Transcriptional Regulation of the ß-Casein Gene by Cytokines: Cross-Talk between STAT5 and Other Signaling Molecules

Dai Chida, Hiroshi Wakao, Akihiko Yoshimura and Atsushi Miyajima

Institute of Molecular and Cellular Biosciences (D.C., A.M.) The University of Tokyo Tokyo 113-0032, Japan.
The Helix Research Institute (H.W.) Chiba 292-0812, Japan
Institute of Life Science (A.Y.) Kurume University Kurume 839-0861, Japan


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The ß-casein promoter has been widely used to monitor the activation of STAT (signal transducer and activator of transcription)5 since STAT5 was originally found as a mediator of PRL-inducible ß-casein expression. However, not only is expression of the ß-casein gene regulated by STAT5 but it is also affected by other molecules such as glucocorticoid and Ras. In this report, we describe the transcriptional regulation of the ß-casein gene by cytokines in T cells. We have found that the ß-casein gene is expressed in a cytotoxic T cell line, CTLL-2, in response to interleukin-2 (IL-2), which activates STAT5. While IL-4 does not activate STAT5, it induces expression of STAT5-regulated genes in CTLL-2, i.e. ß-casein, a cytokine-inducible SH2-containing protein (CIS), and oncostatin M (OSM), suggesting that STAT6 activated by IL-4 substitutes for the function of STAT5 in T cells. IL-2-induced ß-casein expression was enhanced by dexamethasone, and this synergistic effect of Dexamethasone requires the sequence between -155 and -193 in the ß-casein promoter. Coincidentally, a deletion of this region enhanced the IL-2-induced expression of ß-casein. Expression of an active form of Ras, Ras(G12V), suppressed the IL-2-induced ß-casein and OSM gene expression, and the negative effect of Ras is mediated by the region between -105 and -193 in the ß-casein promoter. In apparent contradiction, expression of a dominant negative form of Ras, RasN17, also inhibited IL-2-induced activation of the promoter containing the minimal ß-casein STAT5 element as well as the promoters of CIS and OSM. In addition, Ras(G12V) complemented signaling by an erythropoietin receptor mutant defective in Ras activation and augmented the activation of the ß-casein promoter by the mutant erythropoietin receptor signaling, suggesting a possible role of Ras in Stat5-mediated gene expression. These results collectively reveal a complex interaction of STAT5 with other signaling pathways and illustrate that regulation of gene expression requires integration of opposing signals.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The class I cytokine receptors are a large family of cell surface receptors devoid of any intrinsic kinase activity that includes the receptors for various interleukins (ILs), colony stimulating factors (CSF), erythropoietin (EPO), thrombopoietin (TPO), GH, and PRL. Binding of a cytokine to its cognate receptor induces the activation of a JAK tyrosine kinase that is constitutively associated with the receptor. The activated JAK phosphorylates the receptor at tyrosine residues, and signaling molecules with an SH2 domain are recruited to the phosphorylated receptors. Such signaling molecules include: tyrosine phosphatases, SHP-1 and SHP-2, PI-3 kinase, Vav, STATs, Src family tyrosine kinases, and adapters such as SHC and GRB2 that lead to activation of Ras (1).

STATs (signal transducers and activators of transcription) are SH-2-containing transcription factors that play a major role in the cytokine signaling. Currently, STAT1–STAT6 have been molecularly cloned (2). STAT1 and STAT2 were identified as mediators of interferon signaling. STAT3 was found as a transcription factor for IL-6-induced acute phase protein production and is activated by all the members of IL-6 family cytokines as well as growth factors such as epidermal growth factor (EGF). STAT4 and STAT6 are activated exclusively by IL-12 and IL-4, respectively. STAT5 was originally identified as a transcription factor that is activated by PRL in the mammary gland, but it is now known to be activated by a number of cytokines including GH, IL-2, IL-3, IL-5, granulocyte macrophage (GM)-CSF, EPO, TPO, and EGF (2). Activation of STATs is initiated by phosphorylation of a C-terminal tyrosine that then interacts with the SH2 domain of another STAT molecule to form a dimer. The dimerized STATs translocate to the nucleus and bind to specific regulatory sites in the promoters of target genes. There are multiple steps that regulate the activation of STATs in addition to tyrosine phosphorylation. For example, phosphorylation of a serine residue by mitogen-activated protein kinase (MAPK) was shown to be important for the optimal activation of STAT1 and STAT3 (3, 4). CBP300, a coactivator, has also been implicated in STAT1 function (5). Finally, tyrosine phosphatases may also be involved in the regulation of the STAT activation (6).

Since a cytokine simultaneously activates various intracellular signaling pathways, interactions may occur between different signaling pathways activated by the same receptor. By using various receptor mutants, we previously established that the GM-CSF receptor activates Ras as well as STAT5 (7, 8). While Ras activation is absolutely required for the induction of c-fos, expression of a dominant negative form of STAT5 suppressed the cytokine-inducible expression of c-fos (9). As there are a serum-responsive element (SRE) as well as a potential STAT5 binding site, serum- inducible element (SIE), in the c-fos promoter, the optimum induction of c-fos appears to be induced by a combination of Ras and STAT5 (9). In this paper we describe another mechanism of interaction between STAT5 and Ras pathways in regulation of ß-casein gene expression.

Production of milk proteins in the mammary gland is regulated by the lactogenic hormones such as insulin, glucocorticoids, and PRL (10). Extensive studies have defined multiple cis-acting elements involved in the regulation of milk protein production and identified transcription factors that bind to these elements (11, 12, 13, 14, 15, 16, 17). Among them, the DNA-binding activity of the mammary gland factor MGF (now known as STAT5) is developmentally regulated and plays an essential role in ß-casein expression (11, 18). While STAT5 is activated in various cells by a number of cytokines as listed above, expression of ß-casein was found only in the mammary gland and cytotoxic T cells (CTLs) (19). As the ß-casein promoter was used to identify STAT5, the ß-casein minimum promoter-luciferase construct has been widely used as a reporter to monitor the STAT5 activation by various cytokines in transient expression systems. However, the endogenous ß-casein gene is not usually activated by such cytokines. Even in the mammary gland, PRL alone is not sufficient for ß-casein expression and additional lactogenic hormones, glucocorticoid and insulin, are required for the expression (10). Although the functional consequence remains to be elucidated, the {alpha}-casein gene was identified as the T cell-specific gene by differential screening, and CTLs were found to express ß-casein mRNA (19). Here, we describe the ß-casein gene expression by STAT5 in conjunction with other signaling molecules in an IL-2-dependent mouse cytotoxic T cell line, CTLL-2.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of ß-Casein in T Cells
STAT5 was originally identified and cloned as a mediator of the ß-casein gene expression in the mammary gland. While other cytokines including IL-3, GM-CSF, IL-5, IL-2, EPO, GH, and TPO were subsequently found to utilize STAT5 and transactivate the ß-casein promoter-luciferase reporter, expression of the endogenous ß-casein gene was never induced in Ba/F3 cells in response to IL-3 (data not shown). In contrast, we detected expression of ß-casein mRNA in CTLL-2 cells in response to IL-2, and the expression was augmented by the addition of dexamethasone (Dex), although Dex alone had no effect on the ß-casein expression (Fig. 1Go, A and C). Dex also enhanced the expression of ß-casein in primary thymocytes in the presence of IL-2 (Fig. 1DGo), suggesting that the Dex effect is not specific to the CTLL-2 cell line. While the ß-casein expression in CTL was previously reported (19), the mechanism of induction has not been addressed. We examined the effect of another potent T cell cytokine, IL-4, on the induction of ß-casein in CTLL-2. IL-4 did induce ß-casein mRNA in CTLL-2, although to a lesser extent than IL-2, and Dex again synergistically elevated the expression (Fig. 1Go, A and C). IL-15, another cytokine that utilizes the ß- and {gamma}-subunits of the IL-2 receptor, also induced ß-casein in CTLL-2, and this induction was also enhanced by Dex (data not shown).



