Stat5b Inhibits NF{kappa}B-Mediated Signaling

Guoyang Luo and Li-yuan Yu-Lee

Department of Microbiology and Immunology (G.L., L.-y.Y.-L.), Medicine (L.-y.Y.-L.), and Molecular and Cellular Biology (L.-y.Y.-L.) Baylor College of Medicine Houston, Texas 77030


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Signal transducers and activators of transcription (Stat) are latent transcription factors that participate in cytokine signaling by regulating the expression of early response genes. Our previous studies showed that Stat5 functions not only as a transcriptional activator but also as a transcriptional inhibitor, depending on the target promoter. This report further investigates the mechanism of Stat5b-mediated inhibition and demonstrates that PRL-inducible Stat5b inhibits nuclear factor{kappa}B (NF{kappa}B) signaling to both the interferon regulatory factor-1 promoter and to the thymidine kinase promoter containing multimerized NF{kappa}B elements (NF{kappa}B-TK). Further, PRL-inducible Stat5b inhibits tumor necrosis factor-{alpha} signaling presumably by inhibiting endogenous NF{kappa}B. This Stat5b-mediated inhibitory effect on NF{kappa}B signaling is independent of Stat5b-DNA interactions but requires the carboxyl terminus of Stat5b as well as Stat5b nuclear translocation and/or accumulation, suggesting that Stat5b is competing for a nuclear factor(s) necessary for NF{kappa}B-mediated activation of target promoters. Increasing concentrations of the coactivator p300/CBP reverses Stat5b inhibition at both the interferon-regulatory factor-1 and NF{kappa}B-TK promoters, suggesting that Stat5b may be squelching limiting coactivators via protein-protein interactions as one mechanism of promoter inhibition. These results further substantiate our observation that Stat factors can function as transcriptional inhibitors. Our studies reveal cross-talk between the Stat5b and NF{kappa}B signal transduction pathways and suggest that Stat5b-mediated inhibition of target promoters occurs at the level of protein-protein interactions and involves competition for limiting coactivators.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cellular responses to activation by multiple cytokines depend on the expression of specific cytokine receptors, the complement of signaling molecules, and the stage of differentiation of the responding cells. Cytokines elicit numerous biological responses by activating a variety of latent transcription factors, including signal transducers and activators of transcription (Stats) and nuclear factor of kappa B (NF{kappa}B). Although their activation pathways are different, activated Stats and NF{kappa}B translocate into the nucleus and function either individually or cooperatively in regulating the expression of target genes (1 2 3 ). Interactions among various cytoplasmic as well as nuclear factors, the promoter context of the target gene, and the presence of coactivator complexes determine the expression of the target gene and hence the biological outcome of cytokine actions.

PRL, a pituitary peptide hormone as well as a T cell cytokine, plays a modulatory role in various aspects of the immune response (4 ). The immunomodulatory effect is mediated by the binding of PRL to the PRL-receptor (PRL-R), which is a member of the hematopoietin/cytokine receptor superfamily. The interaction of PRL and PRL-R leads to the activation of the protein tyrosine kinase JAK2 and a number of Stat factors (5 ). Stat1, Stat3, Stat5a, and Stat5b have been shown to be rapidly tyrosine phosphorylated in response to PRL stimulation (6 7 8 ). Activated Stats translocate into the nucleus and bind to the interferon {gamma} activation sequence (GAS) in the promoter region of target genes and regulate the transcription of these genes. Our previous studies have shown that PRL stimulates the transcription of the immediate early gene interferon regulatory factor 1 (IRF-1) in Nb2 T cells (6 9 ). Promoter analysis has shown that the -200 bp promoter proximal region is responsible for mediating PRL induction of the IRF-1 gene (6 ). Further, a GAS element at -112 bp has been shown to function as a PRL-responsive enhancer element in the IRF-1 promoter. Stat1 has been shown to bind to the IRF-1 GAS in a PRL-inducible manner and positively mediates PRL activation of the IRF-1 promoter (10 ). Stat5 has also been demonstrated to bind the IRF-1 GAS in a PRL-inducible manner (11 ). Unexpectedly, Stat5 inhibits PRL induction of the IRF-1 promoter. The inhibitory effect does not require Stat5 to bind DNA, suggesting that the inhibition may be mediated via protein/protein interaction by competing for factor(s) that is necessary for PRL induction of the IRF-1 promoter (11 12 ).

