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Molecular Endocrinology, doi:10.1210/me.2006-0543
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Molecular Endocrinology 21 (6): 1443-1457
Copyright © 2007 by The Endocrine Society

Signal Transducer and Activator of Transcription (Stat) 5b-Mediated Inhibition of Insulin-Like Growth Factor Binding Protein-1 Gene Transcription: A Mechanism for Repression of Gene Expression by Growth Hormone

Mitsuru Ono, Dennis J. Chia, Roxana Merino-Martinez, Amilcar Flores-Morales, Terry G. Unterman and Peter Rotwein

Departments of Biochemistry and Molecular Biology (M.O., D.J.C., P.R.) and Pediatrics (D.J.C.), Oregon Health & Science University, Portland, Oregon 97239; Department of Molecular Medicine and Surgery (R.M.-M., A.F.-M.), Karolinska Institute, 17176 Stockholm, Sweden; and Departments of Medicine and Physiology and Biophysics (T.G.U.), University of Illinois College of Medicine and the Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60612

Address all correspondence and requests for reprints to: Peter Rotwein, Department of Biochemistry and Molecular Biology, Oregon Health & Science University, 3181 Southwest Sam Jackson Road, Mail Code L224, Portland, Oregon 97239. E-mail: rotweinp{at}ohsu.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GH plays a central role in controlling somatic growth, tissue regeneration, and intermediary metabolism in most vertebrate species through mechanisms dependent on the regulation of gene expression. Recent studies using transcript profiling have identified large cohorts of genes whose expression is induced by GH. Other results have demonstrated that signal transducer and activator of transcription (Stat) 5b, a latent transcription factor activated by the GH receptor-associated protein kinase, Jak2, is a key agent in the GH-stimulated gene activation that leads to somatic growth. By contrast, little is known about the steps through which GH-initiated signaling pathways reduce gene expression. Here we show that Stat5b plays a critical role in the GH-regulated inhibition of IGF binding protein-1 gene transcription by impairing the actions of the FoxO1 transcription factor on the IGF binding protein-1 promoter. Additional observations using transcript profiling in the liver indicate that Stat5b may be a general mediator of GH-initiated gene repression. Our results provide a model for understanding how GH may simultaneously stimulate and inhibit the expression of different cohorts of genes via the same transcription factor, potentially explaining how GH action leads to integrated biological responses in the whole organism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE POTENT EFFECTS OF GH on somatic growth and intermediary metabolism in mammals and in other vertebrate species have been recognized for many decades (1, 2). Much is now known about the physiology and biochemistry of GH action in the whole animal, and about the earliest events in GH-initiated intracellular signaling pathways in hormone-responsive cells and tissues (1, 3, 4). Progress also is being made in elucidating the changes in gene expression that are thought to be responsible for the long-term effects of GH in stimulating growth during childhood, promoting tissue regeneration in the adult, and maintaining tissue integrity during aging (5, 6, 7, 8).

A single GH molecule binds sequentially with high affinity to the extracellular domains of two copies of its cognate receptor (4), leading to the rapid activation of the GH receptor-associated intracellular tyrosine protein kinase, Jak2 (1). Jak2 in turn initiates a series of protein phosphorylation steps that culminate in the induction of several intracellular signaling networks, including the Ras-Raf-Mek-Erk and phosphatidyl inositol 3-kinase (PI3-kinase)-Akt pathways (1). Among other molecules acutely activated by the GH receptor and Jak2 are a number of transcription factors (3), including members of the signal transducer and activator of transcription (Stat) family (9), and these proteins in turn are responsible for many of the effects of GH on gene expression (3, 9). Several studies have begun to catalog genes activated by GH under a variety of different experimental situations (5, 6, 7, 8), and efforts have begun to identify the transcription factors responsible for the induction of each GH-regulated gene. In this regard, we have shown recently that Stat5b may be required for only a fraction of genes acutely stimulated by GH (8). Within this cohort is IGF-I, which plays a central role in GH-regulated somatic growth (2, 10, 11). A combination of studies utilizing mice with targeted gene knockouts (12, 13), investigating potentially novel causes of growth defects in children (14, 15), and directly analyzing the molecular mechanisms of GH-regulated IGF-I gene activation (16, 17, 18) have defined Stat5b as the critical mediator of IGF-I gene transcription in response to GH.

GH action also can lead to the inhibition of gene expression (6, 8), although much less is known about the relevant biochemical or molecular mechanisms than about steps responsible for GH-induced gene activation. IGF binding protein-1 (IGFBP-1) is a liver-enriched secreted protein that modulates the short-term bioavailability of IGF-I in response to fasting or feeding (19). In the liver, IGFBP-1 gene expression is stimulated by FoxO transcription factors (20). FoxO proteins comprise a subgroup within the Forkhead box family of transcriptional activators (20, 21, 22) and are major targets of signal transduction pathways activated by insulin (20, 21). Insulin action dominantly inhibits IGFBP-1 gene transcription in the liver through Akt-mediated phosphorylation of FoxO1, which leads to suppression of its transactivation functions and stimulates its nuclear export (20, 21, 23, 24, 25). GH also blocks IGFBP-1 gene transcription (26), causing a rapid reduction in IGFBP-1 mRNA levels in vivo in hormone-treated rodents and in isolated hepatocytes (26, 27, 28), but to date, the mechanisms of its regulation have not been elucidated. Here we demonstrate that GH inhibits IGFBP-1 gene transcription by impairing the actions of FoxO1 on the IGFBP-1 promoter, but uses a pathway that is dependent on Stat5b rather than Akt. Our results provide a model for understanding how GH may simultaneously stimulate and inhibit the expression of different cohorts of genes via the same transcription factor, potentially explaining how GH action leads to integrated biological responses in the whole organism.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GH Inhibits IGFBP-1 Gene Transcription through Stat5b
The expression of IGFBP-1, a liver-enriched secreted protein, is regulated by a series of hormone-activated signaling pathways that act primarily at the transcriptional level to stimulate or inhibit IGFBP-1 mRNA synthesis (25, 29). It has been known for over 15 yr that GH reduces IGFBP-1 gene expression (26), yet the biochemical and molecular mechanisms remain undefined. As depicted in the microarray-based expression measurements shown in Fig. 1AGo, a single systemic pulse of GH potently decreased IGFBP-1 mRNA levels in the liver of GH-deficient rats. As seen in Fig. 1BGo, GH also rapidly and transiently inhibited IGFBP-1 gene transcription, as measured by analysis of nascent nuclear RNA. In these experiments, systemic GH treatment was effective in concurrently stimulating the expression of other genes because both the mRNA abundance (Fig. 1AGo) and transcription rates (Fig. 1BGo) increased for IGF-I and Socs2, two genes that require activation of the Stat5b transcription factor by GH (8, 30). As shown in Fig. 1CGo, GH induced the rapid accumulation of tyrosine-phosphorylated Stat5b in the same hepatic nuclei that were used to measure changes in gene transcription. Based on these results, we examined the effects of delivery to the liver by recombinant adenovirus of constitutively active (CA) Stat5b [Ad-Stat5bCA, in which asparagine 642 was changed to histidine (16)] on IGFBP-1 gene expression in GH-deficient rats. As seen in Fig. 1DGo, Ad-Stat5bCA was as potent as GH in decreasing IGFBP-1 mRNA levels, and like GH, also concurrently enhanced IGF-I and Socs2 gene expression. Based on these latter observations, we postulated that Stat5b, in addition to its role as a transcriptional activator, also might function as a GH-stimulated transcriptional inhibitor for IGFBP-1, and potentially for other genes, and designed a series of experiments to test this hypothesis.