View larger version (43K):
[in this window]
[in a new window]
 
Figure 1. Induction of the Transcripts for ß-Casein, CIS, and OSM in CTLL-2 Cells (A and C), ERT/E2 cells (B and C), and Primary T Cells (D)

A, The induction of transcripts for ß-casein, CIS, and OSM by IL-2 or IL-4 in CTLL-2 cells. After 10 h of IL-2 depletion, cells (1 x 107 cells per point) were stimulated for 4 h with 10 ng/ml hIL-2, 10 ng/ml mIL-4 with or without 1.0 x 10-7 M Dex, or left untreated. Total RNA (10 µg/lane) was separated on an agarose gel containing formaldehyde and subjected to Northern analysis with random primer-labeled DNA probes. B, The induction of transcripts for ß-casein, CIS, and OSM by IL-2 or EPO in ERT/E2 cells. After 10 h of cytokine depletion, ERT/E2 cells (1 x 107 cells per point) were stimulated for 4 h with IL-2, 2 U/ml hEPO with or without Dex, or left untreated. C, Northern blot data in panels A and B were scanned and quantitated using MacBAS V2.31. The relative intensity was calculated as the ratio of intensity. D, Dex-enhanced ß-casein expression. Primary thymocytes were stimulated for 4 h with IL-2 (10 ng/ml) or IL-2 plus Dex (1.0 x 10-8 M) in RPMI-15% FBS.

 
To further examine the cytokine responsiveness of ß-casein induction, we used a cell line derived from CTLL-2, ERT/E2. This cell line was established by transfecting the EPO receptor (EPOR) cDNA into CTLL-2 and selected by EPO responsiveness (20). ERT/E2 expressed ß-casein mRNA in response to either IL-2 or EPO, and the induction was enhanced by the addition of Dex (Fig. 1Go, B and C). Induction of ß-casein by either IL-2 or EPO occurred as early as 1 h and reached the maximum level at 4 h after stimulation (data not shown). We also found that the ß-casein mRNA was induced in response to hGM-CSF in CTLL-2/hGMR{alpha}ß cells that expressed both human GM-CSF receptor {alpha}- and ßc-subunits (21) (data not shown).

Synergistic Effect of Dex Is Specific to ß-Casein
As STAT5 is a key regulator of many genes, we wished to determine whether the synergistic effect of Dex is general or specific to ß-casein. By using a dominant negative STAT5, we previously demonstrated that expression of CIS, OSM, PIM-1, and Id-1 is regulated by STAT5 in the IL-3 dependent Ba/F3 cell line (9). Among these genes, expression of CIS and OSM was induced by IL-2 and IL-4 in CTLL-2 cells, and by IL-2 and EPO in ERT/E2 cells. The addition of Dex did not enhance the expression of CIS and OSM by IL-2, IL-4, and EPO (Fig. 1Go, A–C). These results indicate that the synergistic effect of Dex is specific to the ß-casein gene in T cells.

The Specificity of STAT5 and STAT6
Since expression of ß-casein, CIS, and OSM is regulated by STAT5 (9, 22, 23) and because IL-4 specifically activates STAT6 but not STAT5 (24), our observation that IL-4 induced the expression of these genes was somewhat puzzling. Previous reports have shown that STAT5 and STAT6 bind to distinct sequences, i.e. STAT5 binds to a sequence 5'-TTCxxxGAA-3', which has three spacer nucleotides between TTC and GAA, and STAT6 preferentially binds to 5'-TTCxxxxGAA-3' with four spacer nucleotides (20, 25, 26). In the promoter of the rat ß-casein gene, there are two potential STAT binding sites: the one between -97 and -89 is for STAT5 and another between -144 and -134 is for STAT6. In contrast, whereas STAT5 binding sites are present in the promoters of CIS and OSM, no canonical STAT6 binding sites can be found in these minimum promoters (Fig. 2AGo) (22, 23).




View larger version (72K):
[in this window]
[in a new window]
 
Figure 2. STAT6 Activates the ß-Casein, CIS, and OSM Promoters through STAT5- Binding Boxes

A, Structure of the ß-cas344, ß-cas105, CIS, or OSM promoter-luciferase reporter constructs. STAT-binding sites are shown by an open box and their actual sequences were shown. B, Induction of luciferase activity by ß-cas105, CIS, and OSM promoters and their mutant promoters in response to IL-2 and overexpression of STAT5. CTLL-2 cells were transiently transfected with promoter-luciferase reporter constructs with control vector or STAT5 as described in Materials and Methods. Cytokine-depleted cells were either left unstimulated or stimulated with IL-2 for 6 h, and cell lysates were prepared for luciferase assays. C, Induction of luciferase activity by ß-cas105, CIS and OSM promoters, and their mutant promoters as shown by ß-cas105 mut, CIS mut, and OSM mut in response to IL-4 and overexpression of STAT6. CTLL-2 cells were transiently transfected with reporter-luciferase constructs with control vector or STAT6 and luciferase assays were performed as in panel B. The relative luciferase activity was calculated as the ratio of luciferase activities in cells treated with or without factors. The means ± SEM for three experiments are shown. D, Induction of luciferase activity by ß-cas344 promoters and their mutant promoters in response to IL-2 and overexpression of STAT5. E, Induction of luciferase activity by ß-cas344 promoters and their mutant promoters in response to IL-4 and overexpression of STAT6.

 
To assess the specificity of STAT5 and STAT6, we examined the effect of overexpression of STAT5 or STAT6 on the promoter activities of ß-casein, OSM, and CIS. The luciferase reporter constructs of ß-cas344, ß-cas105, OSM, CIS, and their mutant constructs (Fig. 2AGo) were transiently transfected along with STAT5 or STAT6 cDNA into CTLL-2 cells, and luciferase activity in response to IL-2 and IL-4 was monitored (Fig. 2Go, B and C). As expected, STAT5 overexpression enhanced IL-2-dependent activation of the ß-cas105, CIS, and OSM promoters (Fig. 2BGo), without altering IL-4-dependent activation (data not shown). STAT6 overexpression enhanced the transcriptional activation of the ß-cas344 promoter about 3-fold, and it also enhanced the activation by ß-cas105, CIS, and OSM promoters by approximately 2-fold in response to IL-4 (Fig. 2CGo). Mutation or deletion of the STAT binding sites completely abolished promoter activity (Fig. 2Go, B and C), indicating that the induction requires the STAT binding sites. Since these three promoters lack a canonical STAT6 binding site TTCxxxxGAA, these results strongly suggest that STAT6 activates these promoters through the STAT5 binding sites TTCxxxGAA. Overexpression of STAT5, as well as STAT6, enhanced IL-2-dependent and IL-4-dependent activation of the ß-cas344 promoters (Fig. 3Go, D and E). Although mutations of the STAT5- or STAT6-binding sites decreased the response, they did not completely abolish the response, further supporting the idea that STAT5 function is partly mediated through the STAT6-binding site, and STAT6 action is partly mediated by the STAT5-binding site. This is consistent with our previous finding that IL-4 induced a DNA binding factor that is competed with the STAT5 binding sequence in CTLL-2 cells (20), which turned out to be identical to STAT6 (27). These results collectively indicate that STAT6 activated by IL-4 substitutes the function of STAT5 for expression of certain genes in CTLL-2.