In addition to the GAS element, a number of other recognition sites for DNA-binding proteins, such as NF{kappa}B, are present in the IRF-1 promoter (13 ). Recent studies have shown that IFN{gamma}-activated Stat1 and tumor necrosis factor-{alpha} (TNF{alpha})-activated NF{kappa}B function synergistically at the IRF-1 promoter to induce IRF-1 expression (1 2 ). These studies suggest that IRF-1 gene expression can be cooperatively regulated by both the JAK/Stat signaling pathway and NF{kappa}B signaling pathway. In this report, we examined the negative cross-talk between Stat5b and NF{kappa}B signaling at the IRF-1 promoter. We show that Stat5b inhibits p50/p65 NF{kappa}B signaling to the IRF-1 promoter as well as to the heterologous thymidine kinase (TK) promoter containing multimerized NF{kappa}B elements. Stat5b-mediated inhibitory action requires the presence of the carboxyl terminus and the nuclear translocation of Stat5b, but does not require Stat5b to bind to the GAS element. Stat5b inhibition appears to be mediated by protein/protein interactions, in particular, by squelching of limiting amounts of the p300/CBP coactivators, as one mechanism of Stat5b transcriptional inhibition at target promoters. These results suggest that Stat5b inhibits NF{kappa}B signaling by competing for coactivators necessary for NF{kappa}B-mediated gene transcription.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Stat5b Inhibits NF{kappa}B Induction of the IRF-1 Promoter
Our previous studies have shown that Stat5b inhibits PRL induction of the IRF-1 promoter in transient transfection assays (11 ). This inhibitory effect does not require Stat5b to bind to DNA, suggesting that Stat5b competes for factor(s) necessary for PRL induction of the IRF-1 promoter (11 ). To determine what factor(s) Stat5b may be competing for, we first examined other transcription factors that have potential binding sites at the IRF-1 promoter. One such factor is NF{kappa}B, which has a binding site (-35 to -45 bp) 3' to the PRL-responsive GAS element (-112 bp) in the IRF-1 promoter (Fig. 1Go). This NF{kappa}B element has been shown to mediate TNF{alpha} induction of the IRF-1 promoter in HepG2 and NIH3T3 cells (1 2 ). The TNF{alpha}-mediated induction of the IRF-1 promoter is further synergized by INF{gamma} via activation of Stat1 binding to the GAS element. To determine whether Stat5b may affect NF{kappa}B signaling to the IRF-1 promoter, COS cells were cotransfected with the PRL-R, Stat5b, p50 NF{kappa}B, p65 NF{kappa}B, and the 1.7-kb IRF-1-chloramphenicol acetyltransferase (CAT) constructs. In this reconstituted COS cell transfection system, PRL stimulates a 2- to 3-fold induction of the IRF-1 promoter, which is inhibited by Stat5b (Fig. 1Go) as shown previously (11 ). Cotransfection of p50/65 NF{kappa}B led to a 5- to 6-fold induction of the IRF-1 promoter in the absence of PRL stimulation (Fig. 1Go). PRL stimulation further enhanced IRF-1 promoter activity above that induced by NF{kappa}B, presumably due to the activation of endogenous Stat1 (10 ). In the absence of the PRL stimulation, overexpression of Stat5b did not affect NF{kappa}B-mediated induction of the IRF-1 promoter. In contrast, upon PRL stimulation, Stat5b strongly inhibited both PRL and NF{kappa}B-mediated IRF-1 promoter activity in a dose-dependent manner (Fig. 1Go). These studies show that NF{kappa}B can activate the IRF-1 promoter and this induction is inhibited by Stat5b in a dose-dependent as well as PRL-dependent manner.



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Figure 1. Stat5b Inhibits NF{kappa}B Signaling to the IRF-1 Promoter

COS cells were transiently cotransfected with 1 µg of the Nb2 PRL-R, 0.1 µg of the 1.7-kb IRF-1-CAT, each of p50/p65 NF{kappa}B and Stat5b as indicated, stimulated with 100 ng/ml of PRL for 24 h, and assayed for CAT expression as described in Materials and Methods. The data are summarized from multiple independent experiments (n = 5).

