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Endocrinology Vol. 145, No. 2 867-880
Copyright © 2004 by The Endocrine Society

Cell-Specific Pituitary Gene Expression Profiles after Treatment with Leukemia Inhibitory Factor Reveal Novel Modulators for Proopiomelanocortin Expression

Rula A. Abbud, Robert Kelleher and Shlomo Melmed

Department of Medicine, Division of Endocrinology, Cedars Sinai Research Institute, University of California School of Medicine, Los Angeles, California 90048

Address all correspondence and requests for reprints to: Shlomo Melmed, M.D., Division of Endocrinology, Department of Medicine, Cedars Sinai Medical Center, Los Angeles, California 90048. E-mail: melmeds{at}cshs.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leukemia inhibitory factor (LIF) mediates the hypothalamo-pituitary-adrenal stress response. Transgenic mice overexpressing LIF in the developing pituitary have altered pituitary differentiation with expansion of corticotropes, maintenance of Rathke’s cleft cysts, and suppression of all other pituitary cell types. Affymetrix GeneChips were used to identify modulators of LIF effects in corticotrope (AtT-20) and somatolactotrope (GH3) cells. In addition to genes known to respond to LIF in corticotrope cells [e.g. suppressor of cytokine signaling-3 (SOCS-3), signal transducer and activator of transcription-3, SH2 domain-containing tyrosine phosphatase-1, and proopiomelanocortin (POMC)], corticotrope-specific changes were also observed for genes involved in glycolysis and gluconeogenesis, transcription factors, signaling molecules, and expressed sequence tags. Two transcription factors identified, CCAAT/enhancer-binding protein ß (C/EBPß) and glial cell-derived neurotrophic factor (GDNF)-inducible factor (GIF), dose-dependently induced expression of the rat POMC promoter when overexpressed in AtT-20 cells. LIF further induced POMC transcription with C/EBPß, but not with GIF. C/EBPß also induced expression of the SOCS-3 promoter that was further enhanced by cotreatment with LIF. However, GIF did not affect SOCS-3 expression. These results indicate that C/EBPß and GIF are downstream effectors of LIF corticotrope action. LIF also stimulates the expression of inhibitors of its actions, such as SOCS-3 and SH2 domain-containing tyrosine phosphatase-1. {alpha}2-HS-glycoprotein (AHSG)/fetuin, a secreted protein that antagonizes bone TGFß/bone morphogenic protein signaling, was induced by LIF in a signal transducer and activator of transcription-3-dependent fashion. Pretreatment with AHSG/fetuin blocked LIF-induced expression of the POMC promoter independently of SOCS-3. Thus, using GeneChips, C/EBPß and GIF have been identified as novel mediators and AHSG/fetuin as an inhibitor of LIF action in corticotropes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEUKEMIA INHIBITORY factor (LIF) is a pleiotropic cytokine of the IL-6 cytokine family that is induced by proinflammatory agents or stress in most tissues (1, 2). Aberrant LIF expression has been associated with autoimmune diseases (3), septic shock (4), and cancer (5). LIF exerts different and sometimes opposite roles in different tissues. For example, it inhibits embryonic stem cell differentiation (6), stimulates macrophage differentiation in the M1 murine myeloblastic leukemia cell line (7), promotes the growth of myeloma cells (8), activates hepatic acute phase proteins (9), stimulates osteoclast formation resulting in bone loss (10, 11), promotes cardiac hypertrophy in vitro (12, 13), stimulates cholinergic differentiation of sympathetic neurons (14), facilitates blastocyst implantation (15), and regulates ovarian function (16). Thus, depending on the cell type, LIF could either induce or inhibit cellular differentiation.

In the pituitary, LIF mediates the neuro-immuno-endocrine interface (17, 18). LIF was identified in bovine pituitary folliculostellate cells (19), and LIF and its receptors were demonstrated in both fetal and adult pituitary, where expression was noted in 20–30% of GH- and ACTH-producing cells and in 10–15% of TSH-, PRL-, gonadotropin-, and nonhormone-producing cells (20). Their expression is up regulated by a variety of stressful stimuli, resulting in increased proopiomelanocortin (POMC) gene expression and ACTH secretion (20, 21, 22, 23). LIF acts in synergy with hypothalamic CRH to stimulate ACTH secretion both in vivo and in vitro (22, 24, 25). LIF gene disruption in transgenic mice abrogates stress-mediated ACTH responses (26, 27), whereas LIF overexpression in the developing pituitary alters pituitary cell differentiation, resulting in corticotrope cell expansion, Rathke’s cleft cysts, and suppression of cells of the lhx3 lineage (28, 29). Transgenic mice overexpressing LIF thus exhibited Cushingoid features, including truncal obesity, thin skin, high basal corticosterone, and incomplete dexamethasone suppression. Early expression of LIF altered the expression of transcription factors essential for the development of gonadotropes, thyrotropes, somatotropes, and lactotropes. Gonadotrope and somatotrope hypoplasia resulted in infertility and dwarfism, respectively. These studies suggested that LIF behaves as a soluble differentiation factor in the developing pituitary. When expressed inappropriately, it diverts pituitary cell differentiation toward the corticotrope and ciliated epithelial cell lineages.