View larger version (26K):
[in this window]
[in a new window]
 
Figure 3. Synergistic Effect of Dex Requires the Sequence between -155 and -193 in ß-Casein Promoter

Dex synergistically enhances ß-cas344 promoter-luciferase reporter constructs but not with ß-cas105, CIS, or OSM, and the synergistic effect of DEX requires the sequence between -155 and -193. CTLL-2 cells were transiently transfected with promoter-luciferase reporter constracts as described in Materials and Methods. Cytokine-depleted cells were either left unstimulated or stimulated with Dex, IL-2, or IL-2 plus Dex for 6 h. Cell lysates were prepared and subjected to luciferase assay. The relative luciferase activity was calculated as the ratio of luciferase activities in cells treated with or without factors. The means ± SEM for three experiments are shown.

 
The Synergistic Effect of Dex on the ß-Casein Gene Expression Requires cis-Elements between -193 and -155 in Addition to the STAT5-Binding Site in the ß-Casein Promoter
Dex was shown to synergize with PRL in transactivating the -344/-1 fragment of the rat ß-casein gene promoter in the stably transfected mouse mammary epithelial cell line HC11 (28). We examined the effect of Dex on the promoters for ß-casein, CIS, and OSM in CTLL-2 cells. The luciferase reporter constructs of ß-cas344, ß-cas105, OSM, and CIS were transiently transfected in CTLL-2 cells, and luciferase activity was monitored. IL-2 stimulation resulted in 3-fold induction of the CIS promoter, 4-fold induction of the OSM promoter, and 8-fold induction of ß-cas344 and ß-cas105 promoters. Although we could not detect any synergistic effect of Dex on the ß-cas105, CIS, and OSM promoters, 3-fold enhancement by Dex was observed with the ß-cas344 promoter (Fig. 3Go). This result indicates that the synergy with Dex requires elements between -105 and -344 of the ß-casein promoter. As Dex failed to enhance the activation of the ß-cas105, CIS, and OSM promoters, which have a STAT5-binding site, the effect of Dex appears to be mediated by elements other than STAT5.

We further delineated the cis-regulatory element of ß-casein promoter by deletion analysis of the ß-casein promoter. The luciferase constructs, ß-cas105, ß-cas155, ß-cas193, and ß-cas344, were transiently transfected in CTLL-2 cells, and the luciferase activity in response to Dex, IL-2, or IL-2+Dex was measured (Fig. 3Go). Luciferase activity in response to IL-2 plus Dex was diminished by 5'-deletion of the ß-casein promoter. Although the synergistic effect of Dex was still observed with the ß-cas193 and ß-cas344 promoters, it was lost in the ß-cas155 and ß-cas105 promoters (Fig. 3Go), indicating that a cis-element(s) between -193 and -155 is required for the synergy. Interestingly, deletions up to -155 also resulted in an enhanced response to IL-2 alone, suggesting the presence of a negative regulatory element of the IL-2 response. These results raise an intriguing possibility that Dex synergizes with STAT5 by relieving this negative inhibition.

Negative Regulation of ß-Casein Gene Expression by Ras
Ras is activated by various cytokines including IL-2 (29). We therefore examined the possibility that cytokine-activated Ras may play a role in the negative regulation of the ß-casein expression. We established CTLL-2 transfectants constitutively expressing wild-type (WT) Ras or an activated form of Ras, Ras(G12V). Expression of exogenous Ras was confirmed by Western blotting (Fig. 4BGo). Constitutive expression of Ras(G12V) in CTLL-2 cells resulted in the complete inhibition of ß-casein and OSM gene expression, while expression of WT Ras showed only a slight reduction of ß-casein expression and no inhibition of OSM transcription. In contrast, expression of CIS was not affected by expression of either form of Ras (Fig. 4AGo). Multiple independent transfectants showed the same response, indicating that Ras(G12V) suppresses expression of the ß-casein and OSM genes. These results were consistent with the previous report that expression of oncogenic H-Ras resulted in the inhibition of PRL-induced ß-casein expression in mammary epithelial cells (30, 31).



View larger version (44K):
[in this window]
[in a new window]
 
Figure 4. Ras Negatively Regulates ß-Casein Expression through Distinct Regions from STAT5-Binding Box

A, Ras(G12V) inhibits ß-casein and OSM gene expression in CTLL-2 cells. RNA was extracted from growing CTLL-2 cells or stable transfectants expressing WT Ras, or Ras(G12V), and expression of ß-casein, CIS, and OSM was examined. B, The expression of exogenous WT Ras or Ras(G12V) was confirmed by Western blotting with anti-Ras (Santa Cruz). Total cell lysate was extracted from growing CTLL-2 cells or stable transfectants expressing WT Ras, or Ras(G12V). C, Ras(G12V) inhibits ß-cas344 promoter, while it does not inhibit ß-cas105, CIS, OSM, or c-fos promoters. CTLL-2 cells were transiently transfected with various promoter-luciferase reporter constructs as indicated and either control vector, WT Ras, or Ras(G12V) cDNA as described in Materials and Methods. Cytokine-depleted cells were either left unstimulated or stimulated with IL-2 for 6 h. Cell lysates were prepared and subjected to luciferase assay. D, Inhibition of ß-casein promoter by Ras(G12V) is mediated through the promoter region between -105 and -193. CTLL-2 cells were transiently transfected with ß-cas105, ß-cas155, ß-cas193, or ß-cas344 promoter-luciferase reporter constructs. Cytokine-depleted cells were either left unstimulated or stimulated with IL-2 for 6 h. Cell lysates were prepared and subjected to luciferase assay. The relative luciferase activity was calculated as the ratio of luciferase activities in cells treated with or without factors. The means ± SEM for three experiments are shown.

 
We then attempted to analyze the molecular mechanism of H-Ras-mediated inhibition of ß-casein and OSM gene expression. The cDNA encoding WT Ras or Ras(G12V) was transiently transfected in CTLL-2 cells with ß-cas105, ß-cas344, CIS, OSM, or c-fos luciferase constructs, and luciferase activity in response to IL-2 was monitored (Fig. 4CGo). We used the c-fos promoter as a control because we have previously reported that the optimum induction of c-fos requires both Ras and STAT5, and this synergistic effect may be mediated by SRE and SIE in the c-fos promoter (9). While expression of Ras(G12V) alone slightly elevated the basal luciferase activity, it severely inhibited the luciferase activity of the ß-cas344 construct in response to IL-2, and WT Ras modestly inhibited the luciferase activity of the ß-cas344 promoter. However, expression of WT Ras or Ras(G12V) did not inhibit the luciferase activity of the ß-cas105, CIS, and OSM luciferase constructs (Fig. 4CGo), indicating that the inhibition of OSM promoter activity by Ras is mediated probably through a more upstream region. Expression of Ras(G12V) was sufficient for the activation of the c-fos promoter, and IL-2 has an additive effect on the c-fos promoter. These results indicate that inhibition of ß-casein promoter activity by Ras is mediated through a cis-regulatory element in the ß-casein promoter that is distinct from the STAT5-binding site.

We further delineated the element responsible for the negative regulation by Ras(G12V) using deletion constructs of the ß-casein promoter (Fig. 4DGo). As the negative effect of Ras(G12V) was observed with ß-cas155, ß-cas193, and ß-cas344 promoters but not with ß-cas105, the region between -105 and -155 is negatively regulated by Ras. In addition, as the ß-cas193-induced luciferase activity was more severely inhibited by Ras(G12V) than that of ß-cas155 (Fig. 4DGo), the region between -155 and -193 also represents a second element that is negatively regulated by Ras.