Figure 1
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  		Fig. 1. GH Inhibits IGFBP-1 Gene Transcription via Stat5b

A, GH treatment reduces levels of IGFBP-1 mRNA. Results of duplicate microarray experiments measuring IGFBP-1, IGF-I, and Socs-2 gene expression using liver RNA isolated from hypophysectomized rats treated with recombinant rat GH (1.5 µg/g) for the times indicated. B, GH acutely inhibits IGFBP-1 gene transcription in the liver. Time course of accumulation of nascent nuclear transcripts for IGFBP-1, IGF-I, Socs-2, and ß-actin genes after in vivo GH treatment (1.5 µg/g) for 0–120 min, as assessed by semiquantitative RT-PCR using the oligonucleotide primers depicted in the gene maps to the right. DNA sequences for all primers may be found in Table 3Go. No transcripts were observed in the absence of the reverse transcription step, indicating no contamination with chromosomal DNA. Results are representative of three independent experiments. C, GH acutely stimulates the nuclear accumulation of tyrosine phosphorylated Stat5b. Results of immunoblotting of liver nuclear proteins isolated from the same GH-treated hypophysectomized rats as in B. D, Active Stat5b reduces levels of IGFBP-1 mRNA. Results of duplicate microarray experiments measuring IGFBP-1, IGF-I, and Socs-2 gene expression using liver RNA isolated from hypophysectomized rats infected 2 d earlier with recombinant adenoviruses for EGFP (C) or Stat5bCA.

 
As a first step in assessing the effects of GH treatment on IGFBP-1 gene transcription, we examined a promoter-reporter gene encoding luciferase directed by a 320-bp fragment of the rat IGFBP-1 promoter plus 156 bp of exon 1 (29) that was transiently transfected into Cos-7 cells reconstituted for GH signaling by coexpression of the mouse GH receptor. We performed these experiments in the presence of the transcription factor FoxO1, a strong activator of the IGFBP-1 promoter that is important in the regulation of IGFBP-1 gene expression in the liver (20, 21). In agreement with published studies (24), the IGFBP-1 promoter was minimally active in the absence of cotransfected FoxO1, and FoxO1 strongly stimulated promoter activity (Fig. 2AGo). As also illustrated in Fig. 2AGo, in the absence of Stat5b, GH treatment had little effect on IGFBP-1 promoter activity in the presence of either wild-type (WT) or a CA form of FoxO1 (CA FoxO1) that is not phosphorylated by Akt and is resistant to inhibition by this protein kinase (24). Incubation of cells with GH also did not appreciably alter the abundance of either WT or CA FoxO1 proteins (Fig. 2BGo). In contrast, when an expression vector for WT Stat5b (Stat5bWT) was included in these cotransfection experiments, GH treatment caused a 35–50% decline in IGFBP-1 promoter activity (Fig. 2CGo). Neither GH nor Stat5b altered steady-state levels of either WT or CA FoxO1 (Fig. 2DGo), although in these experiments the abundance of WT FoxO1 was approximately 2-fold higher than CA FoxO1. As also shown in Fig. 2DGo, GH treatment stimulated phosphorylation of WT FoxO1, presumably secondary to activation of the PI3-kinase-Akt pathway (1), but as expected, had no effect on CA FoxO1. Despite the ability of GH to phosphorylate WT FoxO1, in the presence of Stat5b, GH more potently inhibited the transcriptional actions of Akt-resistant CA FoxO1 than WT FoxO1 toward the IGFBP-1 promoter (Fig. 2CGo). Thus, the actions of GH to reduce IGFBP-1 gene transcription are more dependent on Stat5b than on Akt.


Figure 2
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  		Fig. 2. Stat5b Inhibits FoxO1-Dependent IGFBP-1 Promoter Activity

A, Results of luciferase assays (mean ± SEM, n = 5 experiments performed in duplicate) in Cos-7 cells transiently transfected with a rat IGFBP-1 promoter-luciferase reporter gene, and expression plasmids encoding the mouse GH receptor and either WT or CA FoxO1 and incubated with rat GH (40 nM) or vehicle for 18 h. B, Immunoblots showing no effect of GH treatment on steady-state levels of FoxO1 or tubulin. C, Results of luciferase assays (mean ± SEM, n = 4 experiments performed in duplicate) in Cos-7 cells transiently transfected with a rat IGFBP-1 promoter-reporter gene, and expression plasmids encoding the mouse GH receptor, WT Stat5b (Stat5bWT), and either WT or CA FoxO1, and incubated with rat GH (40 nM) or vehicle for 18 h. D, Immunoblots showing no effects of Stat5b or GH treatment on steady-state levels of FoxO1 or tubulin, and demonstrating effectiveness of GH in stimulating tyrosine phosphorylation of Stat5b (pStat5) and serine/threonine phosphorylation of FoxO1 (pFoxO1).

