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(Received for publication, April 24, 1995; and in revised form, June 28, 1995) From the
In numerous studies on mammary epithelial cell lines multiple
factors, added to the medium or contained in the serum, were required
for casein gene expression. It has been shown in these systems that the
mammary gland factor (MGF) is implicated in the activation of the
Prolactin (PRL) ( PRL receptors are widely
distributed in mammalian tissues and immune cells and they belong to
the superfamily of genes encoding cytokines and hematopoietin class I
receptors. This family includes receptors for GH, erythropoietin,
interleukins 2-7 (IL-2-7), granulocyte colony-stimulating
factor, granulocyte-macrophage colony-stimulating factor, leukemia
inhibitory factor, oncostatin M, and ciliary neurotrophic factor. The
more distantly related class II family includes receptors for tissue
factor, interferon- Recent progress has also
been made in understanding the intracellular events which mediate
receptor activated gene expression. For several cytokines it has been
demonstrated that latent cytoplasmic transcription factors, termed
signal transducing factors (STFs), are rapidly translocated to the
nucleus after tyrosine phosphorylation. IFN- It is well established in several species that casein genes are
regulated at the transcriptional level by lactogenic hormones (PRL),
glucocorticoids, insulin, and extracellular matrix. Only small amounts
of caseins are detected in the resting mammary gland. The
transcriptional activation of casein genes by PRL involves a highly
conserved TTCnnnGAA consensus element in the promoters of these genes.
This sequence has been shown to bind MGF, whose activity is tightly
coordinated with mammary gland differentiation. This factor is
essential for the activity of the rat Recent studies from
our laboratory, on rabbit mammary cells, have shown that the induction
of
Figure 1:
Kinetics of MGF
induction by prolactin in the nucleus. Rabbit mammary cells were
incubated for 24 h in a hormone-free synthetic medium and then
submitted to prolactin treatment. Nuclear extracts were prepared at
different times after PRL addition to rabbit mammary epithelial cells
and examined by mobility shift assay with a labeled rabbit
Figure 2:
Effect of EGF, cycloheximide,
dexamethasone, and extracellular matrix on the MGF induction by PRL in
rabbit mammary epithelial cells. Nuclear extracts were prepared and
assayed as described in Fig. 1. A, deinduced mammary
epithelial cells were pretreated for 3 h or 15 min with EGF (10 ng/ml) (lanes 4 and 5 and lanes 6 and 7,
respectively) or with CHX (5 µg/ml) for 1 h (lane3). oPRL (500 ng/ml) was then not added (lanes1, 4, and 6) or added to the culture
medium for 15 min (lanes2, 3, 5,
and 7). B, mammary epithelial cells were cultured
directly on plastic dishes (lanes 1-5). Cells from
several dishes were trypsinized and reseeded on plastic (PL)
support (lane6) or on Engelbreth-Holm-Swarm (EHS) exogenous extracellular matrix (lane7). Cells were treated with oPRL (500 ng/ml) for
indicated periods of time. C, mammary epithelial cells were
not treated (lanes1 and 2) or treated with
dexamethasone (Dex) for 24 h (lanes3 and 4). PRL was then added as described in A to
nontreated cells (lane2) or to dexamethasone-treated
cells (lane4).
The
down-regulation of MGF activity has previously been observed in murine
mammary HC11 cells when they are cultured in EGF for 3 days (35) or most recently for several hours(41) . No
results are available on the early, within minutes, action of EGF on
mammary cells. Since EGF is known to rapidly induce tyrosine
phosphorylation of STAT
proteins(27, 45, 46) , we wanted to determine
whether EGF treatment might affect the activation of MGF in our system
or also activate different GAS-binding factor. Mammary epithelial cells
were incubated in synthetic medium with EGF and PRL for 15 min, or
pretreated with EGF for 3 h prior to PRL stimulation. EGF was neither
able to induce MGF binding activity, nor interfere with the appearance
of MGF activated by PRL (Fig. 2A). This suggests that
EGF does not control the initial activation of MGF and is not directly
involved in the slow down-regulation of MGF activity. ECM proteins
act synergistically with PRL in the induction of genes by PRL and
moreover, only limited casein mRNA synthesis occurs in the absence of
ECM. Furthermore, ECM has been shown to regulate gene expression at the
transcriptional level of several genes including the Since glucocorticoids are also known to
enhance milk production and are generally used in cultures of mammary
cells, we tested the effect of glucocorticoids in the same series of
experiments in serum-free medium (Fig. 2C).
Glucocorticoids appeared to have no effect on PRL-stimulated MGF
activation even after 24 h (Fig. 2C, lanes 3 and 4). The data reported in this section thus
demonstrate that EGF, ECM and glucocorticoids do not directly affect
the activation of MGF.