The signaling cascade activated by LIF in corticotropes has been extensively studied (20, 21, 24, 30, 31, 32, 33, 34, 35). LIF binding to its receptor results in receptor heterodimerization with glycoprotein 130 (gp130), a common transmembrane subunit shared by members of the IL-6 family. Both receptor subunits lack intrinsic kinase activity. The LIF receptor-gp130 complex activates Janus kinase 2 (JAK2) by tyrosine autophosphorylation, which phosphorylates signal transducer and activator of transcription-3 (STAT-3). Phosphorylated STAT-3 forms homo- and heterodimers that are translocated to the nucleus, bind to specific sequences on the POMC promoter, and activate its expression (21, 24, 31, 33, 35). STAT-3 also induces the suppressor of cytokine signaling-3 (SOCS-3), which acts in a negative feedback mechanism to limit LIF-induced POMC expression (30, 36, 37). LIF stimulates other transcription factors, such as c-Fos and JunB, which transduce the synergistic effects of LIF and CRH on POMC (21). c-Fos and JunB activation may be secondary to activation of the JAK-STAT signaling cascade, as both possess STAT-3-responsive sequences in their promoter regions, and SOCS-3 overexpression blocked LIF-induced, but not CRH-induced, c-Fos and JunB expression. Although most studies suggest that LIF acts mainly by activating the JAK-STAT signaling cascade in corticotropes, other signaling cascades could also mediate its effects. Depending on the cell type, LIF may activate other signaling molecules, such as phospholipase C, phosphotyrosine phosphatase D, members of the MAPK pathway (38, 39, 40, 41, 42, 43, 44), phosphoinositol 3-kinase (45), as well as insulin receptor substrate 1 (46), and insulin receptor substrate 2 (47).

Little is known about the differential effects of LIF in the pituitary. Observations in transgenic mice appear to also manifest in vitro; although LIF stimulates ACTH secretion and differentiation of AtT20 cells (48), it inhibits PRL (49, 50) and GH secretion and induces cell proliferation in MtT/SM cells (51). To further clarify differential pituitary effects of LIF, global patterns of gene expression were compared in different pituitary cell lines using the Affymetrix GeneChip system, and differential gene expression patterns were observed in response to LIF in AtT20 and GH3 cell lines. Using this approach, previously reported findings were confirmed, and novel cell-specific LIF targets were identified, including two transcription factors, CCAAT/enhancer-binding protein ß (C/EBPß) and glial cell-derived neurotrophic factor (GDNF)-inducible factor (GIF), and an inhibitor of TGFß/bone morphogenic protein (BMP) cytokine signaling, {alpha}2-HS-glycoprotein (AHSG). C/EBPß and GIF differentially stimulate POMC and SOCS-3 gene expression and may mediate LIF effects. In contrast, AHSG/fetuin acts in an autocrine fashion to inhibit the effects of LIF on POMC expression. Thus, using GeneChips, novel modulators for pituitary LIF signaling have been identified.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
AtT20 cells were plated in DMEM containing low glucose (Invitrogen, Carlsbad, CA), 5% fetal bovine serum, 5% horse serum, and penicillin-streptomycin. GH3 cells were cultured in DMEM containing 15% horse serum, 1% glutamine (Glutamax, Invitrogen), and 1% penicillin-streptomycin. Cells that stably express either wild-type STAT-3 (STAT-3 WT) or STAT-3D were also grown in their appropriate medium (31). Cells were plated at a concentration of 100,000 cells/ml, and most treatments were performed in triplicate in 6-well plates after overnight incubation in DMEM containing 5% charcoal-stripped fetal bovine serum.

Bromodeoxyuridine (BrdU) incorporation experiments
After overnight incubation in charcoal-stripped, serum-containing medium, cells were treated with recombinant mouse LIF (Chemicon International, Temecula, CA; 10 ng/ml). Sixteen hours after LIF treatment, cells were incubated with BrdU (3 mg/ml) for 3 h and harvested by trypsin treatment, followed by incubation in ice-cold 70% ethanol for 30 min and five washes in PBS. Cytospins were generated for immunocytochemical visualization of BrdU. Slides were incubated with mouse anti-BrdU antibody (1:100; Dako, Carpenteria, CA), followed by biotinylated antimouse IgG (1:100; Dako). The avidin-biotin peroxidase complex kit (Dako) was used to further amplify the signal, and diaminobenzidene was used to stain dividing cells.

Affymetrix Genechips
Experimental design.
A pilot experiment was performed using 1, 10, and 100 ng/ml LIF treatment for AtT-20 and GH3 cells. Each treatment was performed in triplicate for each experiment, and total RNA (6 µg in total, with 2 µg from each triplicate) was used to label probe for chip hybridization. Expression profiles were compared with untreated, and the dose of 10 ng/ml was chosen for all subsequent experiments. Four different experiments were performed for AtT-20 and GH3 cells (untreated and treated with 10 ng/ml LIF overnight). Samples from the same experiment were concurrently hybridized and processed on the chips. The mouse Affymetrix MG-U74A [version 1 (n = 2) and version 2 (n = 2) GeneChip (Affymetrix, Inc., Santa Clara, CA; www.affymetrix.com)] was used to study expression pattern changes of 10,028 (version 1) to 12,639 (version 2) mouse genes, including expressed sequence tags (ESTs), in AtT-20 cells, whereas the rat RG-U34A chip (n = 4) containing 8,799 rat sequences was used to study GH3 cells.

Total RNA isolation.
Cells were washed twice with cold PBS, and total RNA was prepared using the RNeasy kit (Qiagen, Chatsworth, CA). Briefly, cells were lysed with buffer RLT containing ß-mercaptoethanol and further homogenized using the Qiashredders (Qiagen). Cell lysate was loaded on RNeasy columns and processed according to the manufacturer’s protocol to obtain total RNA. RNA quality was assessed using the Agilent RNA chips following kit instructions. Only RNA with clean 28S and 18S ribosomal peaks with a ratio in peak size equal to 2 was used for experimental purposes.

RNA labeling for Genechips.
Subsequent procedures, including preparation of double-stranded cDNA from total RNA (Superscript Choice system, Life Technologies, Inc., Grand Island, NY), biotin-labeled cRNA (Enzo Bioarray High Yield RNA Transcript Labeling Kit), cRNA fragmentation, and hybridization to the Affymetrix Genechips (Affymetrix Fluidics Station), were performed according to the recommended protocol provided by Affymetrix. Quality controls were performed to ensure quality RNA, with 5' to 3' end ratios for control genes and internal controls approximating 1.0.