While IL-2 activates Ras, IL-2 efficiently induced luciferase expression from the ß-cas155-luciferase construct. In this case, the Ras-mediated negative regulation, which is activated by IL-2, may be canceled by IL-2-activated STAT5 through the STAT box present in the same region between -105 and -155. Thus, it appears that the balance between the Ras activation and STAT5 activation may be important for the casein expression.

Positive Regulation of STAT5 Activity by Ras
Previous reports demonstrated that full transcriptional activation of STAT1 requires serine phosphorylation by MAPK (4). We examined the possibility that STAT5 activity is also positively regulated by the Ras-MAPK pathway. As IL-2 activates MAPK through Ras, we expressed RasN17 (a dominant negative form of Ras) to test whether Ras has any positive role in STAT5 activity (Fig. 5AGo). Interestingly, expression of RasN17 severely inhibited the luciferase activity driven by the ß-cas105, ß-cas344, CIS, OSM, and c-fos promoters in response to IL-2 (Fig. 5AGo). The role of Ras in STAT5 activation was further examined by the EPOR mutant (EPORH), which lacks the ability to activate Ras (Fig. 5BGo). EGF receptor (EGFR)-EPORH is a chimeric receptor consisting of the extracellular domain of the EGFR and the truncated intracellular domain of the EPOR, which lacks the region responsible for the activation of Ras (32). The truncated cytoplasmic domain of EPOR still has the ability to activate STAT5 and is capable of inducing the transcription of CIS and OSM (22, 33). The cDNA encoding EGFR-EPORH was transiently transfected with the ß-cas105 luciferase construct, which is insensitive to Ras-induced negative regulation, in ERT/E2 cells (Fig. 5BGo). As ERT/E2 cells expressed the WT EPOR, the luciferase activity induced by EGF was normalized to that elicited by EPO. EGF induced the activation of the ß-cas105 promoter 70% of that induced by EPO. Coexpression of Ras(G12V) was sufficient to raise the luciferase activity to the level comparable to EPO stimulation (Fig. 5BGo), while WT Ras failed to complement. Thus, the Ras activation complements the signaling by the EGFR-EPORH mutant receptor, suggesting that Ras contributes to the STAT5 activation.



View larger version (26K):
[in this window]
[in a new window]
 
Figure 5. Ras Positively Regulates STAT5 Activation

A, RasN17 inhibited ß-cas344, ß-cas105, CIS, OSM or c-fos promoter. CTLL-2 cells were transiently transfected with ß-cas105, ß-cas344, CIS, or OSM promoter-luciferase reporter constructs and with control vector or RasN17 cDNA, and cytokine-depleted cells were either left unstimulated or stimulated with IL-2. The relative luciferase activity was calculated as the ratio of luciferase activities in cells treated with or without factors. The means ± SEM for three experiments are shown. B, Ras(G12V) and Ras(G12V/V45E) enhance transactivation of STAT5. ERT/E2 cells were transiently transfected with the ß-cas105 promoter-luciferase reporter construct and EGFR-EPORH with a control vector, WT Ras, Ras(G12V), Ras(G12V/V45E), or {Delta}raf cDNA, and cytokine-depleted cells were either left unstimulated or stimulated with EPO or 10 ng/ml EGF. Promoter activity was calculated by dividing the luminescence activity obtained with the EGF stimulation by that of EPO stimulation.

 
We further analyzed which downstream pathway of Ras might contribute to the activation of STAT5. Ras(G12V/V45E) is a constitutively activated double Ras mutant that activates downstream signaling molecules such as PI-3 kinase but lacks the ability to activate the Raf-MAPK pathway (34, 35). ERT/E2 cells were transiently transfected with the Ras(G12V/V45E), EGFR-EPORH, and ß-cas105 luciferase constructs, and the luciferase activity induced by EGF was compared with that by EPO (Fig. 5BGo). Interestingly, Ras(G12V/V45E) enhanced the luciferase activity to the same level as that enhanced by Ras(G12V), suggesting that the Raf-MAPK pathway does not contribute to the enhancement of STAT5 activation by Ras. This possibility was further supported by expression of an active form of Raf that lacks the N-terminal regulatory domains. The active form of Raf did not enhance the ß-cas105 luciferase activity in response to EGF (Fig. 5BGo). These results indicate that the Raf-MAPK pathway is not responsible for the Ras-mediated enhancement of STAT5 activity.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of the ß-Casein Gene in CTLL-2 Cells
While the ß-casein promoter has been widely used for monitoring the STAT5 activation by various cytokines in various cells using a transient expression system, expression of the endogenous ß-casein was found only in the mammary gland and cytotoxic T lymphocytes (CTL) (19). As ß-casein is a major milk protein, its expression in the mammary gland has been extensively studied, and STAT5 was identified through studies of PRL-dependent expression of ß-casein in the mammary gland. Interestingly, both {alpha}- and ß-casein are also expressed in CTL cells. Caseins are known to form calcium-dependent micelles, and it was proposed that those micelles function as a vehicle to deliver perforins to the surface of target cells (19). However, the physiological role of caseins in CTL remains to be established. Nevertheless, the ß-casein gene provides a useful model to study the mechanism of cytokine signaling that leads to gene expression.

In this study, we have shown that IL-2 and IL-4 induce the expression of the endogenous ß-casein gene in the CTL cell line CTLL-2 (Fig. 1AGo), suggesting that the ß-casein expression may be mediated by IL-2 and IL-4 in the thymus. While IL-2 produced by Th1 cells supports the CTL responses, Th2 cytokines such as IL-4 also support CTL (36). However, signaling pathways activated by IL-2 and IL-4 are quite distinct, e.g. IL-2 activates STAT5 whereas IL-4 activates STAT6. Our results that IL-4-induced STAT6 could induce the genes that are known to be regulated by Stat5 provides an explanation for the overlapping effect of IL-2 and IL-4 on CTL response.

We and others have shown that IL-2 utilizes STAT5 whereas IL-4 activates STAT6 in CTLL-2 (20, 27). STAT5 and STAT6 are known to bind to TTCxxxGAA and TTCxxxxGAA, respectively (20, 25, 26). Nevertheless, both cytokines activated the ß-cas105 promoter that has only a TTCxxxGAA sequence (Fig. 2AGo). Likewise, although the promoters of OSM and CIS genes contain TTCxxxGAA, but not TTCxxxxGAA, IL-4 also activated these promoters (Fig. 2CGo) and induced endogenous CIS and OSM expression in CTLL-2 cells (Fig. 1Go, A and C). Furthermore, expression of IL-4-induced luciferase activity driven by the ß-cas105, CIS, and OSM promoters was enhanced by STAT6 overexpression (Fig. 2CGo). These results, together with our previous finding that IL-4 induced a DNA-binding complex with a canonical STAT5-binding site (20), indicate that STAT6 can bind to a TTCxxxGAA site and induce transcription. However, the induction levels of ß-casein, CIS, and OSM by STAT6 were lower than those by STAT5. This is probably due to the different binding affinity of STAT5 and STAT6 to the STAT-binding sites present in the promoter region. During the preparation of this manuscript, Morriggl et al. (37) reported that IL-4 activates the ß-casein gene expression in response to IL-4 in HC11 cells and Dex synergistically enhanced the ß-cas344 luciferase activity in response to IL-4 in COS cells. While they claimed that IL-4 induced ß-casein expression through the STAT6-binding site between -144 and -134 in the ß-casein promoter, our result indicates that the STAT5-binding site between -97 and -89 of the ß-casein promoter also contributes to the IL-4-induced ß-casein expression in lymphocytes (Fig. 2Go). These results collectively indicate that IL-4-activated STAT6 substitutes for the function of STAT5 for expression of certain genes in CTLL-2.