Figure 3:
Tyrosine phosphorylation of MGF is
necessary for its DNA binding activity. Nuclear extracts from mammary
epithelial cells were analyzed as described in Fig. 1. A, mammary epithelial cells were noninduced (lane1) or induced with 2 µg/ml oPRL for 20 h (lanes
2-7). Protein kinase inhibitors: staurosporine (St,
2 nM, lane3; 20 nM, lane4), genistein (G, 25 µg/ml, lane5), 6-dimethylaminopurine (6D, 2.5 mM, lane6), and H7 (10 µM, lane7) were then added for 4 h. Four micrograms of
nuclear protein extracts from these cells was analyzed in a gel
retardation assay using the rabbit
Figure 4:
Induction of two different
First,
the experiments with cytoskeletal disrupting agents (e.g. cytochalasin D and colchicine) demonstrate that these two
complexes respond differently to this treatment (Fig. 4B). While cytochalasin D and colchicine inhibit
the accumulation of casein mRNA in several
species(52, 53) , they did not affect the formation of
the PV-induced MGF-comigrating band (lanes 3-5) and MGF (lanes7-9). On the contrary, we observed that
these agents both preferentially affected formation of the faster
migrating complex induced by PV (lanes 3-5). It has
been shown recently that STAT5 induced in HC11 cells and STAT1 from
mouse macrophage cell line J774A.1 have different mobility in EMSA
experiments(41) . To further characterize the nature of the
faster migrating complex, we compared it to GAF, activated in rabbit
mammary epithelial cells, using unique properties of our cell system to
respond to PRL as well to IFN
Figure 5:
Activation of cytoplasmic and nuclear
To investigate further these DNA-binding complexes,
competition with several different GAS oligonucleotides was carried
out. MGF and MGF-comigrating rabbit
Figure 6:
Competition profile of different
Figure 7:
PRL and hGH, not bGH or pIFN
Our other results (not
shown) demonstrated that the faster but not the slower migrating
complex effectively competed with the Ly6 GAS, and partially with the
IFP GAS. Moreover, one antisera raised against STAT1 (Ab 1) recognized
faster migrating complex induced by PRL in Nb2 cells (not shown). These
data suggest that lactogenic hormones PRL and hGH, but not bGH or
IFN
Figure 8:
Analysis of cross-reactivity of MGF with
STAT1- and MGF-specific antibodies by Ab interference mobility shift
assay. A, the increasing amounts of the IgG fraction of NIS93
antiserum (lanes 3-6) were incubated for 10 min with 4
µg of nuclear extract prepared from mammary epithelial cells
induced in the synthetic medium for 15 min by oPRL. Labeled rabbit
Figure 9:
Analysis of the DNA-binding complexes
activated by hGH, bGH, pIFN
Our laboratory has developed the culture of rabbit primary
mammary epithelial cells in an effort to study casein gene expression
under physiological conditions. The cells are grown in a synthetic
nutrient medium, which contains no serum factors such as prolactin,
glucocorticoids, or insulin. Under these conditions, we have been able
to demonstrate the casein gene induction in response to PRL alone. This
differs from other systems, which require the addition of several
hormones for casein induction. This has enabled us to study casein gene
induction in response to a single ligand, PRL. Our results provide
clear evidence that the induction of MGF occurs rapidly after the
addition of PRL to the culture medium. The rapid activation of MGF is
consistent with the rapid accumulation of casein mRNA by 1 h, reaching
a high level 4 h after PRL addition to mammary epithelial
cells(44) . The MGF activation occurs in the cytoplasm within 5
min, subsequently accumulating in the nucleus, reaching a maximum level
by 15 min. The rapidity of activation and its resistance to CHX is
similar to what has been observed for other STFs. Our data strongly
support the idea that PRL-R is directly involved in the MGF activation
in mammary epithelial cells enabling it to translocate to the nucleus
and activate target genes. Our results demonstrate that
dexamethasone does not affect MGF activation and that PRL alone is
sufficient for MGF activation. Previous studies on MGF activation,
carried out in HC11 cells, required the presence of glucocorticoids
along with PRL. While these hormones have been shown to activate MGF in
HC11 cells, EGF has been shown to have an inhibitory
effect(35) . As our studies were being completed, rapid
activation of MGF by PRL in murine HC11 cells was
reported(40) . However, the cells were cultured in medium
containing 10% fetal calf serum. Thus, it is not clear from this work
whether other factors may have contributed to PRL activation of MGF.