Data analysis.
The Affymetrix Microarray Suite was used as a first tool for data analysis. This software allows for calculation of the signal and determination of whether each probe set is present. The Data Mining Tool (Affymetrix) and GeneSpring 5.0 (Silicon Genetics) software were used to average results from different replicates and perform statistical analysis to compare between treatments. More than 1.7-fold and less than 0.6-fold changes in gene expression with a statistically significant t test were the cut-offs used for determination of significant changes in expression patterns. Another software that was useful in comparing rat and mouse data were the GenMAPP software (Gladstone Institute) (52) (University of California at San Francisco, San Francisco, CA), allowing for comparison of expression patterns for specific pathways. This software allows color-coding of changes in expression patterns in each cell type, where genes that were not expressed are coded green, genes that increased in response to LIF are coded pink, genes that are suppressed by LIF are coded purple, genes that did not change are coded gray, and genes that were not detected on the chip are not colored.

Western blot.
Differential expression patterns were confirmed at the protein level by Western blot analysis for two genes selected from the microarray data: AHSG/fetuin and C/EBPß. Cells were treated as described above (LIF, 10 ng/ml overnight) and lysed after two washes with cold PBS, using the protein lysis buffer containing dithiothreitol (Tropix Dual-Light Luciferase and ß-Galactosidase Reporter Assay kit, PE Applied Biosystems, Foster City, CA). After freeze-thawing, protein content was measured using a Bradford assay (protein assay, Bio-Rad Laboratories, Hercules, CA). An equal amount of protein was loaded on precast gels for PAGE (4–15% Tris-HCl; Bio-Rad) and transferred to nitrocellulose membrane. Membranes were incubated with either a goat polyclonal fetuin (G-17) antibody (1:200; sc-9668, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or rabbit polyclonal C/EBPß (C-19) antibody (1:200; sc-150, Santa Cruz Biotechnology, Inc.) overnight at 4 C. Secondary antibodies were antigoat antibody conjugated with horseradish peroxidase (1:2000; Sigma-Aldrich Corp., St. Louis, MO) for fetuin and antirabbit peroxidase-conjugated antibody (1:2000; Amersham Pharmacia Biotech, Arlington Heights, IL) for C/EBPß. Chemiluminescent visualization of the specific protein band was obtained using ECL Western Blotting Detection Reagents (Amersham Pharmacia Biotech) and exposure to film. Signal specificity was confirmed by coincubation with an excess of the respective blocking peptide (Santa Cruz Biotechnology, Inc.). Semiquantitative analysis of the Western blot results was performed using Scion Image software (release ß 4.0.2, Scion Corp., Frederick, MD).

DNA plasmids
The expression vectors used are as follows: rat POMC promoter (-706/+64) driving expression of the luciferase gene [POMC-luciferase (Luc)] (33), Rous sarcoma virus (RSV) promoter driving expression of the ß-galactosidase gene (RSV-ßGal), C/EBPß expression vector (pEF-BOS-nuclear factor-IL6, gift from Dr. Shizuo Akira, Osaka University, Osaka, Japan) (53), cytomegalovirus (CMV) promoter driving expression of hygromycin resistance gene (CMV-HYG), human TGFß inducible early gene (TIEG)1 expression vector (gift from Dr. Thomas Spelsberg, Mayo Clinic, Rochester, MN) (54), and SOCS-3 promoter (-2757/+106) driving expression of luciferase (clone 4) (54, 55).

Transfections, luciferase, and ßGal assays
AtT-20 cells were plated in 6-well plates at a concentration of 100,000 cells/ml in DMEM containing 5% charcoal-stripped fetal bovine serum and 1% penicillin-streptomycin (Invitrogen). After overnight incubation, cells were fed with the same medium and transfected using Fugene-6 (3 µl/well) transfection reagent (Roche, Indianapolis, IN) according to the manufacturer’s protocol. In all transfections, RSV-ßGal (0.1 µg/well) was cotransfected for transfection efficiency control. POMC-Luc or SOCS-3-Luc were transfected at 0.9 µg/well, and different amounts of either C/EBPß or TIEG1 were added (0.1, 0.3, or 0.6 µg/well). Control promoter-less luciferase vector (pGL2-Basic, 1 µg/well) and control expression vector (CMV-HYG, 0.6 µg/well) were also included in all experiments. After overnight incubation in DNA and Fugene, cells were incubated with fresh medium with or without LIF (10 ng/ml) for 6 h. At the end of this incubation period, cells were lysed and processed for measurement of luciferase and ßGal activity using the Tropix Dual-Light System (PE Applied Biosystems, Bedford, MA). Each experiment was performed in triplicate and repeated 3–4 times.

Fetuin treatment
Different concentrations of bovine fetuin (Sigma-Aldrich Corp.) were used to determine its effect on POMC expression and STAT-3 phosphorylation. For POMC expression experiments, POMC-Luc and RSV-ßgal were transfected in AtT-20 cells, as described above. Before treatment with LIF, cells were treated with fetuin for either 30 min or 24 h and its effect on luciferase activity was determined. To study the effect of fetuin on STAT-3 phosphorylation, cells were seeded at 100,000 cells/ml of charcoal-stripped containing medium and pre-treated on the following day with fetuin (20 µM) for either 30 min or overnight before incubation with LIF (10 ng/ml) for up to 60 min. Following treatment, cells were lysed and processed for immunoprecipitation with STAT-3 antibody. Transfection experiments were performed in triplicate and repeated 3 times and immunoprecipitations were repeated 4 times.