Cell Type-Specific Gene Expression in Response to Cytokines
In Ba/F3 cells, expression of CIS, OSM, Id-1, and PIM-1 genes was induced in response to IL-3 through STAT5 activation (8), but expression of the endogenous ß-casein gene was never observed in response to IL-3. A similar induction pattern of these transcripts was observed in response to EPO in Ba/F3 cells ectopically expressing EPOR (data not shown) (22, 33). In contrast, ectopic expression of EPOR and hGM-CSF{alpha}ß receptors in CTLL-2 cells resulted in expression of the ß-casein, CIS, and OSM genes, but not the Pim-1 and Id-1 genes, in response to EPO or hGM-CSF (Fig. 1BGo and data not shown). Thus, the gene expression pattern is not solely determined by cytokine receptor signaling, but also depends largely on the program intrinsic to the cell itself. Involvement of tissue- and cell type-specific transcription factors in STAT-regulated gene expression has been suggested by recent reports. The WAP (whey acidic protein) gene expression is regulated by a cooperative interaction between two transcription factors, NF1 and STAT5 (38). More recently, it was shown that both STAT5 and the lymphoid/myeloid-specific Ets family protein, Elf-1, are important for the IL-2-mediated IL-2 receptor {alpha} chain induction (39). In the case of ß-casein, although the ß-cas344 promoter is responsive to cytokine stimulation in various hematopoietic cell lines in a transient expression system, a regulatory element(s) upstream of 344 seems to play an important role for the cell type-specific expression of ß-casein gene. Taken together, it appears that STAT5 controls a set of gene expression in concert with tissue- or cell type-specific transcription factors.

Cross-Talk between STAT5 and Glucocorticoid
Glucocorticoids are potent antiinflammatory substances that exhibit profound and complex effects on the immune system (40). Glucocorticoids exert their diverse effects on the immune system through regulating gene expression including cytokine production. In mammary epithelial cells, PRL and glucocorticoid are required for the maximum induction of ß-casein transcription (10). Using CTLL-2 cells as well as primary thymocytes, we showed that Dex enhances the IL-2-induced ß-casein expression (Fig. 1AGo). The mechanism of Dex action on the ß-casein promoter was studied extensively. One possible explanation for the synergistic action of Dex and PRL on the rat ß-casein promoter is a direct physical interaction between STAT5 and the glucocorticoid receptor (GR) (41, 42, 43). However, our results show that the synergistic action of Dex requires additional elements. First, Dex does not enhance expression of STAT5-regulated endogenous genes such as CIS and OSM (Fig. 1AGo). Second, although Dex enhanced the IL-2-induced luciferase activity from the ß-cas193 and ß-cas344 promoters, it did not enhance luciferase activity driven by the ß-cas105, ß-cas155, CIS, and OSM promoters (Fig. 3AGo). We also found that overexpression of STAT5 does not enhance the synergy (data not shown). These results collectively indicate that the STAT5-GR interaction and the presence of STAT5 binding site in the promoter are not sufficient for the synergistic effect of Dex on STAT5. We also found the synergistic effect of Dex with STAT6 on the ß-cas344 promoter but not on the ß-cas105, CIS, or OSM promoter (data not shown), indicating that a similar mechanism is responsible for the synergistic action of Dex on STAT6. Our results are consistent with the previous report that the 5'-flanking region between -170 and -157 contains a cis-acting sequence required for the synergistic action of Dex in HC11 cells (44). They also showed importance of the potential GR half-palindromic sites in the ß-casein promoter using HC11 and COS cells (45). While GR half-palindromic sites may contribute to the synergy, as there are no GR-binding sites in the 5'-flanking region between -170 and -157, a factor that binds to an element between -170 and -157 in the ß-casein promoter appears to be necessary for the synergistic action of Dex on STAT5. As our deletion analysis of the rat ß-casein promoter in response to IL-2 revealed a negative regulatory element(s) in the promoter region between -155 and -193 (Fig. 3BGo), it is an attractive hypothesis that Dex relieves this negative regulation.

Positive and Negative Role of Ras in ß-Casein Expression
We have shown that expression of Ras(G12V) inhibited expression of the endogenous ß-casein gene as well as expression from the exogenously transfected ß-casein promoter through cis regulatory elements between -105 and -193 (Fig. 4Go, C and D). This result is consistent with a previous report that Ras(G12V) inhibited the expression of ß-casein induced by PRL, Dex, and insulin in HC11 cells (30, 31). The Ras-mediated negative effect appears to be mediated by two distinct regions, one between -155 and -193 and the other between -105 and -155 (Fig. 4DGo). As IL-2 induces the activation of Ras as well as STAT5 and there are both STAT5-binding sites and also Ras-mediated negative regulatory site(s) in the ß-cas155 and ß-cas193 promoters, the balance between the activated Ras and STAT5 may determine the net luciferase activity from these promoters in response to IL-2. We also found that expression of the endogenous OSM gene induced by IL-2 was inhibited by Ras(G12V). The inhibition of OSM transcription is another example of cross-talk between STAT5- and Ras-signaling pathways. Expression of WT Ras or Ras(G12V) did not inhibit the luciferase activity of the OSM promoter (Fig. 4CGo), indicating that the inhibition of OSM promoter by Ras is mediated probably through a more upstream region. However, the mechanism of inhibition by Ras appears to be different from the ß-casein expression, as constitutive expression of Ras(G12V/V45E), an active form of Ras that lacks the ability to activate Raf, effectively inhibits the OSM expression but failed to inhibit the ß-casein expression (D. Chida and A. Miyajima, unpublished results).

Recently, the importance of CAAT enhancer-binding protein-ß (C/EBPß) in the normal development of the mammary gland, e.g. ß-casein expression, was elegantly demonstrated by transplantation of WT ovarian and mammary glands in C/EBPß-deficient mice (46, 47). C/EBP is possibly involved in the regulation of ß-casein expression through the region between -155 and -193, as C/EBP-binding sites were shown to be essential for ß-casein expression in HC11 cells (16). The transcriptional activity of C/EBPß is regulated by glucocorticoids as well as the Ras-MAPK pathway. As GR binds directly to C/EBPß (48), glucocorticoid may regulate the C/EBPß activity directly. Alternatively, as there are C/EBPß isoforms that are either active or dominant negative, glucocorticoids may affect C/EBPß activity by changing the ratio of these isoforms (47, 49). The Ras-MAPK pathway was shown to phospholylate and activate NF-IL6, a human counterpart of C/EBPß (50), apparently contradicting our results that the Ras pathway inhibits ß-casein expression. However, considering the fact that a dominant negative form of C/EBPß that lacks the N-terminal transactivation domain also has a MAPK phospholylation site, it is possible that the Ras-MAPK pathway enhances the negative effect of the truncated form of C/EBPß by phosphorylation, and that transcriptional inhibition by the Ras-MAPK pathway is mediated through the C/EBPß binding site in the ß-casein promoter. These results raise the intriguing possibility that GR and the Ras-MAPK pathway regulate the same transcription factor C/EBPß in ß-casein expression.