 
Insulin inhibits IGFBP-1 gene transcription through insulin receptor-mediated stimulation of Akt, which then phosphorylates and inactivates FoxO1 (20, 21). We mimicked the effects of insulin on FoxO1-stimulated IGFBP-1 gene transcription with inducible Akt (iAkt), a tamoxifen-inducible Akt fusion protein (31). As seen in Fig. 3Go, activated iAkt potently reduced IGFBP-1 promoter activity in the presence of WT FoxO1, but not when Akt-resistant CA FoxO1 was coexpressed. We additionally assessed the ability of iAkt to phosphorylate WT FoxO1 and compared it with CA Stat5b (Stat5bCA). The latter protein contains a substitution of histidine for asparagine 642, and is an active transcription factor even in the absence of GH signaling (16). As depicted in Fig. 3CGo, activated iAkt caused phosphorylation of WT FoxO1, whereas Stat5bCA did not.


Figure 3
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  		Fig. 3. Akt Blocks the Transcriptional Actions of WT FoxO1

A, Results of luciferase assays (mean ± SEM, n = 3 experiments performed in duplicate) in Cos-7 cells transiently transfected with a rat IGFBP-1 promoter-reporter gene, and expression plasmids encoding a iAkt, and either WT or CA FoxO1, and incubated with 4-hydroxy-tamoxifen (4-HT, 1 µM) for 18 h. B, Immunoblots showing no effects of iAkt or 4-HT on steady-state levels of FoxO1 or tubulin. C, Immunoblots showing stimulation of serine/threonine phosphorylation of FoxO1 (pFoxO1) by activated iAkt, but no effect of Stat5bCA.

 
A FoxO1 Binding Site Is Sufficient for Stat5b-Mediated Inhibition of Gene Transcription
We next examined the effects of GH-activated Stat5b on FoxO1-mediated transcription of a reporter gene containing the minimal thymidine kinase (TK) promoter plus three copies of insulin-responsive sequence A (IRSA), the more 5' of the tandem FoxO1 binding sequences found in the proximal IGFBP-1 promoter (24). As shown in Fig. 4AGo, luciferase activity directed by this chimeric gene was increased by 35- to 60-fold by WT or CA FoxO1, respectively, and was reduced by 35–50% by GH plus Stat5b, results nearly identical with those seen with the full IGFBP-1 promoter. Thus, only a FoxO1 binding site appears to be needed for Stat5b-inhibited gene transcription.


Figure 4
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  		Fig. 4. Stat5b Inhibits FoxO1-Mediated Transcription, whereas FoxO1 Enhances IGF-I Promoter Activity via HS7

Results of luciferase assays in Cos-7 cells transiently transfected with expression plasmids for the mouse GH receptor, Stat5bWT, and either WT or CA FoxO1, and incubated with rat GH (40 nM) or vehicle for 18 h. A, Results are illustrated (mean ± SEM, n = 3 experiments performed in duplicate) with a luciferase reporter gene containing three copies of the IGFBP-1 IRSA DNA sequence fused to TK. B, Results are shown (mean ± SEM, n = 4 experiments performed in duplicate) using a luciferase reporter plasmid containing the minimal TK promoter plus an 84-bp sequence spanning GHRE-1 and GHRE-2 of HS7, derived from intron 2 of the rat IGF-I gene (17 ) (*, P < 0.01 vs. no FoxO1).

 
FoxO1 Does Not Inhibit Stat5b-Stimulated Gene Transcription
We next asked whether FoxO1 could interfere with GH and Stat5b-activated gene transcription. We analyzed the effects of concurrent expression of FoxO1 on a GH- and Stat5b-responsive luciferase reporter gene containing the minimal TK promoter plus the Stat5b-dependent HS7 response element that we have identified in the IGF-I gene (17). As seen in Fig. 4BGo, addition of GH triggered a 4-fold increase in reporter gene activity in the absence of FoxO1, similar to what we reported previously (17). When either WT or CA FoxO1 was cotransfected, both basal and GH-activated promoter activity increased by 2- to 4-fold, despite the absence of FoxO1 binding sites in either the TK promoter or HS7. Similar results were observed with IGF-I promoter 1 fused to HS7 (data not shown). Thus, whereas Stat5b inhibited transcription of a FoxO1-dependent gene, FoxO1 did not reciprocally impair GH-stimulated activity of a Stat5b-activated gene, and in fact increased Stat5b-stimulated gene expression. We interpret these results to indicate that Stat5b and FoxO1 are not competing for a potentially limiting supply of transcriptional cofactors within the nucleus, but rather in some circumstances may act synergistically.

Mechanism of Stat5b-Mediated Inhibition of FoxO1-Dependent Gene Transcription
In previous studies, we employed both CA and dominant-negative (DN) versions of Stat5b to establish that this transcription factor was required for GH-induced IGF-I gene activation (16). Here we have used the same expression vectors to elucidate the requirements for inhibition of FoxO1-regulated IGFBP-1 transcription. DN Stat5b (Stat5bDN) contains the substitution of phenylalanine for tyrosine 699 and is unable to be phosphorylated by the GH receptor-associated tyrosine kinase, Jak2, whereas as noted above, Stat5bCA is an active transcription factor even in the absence of GH (16). As seen in Fig. 5AGo, WT Stat5b (Stat5bWT) was able to mediate the inhibitory effects of GH on IGFBP-1 promoter activity. Stat5bCA was also effective in suppressing promoter function, even in the absence of GH, whereas Stat5bDN did not inhibit promoter function even with GH treatment. All three Stat5b isoforms were expressed at relatively equivalent levels in cotransfected Cos7 cells, as was CA FoxO1 (Fig. 5BGo). The low levels of immunoreactive Stat5 detected in cells not transfected with a Stat5b expression plasmid represent Stat5a (data not shown). In addition, as seen in Fig. 5CGo, studies in hyphysectomized rats infected with Ad-Stat5bDN demonstrate that Stat5bDN blocks the ability of GH to suppress IGFBP-1 gene expression in vivo.