Immunoprecipitations
Cell lysates were homogenized and centrifuged to exclude debris. An equal amount of protein was precleared with normal rabbit IgG (Santa Cruz Biotechnology, Inc.) and protein A agarose suspension (Roche) at 4 C for 30 min. Immunoprecipitation was performed using the STAT-3 antibody (rabbit polyclonal H190, sc7179, Santa Cruz Biotechnology, Inc.). A Western blot was subsequently performed on the immunoprecipitates, using a mouse monoclonal P-STAT3 antibody specific for Tyr-705 (1:200 in 1% BSA; sc8059, Santa Cruz Biotechnology, Inc.), and the antigoat peroxidase-conjugated IgG as secondary antibody. After chemiluminescent visualization of phospho-STAT-3, membranes were stripped and incubated with STAT-3 antibody (1:200 in 1% BSA), followed by antirabbit peroxidase-conjugated secondary antibody (1:2000 in 1% BSA). Scion Image software (release ß 4.0.2 Scion Corp.) was used to quantify P-STAT3 and STAT3 signals.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Differential effects of LIF on corticotrope and somatolactotrope cells in vitro
Effect of LIF on cell proliferation was determined in GH3 somatolactotrope cells. Previous studies indicated that ACTH induction by LIF is accompanied by decreased cell proliferation and induction of corticotrope differentiation. Thus, the effects of LIF on AtT-20 and GH3 cell proliferation were compared by BrdU incorporation. LIF has opposite effects on BrdU incorporation in AtT-20 and GH3 cells. Although cell proliferation is suppressed by LIF in AtT-20 cells (48), it is enhanced in GH3 cells. The percentage of BrdU-labeled cells increased from 25.6 ± 1.1% to 35.9 ± 3% in the presence of LIF (10 ng/ml). These observations indicate differential effects of LIF on corticotrope and somatolactotrope cell proliferation.

Differential cell-specific patterns of pituitary gene expression in response to LIF
To further elucidate differential effects of LIF on pituitary cell types, global changes in gene expression were assessed, using Affymetrix GeneChips, after LIF treatment of AtT-20 and GH3 cells. Forty to 45% of the genes spotted on the chip were termed present in both cell types. After treatment with LIF (10 ng/ml) for 24 h, the expression pattern of most genes did not change, but variable expression patterns of some genes were evident. POMC expression was selectively increased in AtT-20, but not in GH3 cells, as expected. The percentage of genes that increased in each experiment ranged from 0.7–1.9%, whereas 1.2–2.1% of genes decreased expression. Averaging the results from all experiments revealed statistical changes in the expression pattern of a smaller number of genes (Tables 1Go and 2Go). The expression of 48 genes, including transcription factors, signaling molecules, and ESTs, statistically increased (P < 0.05) after treatment of AtT-20 cells with LIF. In addition, LIF suppressed the expression of 29 genes in AtT20 cells (Table 2Go). In contrast, the expression pattern of only one transcription factor (JunB) was up-regulated after treatment of GH3 cells. Tables 3Go and 4Go show complete lists of genes that increased (16 genes) and decreased (7 genes) in response to LIF in GH3 cells.


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  		TABLE 1. Genes that increased after LIF treatment (10 ng/ml, overnight) in AtT20 cells

 

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  		TABLE 2. Genes down-regulated by LIF treatment (10 ng/ml, overnight) in AtT20 cells

 

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  		TABLE 3. Genes that were induced by LIF (10 ng/ml, overnight) in GH3 cells

 

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  		TABLE 4. Genes that decreased after LIF treatment of GH3 cells

 
GenMAPP software (Gladstone Institute) was used to compare expression patterns between GH3 and AtT-20 cells for genes known to be involved in previously established intracellular pathways. Figure 1Go shows GenMAPP results for the JAK/STAT signaling cascade for AtT-20 and GH3 cells after treatment with LIF (10 ng/ml). As previously reported, LIF stimulated POMC, STAT-3, SOCS-3, and SH2 domain-containing tyrosine phosphatase-1 (SHP-1) expression in AtT-20 cells. These changes were not observed or did not reach statistical significance in GH3 cells. These results provide further evidence for the differential effects of LIF on AtT-20 and GH3 cells.



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  		FIG. 1. Differential gene expression changes of known LIF signaling pathway mediators in GH3 and AtT-20 cells after treatment with LIF (10 ng/ml). GenMAPP software was used to color-code changes in JAK/STAT pathway gene expression. Genes that were absent in all experiments are green, those that increased are colored pink, those that decreased are purple, those that did not change are gray, and those that are not found on the chip are white. The fold change ± SEM are shown for each gene.

 
Differential effects of LIF on cellular metabolism in GH3 and AtT-20 cells
Changes in cellular proliferation are linked to changes in cellular metabolism; in particular, growth-promoting agents stimulate glycolysis (56). The expression pattern of enzymes involved in glycolysis and gluconeogenesis after LIF treatment in AtT-20 and GH3 cells is depicted in Fig. 2Go. GenMAPP was used to map changes in the expression of the rate-limiting enzymes of glycolysis and gluconeogenesis. In AtT-20 cells, the expression of hexokinase II was decreased. Fructose-1,6-bisphosphatase-1 was undetectable in untreated cells and appears upon LIF treatment (P = 0.03). In GH3 cells, LIF induced the expression of 6-phosphofructokinase C, the rate-limiting enzyme in glycolysis. These observations suggest opposite effects of LIF on glycolysis and gluconeogenesis in AtT-20 and GH3 cells. The increase in glycolysis rate-limiting enzyme expression in GH3 cells correlates with increased cell proliferation.



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  		FIG. 2. Differential gene expression changes in glycolysis and gluconeogenesis rate-limiting enzymes. Shown is a glycolysis and gluconeogenesis GenMAPP adapted from N. Fidelman and K. Dahlquist (Gladstone Institutes). Genes that were absent in all experiments are green, those that increased are colored pink, those that decreased are purple, those that did not change are gray, and those that are not found on the chip are white. The fold change ± SEM are shown for each gene.