Additionally, the rapid effect of EGF on MGF activity was not observed
in our system (i.e. mammary epithelial cells). This is
consistent with recent data indicating that the inhibitory action of
EGF was only observed after several hours of incubation of cells with
this growth factor and was half-maximal after 10 h(41) . This
suggests that the inhibitory effect observed in HC11 cells is therefore
most likely indirect and does not interfere with the mechanism leading
to the rapid activation of MGF by prolactin. Furthermore, present
studies suggest that the effect of glucocorticoids on milk gene
expression over 24 h of action on cells is not exerted at the level of
MGF activation. This result is consistent with recent data describing
the activation of MGF by PRL, observed in HC11 cells, cultured for 5
days at confluence in the absence of glucocorticoids(41) .
Although it is not clear why a very long culture conditions were used
to induce MGF activity in the absence of glucocorticoids in HC11 cells
in contrast with our cell system, these observations indicate that
other mechanisms mediate transcriptional effect of glucocorticoids on
casein gene expression. A promoter element, BCE-1, which is thought
to respond to ECM, has been described in the distal region of bovine
Data presented here provide
evidence that the activation of endogenous MGF involves tyrosine
phosphorylation. The activation is blocked by tyrosine kinase
inhibitors, and GAS binding activity is sensitive to treatment with a
tyrosine-specific phosphatase or with anti-phosphotyrosine antibody.
This is consistent with recent work demonstrating that tyrosine
phosphorylation of MGF is necessary for its DNA binding and recent
reports indicating a role for the Jak2 kinase in activation of STAT5 in vitro(33, 57) . Moreover, the kinetics of
MGF activation described here correlate well with the kinetics of Jak2
activation reported by others(10) . Thus, the modulation of
receptor-associated protein-kinase Jak2 activity may determine the MGF
activity in response to PRL. Other recent data indicate that Jak1 and
Jak3 can also be involved in STAT5 activation in response to IL-2 in
CTLL-2 cells(78) . As our manuscript was being revised, another
group reported that, although STAT3 DNA binding is not blocked by H7,
transcriptional activation is blocked. In contrast, our results with
PKC inhibitors H7 and GF 109203X indicate that PKC does not play a role
in Although
termination of protein-tyrosine kinase signals most likely involves a
tyrosine dephosphorylation, it is not known if protein-tyrosine
phosphatases regulate PRL transduction. A number of studies have
demonstrated that activation of STAT proteins occurs after the addition
of phosphotyrosine phosphatase inhibitors to
cells(73, 74) . We present data which
suggest that PV-induced complexes correspond to MGF and
IFN Our
other results demonstrate that PV alone is unable to induce casein mRNA
in mammary explants after 8 h of treatment and even show some
inhibitory effect on prolactin action in this system. Interestingly,
some enhancement of existing basal level of casein gene transcription,
generally observed in mammary explants, is detected after 24 h of PV
treatment(71) . These data strongly implicate other factors
involved in PV activity, such as positive or negative regulators. For
instance, the relief of transcriptional repression by another factors
regulated by lactogenic hormones, single-stranded DNA-binding
transcriptional regulators, may be necessary to activate casein gene
expression(63) . Thus, PV treatment that activates MGF may be
not sufficient to relieve the repression by single-stranded DNA-binding
transcriptional regulator, or activate another essential factor. We
cannot exclude the hypothesis that, additionally, PME Our
results demonstrate the activation of different STFs in mammary
epithelial cells after a number of stimuli. Interestingly, hGH and, to
a much lower extent, bGH appear to induce MGF in mammary epithelial
cells. These factors show the same competition pattern, are recognized
by the same antiserum NIS93, and comigrate with MGF. Furthermore, hGH
is known to be a ligand for PRL-R and to induce lactation in the
rabbit, whereas bGH shows only a low affinity for rabbit PRL-R with
little lactogenic activity(54, 55) . Thus, we suggest
that hGH, and to a much lesser degree bGH, interact with the rabbit
PRL-R to activate MGF. This low activation of MGF by bGH may explain
why this hormone is not able to induce casein gene expression. We
cannot exclude, however, the possibility that GH may also activate MGF
through interactions with the GH receptor. We compared the PRL and
other cytokines signaling in highly differentiated mammary epithelial
cells and Nb2 cells whose proliferation is PRL- or hGH-dependent.
PRL-dependent tyrosine phosphorylation of several proteins in these
cells has been reported(8, 9) . Our results
demonstrate that two GAS-binding complexes were detected in cytoplasmic
and nuclear fractions of PRL- and hGH-treated cells. The faster complex
comigrated with IFN
Volume 270,
Number 36,
Issue of September 08, pp. 20952-20961, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
, Growth Hormones, and a
Tyrosine Phosphatase Inhibitor in Rabbit Primary Mammary Epithelial
Cells (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-casein gene promoter. In the present study, we determined the
relationship between known agents that affect casein gene expression
and MGF activity using the properties of rabbit primary mammary
epithelial cells to respond to PRL alone, when cultured in chemically
defined medium. We demonstrate that MGF is rapidly activated by PRL
alone or by human growth hormone, a natural ligand of many PRL
receptors (PRL-Rs), in the cytoplasm and accumulated in the nucleus.