Expression of a dominant negative form of Ras, RasN17, inhibits STAT5-mediated expression of luciferase (Fig. 5AGo), suggesting that Ras plays a positive role in STAT5-mediated transactivation. This possibility is further supported by the finding that coexpression of Ras(G12V) with the EGFR-EPORH mutant chimeric receptor defective in the Ras activation complemented the EGF-dependent luciferase expression by the minimum ß-casein promoter ß-cas105 (Fig. 5BGo). Although the mechanism of STAT5 activation by Ras is not clear, a recent finding that STAT proteins are phosphorylated at the serine residues, in addition to tyrosine residue, suggests the possibility that Ras enhances STAT5 activity by serine phosphorylation (51, 52, 53). Phosphorylation of MAPK consensus phosphorylation sites in the C-terminal region of STAT1 and STAT3 was demonstrated in vivo and in vitro (4). In accordance with this observation, previous studies have demonstrated a positive role of serine phosphorylation of STAT5 (51, 53, 54, 55). However, the role of MAPK in the STAT5 activation is a controversial issue. Pircher et al. (55) reported that PD98059, a specific inhibitor of MAPK, partially inhibited the transcriptional activity of STAT5a, but not STAT5b, by GH (55), while others described that PD98059 had no effect on the transcriptional activity of STAT5 (51, 53, 54). It is thus possible that a different serine kinase is responsible for serine phosphorylation of STAT5 in different cell types or cytokine receptors. We found that expression of an activated form of Raf, a Ras-regulated kinase, failed to complement the defect of EGFR-EPORH, whereas Ras(G12V/E45) was capable of complementing the defect of EGFR-EPORH (Fig. 5BGo). It is therefore likely that Ras enhances the STAT5 activity through a Ras effector molecule other than Raf. As Ras(G12V/V45E) interacts with PI-3 kinase, a kinase downstream of PI-3 kinase may be responsible for the enhancing activity. However, as we failed to detect any effects of wortmanin, a specific inhibitor of PI3K, on the ß-casein gene expression, some other kinases may be responsible for this effect. Previously we reported that the optimum induction of c-fos requires both Ras and STAT5, and this synergistic effect may be mediated by SRE and SIE in the c-fos promoter (9). However, the results presented here have added an additional mechanism of synergistic interaction between Ras and STAT5.

In this report we have described that Ras affects ß-casein gene expression in two opposing ways; one is to enhance STAT5 activation, and another is to suppress the transcription through the promoter region between -105 and -193 (Fig. 6Go). As exemplified in the activation of the ß-casein promoter by cytokines, gene expression is determined by integrating various intracellular signaling events.



View larger version (22K):
[in this window]
[in a new window]
 
Figure 6. A Model of the Regulatory Mechanisms of ß-Casein Gene Expression

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cells
CTLL-2 cells were cultured in RPMI 1640 medium containing 8% FBS, 50 µM ß-mercaptoethanol, 50 µg/ml gentamycin, and 10 ng/ml hIL-2 (Ajinomoto, Tokyo, Japan). ERT/E2 cells and CTLL-2 Ras-expressing transfectants were cultured in the same medium plus G418 (0.5 mg/ml). Thymocytes were harvested from thymi of BALB/c mice (Sankyo Laboratory, Tokyo, Japan), and the cells were strained through a 70-µm cell filter (Becton Dickinson).

Plasmid
The (-344/+1) ß-casein promoter luciferase reporter construct (pZZ1, ß-cas344Luc) and the (-105/+1) ß-casein promoter luciferase reporter construct (pZZ2, ß-cas105Luc) were described previously (56). Two additional 5'-deletion plasmids (ß-cas193Luc, ß-cas155Luc) were constructed by PCR with the following primers: the ß-193 primer, 5'-cgggatccttcaccagcttctgaattgc-3', corresponding to the sequence between -193 to -174 with a BamHI site at the 5'-end, the ß-155 primer, 5'-cgggatcccccagaatttcttgggaaag-3', corresponding to -155 to -133 with a BamHI site at the 5'-end, and the ß-1 primer, 5'-ccgctcgaggtctatcagactctgtgac-3', corresponding to -19 to -1 with a XhoI site at the 5'-end, were used with the template DNA; the PCR-generated ß-casein promoter fragments were substituted with the BamHI and XhoI fragment in the original ß-casein (-344/+1) promoter luciferase reporter construct. Mutation of the Stat5-binding site and the Stat6-binding site in the ß-casein promoter were introduced by PCR-mediated site-directed mutagenesis using the following primers: the ß-344 primer; 5'-cgggatcctctctaaagcttgtgaat-3', the Stat5-sense; 5'-gtgaacttcttttaattaag-3', Stat5-antisense; 5'-cttaattaaaagaagttcac-3', Stat6-sense; 5'-ccagaatttcttgttaaaga-3', Stat6-antisense; 5'-tctttaacaagaaattctgg-3', the ß-1 primer. The PCR fragment produced by the ß-344 primer and the Stat5-antisense primer (or the Stat6-antisense primer) and the PCR fragment produced by the Stat5-sense primer (or the 6-sense primer) and the ß-1 primer were used as a template for the second-round PCR using the ß-344 and the ß-1 primers. Mutations were confirmed by sequencing. pME18S/STAT6 was constructed by inserting the STAT6 cDNA, kindly provided by Dr. K. Yamamoto (Tokyo Medical and Dental University, Tokyo, Japan) and Dr. J. Ihle (St. Jude Research Hospital, Memphis, TN), downstream of the SR{alpha} promoter of pME18S. pLNC-{Delta}raf was kindly provided by Dr. T. Satoh (Tokyo Institute of Technology). Other constructs were described previously (9, 22, 32, 33, 34).

Generation of Stable Transfectants
pCMV containing Ras(G12V) or WT Ras cDNA was cotransfected with pME18S Neo by electroporation (960 µFarads, 330 V), and transfectants were isolated by limiting dilution in the presence of 1.0 mg/ml of G418. Expression of the transfected Ras gene in the transfectants was confirmed by Western blotting using the anti-Ras antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Northern Blot
For Northern blot analysis, total RNA (10 µg) was separated on an agarose gel containing 1.0% formaldehyde and transferred to a positively charged nylon membrane (Boehringer Mannheim, Indianapolis, IN). After UV cross-linking, the membrane was hybridized with random primer-labeled DNA probes.

Transient Transfection and Luciferase Assay
CTLL-2 cells were transiently transfected by electroporation (960 µF, 270 V) with reporter plasmids containing the luciferase gene linked to the ß-casein, OSM, CIS, and c-fos promoters (22, 23, 56). After 12 h of culture in RPMI containing 8% FBS and IL-2, cells were washed to remove IL-2 and starved for 12 h in RPMI + 8% FCS without cytokines. Cells were then challenged with Dex, IL-2, or IL-2 plus Dex. After 6 h of incubation, proteins were extracted and assayed for luciferase activity (Promega, Madison, WI) (56).


    ACKNOWLEDGMENTS
 
We thank Dr. Alice Mui for critical reading of the manuscript, Dr. M. Shirouzu, and Dr. S. Yokoyama for Ras expression vector, Dr. Toshio Kitamura for CTLL-2/hGM{alpha}ß cells, Kirin Brewery Co. Ltd. (Tokyo, Japan) for recombinant human EPO, and Ajinomoto Co. Ltd. for recombinant human IL-2.


    FOOTNOTES
 
Address requests for reprints to: Dr. Atsushi Miyajima, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1–1-1 Yayoi Bunkyo-ku, Tokyo, Japan 113-0032. E-mail: miyajima{at}ims.u-tokyo.ac.jp

D.C. is supported by Fellowships in Cancer Research from the Japan Society for the Promotion of Science for Young Scientists. This work was supported in part by grants from the Ministry of Education, Culture, Sports, and Science (Monbushou), Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Corporation, and the Toray Research Foundation.