 
Transcription factors selectively affected by LIF in AtT-20 cells
LIF induced cell-specific changes in the expression patterns of several signaling molecules (Table 1Go). Transcription factors that were selectively induced in AtT-20 cells included: C/EBPß (mean fold change ± SEM, 2.1 ± 0.2), GIF (2.1 ± 0.2), STAT3 (1.7 ± 0.2), E74-like factor 3 (ELF3) (1.7 ± 0.1), interferon regulatory factor 1 (Irf1) (1.7 ± 0.03), and an EST that has homology to X box-binding proteins and basic leucine zipper transcription factors (2 ± 0.1).

The relevance of increased expression of two transcription factors, C/EBPß and GIF, was further elucidated. Figure 3AGo shows a representative Western blot (n = 3) for C/EBPß comparing protein levels in AtT20 and GH3 cells with and without LIF. C/EBPß has three isoforms: full-length (FL; 45 kDa), liver-activated peptide (LAP; 38 kDa), and liver inhibitor peptide (LIP; 20 kDa) (57, 58). In untreated cells, both FL and LAP are readily detected in AtT-20 and GH3 cells. LIF treatment increases levels of LAP and LIP in AtT20, but not GH3, cells.



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  		FIG. 3. C/EBPß is induced by LIF and stimulates POMC gene expression. A, Western blot showing that LIF (10 ng/ml, overnight) induces LAP (38 kDa), and LIP (20 kDa) forms of C/EBPß in AtT-20, but not GH3, cells (n = 3). FL (45 kDa) C/EBPß did not change significantly in either cell type. a, P < 0.05 compared with no LIF. B, Overexpression of C/EBPß stimulates and enhances the effects of LIF on rat (-706/+64) POMC-Luc expression in AtT20 cells. The fold change in Luc/ßGal relative to control (n = 9–12). The control consisted of signal obtained from cells transfected with CMV-HYG and RSV-ß-Gal, and not treated with LIF. Cells were treated with LIF (10 ng/ml for 6 h). a, P < 0.05 vs. control; b, P < 0.05 LIF-treated vs. no treatment.

 
To further understand the significance of increased C/EBPß expression in AtT-20 cells after LIF treatment, the effect of C/EBPß overexpression was studied on POMC promoter expression. For these experiments, the rat POMC promoter (-706/+64) driving expression of the luciferase gene (POMC-Luc) was cotransfected with a C/EBPß expression vector and RSV-ßGal in AtT-20 cells. Figure 4BGo shows the fold change in POMC expression relative to the control, which consisted of cells transfected with POMC-Luc, RSV-ßGal, and CMV-HYG. These cells did not receive LIF treatment. As expected, LIF stimulated POMC promoter expression. Overexpression of C/EBPß induced basal POMC promoter expression in a dose-dependent fashion, with maximal stimulation at 0.3 µg expression vector. LIF caused further stimulation of the POMC promoter. In the absence of C/EBPß, LIF induced a 2.4-fold increase in POMC promoter activity. Addition of 0.1 µg C/EBPß resulted in a synergistic response to LIF, leading to a 4.5-fold increase in POMC expression. However, when higher amounts of C/EBPß were transfected, the response to LIF ranged between 1.8- and 2.8-fold in the presence of 0.3 and 0.6 µg C/EBPß, respectively. These results demonstrate that C/EBPß regulates transcription of the POMC promoter. In addition, C/EBPß may mediate some of the effects of LIF on POMC expression. LIF further stimulates POMC promoter expression with maximal C/EBPß stimulation (0.6 µg), suggesting the involvement of other transcription factors and signaling molecules in mediating LIF effects on POMC promoter expression.



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  		FIG. 4. TIEG1 dose-dependently stimulates basal (0), but not LIF-induced, POMC promoter expression in AtT-20 cells. Overexpression of TIEG1 stimulates and enhances the effects of LIF on rat (-706/+64) POMC-Luc expression in AtT20 cells. The fold change in Luc/ßGal relative to control (n = 12). The control consisted of signal obtained from cells transfected with CMV-HYG and RSV-ß-Gal and not treated with LIF. a, P < 0.05 vs. control; b, P < 0.05, LIF-treated vs. no treatment.

 
To determine the functional significance of increased GIF expression after LIF treatment in AtT-20 cells, human TIEG1 expression vector was cotransfected with POMC-Luc and RSV-ßGal in AtT20 cells. POMC expression with and without 6 h of LIF treatment was determined. Figure 4Go shows the fold change relative to controls transfected with CMV-HYG. Cotransfection with TIEG1 stimulated basal POMC promoter expression in a dose-dependent fashion. However, POMC promoter induction by LIF was attenuated in the presence of TIEG1 vector. These results suggest a role for TIEG1/GIF in the stimulation of POMC expression and modulation of LIF effects.

As LIF also induces the expression of the suppressor of cytokine signaling (SOCS-3) (36), SOCS-3 promoter (-2757/+106) driving the expression of luciferase was used instead of POMC-Luc in cotransfection experiments with C/EBPß and TIEG1 (Fig. 5Go). Although C/EBPß induced dose-dependent stimulation of SOCS-3 expression that was further stimulated with LIF, TIEG1 had no effect on either basal or LIF-induced SOCS-3 expression. These results suggest that C/EBPß may also be partially involved in mediating the effects of LIF on SOCS-3 gene expression, whereas TIEG1 is specific for POMC and has no effect on SOCS-3.