 
The IRF-1 GAS Element Does Not Mediate Stat5b Inhibition of NF{kappa}B Signaling to the IRF-1 Promoter
Next, we determined whether Stat5b inhibition of NF{kappa}B signaling requires the presence of a functional GAS element at the IRF-1 promoter. An intact IRF-1 GAS element is critical for PRL induction of the IRF-1 promoter (Fig. 2Go), as site-directed mutations in the GAS element abolished PRL activation, as was shown previously (10 ). However, mutation of this GAS element did not affect p50/p65 NF{kappa}B-mediated activation of the IRF-1 promoter, confirming that NF{kappa}B activates the IRF-1 promoter independently of a functional GAS element. This NF{kappa}B-mediated signaling to the mutant GAS IRF-1 promoter was still inhibited by Stat5b in a PRL-dependent manner. These results clearly show that Stat5b inhibition of NF{kappa}B signaling to the IRF-1 promoter does not require Stat5b to interact with the GAS element.



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Figure 2. Stat5b Inhibition of NF{kappa}B Signaling Is Independent of the GAS Element

COS cells were transiently cotransfected as described in Fig. 1Go, except that the 1.7-kb mutant GAS IRF-1-CAT reporter was used (n = 3).

 
Stat5b Inhibits NF{kappa}B Activation of the Heterologous NF{kappa}B-TK Promoter
To further determine whether Stat5b-mediated inhibitory effects can be demonstrated at a promoter that only contains NF{kappa}B elements, the heterologous TK promoter containing two copies of the NF{kappa}B element was examined (14 ). As expected, the NF{kappa}B-TK promoter was not responsive to PRL stimulation, as it does not have a GAS element (Fig. 3Go). p50/p65 NF{kappa}B mediated greater than 10-fold induction of the NF{kappa}B-TK promoter, which again is not further inducible by PRL stimulation. Interestingly, Stat5b inhibited the NF{kappa}B-mediated activation of this heterologous TK promoter, in a PRL- as well as DNA dose-dependent manner (Fig. 3Go). These results demonstrate that Stat5b can inhibit NF{kappa}B signaling to promoters that do not contain a GAS element. These results further support our hypothesis that Stat5b-mediated inhibition of NF{kappa}B signaling is not mediated by Stat-DNA interactions but is mediated by protein-protein interactions.



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Figure 3. Stat5b Inhibits NF{kappa}B Signaling to the NF{kappa}B-TK Promoter

COS cells were transiently cotransfected as described in Fig. 1Go, except that the NF{kappa}B-TK-CAT reporter was used (n = 4).

 
Stat5b-Mediated Inhibition Requires Its Nuclear Localization
To further understand how Stat5b inhibits NF{kappa}B-mediated signaling to both the IRF-1 and NF{kappa}B TK promoters, a Stat5b DNA binding mutant, Stat5b VVVI (11 ), was tested for its ability to inhibit NF{kappa}B signaling to either promoter. Previous studies have shown that the Stat5b VVVI mutant strongly inhibits PRL signaling to the IRF-1 promoter (11 ). First, immunofluorescence microscopy was used to examine the intracellular localization of Stat5b and Stat5b VVVI mutant after PRL stimulation in transfected COS cells. Using deconvolution confocal microscopy, wild-type Stat5b immunofluorescence staining (red) was detected throughout the transfected COS cell in the absence of PRL stimulation (Fig. 4AGo). Others have also observed the general staining of inactive Stats in the nucleus of transfected cells (15 16 ). However, upon PRL stimulation, Stat5b exhibited a punctate staining pattern in the nucleus, indicating PRL-inducible nuclear translocation of Stat5b (Fig. 4BGo). Similarly, in unstimulated cells, Stat5b VVVI mutant also showed general staining throughout the cytoplasm with a low level of staining detected in the nucleus (Fig. 4CGo). In contrast, with PRL stimulation, the Stat5b VVVI mutant remained primarily in the cytoplasm, often with increased staining around the perinuclear region (Fig. 4DGo). Although a low level of Stat5b VVVI staining was found in the nucleus, the general staining pattern of the Stat5b VVVI mutant is consistently distinct from the clear punctate nuclear staining of wild-type Stat5b in response to PRL stimulation (compare Fig. 4DGo with Fig. 4BGo). These results suggest that Stat5b VVVI mutant does not translocate into or accumulate effectively in the nucleus upon PRL stimulation.



