excess of unlabeled GTII probe (Fig. 2EGo, lane 2). In contrast, binding activity of a general transcription factor, Octamer binding protein 1, to its recognition motif was not affected by TRH stimulation (data not shown). To determine whether binding activity from GH3 cells reflected a specific interaction between the putative nucleoprotein and the radiolabeled probe, we used the supershift assay using a specific antibody against IB1. Results in Fig. 2FGo showed that the protein-DNA complex was supershifted (lane 2) by the addition of anti-IB1 antibody but not by preimmune antibody (lane 1). Next we tested whether TRH induction of IB1/JIP-1 expression was transcriptionally regulated. For these studies, we linked the IB1 promoter to the luciferase reporter gene (pIB1-LUC) and transfected this construct into GH3 cells treated with or without TRH. Results (Fig. 2GGo) showed that TRH induced a 3.5-fold increase in IB1 promoter activity in the transfected GH3 cell. Together, these findings showed that IB1 is expressed in the pituitary cell line, GH3. In these cells, IB1 mRNA and promoter activity are induced by TRH.



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  		FIG. 2. TRH and 8-bromo-cAMP regulation of IB1/JIP-1 expression. A, TRH increases IB1/JIP-1 mRNA expression. The abundance of IB1/JIP-1 and ß-actin mRNA levels in 20 µg of total RNA were measured using Northern blot hybridization and an Bioimaging Analyzer, and the ratio of IB1/JIP-1 mRNA to ß-actin mRNA is shown as the percentage of control in the figure. Numbers in parentheses indicate numbers of observations in each group. Control, no treatment; TRH, 10 nM TRH. The asterisk denotes a significant difference (P < 0.01) between treated and control cells. B, TRH increases IB1/JIP-1 protein expression. Rat pituitary cells were exposed to 10 nM TRH for 24 h, and IB1/JIP-1 protein in total cell lysate was detected using Western blot analysis probed with an anti-IB1 antibody. Abundance of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a control and is shown on the bottom of each lane. An independently performed identical experiment gave similar results. Cont, Control; cAMP, 1 µM 8-bromo-cAMP; TRH, 10 nM TRH. The asterisk denotes a significant difference (P < 0.01). C, Dose-dependent induction of IB1/JIP-1 protein by 8-bromo-cAMP or TRH. GH3 cells (1 x 104) were seeded in six-well plates and exposed to the indicated amounts of 8-bromo-cAMP (C-1) or TRH (C-2) for 24 h. IB1/JIP-1 in total cell lysate was detected using Western blot analysis probed with an anti-IB1 antibody. Abundance of GAPDH served as a control and is shown on the bottom of each lane. The ratio of IB1 to GAPDH is shown as the percentage of control in the figure. Each data point shows the mean and SEM (n = 3) of separate experiments. The asterisk denotes a significant difference (P < 0.01). D, TRH increases IB1 protein expression in nuclear protein. Nuclear protein was extracted from GH3 cells treated with TRH. Cont, Control vehicle; TRH, 10 nM TRH. Abundance of TFIID served as a control and is shown on the bottom of each lane. An independently performed identical experiment gave similar results. E, TRH enhances GTII binding activity. Cells were exposed to varying concentrations of TRH (noted in the figure) for 12 h before preparation of nuclear extracts. The binding activity of the GTII motifs was examined using EMSA. Lane 1, probe only; lane 2, 0.1 nM TRH with 50x unlabeled GTII probe; lane 3, 0.1 nM TRH; lane 4, 1 nM TRH; lane 5, 10 nM TRH; lane 6, 0 nM TRH (vehicle). F, Supershift assay with the antiserum against IB1. EMSA was carried out using radiolabeled GTII oligonucleotide as the probe. For lane 1, 1 µl of preimmune serum was added to the EMSA reaction. Lane 2 contained 1 µl of antiserum raised against IB1. Arrow indicates IB1 complex; asterisk indicates IB1/antibody complex. G, TRH stimulates IB1 promoter activity. GH3 cells were transfected with pIB1-LUC and then treated with vehicle or TRH for 24 h before cell harvest. The results are expressed as relative luciferase activity compared with control cells arbitrarily set at 100. Each data point shows the mean and SEM (n = 4) of separate transfections. Lane 1, control; lane 2, TRH treated. The asterisk denotes a significant difference (P < 0.01).

 
Effect of IB1 on TSH promoter activity in response to TRH
The GH3 cell line serves as a useful model for understanding TSH expression in response to TRH. Because IB1 expression is stimulated by TRH, we wondered whether IB1 participates in controlling TSH gene activity. Therefore, we measured the transcriptional activity of TSH-ß promoter in the GH3 cells. A construct, pTSH-LUC, containing the full-length rat TSH promoter was cotransfected along with an IB1 expression vector into GH3 cells. Results (Fig. 3AGo) showed that the presence of IB1 induced a 4-fold increase in TSH promoter activity. To further support this evidence, the role of IB1 in TSH-ß gene regulation could arise from the use of a dominant negative isoform of the protein. We have previously described a IB1 point mutant (IB1S59N) isolated from a patient with diabetes mellitus (10). To further demonstrate the involvement of IB1 in the TRH induction of the TSH promoter activity, we cotransfected GH3 cells with pTSH-LUC along with an expression plasmid encoding the mutant IB1 or empty vector as control. Consistent with preceding studies, there was a 3-fold rise in activity of pTSH-LUC after TRH stimulation (Fig. 3BGo, compare columns 1 and 2). In contrast, expression of IB1S59N inhibited TRH induction of pTSH-LUC activity (Fig. 3BGo, column 3) in a dominant negative fashion. Together, these findings support the idea that IB1 is required for TRH induction of TSH expression.