Figure 5
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  		Fig. 5. GH-Regulated Inhibition of IGFBP-1 Promoter Activity Requires Transcriptionally Active Stat5b

A, Results of luciferase assays (mean ± SEM, n = 3 experiments performed in duplicate) in Cos-7 cells transiently transfected with expression plasmids for the mouse GH receptor, CA FoxO1, and either WT, CA, or DN Stat5b, and incubated with rat GH (40 nM) or vehicle for 18 h (*, P < 0.002; **, P < 0.001 vs. other groups). B, Immunoblots showing no effects of Stat5b or GH treatment on steady-state levels of CA FoxO1 (ns, nonspecific protein band). C, Stat5bDN prevents GH-mediated inhibition of IGFBP-1 gene expression. Results of duplicate microarray experiments measuring IGFBP-1 mRNA using liver RNA isolated from hypophysectomized rats infected 2 d earlier with recombinant adenoviruses for EGFP (C) or Stat5bDN, followed by injection with recombinant rat GH (1.5 µg/g) for 2 h. D, Stat5b does not alter the nuclear expression of FoxO1. Immunoblots showing time-course studies examining accumulation of Stat5b and FoxO1 in nuclear protein extracts from Cos-7 cells transiently transfected with expression plasmids for the mouse GH receptor, CA FoxO1, and Stat5bWT, and incubated with rat GH (40 nM) for up to 2 h. E, Stat5b does not alter nuclear accumulation of FoxO1. Immunocytochemical images for Stat5b and FoxO1 of Cos-7 cells transiently transfected with expression plasmids for the mouse GH receptor, CA FoxO1, and Stat5bWT, and incubated with rat GH (40 nM) for 0 or 60 min. Nuclei have been stained with Hoechst 33258 dye. F, Stat5b does not block binding of FoxO1 to its DNA response element. Results of gel-mobility shift assays using IR-labeled double-stranded oligonucleotides for either the FoxO1 binding site in the IGFBP-1 promoter (IRS A + B, top panel), or Sp1 (bottom panel), and nuclear protein extracts from Cos-7 cells transfected with expression plasmids encoding the mouse GH receptor, CA FoxO1, and Stat5bWT, and incubated with GH or vehicle for 1 h. Larger arrows, Protein-DNA complexes; smaller arrows, free probe (ns, nonspecific DNA-protein complex).

 
We next looked at whether the accumulation of Stat5b in the nucleus altered the abundance of FoxO1 in this subcellular compartment by analyzing Cos-7 cells cotransfected with the GH receptor, Stat5bWT, and CA FoxO1, and incubated with GH for up to 2 h. As seen in Fig. 5Go, D and E, the GH-induced appearance of Stat5b in the nucleus, which occurred within 30 min after addition of hormone and was sustained for at least 120 min, did not reduce the level of nuclear CA FoxO1 protein. Similarly, activated Stat5b did not inhibit the ability of CA FoxO1 in nuclear protein extracts to bind to its cognate DNA element by gel-mobility shift assay (Fig. 5FGo).

The results in Fig. 5Go rule out the possibility that active Stat5b stimulates the degradation of FoxO1, alters its nuclear abundance, or interferes with its ability to bind to DNA. We next looked at whether Stat5b and FoxO1 physically interacted in the nucleus of cotransfected Cos-7 cells under conditions in which Stat5bCA inhibited CA FoxO1-activated IGFBP-1 gene transcription by 70% (Fig. 6AGo), but did not reduce its nuclear expression (Fig. 6BGo). Under these experimental conditions we were unable to detect a protein-to-protein interaction between FoxO1 and Stat5b by coimmunoprecipitation assays (data not shown). We next used an avidin-biotin complex DNA binding (ABCD) assay to determine whether FoxO1 and Stat5b associated with each other only on DNA. As pictured in Fig. 6CGo, CA FoxO1 was able to bind to a biotinylated double-stranded oligonucleotide derived from a high-affinity FoxO1 site in the peroxisome proliferator-activated receptor-{gamma} coactivator-1 (PGC-1) gene promoter (32), but Stat5bCA was not found in this protein-DNA complex (lanes 1 vs. 2). Conversely, as seen in lanes 5 and 6, Stat5bCA could bind to its specific DNA element derived from a high-affinity site in the IGF-I gene (18). FoxO1 appeared able to associate with this latter oligonucleotide, even in the absence of Stat5b, but very weakly (lanes 4 and 5). Taken together in conjunction with Fig. 5FGo, these results do not support the idea that Stat5b interferes with FoxO1-mediated gene transcription either by directly interacting with FoxO1 or by modifying its ability to bind to its specific DNA element in a target gene.


Figure 6
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  		Fig. 6. Active Stat5b Inhibits the Transcriptional Actions of FoxO1 without Altering Its Nuclear Localization or DNA Binding Activity

A, Results of luciferase assays (mean ± SEM, n = 3 experiments performed in duplicate) in Cos-7 cells transiently transfected with a rat IGFBP-1 promoter-reporter gene, and expression plasmids encoding CA FoxO1 and Stat5bCA (*, P < 0.01). B, Immunoblots showing the expression in nuclear protein extracts from Cos7 cells transfected in A of FoxO1, Stat5bCA, and CREB. C, Immunoblots showing results of ABCD assays using double-stranded oligonucleotides for either the FoxO1 binding site in the PGC-1 promoter (lanes 1–3) (32 ) or the Stat5b site from the far 5' region of IGF-I (lanes 4–6) (18 ), and nuclear protein extracts from Cos-7 cells transfected with expression plasmids CA FoxO1 and Stat5bCA.

 
Identifying GH- and Stat5b-Inhibited Genes in the Liver
We recently reported results of a series of microarray experiments designed to identify genes whose rapid activation by GH in the liver of hypophysectomized rats was dependent on Stat5b (8). We have used the same model system to identify hepatic genes whose expression was acutely diminished by GH, and have compared these results to genes whose expression was decreased after infection with Ad-Stat5bCA but not with Ad-EGFP. Using Significance Analysis of Microarrays statistics and a FDR of 5%, we detected 97 genes whose mRNA abundance was reduced to 60% or less of starting values within 2 h of a single GH pulse, and 333 whose expression was decreased by Ad-Stat5bCA but not Ad-EGFP. A total of 89 transcripts were reduced by a comparable extent by both GH and Ad-Stat5bCA (Table 1GoGo and Fig. 7AGo).