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Figure 4. Stat5b Inhibition of NF{kappa}B Signaling Requires Its Nuclear Translocation

COS cells were transiently cotransfected with the Nb2 PRL-R and either wild-type Stat5b or Stat5b VVVI mutant, stimulated without (panels A and C) or with 100 ng/ml of PRL for 30 min (panels B and D), fixed and immunostained as described in Materials and Methods. Images were obtained using DeltaVision Deconvolution Confocal Microscopy. Blue, DAPI-stained DNA; red, immunofluorescence staining of Stat5b. E, Stat5b VVVI lost its ability to inhibit NF{kappa}B signaling to the IRF-1 promoter. Stat5b VVVI mutant was transiently cotransfected into COS cells as described in Fig. 1Go (n = 3). The bars depicting –PRL vs. +PRL treatment (see asterisk) in Stat5b VVVI cotransfected cells are not statistically significant (by Student’s t test). F, Stat5b VVVI lost its ability to inhibit NF{kappa}B signaling to the NF{kappa}B-TK promoter. Stat5b VVVI mutant was transiently cotransfected into COS cells as described in Fig. 3Go (n = 3).

 
The defect in nuclear accumulation of Stat5b VVVI mutant is correlated with the inability of this mutant to inhibit NF{kappa}B signaling to either the IRF-1 (Fig. 4EGo) or NF{kappa}B-TK (Fig. 4FGo) promoter. Thus, wild-type Stat5b still inhibits NF{kappa}B signaling to both the IRF-1 and NF{kappa}B TK promoters (Fig. 4Go, E and F), but the Stat5b VVVI mutant fails to inhibit NF{kappa}B signaling to these promoters. Note that the Stat5b VVVI mutant was still capable of inhibiting that portion of IRF-1 promoter activity that is inducible by PRL, but not by NF{kappa}B (open and solid bars are not significantly different in the Stat5b VVVI transfected cells in Fig. 4EGo). These results show that Stat5b-mediated inhibition of NF{kappa}B signaling requires its nuclear translocation. Further, these results imply that Stat5b-mediated inhibition of PRL-inducible Stat1 signaling can occur at both the nuclear as well as extranuclear levels.

Stat5b Inhibits TNF{alpha} Signaling
We next examined whether Stat5b can inhibit endogenous NF{kappa}B activity as stimulated via TNF{alpha}, in the absence of p50/p65 NF{kappa}B overexpression. COS cells were cotransfected with the PRL-R, Stat5b, or two Stat5b mutants, Stat5b{Delta}40C or Stat5b VVVI, and either 1.7-kb IRF-1-CAT or NF{kappa}B TK-CAT constructs. PRL or TNF{alpha} individually stimulated 3- and 2-fold induction, respectively, of IRF-1 promoter activity (Fig. 5AGo). TNF{alpha} plus PRL together stimulated 5- to 6-fold induction of the IRF-1 promoter in vector-transfected control cells (Fig. 5AGo), presumably due to the activation of endogenous NF{kappa}B and endogenous Stat1, respectively. In the presence of Stat5b, PRL stimulation resulted in a reduction in TNF{alpha}-inducible IRF-1 promoter activity. These results show that PRL-inducible Stat5b can inhibit TNF{alpha}-inducible NF{kappa}B signaling to the IRF-1 promoter. Furthermore, PRL-inducible Stat5b also inhibited the large 7- to 8-fold TNF{alpha}-inducible NF{kappa}B signaling to the heterologous NF{kappa}B-TK promoter (Fig. 5BGo).



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Figure 5. Stat5b Inhibits TNF{alpha} Signaling

COS cells were transiently cotransfected with the Nb2 PRL-R, Stat5b, Stat5b{Delta}40C, or Stat5b VVVI mutant, and either the 1.7-kb IRF-1-CAT (panel A, n = 3) or the NF{kappa}B-TK-CAT (panel B, n = 3) as described in Fig. 1Go, except that between 0.2 and 0.3 µg of the various Stat5b constructs were employed. Cells were stimulated with or without 100 ng/ml PRL, 15 ng/ml human TNF{alpha}, or PRL plus TNF{alpha} for 24 h.

 
Previous studies have shown that the Stat5b{Delta}40C mutant, which lacks the carboxyl-terminal 40 amino acids, fails to inhibit Stat1-mediated PRL signaling to the IRF-1 promoter (11 ). The Stat5b{Delta}40C mutant also failed to inhibit TNF{alpha} signaling to both the IRF-1 (Fig. 5AGo) and NF{kappa}B-TK promoters (Fig. 5BGo), suggesting that the carboxyl terminus of Stat5b is involved in inhibition at these promoters. On the other hand, the Stat5b VVVI mutant, which does not accumulate significantly in the nucleus, clearly inhibited PRL signaling to the IRF-1 promoter (Fig. 5AGo), but failed to inhibit TNF{alpha} signaling to either the IRF-1 (Fig. 5AGo) or NF{kappa}B-TK (Fig. 5BGo) promoter. In agreement with the data on overexpression of p50/p65 NF{kappa}B (Fig. 4Go), these results suggest that Stat5b inhibits TNF{alpha} signaling by inhibiting endogenous NF{kappa}B, and that this inhibition involves the carboxyl terminus of Stat5b and requires Stat5b to be translocated into the nucleus.