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  		FIG. 3. Effect of IB1/JIP-1 on TSH promoter activity. A, GH3 cells were transfected with the reporter pTSH-LUC plus an empty (control), wild-type (wild-type IB1), or IB1S59N expression vector. The results are expressed as relative luciferase activity compared with control cells arbitrarily set at 100. Each data point shows the mean and SEM (n = 4) of separate transfections. The asterisk denotes a significant difference (P < 0.01). NS, No significant difference. B, GH3 cells were transfected with the reporter pTSH-LUC plus an empty (control or TRH) or IB1S59N (TRH+IB1S59N) expression vector and then treated with vehicle (control) or TRH (TRH or TRH+IB1S59N) for 24 h before cell harvest. The results are expressed as relative luciferase activity compared with control cells arbitrarily set at 100. Each data point and adjacent bars shows the mean and SEM (n = 4) of separate transfections. The asterisk denotes a significant difference (P < 0.01).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IB1/JIP-1 is recognized as a nuclear protein and believed to part of the scaffolding structure (5). This protein plays an important role in the transcriptional regulation of the GLUT2 gene (5). IB1 transcripts are found in the pancreas, and a strong signal is also present in brain tissue. In this study, we have used antiserum against recombinant IB1 and shown that the protein is present in the nuclei of primary cultured rat pituitary and GH3 cells (Fig. 1Go). Although it is conceivable that the antibody cross-reacted with a protein that was not IB1, several features argue against this possibility. First, the apparent size of the protein was 120 kDa, and the mass corresponded to that predicted for IB1. Second, IB1 mRNA was expressed in both pituitary and GH3 cells (Fig. 1Go). In the same cells, a supershift assay confirmed localization of IB1 to the nucleoprotein fraction extracted from GH3 cells. These findings strongly suggest that the protein recognized by the antibody represents endogenous IB1 and that this is a nuclear protein found in pituitary and GH3 cells.

Pituitary TSH production is stimulated physiologically by the actions of the hypothalamic peptide TRH (1). TRH acts via binding to a seven-transmembrane domain receptor, which in turn stimulates phosphorionositide turnover. This step increases the cellular free Ca2+ concentration and subsequently activates the PKC cascade. In other studies, TSH synthesis and secretion may also be induced by agents that act via the cAMP/PKA cascade (18). There is growing evidence that the TSH-ß promoter is positively regulated by both the PKC/Ca2+- and PKA/cAMP-mediated pathways (1, 2). Consistent with this idea, our studies show that both TRH and a cAMP analog stimulate the expression of IB1 (Fig. 2Go); these agents act via the PKC and PKA pathways, respectively.

Results of the previously mentioned studies show that TRH stimulates the expression of IB1 protein, mRNA, and transcription of the gene. In addition, the transient transfection studies show that TRH increases IB1 promoter activity. On the other hand, it is well known that T3 negatively regulates the expression of TSH; however, there is no effect of T3 on IB1 expression (Abe, H. and K. Murao, unpublished data). Our DNA sequence search suggested that a motif identical to the cAMP response element is present in the –563 to –556 fragment of the IB1 promoter. Further experiments will be needed to clarify the transcriptional regulation of IB1/JIP-1.

IB1 was initially identified by studies of the transcriptional mechanisms that underlie the pancreatic cell-specific control of the GLUT2 gene (5, 19, 20). Recent data show that a single missense mutation in the IB1 gene is associated with accelerated cell death and secondary diabetes mellitus in humans (5, 10). In the brain, IB1 is localized to neurons and interacts functionally with p190 RhoGEF, the ApoE receptor 2, and the low-density lipoprotein-related receptor protein, megalin (11, 21, 22). A recent report from our laboratory suggested a role of IB1/JIP-1 in Alzheimer’s disease (23, 24). Data arising from the current studies show that IB1 is expressed in the anterior pituitary and that it is likely involved in regulating activity of the TSH-ß promoter. Evidence that supports this idea comes from the expression of the mutant IB1. This mutant acts in a dominant negative fashion (IB1S59N) to block TRH activation of the TSH promoter. This finding suggests that IB1 plays an important role in mediating TRH induction of TSH-ß gene activity.

In summary, we examined the role of IB1 in mediating transcriptional regulation of the TSH gene expression in response to the hypothalamic peptide, TRH, in pituitary cells and the cell line GH3. The results indicate that IB1 stimulates TSH gene transcription and thus suggest that IB1 participates in TRH-induced TSH transcription in the anterior pituitary.


   Footnotes
 
Abbreviations: GLUT, Glucose transporter; IB1, islet-brain-1; JIP-1, c-Jun N-terminal kinase interacting protein 1; JNK, c-Jun amino-terminal kinase; PKC, protein kinase C; TFIID transcriptional factor IID.

Received May 18, 2004.

Accepted for publication August 26, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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