The MGF activation by PRL occurred in the absence of endogenous
extracellular matrix, a condition where casein synthesis is known to be
markedly reduced. Different inhibitors of protein-tyrosine kinases,
which have been shown to reduce casein mRNA synthesis, but not of
protein kinase C, decrease the MGF activity. A tyrosine phosphatase
inhibitor, sodium pervanadate, induced two GAS-binding complexes
related to MGF and STAT1. Our data show that MGF is a latent
cytoplasmic factor rapidly activated in mammary epithelial cells, by a
mechanism involving a tyrosine kinase and a tyrosine phosphatase.
)is a pleiotropic hormone that
mediates a wide range of biological effects. PRL regulates diverse
physiological mechanisms such as lactation, reproduction, cell growth,
osmoregulation, steroidogenesis, and immune
response(2, 3, 4) . PRL and growth hormone
(GH) are members of family of polypeptide hormones produced in the
anterior pituitary gland and share similarities in their primary
structure(4, 5) . Human growth hormone (hGH) is
particular since it binds to both the GH receptor or PRL-R and is
considered as a genuine lactogenic hormone.
(IFN), and interferon- (IFN
)
receptors(4, 6, 7) . The
cytokine/hematopoietic receptors do not contain intrinsic tyrosine
kinase activity in their cytoplasmic domain. However, many studies
suggest that members of the Janus family of tyrosine kinases mediate
this activity for these receptors, including the
PRL-R(8, 9, 10, 11) . Studies of
mutant cell lines have demonstrated that IFN
/ receptor
signaling requires coexpression of Jak1 and Tyk2, whereas Jak1 and Jak2
were essential for IFN receptor
signaling(12, 13, 14) . Furthermore, these
kinases have also been shown to be associated with the cytokine
receptors(15, 16, 17, 18, 19, 20, 21) .
Both tyrosine-specific Jak2 and serine/threonine-specific Raf-1 kinases
have recently been shown to be associated with PRL-R and to be
activated by PRL in the PRL-dependent rat T-cell line Nb2 (22, 23, 24) .
for example,
stimulates the rapid tyrosine phosphorylation of STAT1 (p91). Activated
p91 homodimerizes forming STF-IFN
(STAT1:1), which is competent
for nuclear translocation and binding to a GAS element (reviewed in (25) ). The DNA-binding sites for factors activated by
epidermal growth factor (EGF), platelet-derived growth factor, IL-4,
and GH are also closely related to the GAS consensus, and can in many
cases bind
STF-IFN
(17, 26, 27, 28, 29, 30) .
The molecular cloning of transcription factors induced by interferons
and other cytokines has revealed the existence of a growing family of
signal transducing factors, STF/STATs. Recently cloned members of the
family include STAT1
, STAT1, STAT2, STAT3, STAT4, STAT5,
STAT5, and IL-4
STAT(31, 32, 33, 46, 60, 75, 76, 77) . -casein gene
promoter(34, 35) . As these studies were being
completed, Wakao et al.(33) reported the cloning of
MGF and demonstrated that tyrosine phosphorylation is required for the
DNA binding activity of the recombinant factor. It has been suggested
that MGF be renamed STAT5, based on its homology to other STAT
factors(60) . More recently, when our paper was ready for
submission, tyrosine phosphorylation of STAT5 by prolactin in
transfected COS cells and in vitro phosphorylation of STAT5 by
tyrosine kinase Jak2 was described(57) .S1- and -casein genes by PRL is abrogated by protein
kinase inhibitors, suggesting that the phosphorylation of transcription
factors may be crucial for milk gene activation(1) . In the
present study, we have examined the events leading to MGF activation by
PRL and GH, and compared it to the induction of STFs by IFN in
rabbit mammary epithelial cells (referred to throughout this report as
mammary epithelial cells). The response to PRL of Nb2 cells, used
before as a general model to evaluate lactogenic properties of PRL and
GH from different species, has also been examined. Our data support a
model where receptor-associated tyrosine kinases rapidly mediate PRL
signaling to the nucleus by the activation of a latent cytoplasmic
transcription factor, MGF in mammary epithelial cells, but not in Nb2
cells, where a different STF-IFN
-related factor could be involved.
These findings support the idea that biological differences in hormonal
action of PRL in different cell systems are supported at the molecular
level by activation of different latent cytoplasmic factors.