Exogenous p300/CBP Coactivators Reverse Stat5b-Mediated Inhibition at Target Promoters
Recent studies have shown that the activities of Stat factors can be modulated by their interactions with other DNA-binding proteins and non-DNA-binding proteins such as coactivators. p300/CBP has been shown to interact with Stat1 (17 18 19 ), Stat2 (20 ), and Stat5a (21 ) as well as with NF{kappa}B (22 23 24 ) to enhance target gene expression. For example, IFN{alpha}-inducible Stat2 appears to compete with TNF{alpha}-inducible NF{kappa}B for the coactivator p300 as one mechanism for competitive transcriptional regulation of a target gene (24 ). We, therefore, examined whether p300/CBP, which interacts with both Stats and NF{kappa}B, might be a target of Stat5b inhibition. First, COS cells were cotransfected with the PRL-R, p300, and the IRF-1-CAT (Fig. 6AGo). Exogenous p300 further enhanced PRL signaling, suggesting that p300 is limiting in the COS transfection system, and that p300 enhances PRL-inducible Stat1-mediating signaling to the IRF-1 promoter. To determine whether increased expression of p300 would reverse Stat5b inhibition, increasing concentrations of p300 were cotransfected with Stat5b and either the 1.7-kb IRF-1 (Fig. 6BGo) or NF{kappa}B-TK (Fig. 6CGo) promoter. p300 did not affect TNF{alpha} signaling to either the IRF-1 or NF{kappa}B-TK promoter. However, p300 reversed Stat5b-mediated inhibition at the IRF-1 promoter in a dose-dependent manner, not only in response to PRL but also to PRL plus TNF{alpha} stimulation (Fig. 6BGo). Similarly, p300 also reversed Stat5b-mediated inhibition at the NF{kappa}B-TK promoter in a dose-dependent manner (Fig. 6CGo). These results support our interpretation that Stat5b-mediated inhibition most likely occurs via a mechanism in which Stat5b competitively squelches limiting amounts of the coactivator p300/CBP, and thereby functionally antagonizes Stat1- and NF{kappa}B-mediated signaling to target promoters.



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Figure 6. p300/CBP Reverses Stat5b-Mediated Promoter Inhibition

COS cells were transiently cotransfected with the Nb2 PRL-R, 1.0 µg p300, and the 1.7-kb IRF-1-CAT (A, representative of n = 3 experiments) construct as described in Fig. 1Go, and stimulated with 100 ng/ml PRL for 24 h. In panels B and C, increasing concentrations of p300 were transfected as in panel A, except that the 1.7-kb IRF-1-CAT (n = 3) was used in panel B, and the NF{kappa}B-TK-CAT (n = 1) was used in panel C. Cells were stimulated with or without either 100 ng/ml PRL, 15 ng/ml human TNF{alpha}, or PRL plus TNF{alpha} for 24 h.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Stats are a family of latent transcription factors that participate in cytokine signaling by controlling early gene transcription. When activated, Stat factors can function to not only stimulate but also inhibit gene transcription (11 ). Recent studies suggest that the activity of Stat factors is modulated by their interaction with other transcription factors (25 ) and/or coactivators (26 ). These protein-protein interactions lead to synergistic (25 ) or antagonistic (11 24 25 ) effects on gene transcription. Our previous observations show that Stat5b inhibits PRL signaling to the IRF-1 promoter (11 ). Our present studies further show that PRL-inducible Stat5b inhibits NF{kappa}B signaling to the IRF-1 promoter and to a TK promoter containing only NF{kappa}B elements. Our studies illustrate that one mechanism by which Stat5b inhibits transcriptional responses is by sequestering limiting coactivators at target promoters.