Received for publication December 4, 1997. Revision received July 30, 1998. Accepted for publication August 7, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Hara T, Miyajima A 1996 Function and signal transduction mediated by the interleukin 3 receptor system in hematopoiesis. Stem Cells 14:605–618[Abstract]
  2. Ihle JN 1996 STATs: Signal transducers and activation of transcription. Cell 84:331–334[Medline]
  3. Boulton TG, Zhong Z, Wen Z, Darnell Jr JE, Stahl N, Yancopoulos GD 1995 STAT3 activation by cytokines utilizing gp130 and related transducers involves a secondary modification requiring an H7-sensitive kinase. Proc Natl Acad Sci USA 92:6915–6919[Abstract]
  4. Wen Z, Zhong Z, Darnell Jr JE 1995 Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell 82:241–250[Medline]
  5. Zhang JJ, Vinkemeier U, Gu W, Chakravarti D, Horvath CM, Darnell Jr JE 1996 Two contact regions between Stat1 and CBP/p300 in interferon gamma signaling. Proc Natl Acad Sci USA 93:15092–15096[Abstract/Free Full Text]
  6. Haspel RL, Salditt-Georgieff M, Darnell Jr JE 1996 The rapid inactivation of nuclear tyrosine phosphorylated Stat1 depends upon a protein tyrosine phosphatase. EMBO J 15:6262–6268[Abstract]
  7. Satoh T, Nakafuku M, Miyajima A, Kaziro Y 1991 Involvement of ras p21 protein in signal-transduction pathways from interleukin 2, interleukin 3, and granulocyte/macrophage colony- stimulating factor, but not from interleukin 4. Proc Natl Acad Sci USA 88:3314–3318[Abstract]
  8. Mui AL, Wakao H, O’Farrell AM, Harada N, Miyajima A 1995 Interleukin-3, granulocyte-macrophage colony stimulating factor and interleukin-5 transduce signals through two STAT5 homologs. EMBO J 14:1166–1175[Abstract]
  9. Mui AL, Wakao H, Kinoshita T, Kitamura T, Miyajima A 1996 Suppression of interleukin-3-induced gene expression by a C-terminal truncated Stat5: role of Stat5 in proliferation. EMBO J 15:2425–2433[Abstract]
  10. Groner B, Gouilleux F 1995 Prolactin-mediated gene activation in mammary epithelial cells. Curr Opin Genet Dev 5:587–594[CrossRef][Medline]
  11. Schmitt-Ney M, Doppler W, Ball RK, Groner B 1991 Beta-casein gene promoter activity is regulated by the hormone-mediated relief of transcriptional repression and a mammary-gland-specific nuclear factor. Mol Cell Biol 11:3745–3755[Medline]
  12. Altiok S, Groner B 1993 Interaction of two sequence-specific single-stranded DNA-binding proteins with an essential region of the beta-casein gene promoter is regulated by lactogenic hormones. Mol Cell Biol 13:7303–7310[Abstract]
  13. Altiok S, Groner B 1994 beta-Casein mRNA sequesters a single-stranded nucleic acid-binding protein which negatively regulates the beta-casein gene promoter. Mol Cell Biol 14:6004–6012[Abstract]
  14. Meier VS, Groner B 1994 The nuclear factor YY1 participates in repression of the beta-casein gene promoter in mammary epithelial cells and is counteracted by mammary gland factor during lactogenic hormone induction. Mol Cell Biol 14:128–137[Abstract]
  15. Raught B, Khursheed B, Kazansky A, Rosen J 1994 YY1 represses beta-casein gene expression by preventing the formation of a lactation-associated complex. Mol Cell Biol 14:1752–1763[Abstract]
  16. Doppler W, Welte T, Philipp S 1995 CCAAT/enhancer-binding protein isoforms beta and delta are expressed in mammary epithelial cells and bind to multiple sites in the beta-casein gene promoter. J Biol Chem 270:17962–17969[Abstract/Free Full Text]
  17. Saito H, Oka T 1996 Hormonally regulated double- and single-stranded DNA-binding complexes involved in mouse beta-casein gene transcription. J Biol Chem 271:8911–8918[Abstract/Free Full Text]
  18. Schmitt-Ney M, Happ B, Ball RK, Groner B 1992 Developmental and environmental regulation of a mammary gland-specific nuclear factor essential for transcription of the gene encoding beta-casein. Proc Natl Acad Sci USA 89:3130–3134[Abstract]
  19. Grusby MJ, Mitchell SC, Nabavi N, Glimcher LH 1990 Casein expression in cytotoxic T lymphocytes. Proc Natl Acad Sci USA 87:6897–6901[Abstract]
  20. Wakao H, Harada N, Kitamura T, Mui AL, Miyajima A 1995 Interleukin 2 and erythropoietin activate STAT5/MGF via distinct pathways. EMBO J 14:2527–2535[Abstract]
  21. Kitamura T, Miyajima A 1992 Functional reconstitution of the human interleukin-3 receptor. Blood 80:84–90[Abstract]
  22. Yoshimura A, Ichihara M, Kinjyo I, Moriyama M, Copeland NG, Gilbert DJ, Jenkins NA, Hara T, Miyajima A 1996 Mouse oncostatin M: an immediate early gene induced by multiple cytokines through the JAK-STAT5 pathway. EMBO J 15:1055–1063[Abstract]
  23. Matsumoto A, Masuhara M, Mitsui K, Yokouchi M, Ohtsubo M, Misawa H, Miyajima A, Yoshimura A 1997 CIS, a cytokine inducible SH2 protein, is a target of the JAK-STAT5 pathway and modulates STAT5 activation. Blood 89:3148–3154[Abstract/Free Full Text]
  24. Hou J, Schindler U, Henzel WJ, Ho TC, Brasseur M, McKnight SL 1994 An interleukin-4-induced transcription factor: IL-4 Stat. Science 265:1701–1706[Medline]
  25. Mikita T, Campbell D, Wu P, Williamson K, Schindler U 1996 Requirements for Interleukin-4-induced gene expression and functional characterization of STAT6. Mol Cell Biol 16:5811–5820[Abstract]
  26. Seidel HM, Milocco LH, Lamb P, Darnell JE, Jr, Stein RB, Rosen J 1995 Spacing of palindromic half sites as a determinant of selective STAT (signal transducers and activators of transcription) DNA binding and transcriptional activity. Proc Natl Acad Sci USA 92:3041–3045[Abstract]
  27. Quelle FW, Shimoda K, Thierfelder W, Fischer C, Kim A, Ruben SM, Cleveland JL, Pierce JH, Keegan AD, Nelms K, Paul WE, Ihle JN 1995 Cloning of murine Stat6 and human Stat6, Stat proteins that are tyrosine phosphorylated in responses to IL-4 and IL-3 but are not required for mitogenesis. Mol Cell Biol 1555:3336–3343
  28. Doppler W, Groner B, Ball RK 1989 Prolactin and glucocorticoid hormones synergistically induce expression of transfected rat beta-casein gene promoter constructs in a mammary epithelial cell line. Proc Natl Acad Sci USA 86:104–108[Abstract]
  29. Taniguchi T 1995 Cytokine signaling through nonreceptor protein tyrosine kinases. Science 268:251–255[Medline]