Cell Culture
Primary mammary epithelial cells
were isolated from mid-pregnant rabbits by enzymatic digestion of
mammary gland essentially as described (1) with some
modifications. Cells were maintained for 1 day in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum
and 2 or 3 days in a serum- and a hormone-free synthetic medium
(Dulbecco's modified Eagle's medium, 4% Ultroser SF; IBF,
France). Ovine prolactin (NIH PS13), bovine GH (recombinant bGH
provided by G. Kann), human GH (recombinant hGH provided by J. Djiane),
porcine IFN (recombinant protein kindly provided by C. La
Bonnardière), protein kinase inhibitors, protein
phosphatase inhibitors, and cytoskeleton disrupting agents were added
to the medium prior to collecting cells for cytoplasmic and nuclear
extracts preparation. The Nb2-11C cells (provided by A. Gertler) were
grown in RPMI 1640 supplemented with 5% fetal calf serum. They were
factor-starved for 20-24 h in the above medium containing 5%
gelded horse serum (not containing PRL). All experimental procedures
with U937 cells and M12.4.1 (M12) cells stimulated by IFN
and
HepG2 cells stimulated by IL-6 were as described
previously(28) .
Antibodies
Monoclonal anti-phosphotyrosine
antibody PT 66 was from Sigma. Rabbit NIS93 antiserum prepared from a
nonimmunized rabbit was unexpectedly found to be able to recognize MGF.
Polyclonal rabbit antiserum (Ab 1-4) were generated against
various domains of STAT1 (p91) and used as described(28) . Ab 1
recognizes p91 amino acids 609-716, Ab 2 recognizes p91 amino
acids 715-750, Ab 3 recognizes p91 amino acids 2-66, and Ab
4 recognizes amino acids 515-607 of the denatured form of STAT1.Electrophoretic Mobility Shift Assay (EMSA) and Cell
Extracts
Mammary tissue was frozen in liquid nitrogen and
reduced to powder prior to making extract. Extracts from rabbit PME and
Nb2 cells were prepared using the solutions containing MOPS instead
HEPES and nuclei were separated from cytoplasmic fraction on the
sucrose cushion as described previously(36) . To avoid
individual differences between rabbits which are not isogenic, each
experiment described in this paper has been repeated generally several
times. Cytoplasmic, nuclear, or whole cell extracts were prepared
essentially as described previously(37, 38) . Protein
concentrations were estimated using the Bio-Rad assay kit. Standard
shift reactions were carried out with 4 µg of crude extract
incubated with 0.1 ng of labeled double-stranded probe for 20 min at 25
°C. Oligonucleotides were labeled by filling in 5`-protruding ends
with Klenow enzyme, using [-P]dCTP (3000
Ci/mmol). Rabbit
-CAS GAS probe was a double-stranded
oligonucleotide representing the sequence -81 to -106 in
the rabbit -casein gene promoter(39) :
CATATGGAGTTCTTGGAATTGAAACT. The sequence of the mutated probe was
CATATGGAGTTGTTACAATTGAAACT. For the preparation of the double-stranded
probes, the upper strand was hybridized to a primer (agtttcaatt) and
filled in with Klenow enzyme. Oligonucleotide NTAT was designed as
palindrome agctAGAAATTAATTTCT, containing single-stranded 5` overhangs
at both ends and filled in with Klenow enzyme after annealing. Rabbit
-CAS GAS probe was used in EMSA analyzing the shifts in 1
TAE, 5% acrylamide (39:1) gels. The following probes: the rat
-CAS
GAS (gatcGACTTCTTGGAATT), the IRF-1 GAS (gatcCGATTTCCCCGAAAT), the IFP
GAS (gatcCAGATTCTCAGAAA), and the Ly6 GAS (gatcATATTCCTGTAAGT) were
prepared and used in EMSA in 0.22 TBE, 4.5% acrylamide (29:1)
gels as described previously(28) . Phosphotyrosine phosphatase
PTP 1B coupled to agarose beads (Upstate Biotechnology Inc.) was
preincubated with extracts for 1 h at 37 °C before adding labeled
probe. When needed, the tyrosine phosphatase inhibitor sodium vanadate
(1 mM) was added to the reaction mixture. To perform antibody
supershift assay, antisera were added to the binding reaction in a
final dilution of 1:20 unless otherwise indicated.
MGF Is Rapidly Activated by PRL and Down-regulated
during Casein Synthesis in Primary Mammary Epithelial Cells
Independently of PRL Concentration
It has been shown previously
that MGF binding activity can be detected in HC11 line of mouse mammary
epithelial cells after several days of combined PRL and glucocorticoids
treatment or 24 h after PRL treatment in COS cells cotransfected with
cDNAs for MGF and PRL-R(33, 35) . As our studies were
being completed, our observation of a very rapid activation of MGF in
HC11 cells was independently corroborated(40, 41) .
HC11 cells, derived from COMMA-1D cell line, have been used for studies
on the transcriptional regulation of -casein gene expression.