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  		FIG. 5. C/EBPß, but not TIEG1, dose-dependently stimulates basal (No LIF) and LIF-induced SOCS-3 promoter expression. The fold change in SOCS-3 luciferase/RSV-ßGal relative to control is shown. Control cells were transfected with CMV-HYG, rather than C/EBPß or TIEG1, expression vectors and did not receive LIF treatment. Cells were treated with LIF (10 ng/ml) for 6 h. a, P < 0.05 vs. control; b, P < 0.05, LIF vs. no LIF. n = 6.

 
AHSG/fetuin is induced by LIF and acts as an inhibitor of LIF-induced POMC expression
One of the genes markedly induced by LIF in corticotrope, but not somatolactotrope, cells was an EST (cluster AW230891). Sequence analysis suggested 63% homology to the homo sapiens leucine-rich AHSG/fetuin (59). To confirm that AHSG/fetuin protein levels are induced by LIF, a Western blot was performed using a goat antimouse AHSG/fetuin antibody (Santa Cruz Biotechnology, Inc.). As shown in Fig. 6Go, AHSG/fetuin levels were low in AtT20 cells and were induced after treatment with LIF. In GH3 cells, AHSG/fetuin levels were undetectable with or without LIF treatment (data not shown). To determine whether AHSG/fetuin induction in AtT20 cells is dependent on STAT-3 activation, its expression was assessed in cell lines overexpressing either STAT-3 WT or a dominant negative STAT-3 mutant that is unable to bind DNA (STAT-3D) (31). Figure 6Go shows that overexpression of either STAT-3 WT or STAT-3D stimulated basal AHSG/fetuin expression. This effect was not due to a nonspecific effect of the stable transfection of AtT-20 cells, as cells that were stably transfected with SHP-1 wild-type or mutant expression vectors (30) did not exhibit increased basal AHSG/fetuin expression (data not shown). These observations suggest that STAT-3 stimulates AHSG/fetuin expression by a mechanism that does not require DNA binding.



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  		FIG. 6. STAT-3-dependent induction of fetuin/AHSG by LIF in AtT-20 cells. A, Western blot was performed using cell lysates from untransfected AtT-20 cells or AtT-20 cells that were stably transfected with either a wild-type STAT3 expression vector (STAT3-WT) or a mutant STAT3 that cannot bind to DNA (STAT3-D). Responses were compared in untreated (0) and LIF (10 ng/ml)-treated cells. B, Quantitative analysis of fetuin/AHSG by Western blot (n = 3) was performed using Scion Image software (release ß 4.0.2), and data are expressed relative to signal obtained in AtT-20 cells with no added LIF. a, P < 0.05 compared with no LIF.

 
To determine whether AHSG/fetuin regulates POMC expression, AtT-20 cells were cotransfected with a -706/+64 POMC promoter-luciferase and RSV promoter-ßGal vectors and pretreated with bovine fetuin, either overnight or for 30 min before LIF addition. As shown in Fig. 7AGo, pretreatment with fetuin (10 and 20 µM) blocked LIF-induced POMC promoter expression. This inhibitory effect of fetuin was independent of SOCS-3 expression, as LIF-induced expression of the SOCS-3 promoter was unaltered in the presence of fetuin (Fig. 7BGo). However, LIF-induced STAT-3 phosphorylation, as assessed by immunoprecipitation, was diminished in the presence of fetuin (Fig. 8Go). Usually, a peak in the P-STAT-3:STAT-3 ratio was observed within 10 min of LIF treatments. Pretreatment with AHSG/fetuin did not completely suppress STAT-3 phosphorylation, but blocked its further increase at 10 min post-LIF treatment (Fig. 8Go). These results suggest that AHSG/fetuin is induced by LIF in a STAT-3 dependent fashion and acts in an autocrine fashion to antagonize the effects of LIF on POMC, but not SOCS-3, gene expression.



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  		FIG. 7. A, LIF-induced POMC promoter expression is dose-dependently blocked by fetuin. AtT-20 cells (n = 9) were transfected with rat (-706/+64) POMC-Luc and RSV-ßGal. The following day, cells were treated with increasing doses of bovine fetuin for 30 min before a 6-h treatment with LIF (10 ng/ml). Control cells did not receive fetuin or LIF. B, LIF-induced SOCS-3 promoter expression is unaffected by fetuin. AtT-20 cells (n = 6) were transfected with mouse (-2757/+106) SOCS-3-Luc and RSV-ßGal, followed by fetuin and LIF treatment. a, P < 0.05, LIF vs. no LIF; b, P < 0.05 vs. LIF-treated control.

 


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  		FIG. 8. LIF-induced phosphorylated STAT3 (P-STAT3) is attenuated by pretreatment with fetuin. AtT-20 cells were pretreated with 20 µM bovine fetuin for 30 min, followed by LIF (10 ng/ml) for 3–60 min. Equal amounts of proteins were immunoprecipitated with STAT3 antibody, and Western blots were performed with a goat polyclonal P-STAT3 antibody, followed by antigoat peroxidase-conjugated IgG as secondary antibody. After chemiluminescent visualization of P-STAT3, membranes were stripped and incubated with STAT3 antibody, followed by antirabbit peroxidase-conjugated secondary antibody. Shown are representative results from one experiment (n = 4) and a graph of the average P-STAT-3:STAT-3 ratio obtained using Scion Image software. a, P < 0.05 compared with LIF treatment alone; b, P < 0.05 compared with time zero.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
LIF is an essential modulator of pituitary POMC expression and binds to the LIF receptor that dimerizes with the common cytokine receptor subunit (gp130) to activate the JAK/STAT signaling cascade. The effects of LIF are dependent on phosphorylation of STAT-3 on Tyr-705 (20, 31), increased expression of c-Fos and JunB, and phosphorylation of STAT1{alpha} and -ß (21, 31). It has been proposed that up-regulation of c-Fos and JunB mediates the synergistic effects of CRH and LIF (21). Both transcription factors act by binding to an activating protein-1 site (+41/+47) in the first exon, which also binds cAMP response element-binding protein (CREB). The CRH response also depends on binding of the Nur-orphan nuclear receptor to two sites in the promoter: the distal NurRE (-71/-64) and the proximal Nur77-binding response element located at -404/-383 (60, 61, 62, 63, 64, 65). STAT-3 also binds to a sequence overlapping the Nur77-binding response element (-399/-379) and dimerizes with STAT-1 (35). Other LIF-responsive regions in the rat POMC promoter located at -293/-414 and -166/-96 could also mediate LIF effects (35). The -293/-414 region contains sequences that bind several transcription factors involved in basal POMC expression: Tbx19/Tpit (66, 67), Pitx (68, 69), Nurr77 (63), STAT-1, and STAT-3 (21, 31, 33, 35). The -166/-96 region lacks any STAT-3- or STAT-1-binding sites and may mediate the effects of LIF by binding other transcription factors. Thus, LIF action on POMC gene expression does not appear to be solely mediated by activation of the JAK/STAT signaling cascade. Another important step in the POMC response to LIF is suppression of LIF signaling mediated by SOCS-3 (36, 37, 55, 70, 71).