Activation of NF{kappa}B, either by overexpression or by TNF{alpha} stimulation, can induce IRF-1 promoter activity (Figs. 1Go, 2Go, 4EGo, and 5AGo). PRL stimulation further enhances NF{kappa}B-mediated induction of the IRF-1 promoter, presumably due to PRL activation of endogenous Stat1 in COS cells (Figs. 1Go, 2Go, 4EGo, and 5AGo). These results agree with recent observations that Stat1 and NF{kappa}B synergistically activate the IRF-1 promoter, via the GAS and NF{kappa}B elements, respectively, in response to IFN{gamma} and TNF{alpha} stimulation (1 2 ). Interestingly, both Stat1- and NF{kappa}B-mediated induction of the IRF-1 promoter are inhibited by Stat5b (Figs. 1Go, 2Go, 4EGo, and 5AGo). This Stat5b-mediated inhibition is dependent upon PRL stimulation, as this leads to Stat5b tyrosine phosphorylation, dimerization, and nuclear translocation. This inhibitory activity of Stat5b does not require Stat5b to interact with DNA (Figs. 2Go and 3Go) but does require a functional carboxyl terminus (Fig. 5Go), presumably so that Stat5b can engage in protein-protein interaction with a factor that is required for both Stat1- and NF{kappa}B-mediated promoter activation. This is corroborated by a Stat5b DNA-binding mutant, Stat5b VVVI, which is as effective as wild-type Stat5b in inhibiting PRL signaling to the IRF-1 promoter (Figs. 4Go and 5Go), by Stat5b inhibition of a mutant IRF-1 promoter that lacks a functional GAS element (Fig. 2Go), and by Stat5b inhibition of the TK promoter that contains only NF{kappa}B elements (Fig. 3Go). Together, these results strongly support our interpretation that Stat5b inhibition is mediated by protein-protein interactions.

What proteins might be the target of Stat5b inhibition? Stat1 does not directly interact with Stat5 as determined by biacore competition assays, thus ruling out direct complex formation between Stat1 and Stat5 (27 ). Although other Stat proteins have been found to directly interact with NF{kappa}B (3 ), Stat5b does not appear to interact directly with NF{kappa}B as assessed by gel shift assays, glutathione-S-transferase interaction assays, and coimmunoprecipitation experiments (data not shown) (28 ). Thus, neither Stat1 nor NFkB are direct targets of Stat5b inhibition at the IRF-1 promoter. Our studies show that the coactivator p300 can functionally reverse Stat5b inhibition at the IRF-1 and NF{kappa}B-TK promoters (Fig. 6Go), suggesting that the coactivator p300 is one target of Stat5b inhibition at these promoters. Multiple contacts sites between Stat1 and p300/CBP (17 ) and between p65 NF{kappa}B and CBP (22 23 ) have been described. Stat5a and Stat1 interact with an overlapping site in p300, which also interacts with p65 NF{kappa}B (21 ), suggesting that competition for p300 binding might form a basis of their functional antagonism at the IRF-1 promoter. How the Stats and NF{kappa}B interact with p300, which domains of these proteins are involved, and how their transcriptional activities are either enhanced or diminished by interactions with p300 are currently under analysis.

The carboxyl terminus of Stat5a is critical for interaction with p300 to up-regulate the PRL-responsive ß-casein promoter (21 ). Our studies show that the carboxyl terminus of the highly related Stat5b is critical in mediating inhibition of the IRF-1 promoter, presumably by competing with Stat1 for binding to p300. How can these Stat5/p300 interactions be stimulatory at the ß-casein promoter but inhibitory at the IRF-1 promoter? One major difference is that Stat5b needs to bind to the ß-casein GAS element for transcriptional induction (11 21 ) while Stat5b does not need to interact with the IRF-1 GAS element for transcriptional repression (Figs. 2Go, 4Go, and 5Go and Ref. 11 ). How the coactivator p300/CBP integrates the activities of Stats and other promoter-specific DNA-binding proteins may be distinct and may contribute to differences in the transcriptional activities of the Stat5/p300 complex at the two promoters (26 29 ). Furthermore, recent studies show that distinct coactivator complexes are recruited by Stat (17 20 26 ) and by NF{kappa}B (30 31 ) to regulate gene transcription. It is possible that in addition to p300/CBP, other coactivators such as SRC-3 (29 ) and SRC-1 (30 31 ) may also be involved in PRL-inducible Stat1 or TNF{alpha}-inducible NF{kappa}B signaling, respectively, to the IRF-1 promoter. In this regard, it would be interesting to determine whether SRC-1 in combination with p300 will fully reverse Stat5b inhibition of NF{kappa}B signaling to the NF{kappa}B-TK promoter (Fig. 6CGo). These observations support our findings that Stat5b inhibition of Stat1 or NF{kappa}B signaling is not mediated by Stat-DNA interactions but is mediated by Stat/p300/CBP interactions at the target promoters.