However, they have some limitations in their use because of their
relatively poor response to PRL. Furthermore, the induction of MGF by
PRL must be accompanied by additional hormonal stimulation by
glucocorticoids and insulin at confluence to activate -casein gene
expression(61, 63) . Additionally, it was observed
that the activation of MGF in HC11 cells is not sufficient to stimulate
the transcription from the promoter composed of multimeric MGF sites,
whereas the same promoter is activated by IL-6 in HepG2
cells(40) . Moreover, all studies on this cell line were
conducted in the presence of serum. Another cell model have been used
by B. Gröner and co-workers. They used COS cells
overexpressing the PRL-R and STAT5(57) . Here also, authors
used a serum-containing medium, thus not avoiding possible
contamination by factors and hormones interfering with milk genes
expression. Thus it is not clear from all these studies if MGF DNA
binding and its transactivation potential are regulated by PRL alone or
require synergistic action with insulin, glucocorticoids, and seric
factors. We have examined the time course of MGF activation by oPRL in
rabbit mammary epithelial cells, where the induction of milk genes can
be mediated by PRL alone in chemically defined medium. When cells were
incubated in a hormone-free synthetic medium for 24 h, MGF was then not
detectable (Fig. 1, lane2). However, nuclear
MGF was rapidly detected by EMSA in nuclear extracts (NE) prepared from
PRL-treated mammary cells. This GAS binding activity peaked at 15 min
and then began to decrease by 1 h, reaching a stable level between 4
and 24 h post-PRL treatment (Fig. 1, lanes 3-7).
The MGF complex in NE from cultured cells has the same electrophoretic
mobility as the MGF complex prepared from lactating mammary gland (lane1). In all studies on the MGF activation
published elsewhere, PRL was used at supraphysiological concentrations, e.g. 1-5 µg/ml. It has been shown previously in our
laboratory that there exists a reciprocal correlation between the rate
of casein synthesis, casein mRNA concentration, and the down-regulation
of the PRL receptors as a function of PRL concentration in cultures of
rabbit mammary gland explants(70) . To address the question of
PRL concentration and the down-regulation of MGF activity, another set
of cells from different animal were cultured with physiological
concentrations of PRL (50 ng/ml). Nuclear MGF binding activity was
detected as early as 5 min after stimulation with PRL, peaked again at
15 min, and then decayed slowly between 1 and 4 h (Fig. 1, lanes 8-11). This correlates well with the measurement
of PRL-stimulated casein secretion (using a radioimmunoassay) in
mammary epithelial cells cultured in synthetic medium, where no
significant differences are detected between the two different
concentrations of PRL (not shown). These results clearly demonstrate
that PRL alone is sufficient to activate MGF and suggest that the
down-regulation of MGF activity occurs independently of PRL
concentration.
-CAS
GAS probe in a 5.0% polyacrylamide gel. PRL-stimulated nuclear extracts
were prepared from mammary epithelial cells, untreated (lane2) or treated with oPRL added at concentrations of 2
µg/ml (lanes 3-7) and 50 ng/ml (lanes8-11) for indicated time. LMG, nuclear
extract from lactating rabbit mammary gland (lane1).
MGF complex is indicated with arrow.
The Activation of MGF by PRL Occurs at the
Post-translational Level and Is Not Affected by Extracellular Matrix,
Glucocorticoids, or EGF
The rapid activation of MGF by PRL is
similar to the rapid activation of the other STAT transcription factors (17, 37, 42, 43) and suggests that a
post-translational activation mechanism is involved. If PRL activates a
preformed factor, its induction should not require de novo protein synthesis and should therefore be resistant to
cycloheximide (CHX). We therefore examined the ability of PRL to
activate MGF in the presence or absence of CHX in mammary epithelial
cells. The inability of CHX to block MGF activation (Fig. 2A) is consistent with the fact that PRL
stimulates the accumulation of casein mRNA in mammary epithelial cells
for at least 2 h in the presence of CHX(44) .