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  		Table 1. Genes with Decreased Expression after Infection with Ad-Stat5bCA or Acute Systemic GH Treatment

 

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  		Table 1A. Continued

 

Figure 7
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  		Fig. 7. Regulation of Liver Gene Expression by Stat5bCA and GH

A, Venn diagram showing genes whose expression was reduced by GH (97 total) or by Stat5bCA (333 total) in livers of GH-deficient rats either injected with recombinant rat GH (1.5 µg/g) or vehicle for 2 h, or in separate experiments infected with Ad-Stat5bCA or Ad-EGFP for 2 d. A total of 89 genes showed a shared decline in abundance after either GH treatment or expression of Stat5bCA. B, Phylogenetic footprinting for FoxO1 binding sites. Bar graph showing the number of control, and GH- and Stat5b-down-regulated genes analyzed, and the fraction with putative FoxO1 DNA binding sites within 10 kb of the gene transcription start site. See text for details.

 
Identifying Potential FoxO1 DNA Elements in Stat5b-Inhibited Genes by Phylogenetic Promoter Analysis
We next addressed the hypothesis that the cohort of 89 genes whose expression was diminished both by GH and Ad-Stat5bCA were direct transcriptional targets of FoxO1. We used a computational approach to analyze each of these genes for the presence of FoxO1 binding sites within phylogenetically conserved areas between the rat and human orthologues. For each gene, genomic sequences containing 10 kb of DNA 5' to the transcription initiation site and 1 kb of the first intron were extracted from the UCSC rat genomic database and aligned with the orthologous human sequences using LAGAN, a program for rapid global alignment of two homologous DNA sequences (33). The presence of FoxO1 binding sites was assessed using a weight matrix built from alignments of known binding sites, using a consensus sequence: 5'-TG/ATTT-3' (20). Using this approach, we identified a total of 19 genes with putative FoxO1 binding sites out of 89 genes whose expression was reduced by both GH and Stat5bCA. This corresponds to 21% of the genes (Fig. 7BGo). Some of the sites in genes such as IGFBP-1 (24), glucose-6-phosphatase (34), and tyrosine aminotransferase (35) have been characterized previously to bind forkhead proteins (see Table 2Go), thereby providing an independent validation of our predictions. We performed a similar analysis with a set of 322 randomly selected genes with mRNA expression that was not regulated by GH or by Stat5b in our experiments. As also seen in Fig. 7BGo, 19% of the control genes appeared to contain conserved FoxO1 binding sequences despite no effect of GH or Stat5bCA. Thus, we found no statistically significant differences in the frequency of FoxO1 binding sites in the two sets of genes, indicating that impairing FoxO1 accounts at best for only a percentage of the inhibitory effects of GH on gene expression.


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  		Table 2. Genes with Predicted Phylogenetically Conserved FoxO1 Binding Sites

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The long-term effects of GH on somatic growth, and tissue regeneration and repair, require changes in gene expression that are regulated by sequence-specific transcription factors activated through GH-stimulated signal transduction pathways. Because GH activates several signaling pathways, it is not surprising that a variety of transcription factors serve as mediators of GH action under different conditions. The most important GH-stimulated transcription factors are members of the Stat family. Stat proteins were identified originally as components of interferon signaling pathways (36), and now appear to be activated in response to many cytokines and growth factors including GH (1, 9). GH treatment induces Stats 1, 3, 5a, and 5b (9) by stimulating phosphorylation of tyrosine residues through GH receptor-associated Jak2 (1, 36). Once activated, these factors form homodimers (also heterodimers for Stats 1 and 3) that interact with response elements on different target genes (36), although the exact biochemical mechanisms by which Stat5 proteins activate gene transcription subsequent to DNA binding remain incompletely understood. Early studies established that the COOH-terminal region of Stat5a or Stat5b could interact directly with transcriptional coactivators CBP and p300 (37), a process that led to chromatin modifications including histone acetylation (37), and that potentially was facilitated by another interacting factor, Nmi (N-Myc interactor) (38). CPAP, a widely expressed protein, was identified as a second coactivator of Stat5 that also physically interacted with its COOH terminus (39). Other studies have focused on the surprising observation that upon binding of activated Stat5a to regulatory sites on selected target genes, an associated histone deacetylase activity was enhanced (40). In agreement with this idea, in one group of experiments, the deacetylase inhibitor TSA globally blocked cytokine-induced activation of Stat5-dependent genes (40).

We have used adenoviral-mediated gene transfer to rapidly manipulate levels of active or dominant-negative Stat5b in GH-deficient rats (16, 30). With this approach, we have shown recently by transcript profiling that Stat5b appears to be required for the induction of approximately 20% of the genes acutely stimulated by GH in vivo in the liver (8). In the same study, we noted that GH or Stat5bCA caused a rapid decline in the expression of other genes (8). One of the genes that GH and Stat5b coordinately inhibited was IGFBP-1, a liver-enriched modulator of the bioavailability of IGF-I (19, 41). It has been known for over 15 yr that GH represses IGFBP-1 gene transcription (26), although the molecular mechanisms have not been elucidated previously. As we now show by several lines of evidence, reduction of IGFBP-1 gene transcription by GH appears to be dependent on Stat5b. GH thus relies on mechanisms distinct from those used by insulin, which activates Akt to inhibit induction of the IGFBP-1 promoter by the FoxO1 transcription factor (20), even though GH also stimulates the PI3-kinase-Akt pathway (1). As we describe in this report, Stat5bCA or GH decreased IGFBP-1 transcript abundance in the liver (Fig. 1Go), whereas Stat5bDN prevented this acute effect of GH (Fig. 5CGo). In Cos-7 cells reconstituted for GH signaling, GH also required Stat5b to inhibit IGFBP-1 gene transcription, and a robust reduction in IGFBP-1 promoter activity was seen with Stat5bCA, even in the absence of GH, whereas GH had no effect on promoter function in the presence of Stat5bDN (Figs. 2Go and 5Go).