Immunofluorescence studies show that Stat5b is translocated into the nucleus upon PRL stimulation (Fig. 4BGo). In contrast, Stat5b VVVI mutant remains primarily cytoplasmic and does not accumulate significantly in the nucleus even after PRL stimulation (Fig. 4DGo). The lack of nuclear accumulation of the Stat5b VVVI mutant explains in part the inability of Stat5b VVVI mutant to inhibit NF{kappa}B signaling to either the IRF-1 promoter (Fig. 4EGo) or the NF{kappa}B TK promoter (Fig. 4FGo). Yet, the Stat5b VVVI mutant is still capable of inhibiting Stat1-mediated IRF-1 promoter activity (Figs. 4EGo and 5AGo). Our hypothesis is that Stat5b inhibition of NF{kappa}B signaling requires its nuclear localization. However, Stat5b inhibition of Stat1 signaling may occur not only at the transcriptional level, as is the case for wild-type Stat5b, but also at an extranuclear level for the Stat5b VVVI mutant. A potential mechanism may be that the Stat5b VVVI mutant competes for a cytoplasmic factor that can modulate gene transcription in the nucleus. Two such factors have recently been described. N-myc interacting protein, Nmi (33 ), has been shown to interact with Stat1 and Stat5, and Nmi-Stat interaction was shown to stabilize the Stat-CBP complex and to enhance Stat-mediated gene transcription. The growth factor and cytokine receptor adaptor protein, CrkL, has been shown to interact with Stat5 and participate in Stat5 binding to DNA (34 35 ). Whether these factors participate in PRL signaling to the IRF-1 promoter, and whether they are targets of Stat5b and/or Stat5b VVVI inhibition are currently under investigation.

Alternatively, Stat5b VVVI mutant may inhibit Stat1 nuclear translocation and thereby impede Stat1 function at the transcriptional level. This interpretation is consistent with the observation that Stat5b VVVI mutant accumulates in the cytoplasm and the perinuclear region in PRL-stimulated cells and may suggest problems in its transport across the nuclear membrane (36 37 ). Studies to examine Stat1 nuclear translocation in the presence of wild-type and Stat5b VVVI mutant will test this hypothesis. Previous studies have also demonstrated that the Stat5b VVVI mutant remained cytoplasmic even after 1 h of GH stimulation of several different cell types (38 ) and is not specific to transfected COS cells.

Together, these studies reveal novel features of Stat regulation of gene expression. First, Stats can act to repress gene transcription. Recent studies of the Stat5a/Stat5b double knockout animals suggest that Stat5 may act as a transcriptional repressor in vivo (32 ). Second, signaling pathways that activate Stat factors can inhibit signaling pathways that activate NF{kappa}B, depending on the stimuli and Stat factors involved. For example, Stat1 synergizes while Stat5b inhibits NF{kappa}B signaling to the IRF-1 promoter (this work), and Stat2 inhibits NF{kappa}B signaling to the HIV LTR (24 ). Third, Stat regulation of gene expression may occur not only at the level of gene transcription (39 ) but also at an extranuclear level, perhaps involving nuclear transport. This hypothesis is consistent with the observation that Stat factors reside primarily in the cytoplasm and are activated to enter the nucleus for a limited time after which they appear to recycle back into the cytoplasm by the importin {alpha} transport pathway (37 ). Studies are underway to examine the kinetics of nuclear translocation of Stat5b, Stat1, and NF{kappa}B in response to PRL and TNF{alpha} stimulation. The negative cross-talk between Stat5b and NF{kappa}B may elucidate how lactogenic hormones can inhibit TNF{alpha} signaling and provide protection against septic shock (40 ) and hemorrhagic shock (41 ). These results may also elucidate more general mechanisms involving competing cytokine regulation of target genes via the activation of competing Stat factors as occurs during a Th1 vs. Th2 immune response (42 ).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DNA Constructs
The Nb2 PRL-R (pECE), Stat5b, Stat5b{Delta}40C, and Stat5bVVVI mutants [pcDNA3.1(-)], wild-type 1.7-kb IRF-1-CAT, and GAS mutant 1.7-kb IRF-1-CAT constructs were described previously (10 11 43 ). Briefly, Stat5b{Delta}40C is missing the most carboxyl 40 amino acids, which contain the transactivation domain (gift of Dr. Georg H. Fey, Friedrich-Alexander University, Erlangen, Germany) (44 ). Stat5b VVVI was generated by site-directed mutagenesis, in which alanines replaced the highly conserved VVVI residues in the DNA-binding domain (11 ). pCMVp65 and pCMVp50 NF{kappa}B plasmids were provided by Dr. Tse-Hua Tan (45 ). p6TKCAT plasmid containing two copies of the NF{kappa}B elements was provided by Dr. Paula M. Pitha (14 ).