-casein in
mammary epithelial cells (reviewed in (47) ). An enhancer
element, possibly responding to the ECM and PRL, has been localized in
distal region of bovine -casein gene promoter. Although ECM and
PRL can activate the enhancer independently, the ECM response has not
been differentiated from the PRL response in terms of sequence
requirements(48, 49) . Curiously, this DNA fragment
contains MGF consensus sequence TTC TCA GAA, raising the possibility of
the involvement of MGF in ECM response. To determine if ECM proteins
play a role in MGF activation, mammary cells were trypsinized and
reseeded on plastic support to eliminate the endogenous ECM present in
primary cultures. In these conditions caseins are not detected in the
culture medium of primary cells reseeded on plastic and stimulated by
PRL as we have previously demonstrated(1) . Nuclear extracts
were prepared from both cells replated in the absence and presence of
exogenous matrix and no difference was observed in MGF binding activity (Fig. 2B).MGF Activation by PRL Is Affected by the Protein-tyrosine
Kinase Inhibitors Involved in the Down-regulation of Casein
mRNA
It have been shown that tyrosine phosphorylation event is
essential for DNA binding activity of MGF purified from mammary gland
of lactating sheep (33) or overexpressed in COS
cells(57) . The situation is not clear for mammary epithelial
cell line HC11. While Welte et al.(41) shown that the
treatment of HC11 cells with genistein, or incubation of NE from 15-min
PRL-stimulated cells with anti-phosphotyrosine antibody reduce the DNA
binding activity of MGF, Marte et al.(69) described
that a staurosporine derivative CGP 41251, a potential inhibitor of
protein kinase C (PKC), reduced the DNA binding of MGF induced by 24 h
of combined PRL/insulin/glucocorticoid treatment of HC11 cells. Thus it
is not still clear if the MGF activity depend essentially on tyrosine
phosphorylation event or serine/threonine phosphorylations could also
be required. Therefore, we used several protein-tyrosine or
serine/threonine kinase inhibitors, which have been shown recently to
abrogate or not to abrogate the induction of rabbit S1- and
-casein genes in cultures of mammary epithelial
cells(1, 71) . MGF activity was evaluated in cells
cultured in synthetic medium and induced by PRL for 20 h, followed by
treatment with inhibitors (Fig. 3A). Similar results
were obtained when cells were pretreated with inhibitors for 4 h, prior
to a 15-min stimulation with PRL (Fig. 3B). This
suggests that the same kinases are involved in both the rapid and
prolonged activation of MGF. Staurosporine, 6-dimethylaminopurine,
which are broad spectrum kinase inhibitors, and genistein, a more
specific tyrosine kinase inhibitor (see (1) , and references
therein), prevented the PRL-induced activation of MGF (Fig. 3, panel A, lanes 4-6, and panelB, lanes3-6), while a potent
EGF-R tyrosine kinase inhibitor, tyrphostin 25, was ineffective (Fig. 3B, lane7). Interestingly, the
inhibitory effect of staurosporine on the MGF activity was
dose-dependent, analogous to its dose-dependent inhibition of casein
gene expression. Furthermore, a significant inhibitory effect on the
MGF binding activity was observed in extracts prepared from cells
pretreated with staurosporin for either 15 min or 1 h, followed by a
15-min PRL stimulation. To address the involvement of PKC, we used a
well known PKC inhibitor, H7, and an additional very selective
inhibitor of most of PKC isoforms, bisindolylmaleimide GF
109203X(50) . This compound has been shown to inhibit
completely PKC in HC11 cells and in primary rabbit mammary cells
without altering the induction of casein gene expression by
prolactin(72) . (
)GF 109203X only marginally
decreased the intensity of MGF shift (Fig. 3B, lane8), and H7 did not affect the MGF activity (Fig. 3A, lane7). The fact that
serine/threonine kinase inhibitors neither reduce STAT5 activity over
15 min or 24 h of induction with PRL nor abolish the casein mRNA
synthesis suggests the essential role of tyrosine phosphorylations in
MGF activity. To further confirm the role of tyrosine kinases in STAT5
activation in mammary cells, nuclear extracts prepared from
PRL-stimulated cells were treated with human PTP 1B, a
tyrosine-specific phosphatase (Fig. 3C, lanes
2-5) or with a tyrosine-specific monoclonal antibody (lanes6 and 7). As expected, PTP 1B
abolished STAT5 DNA binding (lanes2 and 4)
and vanadate, a phosphatase poison, prevented the loss of binding (lanes3 and 5). The ablation of STAT5 DNA
binding by monoclonal anti-phosphotyrosine antibody (lanes6 and 7) and not by control ascites (lanes8 and 9) further confirms that a phosphotyrosine
residue is critical for STAT5 binding. These observations, along with
the kinase inhibitor results, are consistent with a model in which PRL
activation of MGF, like other STFs, requires a tyrosine phosphorylation
event.
-CAS GAS as probe. B,
mammary epithelial cells were incubated for 4 h in the presence of
protein kinase inhibitors staurosporine (20 nM, lane3; 200 nM, lane4), 6-dimethyl
aminopurine (2.5 mM, lane5), genistein (25
µg/ml, lane6), tyrphostin 25 (TP, 10
µM, lane7), and GF 109203X (GF, 10 µM, lane8), and then
induced by oPRL (500 ng/ml) for 15 min. C, effect of
protein-tyrosine phosphatase and monoclonal anti-phosphotyrosine
antibody on the MGF DNA binding activity. Nuclear extracts from
PRL-induced mammary epithelial cells (at 500 ng/ml for 15 min) were
preincubated for 1 h at 37 °C with increasing amounts of
recombinant PTP 1B (Upstate Biotechnology Inc.) (lanes2-5) or with monoclonal anti-phosphotyrosine
antibody (-pY) PT66 (lanes6 and 7) and control (C) ascites (lanes8 and 9). PV (1 mM) were added to inhibit the
action of PTP1B in the reaction mixture (lanes3 and 5). Labeled -CAS GAS probe was then added for an
additional 15-min incubation and analyzed in gel retardation
assay.