In this paper we identify modulators of LIF effects by microarray-based gene expression profiling. Using Affymetrix Genechips, cell-specific pituitary targets of LIF were identified by comparing expression patterns in pituitary cell lines. The use of cell lines allowed for the study of uniform population of cells and is the best in vitro model available to identify differentially regulated genes in the pituitary. Similar to in vitro models, results from such experiments may not necessarily apply in vivo. As the pituitary is a complex organ, in vivo studies are more complicated and may result in false positives. Thus, using differentiated cell lines we can identify differential patterns of gene expression that can be further validated in vivo. One of the problems with pituitary cell lines is that they are not generated in the same species: AtT20 are murine, whereas GH3 are rat cells. To compare results obtained from two different species, we relied on GenMAPP and GeneSpring software. We observed cell-specific changes in the expression of genes involved in the JAK/STAT pathway and genes involved in the rate-limiting steps of glycolysis and gluconeogenesis, possibly reflecting opposing effects of LIF on the differentiation and proliferation of these different pituitary cell types. As these effects occur 24 h after LIF treatment, the observed changes in gene expression reflect long-term rather than immediate targets of LIF. Furthermore, several of the genes that were selectively up-regulated in corticotrope cells were transcription factors probably involved in long-term mediation of LIF effects on POMC. We further pursued the role of two transcription factors: C/EBPß and TIEG1, and the role of a potential inhibitor of cytokine signaling, AHSG/fetuin. These results are based on the Affymetrix mouse and rat A chips, which do not contain all ESTs. Thus, it is likely that other genes, present on the B and C chips, may also be regulated by LIF. For example, Tbx19, a gene known to be essential for corticotrope differentiation, is not spotted on the MGU74A chip, and we cannot make any comments on its regulation by LIF.

Although C/EBPß mRNA levels were readily detectable in AtT20 cells, they were considered absent in GH3 cells by Affymetrix Microarray Suite software. However, Western blot analysis suggested that GH3 cells do express the FL and LAP forms of C/EBPß. C/EBPß protein levels are increased after corticotrope, but not somatolactotrope, treatment with LIF. It is likely that the low levels of proteins found in somatolactotrope cells are not regulated and may not require induced gene transcription. This may explain the discrepancy between mRNA and protein expression in these cells. It is also likely that the C/EBPß transcript is expressed in GH3 cells at extremely low levels beyond the GeneChip sensitivity. C/EBPß overexpression stimulated both POMC and SOCS-3 basal promoter expression, and LIF causes further POMC and SOCS-3 promoter expression. Although there have been no reports of regulation of the POMC promoter by C/EBPß, three putative C/EBPß-binding sites in the rat POMC -706/+63 promoter sequence can be identified using MatInspector V2.2. One of these sites has one mismatch and is located in the previously described LIF responsive region (-293/-414). The other two sites are located at -433/-447 and -600/-614. In addition, analysis of the SOCS-3 promoter revealed 13 putative C/EBPß-binding sites.

C/EBPß is a member of the basic leucine zipper family of transcription factors (53, 57, 58, 72). It is also known as nuclear factor-IL-6 or IL-6DBP because of its involvement in IL-6-mediated activation of acute inflammatory response genes (53, 73, 74, 75). The effects of IL-6 are mediated via the JAK-STAT signaling cascade, mainly through STAT3 and the Ras/MAPK pathway to stimulate C/EBPß. STAT3 also contributes to C/EBPß activation by IL-6 (75, 76). In addition, LIF was shown to stimulate C/EBPß expression in preadipocytes via activation of the extracellular signal-regulated kinase pathway and CREB/activation transcription factor-1 binding to the C/EBPß promoter (77, 78). It is unlikely that C/EBPß would activate POMC expression in a similar mechanism, because CREB overexpression resulted in lower POMC induction than C/EBPß (data not shown). C/EBPß also interacts with other transcription factors, including the glucocorticoid receptor (79). This may explain the further induction by LIF when C/EBPß is overexpressed.

C/EBPß knockout mice have defective carbohydrate metabolism and lipid storage (80, 81). They are immunodeficient, with defective Th1 and macrophage phagosome responses (82). They have elevated IL-6 and develop Castleman-like lymphoproliferative disease that can be blocked in C/EBPß and IL-6 double-knockout mice (83, 84). Female knockout mice are infertile because of the inability of ovarian follicles to respond to LH (85). Thirty-five percent of homozygous mice die shortly after birth (83, 86). The hypothalamo-pituitary-adrenal axis has not been studied in these animals, and it is possible that deficiencies in hypothalamo-pituitary-adrenal function could contribute to this phenotype. We have recently observed low levels of corticosterone in some of these knockout animals that survived to 3 months of age (data not shown). We expect their responses to stress to be compromised, resulting in failure to thrive.