Transient Transfection and CAT Assays
COS-1 cells (2 x 105/well) were seeded in six-well tissue culture plates overnight in DMEM containing 10% FBS (JRH Biosciences, Lenexa, KS) (11 ). Transient transfections were performed using LipofectAMINE (Life Technologies, Inc., Inc., Gaithersburg, MD) as described previously (11 ). Plasmid DNA concentrations used for transfection are: 1 µg of Nb2 PRL-R, between 0.05 µg and 0.5 µg of Stat5b, Stat5b{Delta}40C, or Stat5b VVVI, 0.1 µg each of p50 NF{kappa}B and p65 NF{kappa}B, and 0.1 µg of the various CAT reporter constructs. Empty vectors (pcDNA3.1 for Stat5 or pCMV for NF{kappa}B) were used as controls as well as for adjusting total DNA concentration in dose-response experiments. After transfection, cells were maintained for 24 h in 3 ml of DMEM with 1% horse serum (ICN-Flow Laboratories, MacLean, VA) and were stimulated with either 100 ng/ml ovine PRL (NIDDK-oPRL-20) or 15 ng/ml human TNF{alpha} (1 x 107 U/mg) (R&D Systems Inc., Minneapolis, MN) for 24 h. Cells were lysed in 600 µl/well of reporter lysis buffer (Promega Corp., Madison, WI), and 40 µl of cell extracts, 5 µl of 5 mg/ml n-butyryl-coenzyme A (Promega Corp.), and 3 µl of [14C]chloramphenicol (50 mCi/mmol, NEN Life Science Products, Boston, MA) were assayed for 4 h at 37 C as described previously (11 ). CAT activity was analyzed by liquid scintillation counting and normalized to counts per µg of protein assayed. Each experiment was set up in triplicate. Error bars represent SEM derived from three to five independent experiments. Data were plotted by using Origin 4.0 (Microcal Software, Inc., Northampton, MA).

Immunofluorescence
COS cells were cultured on glass coverslips coated with poly-D-lysine (1 mg/ml, 70,000–150,000 Da, Sigma, St. Louis, MO). Transient transfections were performed by the calcium phosphate precipitation method using a mammalian cell transfection kit (Specialty Media Inc., Lavallette, NJ). PRL-R (2 µg) was cotransfected with either 2 µg of Stat5b or Stat5b VVVI. After transfection, cells were maintained in DMEM with 1% horse serum for 24 h before stimulation with 100 ng/ml of PRL for 30 min. The cells were then rinsed twice with ice-cold PBS and fixed with 4% paraformaldehyde (Polysciences Inc., Warrington, PA) in PEM buffer (80 mM PIPES, pH 6.9, 1 mM EGTA, 1 mM MgCl2) for 30 min, followed by permeabilization with 0.5% Triton X-100 in the same buffer for 20 min. The cells were blocked in 5% milk in TBS-T (20 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20) containing 0.2% sodium azide overnight at 4 C, followed by incubation first with affinity-purified anti-Stat5b antibodies (11 ) at 1:500 dilution for 1 h at room temperature, and then with goat antirabbit IgG conjugated with Texas Red (Molecular Probes, Inc., Eugene, OR) at 1:1000 dilution for 30 min. The cells were then washed five times with TBS-T and stained by 4,6-diamidino-2-phenylindole using VECTASHIELD mounting media (Vector Laboratories, Inc.,, Burlingame, CA) on glass slides. Images were obtained using DeltaVision Deconvolution Confocal Microscopy (Integrated Microscopy Core, Baylor College of Medicine, Houston, TX).


    ACKNOWLEDGMENTS
 
We thank Dr. Tse-Hua Tan for the NF{kappa}B constructs, Dr. Paula Pitha for the 6tk-CAT constructs, Dr. Elena Kabotyanski for assistance with confocal microscopy, and Dr. Sophia Tsai and Dr. Jeff Rosen for critical comments.


    FOOTNOTES
 
Address requests for reprints to: Li-yuan Yu-Lee, Department of Medicine, Baylor College of Medicine, Houston, Texas 77303.

This work was supported by a Molecular Endocrinology Training Grant T32-K07696 (G. L.) and by a grant from the NIH RO1-DK-44625 (L.-y. Y.-L.).

Received for publication March 3, 1999. Revision received September 15, 1999. Accepted for publication September 20, 1999.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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