Previous studies by Lu et al. (42) using IGFBP-1 promoter-luciferase reporter fusion genes transfected into primary rat hepatocytes, suggested that a negative GH-response element was located in the proximal IGFBP-1 promoter between 930 and 277 bp 5' to the transcription initiation site. Subsequent experiments designed to identify hepatic nuclear proteins that bound to this DNA segment were not successful (42). Our results demonstrate that a more proximal part of the promoter than was mapped by Lu et al. (42) (–320 to +156 with respect to the transcriptional start site) also is susceptible to GH-mediated inhibition. This critical region contains tandem FoxO1 binding sites, and a reporter plasmid with a minimal promoter and several copies of one of these sites also was repressed by GH (Fig. 3Go). We additionally showed that the transcriptional activity of an Akt-resistant form of FoxO1 was decreased by GH via Stat5b and by Stat5bCA in the absence of GH, further indicating that the PI3-kinase-Akt pathway does not play a major role in GH-mediated suppression of IGFBP-1 gene expression.

We were not able to discern the precise biochemical steps by which Stat5b impaired the transcriptional actions of FoxO1, and could not detect direct interactions between these two proteins, either by coimmunoprecipitation experiments or by their coassociation on DNA (Fig. 6Go, and data not shown). Kortylewski et al. (43) also could not demonstrate direct binding between Stat5a and FoxO1. However, we were able to determine that unlike Akt, Stat5b did not increase the phosphorylation of FoxO1 or decrease its abundance in the nucleus. Stat5b also did not interfere with the ability of FoxO1 to bind to its cognate DNA recognition element in vitro, at least as measured by gel-mobility shift and ABCD assays (Figs. 5Go and 6Go). We interpret these results to indicate that Stat5b uses steps distinct from those of activated Akt to interfere with the actions of FoxO1. Future experiments will focus on elucidating the molecular mechanisms of inhibition of FoxO1 by Stat5b.

Several other reports have established conditions under which Stat5b was able to function as an inducible transcriptional repressor. Zhou and Waxman (44, 45) showed that GH-activated Stat5b could reduce the transcriptional effects of several nuclear receptors, including PPAR{alpha}, {gamma}, and {delta}, and the thyroid hormone receptor. In contrast to our results, they found that Stat5bCA was an ineffective inhibitor (45). As in our studies with FoxO1, Stat5b did not appear to interact directly with PPAR{alpha}, and the authors were not able to identify the mechanisms of transcriptional repression. Luo and Yu-Lee (46) demonstrated that prolactin could stimulate Stat5b to inhibit transcription of the IRF-1 gene promoter by blocking the actions of the nuclear factor-{kappa}ß transcription factor through competition for limiting amounts of the transcriptional coactivator, p300. In preliminary studies, we found that added p300 did not rescue inhibition of FoxO1-mediated IGFBP-1 gene transcription by Stat5b (data not shown). This may not be surprising because, even though p300 and the related protein CBP, may act as coactivators for FoxO proteins under some circumstances, acetylation of FoxO1 by these cofactors also can limit transactivation and modulate its function (22). Stat5b and the related protein Stat5a have been shown in certain contexts to block the transcriptional actions of the glucocorticoid receptor (47, 48). Remarkably, on other genes, the glucocorticoid receptor may function as a transcriptional coactivator for Stat5b (49). FoxO1 may behave similarly. In fact, FoxO1 has been found to enhance the transcriptional actions of Stat3 on the {alpha}2-macroglobulin gene promoter (43). Because GH also stimulates the activity of Stat3 (9), it is possible that FoxO1 may increase the transcription rates of some GH-induced genes, whereas GH via Stat5b concurrently represses other FoxO1-dependent genes.

We identified 89 of 97 transcripts whose abundance was acutely decreased by GH in the liver as also being reduced by Stat5bCA in GH-deficient rats. Of the genes encoding these 89 mRNAs, only 19 were found to have phylogenetically conserved potential FoxO1 binding sites in their promoter-regulatory regions. Thus, Stat5b appears to play a major role in mediating the repressive effects of GH on gene expression, but interference with the transcriptional actions of FoxO1 is only one of several potential inhibitory mechanisms. Recently, Clodfelter et al. (50) studied effects of loss of Stat5b in the liver by comparing transcripts in mice lacking this transcription factor with patterns of gene expression in WT animals. They identified 676 mRNAs in male mice and 149 in females that were increased in abundance in the absence of Stat5b (50), thus demonstrating indirectly that Stat5b can inhibit gene expression. From another perspective, Zhang et al. (51) characterized 121 genes whose expression was increased in transgenic mice overexpressing CA FoxO1 in the liver compared with control mice. In our study, GH and Stat5bCA rapidly reduced the abundance of only seven of these 121 genes. One reason for the discordance between our results and those of Zhang et al. (51) may be a difference in experimental design: chronic overexpression of active FoxO1 in one model vs. either treatment with GH for 2 h or targeted expression of Stat5bCA for 2 d in the other.

Finally, our results suggest an inherent complexity in the acute response to GH at the level of gene expression in the liver. In our recent observations only 20% of the 52 genes whose expression was rapidly induced by GH appeared to be regulated by Stat5b (8), indicating that other transcription factors, for example Stat1 and Stat3, and posttranscriptional mechanisms affecting mRNA stability must be involved in these early GH-mediated events. By contrast, as demonstrated in this report, the vast majority of the 97 transcripts whose abundance was acutely diminished by GH appeared to be dependent on Stat5b for their down-regulation. For some members of this group of genes, such as IGFBP-1, GH and Stat5b may inhibit their expression by interfering with the actions of transcriptional activators. In other cases, Stat5b may act more indirectly, potentially either by inducing transcriptional inhibitors, or by other mechanisms, such as stimulating the production of micro-RNAs that target and diminish mRNA abundance. Additional study will be needed to identify and characterize all of the molecular mechanisms that define the early events in GH-mediated gene expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Fetal bovine serum was purchased from Hyclone (Logan, UT), and trypsin from Invitrogen (Carlsbad, CA). DMEM and PBS were from Mediatech-Cellgrow (Herndon, VA). The luciferase assay system was purchased from Promega (Madison, WI) and Immobilon-FL polyvinylidene difluoride was from Millipore Corp. (Billerica, MA). Restriction enzymes, buffers, ligases, and polymerases were from Roche Applied Sciences (Indianapolis, IN) and Fermentas (Hanover, MD). Hoechst 33258 nuclear dye was from Polysciences (Warrington, PA). Antibodies were obtained from the following vendors: anti-Stat5 (C17X) and anti-CREB (cAMP response element binding protein (SC186), Santa Cruz Biotechnology (Santa Cruz, CA); anti-FoxO1, anti-phospho-FoxO1/O3, and anti-Akt, Cell Signaling Technology (Beverly, MA); anti-Flag (M2) and anti-{alpha}-tubulin, Sigma (St. Louis, MO); anti-T7, Novagen (San Diego, CA); phospho-Stat5 (Upstate Cell Signaling, Lake Placid, NY). Recombinant rat GH was purchased from the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIH). Oligonucleotides were synthesized at the Oregon Health & Science University (OHSU) Core Facility in the Department of Molecular Microbiology and Immunology. Transit-LT1 was from Mirus (Madison, WI). All other chemicals were reagent grade and were purchased from commercial suppliers.

