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
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ABSTRACT
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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.
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INTRODUCTION
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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, STAT1STAT6 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
-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.
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RESULTS
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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. 1
, A
and C). Dex also enhanced the expression of ß-casein in primary
thymocytes in the presence of IL-2 (Fig. 1D
), 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. 1
, A and C). IL-15, another cytokine that utilizes the ß- and
-subunits of the IL-2 receptor, also induced ß-casein in CTLL-2,
and this induction was also enhanced by Dex (data not shown).

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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.
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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. 1
, 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
ß cells that expressed both human GM-CSF receptor
-
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. 1
, AC). 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. 2A
) (22, 23).


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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.
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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. 2A
) 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. 2
, B and C). As expected,
STAT5 overexpression enhanced IL-2-dependent activation of the
ß-cas105, CIS, and OSM promoters (Fig. 2B
), 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. 2C
). Mutation or deletion of the STAT binding sites completely
abolished promoter activity (Fig. 2
, 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. 3
, 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.

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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.
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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. 3
). 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. 3
). 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. 3
), 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. 4B
). 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. 4A
). 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).

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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.
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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. 4C
). 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. 4C
), 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. 4D
). 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. 4D
),
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. 5A
). 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. 5A
). The role of Ras in STAT5 activation was further
examined by the EPOR mutant (EPORH), which lacks the ability to
activate Ras (Fig. 5B
). 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. 5B
). 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. 5B
), 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.

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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
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.
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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. 5B
).
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. 5B
). These results indicate that the Raf-MAPK
pathway is not responsible for the Ras-mediated enhancement of STAT5
activity.
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DISCUSSION
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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
- 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. 1A
), 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. 2A
). Likewise, although the promoters of OSM
and CIS genes contain TTCxxxGAA, but not TTCxxxxGAA, IL-4 also
activated these promoters (Fig. 2C
) and induced endogenous CIS and OSM
expression in CTLL-2 cells (Fig. 1
, 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. 2C
). 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. 2
).
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
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. 1B
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
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. 1A
). 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. 1A
). 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. 3A
). 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. 3B
), 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. 4
, 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. 4D
). 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. 4C
),
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. 5A
), 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. 5B
). 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. 5B
). 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. 6
).
As exemplified in the activation of the ß-casein promoter by
cytokines, gene expression is determined by integrating various
intracellular signaling events.
 |
MATERIALS AND METHODS
|
---|
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
promoter of pME18S. pLNC-
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
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, 11-1 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.
 |
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