  30. Happ B, Hynes NE, Groner B 1993 Ha-ras and v-raf oncogenes, but not int-2 and c-myc, interfere with the lactogenic hormone dependent activation of the mammary gland specific transcription factor. Cell Growth Differ 4:9–15[Abstract]
  31. Jehn B, Costello E, Marti A, Keon N, Deane R, Li F, Friis RR, Burri PH, Martin F, Jaggi R 1992 Overexpression of Mos, Ras, Src, and Fos inhibits mouse mammary epithelial cell differentiation. Mol Cell Biol 12:3890–3902[Abstract]
  32. Maruyama K, Miyata K, Yoshimura A 1994 Proliferation and erythroid differentiation through the cytoplasmic domain of the erythropoietin receptor. J Biol Chem 269:5976–5980[Abstract/Free Full Text]
  33. Yoshimura A, Ohkubo T, Kiguchi T, Jenkins NA, Gilbert DJ, Copeland NG, Hara T, Miyajima A 1995 A novel cytokine-inducible gene CIS encodes an SH2-containing protein that binds to tyrosine-phosphorylated interleukin 3 and erythropoietin receptors. EMBO J 14:2816–2826[Abstract]
  34. Shirouzu M, Koide H, Fujita-Yoshigaki J, Oshio H, Toyama Y, Yamasaki K, Fuhrman SA, Villafranca E, Kaziro Y, Yokoyama S 1994 Mutations that abolish the ability of Ha-Ras to associate with Raf-1. Oncogene 9:2153–2157[Medline]
  35. Kinoshita T, Shirouzu M, Kamiya A, Hashimoto K, Yokoyama S, Miyajima A 1997 Raf/MAPK and rapamycin-sensitive pathways mediate the anti-apoptotic function of p21Ras in IL-3-dependent hematopoietic cells. Oncogene 14:619–627[CrossRef]
  36. Tepper RI 1993 The anti-tumour and proinflammatory actions of IL4. Res Immunol 144:633–637[Medline]
  37. Moriggl R, Berchtold S, Friedrich K, Standke GJ, Kammer W, Heim M, Wissler M, Stocklin E, Gouilleux F, Groner B 1997 Comparison of the transactivation domains of Stat5 and Stat6 in lymphoid cells and mammary epithelial cells. Mol Cell Biol 17:3663–3678[Abstract]
  38. Li S, Rosen JM 1995 Nuclear factor I and mammary gland factor (STAT5) play a critical role in regulating rat whey acidic protein gene expression in transgenic mice. Mol Cell Biol 15:2063–2070[Abstract]
  39. John S, Robbins C, Leonard W 1996 An IL-2 response element in the human IL-2 receptor {alpha} chain promoter is a composite element that binds STAT5, Elf-1, HMG-1(Y) and GATA family protein. EMBO J 15:5627–5635[Abstract]
  40. Cupps TR, Fauci AS 1982 Corticosteroid-mediated immunoregulation in man. Immunol Rev 65:133–155[Medline]
  41. Stocklin E, Wissler M, Gouilleux F, Groner B 1996 Functional Interactions between STAT5 and the glucocorticoid receptor. Nature 383:726–728[CrossRef][Medline]
  42. Stoecklin E, Wissler M, Moriggl R, Groner B 1997 Specific DNA binding of Stat5, but not of glucocorticoid receptor, is required for their functional cooperation in the regulation of gene transcription. Mol Cell Biol 17:6708–6716[Abstract]
  43. Cella N, Groner B, Hynes NE 1998 Characterization of Stat5a and Stat5b homodimers and heterodimers and their association with the glucocortiocoid receptor in mammary cells. Mol Cell Biol 18:1783–1792[Abstract/Free Full Text]
  44. Lechner J, Welte T, Doppler W 1997 Mechanism of interaction between the glucocorticoid receptor and Stat5: role of DNA-binding. Immunobiology 198:112–123[Medline]
  45. Lechner J, Welte T, Tomasi JK, Bruno P, Cairns C, Gustafsson J, Doppler W 1997 Promoter-dependent synergy between glucocorticoid receptor and Stat5 in the activation of beta-casein gene transcription. J Biol Chem 272:20954–20960[Abstract/Free Full Text]
  46. Robinson GW, Johnson PF, Hennighausen L, Sterneck E 1998 The C/EBPbeta transcription factor regulates epithelial cell proliferation and differentiation in the mammary gland. Genes Dev 12:1907–1916[Abstract/Free Full Text]
  47. Seagroves TN, Krnacik S, Raught B, Gay J, Burgess-Beusse B, Darlington GJ, Rosen JM 1998 C/EBPbeta, but not C/EBPalpha, is essential for ductal morphogenesis, lobuloalveolar proliferation, and functional differentiation in the mouse mammary gland. Genes Dev 12:1917–1928[Abstract/Free Full Text]
  48. Nishio Y, Isshiki H, Kishimoto T, Akira S 1993 A nuclear factor for interleukin-6 expression (NF-IL6) and the glucocorticoid receptor synergistically activate transcription of the rat alpha 1-acid glycoprotein gene via direct protein-protein interaction. Mol Cell Biol 13:1854–1862[Abstract]
  49. Raught B, Liao WS, Rosen JM 1995 Developmentally and hormonally regulated CCAAT/enhancer-binding protein isoforms influence beta-casein gene expression. Mol Endocrinol 9:1223–1232[Abstract]
  50. Nakajima T, Kinoshita S, Sasagawa T, Sasaki K, Naruto M, Kishimoto T, Akira S 1993 Phosphorylation at threonine-235 by a ras-dependent mitogen-activated protein kinase cascade is essential for transcription factor NF-IL6. Proc Natl Acad Sci USA 90:2207–2211[Abstract]
  51. Beadling C, Ng J, Babbage JW, Cantrell DA 1996 Interleukin-2 activation of STAT5 requires the convergent action of tyrosine kinases and a serine/threonine kinase pathway distinct from the Raf1/ERK2 MAP kinase pathway. EMBO J 15:1902–1913[Abstract]
  52. Kirken RA, Malabarba MG, Xu J, Liu X, Farrar WL, Hennighausen L, Larner AC, Grimley PM, Rui H 1997 Prolactin stimulates serine/tyrosine phosphorylation and formation of heterocomplexes of multiple Stat5 isoforms in Nb2 lymphocytes. J Biol Chem 272:14098–14103[Abstract/Free Full Text]
  53. Kirken RA, Malabarba MG, Xu J, DaSilva L, Erwin RA, Liu X, Hennighausen L, Rui H, Farrar WL 1997 Two discrete regions of interleukin-2 (IL2) receptor beta independently mediate IL2 activation of a PD98059/rapamycin/wortmannin-insensitive Stat5a/b serine kinase. J Biol Chem 272:15459–15465[Abstract/Free Full Text]
  54. Wartmann M, Cella N, Hofer P, Groner B, Liu X, Hennighausen L, Hynes NE 1996 Lactogenic hormone activation of Stat5 and transcription of the beta- casein gene in mammary epithelial cells is independent of p42 ERK2 mitogen-activated protein kinase activity. J Biol Chem 271:31863–31868[Abstract/Free Full Text]
  55. Pircher TJ, Flores-Morales A, Mui AL, Saltiel AR, Norstedt G, Gustafsson JA, Haldosen LA 1997 Mitogen-activated protein kinase kinase inhibition decreases growth hormone stimulated transcription mediated by STAT5. Mol Cell Endocrinol 133:169–176[CrossRef][Medline]
  56. Gouilleux F, Wakao H, Mundt M, Groner B 1994 Prolactin induces phosphorylation of Tyr694 of Stat5 (MGF), a prerequisite for DNA binding and induction of transcription. EMBO J 13:4361–4369[Abstract]