Recombinant Plasmids
The following recombinant mammalian expression plasmids have been described: mouse GH receptor in pcDNA3 (16), iAkt in pcDNA3 (31), WT, CA, and DN Flag-tagged rat Stat5b in pcDNA3 (16), and WT and CA Flag-tagged rat FoxO1 in pFlag-CMV (24). An expression plasmid for WT Stat5b with an NH2-terminal T7 epitope tag was generated in pcDNA3 using standard molecular biological methods. The following luciferase reporter plasmids have been described: rat IGFBP-1 promoter (coordinates –320 to +156) (29), the HS7 region of the rat IGF-I gene fused to a minimal TK promoter (17), and three copies of the IRSA sequence from the rat IGFBP-1 promoter fused to TK (24).

Cell Culture, Transient Transfections, and Reporter Gene Assays
Cos-7 cells (ATCC CRL-1651) were incubated in antibiotic-free DMEM containing 10% fetal bovine serum. Cells were cotransfected on 10 cm tissue culture dishes with various combinations of expression plasmids (250 ng of DNA for each), using Transit-LT1. After incubation for 24 h, serum-free media were added containing 1% BSA for 4 h, followed by addition of recombinant rat GH (40 nM final concentration) or vehicle for up to 120 min. Cells were then harvested, and either whole cell extracts or nuclear and cytoplasmic proteins were isolated. For reporter gene assays, cells on six-well tissue culture dishes were cotransfected with mouse GH receptor (100 ng of DNA) or inducible Akt (250 ng), combinations of other expression plasmids or empty vector DNA (250 ng each), and one of the promoter-reporter plasmids indicated above (250 ng). Cells were incubated for 24 h, followed by addition of serum-free media containing 1% BSA and either vehicle, 4-hydroxy-tamoxifen (1 µM), or rat GH (40 mM) for 18 h. Cells were then harvested and lysates used for luciferase assays, which were measured with a Veritas microplate luminometer (Turner Biosystems, Sunnyvale, CA). All results were normalized for total cellular protein concentration. Typical luciferase values for the IGFBP-1 promoter in the presence of FoxO1 ranged from 1–4 x 104 light units/sec/µg of cellular protein.

Immunoblotting
Whole cell protein lysates were prepared after washing cells with PBS, and incubating on ice for 15 min in RIPA Buffer [50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1.25 mM EDTA, 0.1% sodium dodecyl sulfate, 0.5% Na-deoxycholate, 1% IGEPAL CA-630, 1 mM dithiothreitol, 1x protease inhibitor cocktail (Roche), 0.25 mM phenylmethylsulfonyl fluoride] followed by passage through a 22-gauge needle and centrifugation at 15,000 RPM at 4 C. Protein concentrations were determined using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Nuclear and cytoplasmic proteins were isolated as described (17, 18). Protein samples (30 µg for whole cell protein extracts, 30 µg for cytoplasmic proteins, and 10 µg for nuclear proteins) were separated by SDS-PAGE, transferred to Immobilon membranes, and incubated with antibodies as described (18), followed by detection with a LiCoR Odyssey infrared imager and version 1.2 analysis software (LiCoR Biosciences, Lincoln, NE). Antibodies were used at the following dilutions: Flag M2 1:2000, T7 1:2000, FoxO1 1:1000, phospho-FoxO1/O3 1:1000, Stat5b 1:5000, phospho-Stat5 1:2000, Akt 1:1000, {alpha}-tubulin 1:2000, mouse IR680 (LiCoR) 1:5000, and rabbit IR800 (LiCor) 1:5000.

Immunocytochemistry
Cos-7 cells in six-well plates were transiently transfected with expression plasmids for T7-tagged Stat5bWT and FoxO1CA. After 48 h, cells were fixed in 4% paraformaldehyde for 15 min at 20 C and permeabilized with a 50:50 mixture of methanol and acetone for 2 min before blocking in 0.25% normal goat serum for 1 h at 20 C. After addition of T7 monoclonal antibody (1:2000 dilution) and FoxO1 polyclonal antibody (1:500) in blocking buffer overnight, followed by a washing step, and incubation in goat antimouse-Alexa 488 (1:1000 dilution) and goat antirabbit-Alexa 594 (1:1000) in blocking buffer for 2 h, images were captured with a Roper Scientific Cool Snap FX charge-coupled device camera attached to a Nikon Eclipse T300 fluorescent microscope using IP Labs 3.5 software (Scanalytics, Rockville, MD).

Animal Studies
Male Sprague Dawley rats, hypophysectomized by a transauricular route at age 7 wk, were purchased from Harlan Sprague Dawley (Indianapolis, IN). Animals were housed at the OHSU Animal Care Facility on a 12-h light, 12-h dark schedule with free access to food and water, and received care according to NIH guidelines. Replacement doses of glucocorticoids (cortisol phosphate, 400 µg/kg·d) and T4 (10 µg/kg·d) were given daily by sc injection, and GH deficiency was confirmed by failure to grow during a 2-wk observation period. After this interval, in one group of experiments, rats were injected iv via tail vein with 0.2 ml of sterile PBS containing 2 x 1010 plaque-forming units (pfu) of recombinant adenoviruses encoding EGFP (Ad-EGFP), Stat5bDN (Ad-Stat5bDN), or Stat5bCA (Ad-Stat5bCA), and 2 x 109 pfu of helper virus Ad-tTA, as described (16), and were injected ip 2 d later with 1.5 µg/g of recombinant rat GH (16). In other studies, rats were injected with either vehicle (saline) or 1.5 µg/g of recombinant rat GH. In both groups of experiments, after an additional 30–120 min, rats were anesthetized with pentobarbital (50 mg/kg given ip), and killed. Liver nuclear proteins and total and nuclear RNA were isolated as described (16). The OHSU Committee on Animal Care and Use approved all experiments involving rats.