Another transcription factor induced by LIF is GIF. Murine GDNF-inducible factor is the mouse homolog of TIEG1 (87). TIEG1 is a member of the Sp-1-like family of transcription factors that contains three zinc finger motifs at the C-terminal end and binds GC-rich sequences (54). Overexpression of human TIEG1 stimulates basal POMC, but does not allow for further stimulation by LIF. However, TIEG does not alter SOCS-3 promoter expression. TIEG was first identified by differential display as a TGFß-inducible early gene in human fetal osteoblasts (88, 89). Two novel Sp1-like proteins, TIEG1 and TIEG2, have since been identified. They bind GC-rich sequences and repress transcription via three independent repressor domains conserved within the amino terminus (90, 91, 92, 93).

The results shown here support the idea that GIF overexpression stimulates POMC promoter activity. However, the inability of LIF to further enhance this effect is intriguing and may involve GIF repressor domains. It is also likely that the inability of GIF to stimulate SOCS-3 may result in sustained activation of the JAK/STAT signaling cascade that needs to be inactivated for further effects of LIF to be observed. It is also likely that GIF interacts with other transcription factors mediating LIF effects that could become limiting upon treatment with LIF. We found three putative Sp1 sites in the POMC promoter. One of these sites is adjacent to one of the C/EBPß sites located at -422/-432. Thus, it is likely that C/EBPß and GIF could interact in the regulation of POMC gene expression.

Another gene induced by LIF is AHSG/fetuin. The results shown here suggest that AHSG/fetuin is a novel LIF-induced autofeedback mechanism that acts in an autocrine fashion to down-regulate LIF signaling. LIF stimulates AHSG/fetuin expression in a STAT-3-dependent fashion that may not require STAT-3 binding to DNA. Treatment of AtT-20 cells with bovine fetuin inhibits LIF stimulation of POMC, but not SOCS-3 expression. Thus, the effects of fetuin are independent of SOCS-3. Studies of STAT-3 phosphorylation suggest that fetuin acts by diminishing phosphorylation of STAT-3.

AHSG/fetuin is a secreted protein synthesized in liver, bone, and developing brain and pituitary (94, 95, 96). Human AHSG has high nucleotide homology to bovine fetuin. Despite species-specific differences, this protein exhibits similar functions in different species, such as playing a role in inflammation (97), lipid metabolism (98), and bone growth and remodeling (99, 100, 101, 102).

Although AHSG has been proposed to be a reverse acute phase reactant (103), its precise role in the regulation of inflammatory cytokine action is not clear. In rat hepatoma cell lines, IL-1ß and TNF{alpha}, but not IL-6, diminished fetuin gene expression. The phosphorylated form of AHSG/fetuin (phosphofetuin) has been implicated in liver regeneration under inflammatory conditions, such as hepatitis (104, 105). Dennis et al. (99) also suggested studies in which AHSG knockout mice are shown to be more resistant to endotoxin-induced inflammation than wild-type mice.

In bone, AHSG/fetuin blocks cytokine action by binding members of the TGFß/BMP family and antagonizing their osteogenic effects (101, 102). Indeed, AHSG knockout mice have growth plate defects, increased formation of bone with age, and potentiated osteogenesis in response to cytokines (99, 106).

Fetuin has also been recognized as an antagonist of insulin receptor tyrosine kinase activity, without affecting insulin metabolic effects. Subsequent studies in adipose cells revealed that AHSG inhibited insulin-and platelet-derived growth factor-stimulated Elk-1 phosphorylation (105, 107, 108, 109, 110, 111). Improved insulin sensitivity and resistance to weight gain in mice lacking AHSG/fetuin further support a role for AHSG/fetuin in insulin action (112).

However, little is known about the role of fetuin in LIF signaling. The results shown here support a novel role for AHSG/fetuin in modulating pituitary LIF signaling. As TGFß/BMP gradients are essential for proper pituitary development (113, 114, 115), we speculate that AHSG/fetuin may play a greater role than inhibition of LIF corticotrope signaling. This role may be similar to the effects of other cytokine antagonists, such as noggin, which dramatically altered Rathke’s pouch development upon overexpression in transgenic mice (113).


   Acknowledgments
 
We thank Dr. Philip Koeffler, Dr. Adrian Gombart, Dr. Makoto Matsumoto, Dr. Shizuo Akira, Dr. Thomas Spelsberg, Dr. Ida Chen, Dr. Song-Guang Ren, Mr. Kenny Lien, and Ms. Lin Ota for providing the necessary support for different aspects of this work.


   Footnotes
 
This work was supported by NIH Grant CA-075979 and the Cedars Sinai Research Institute.

Abbreviations: AHSG, {alpha}2-HS-glycoprotein; BMP, bone morphogenic protein; BrdU, bromodeoxyuridine; C/EBPß, CCAAT/enhancer-binding protein ß; CMV, cytomegalovirus; CREB, cAMP response element-binding protein; EST, expressed sequence tag; FL, full length; ßGal, ß-galactosidase; GIF, glial cell-derived neurotrophic factor-inducible factor; gp130, glycoprotein 130; HYG, hygromycin; JAK, Janus kinase; LAP, liver-activated peptide; LIF, leukemia inhibitory factor; LIP, liver inhibitor peptide; Luc, luciferase; POMC, proopiomelanocortin; RSV, Rous sarcoma virus; SHP-1, SH2 domain-containing tyrosine phosphatase-1; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; TIEG, TGFß inducible early gene; WT, wild-type.

Received July 18, 2003.

Accepted for publication October 14, 2003.


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 Introduction
 Materials and Methods
 Results
 Discussion
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