Expression Profiling Using Microarrays
Microarrays containing 70-oligomer oligonucleotide probes for 7000 rat protein-coding genes were fabricated as described (5). Total liver RNA (5 µg) was incubated with deoxyribonuclease I (Promega, Madison, WI) according to the supplier’s instructions, and was labeled by reverse transcription using the Pronto labeling kit (Corning Inc., Corning, NY) in the presence of Cy5-CTP. An equal amount of the Universal rat RNA reference (Stratagene, La Jolla, CA) was labeled with Cy3-CTP, mixed with the Cy5-labeled target cDNA, purified and added to 40 µl of hybridization buffer supplied with the Pronto System. Labeled cDNA was incubated with the array in a sealed hybridization chamber (Corning) for 15–18 h at 65 C. After washing, the dried arrays were scanned immediately using a GenePix scanner (Axon Instruments, Union City, CA). Image analysis was performed using GenePix Pro 6.0 software (Axon Instruments). Absent or very weak spots were identified by automatic flagging and were excluded from further analysis. Fluorescence (Cy5/Cy3) ratios were normalized as described previously (52) using the Locally Weighted Scatter Plot Smoother method found in the Statistics for Microarray software package (www.bioconductor.org). Both data analysis and statistical analysis and statistical evaluation have been described previously (8). Differentially expressed genes were identified with a false discovery rate of less than 5%.

Prediction of FoxO1 Binding Sites
To analyze the presence of FoxO1 binding sites in the promoter regions of the genes identified by the microarray analysis, we used the Prometheus software application (8). Prometheus was designed to predict aspects of promoter architecture for genes that may be regulated by a common mechanism. It uses weight matrices that describe transcription factor binding sites (TFBS) to find putative cis elements within conserved regions between different genomes. The weight matrix for FoxO1 was built by aligning genomic sequences known to bind this transcription factor, as identified in the published literature (53). Genomic sequences for human and rat were uploaded from the UCSC Genome Informatics data repository. Sequence alignments were performed using the pair-wise alignment program, LAGAN (33). Because tests where multiple genes are analyzed are computationally intensive, the algorithm is implemented to run in a computer GRID. Two runtime environments have been set up on NorduGrid (www.NorduGrid.org) to execute the alignment and TFBS finding algorithms: /APPS/BIO/LAGAN-1.2 and /APPS/BIO/TFBS-0.5.0. Both algorithms run in parallel (for each accession number) using NorduGrid as virtual organization. Once a set of lists is processed and TFBS are found, a statistical analysis, based on the Chi square test, is performed. This analysis compares the frequency of appearance of specific TFBS in the two sets of genes.

DNA-Protein Binding Studies
EMSAs were performed as indicated (18), with Cos-7 nuclear proteins and 5'-IRDye 700-labeled double-stranded oligonucleotides from derived from the rat IGFBP-1 insulin response sequence (IRS) A and B (23) (top strand, 5'-CTCACAAGCAA AACAAACTTATTTTGAACACGGG-3'; IRSA and B underlined), and from a site recognized by the transcription factor Sp-1 (18) (top strand, 5'-ATTCGATCGGGGCGGGGC GAGC-3'). After incubation of proteins and DNA for 30 min at 4 C, products were separated by electrophoresis through nondenaturing 5% polyacrylamide gels in TBE buffer [90 mM Tris, 90 mM boric acid, and 2 mM EDTA (pH 8.3)] at 200 V for 25–35 min at 20 C. Results were analyzed using an Odyssey infrared imaging system and version 1.2 analysis software. ABCD assays were performed as described (32), using Cos7 nuclear proteins and the following double-stranded oligonucleotides, labeled with biotin at their 5' ends: rat IGF-I distal region RE-1 (18) (top strand, 5'-GGGCCTTCCTGGAAGAAA-3'), human PGC-1 IRS3 site (32) (top strand, 5'-GCCACTTGCTTGTTTTGGAAGGAAAAT-3'). Protein extracts (200 µg) were incubated with a biotinylated double-stranded oligonucleotide that had been precomplexed to streptavidin-conjugated beads in the presence of poly-deoxyinosine-deoxycytosine. After a washing step, the beads were incubated in SDS-PAGE sample buffer. Proteins were separated by SDS-PAGE, and detected by immunoblotting, as described above.

Analysis of Gene Transcription
Nuclear RNA (5 µg) was reverse transcribed with random hexamers in a final volume of 20 µl using a RT-PCR kit (Invitrogen). PCRs were then performed with 0.5 µl of cDNA (30). Primer sequences are listed in Table 3Go. The linear range of product amplification was established in pilot studies for each primer pair, and the cycle number that reflected the approximate midpoint was used in final experiments. This varied from 24–28 cycles. Results were quantified by densitometry after electrophoresis through 1.5% agarose gels. All experiments were performed on at least three separate occasions with comparable results.


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  		Table 3. Primers Used for RT-PCR of Nuclear RNA

 


   ACKNOWLEDGMENTS
 
This paper is dedicated to the memory of our colleague and friend Liam J. Murphy.


   FOOTNOTES
 
This work was supported by grants from the National Institutes of Health [RO1 DK69703 (to P.R.) and RO1 DK41430 (to T.G.U.)], from the Department of Veterans Affairs Merit Review Program to T.G.U., and from the Swedish Research Council, Wallenberg Foundation, and the Swedish Institute (to A.F.-M.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 10, 2007

Abbreviations: ABCD, Avidin-biotin complex DNA binding; CA, constitutively active; CREB, cAMP response element binding protein; DN, dominant negative; iAkt, inducible Akt; IGFBP-1, IGF binding protein-1; IRSA, insulin-responsive sequence A; PGC-1, peroxisome proliferator-activated receptor-{gamma} coactivator-1; PI3-kinase, phosphatidyl inositol 3-kinase; Stat, signal transducer and activator of transcription; WT, wild type.

Received for publication December 18, 2006. Accepted for publication April 5, 2007.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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