The Tyrosine Phosphatase Inhibitor Sodium Pervanadate
Activates Two Distinct DNA-binding Complexes in Primary Mammary
Epithelial Cells
Signaling through PRL receptor is promoted by
the activation of one or more phosphotyrosine kinases and presumably
down-regulated by one or more phosphotyrosine phosphatases. No data are
known on the involvement of phosphatases in the regulation of PRL
signaling. To assess the possible role of endogenous tyrosine
phosphatases in STAT5 activation, we treated mammary epithelial cells
with sodium pervanadate (PV), a known protein-tyrosine phosphatase
inhibitor(51) . In these studies we employed the peroxy
derivative of sodium vanadate, which penetrates intact cells more
readily. A 15-min incubation of mammary epithelial cells with
pervanadate resulted in the appearance of two GAS-binding complexes,
one of which comigrated with MGF induced by PRL. The maximal
stimulation was observed after 1 h of treatment, comparable to the
intensity of MGF complex after 1 h of PRL induction (Fig. 4A, lanes3, 4, and 8). The formation of both complexes was inhibited by
staurosporine, added 1 h before PV treatment (lane5)
but not by the serine/threonine inhibitor GF 109203X (lane7), suggesting that the inhibitory effect of
staurosporine is tyrosine-specific. Additionally, PRL was not able to
restore MGF activity in cells treated by staurosporine and then induced
by PV (Fig. 4A, lane6). The
induction of two GAS-binding complexes by PV is an intriguing result.
Recently the existence of two APRF (STAT3) forms, which differ in
electrophoretic mobility(67) , has been demonstrated. Most
recently, the slower migrating form of APRF was identified as
functionally different, serine-phosphorylated form of STAT3,
transcriptionally active(68) . Welte et al.(41) also have recently described the presence of three
MGF complexes in HC11 cells, but these results were not confirmed by
other groups. Although, our results show for the first time that a
tyrosine phosphatase inhibitor can induce two GAS-binding complexes, we
addressed a question about their identity. Several lines of evidence
demonstrate that one complex is STAT5 and the second complex, migrating
faster, is a different factor but not a converted form of MGF as a
result of post-transcriptional modifications induced by PV.
-CAS
GAS-binding complexes by a phosphatase inhibitor, sodium pervanadate.
Gel retardation assay was performed with 4 µg of nuclear extracts
incubated with the labeled rabbit -CAS GAS probe as described in Fig. 1. A, mammary epithelial cells were not treated
with inhibitors (lanes 1-4 and 8) or pretreated for 1 h
with the protein kinase inhibitor staurosporine (200 nM, lanes5 and 6) and with GF 109203X (10
µM, lane7) and then nonstimulated (lane1) or stimulated with 500 ng/ml oPRL for 15 min (lane2) or 1 h (lanes6 and 8) or with 10 µM PV for 15 min (lane3) or 1 h (lanes 4-7). B, effect
of cytoskeleton-disrupting agents on MGF and PV-induced factors.
Mammary epithelial cells were submitted to colchicine (Col, 1
µM, lanes3 and 7) or
cytochalasin D (CD, 2 µM, lanes4, 5, 8, and 9) treatment
before induction with 10 µM PV for 1 h (lanes
2-5) or with 500 ng/ml oPRL for 15 min (lanes
6-9). C, comparison of electrophoretic mobility of
PV-, PRL-, and IFN-induced
-CAS GAS binding activities.
Mammary epithelial cells were not treated (lanes1, 2, 4, 6, and 8) or pretreated with
200 nM staurosporine for 15 min (lanes3, 5, and 7) and then induced with 500 ng/ml oPRL for 15
min (lanes2 and 3), with PV at 10
µM for 1 h (lanes4 and 5),
with pIFN at 500 ng/ml for 15 min, or with combined action of PV
for 45 min followed by pIFN
treatment for additional 15 min (lane8).
. Cells were treated with oPRL and
porcine IFN
(pIFN
) for 15 min, or with PV for 1 h. As shown
in Fig. 4C, the pIFN
-induced complex (referred to
as PME
GAF) comigrates with the faster PV complex. PMEGAF
was induced in a dose-dependent manner, with a detectable signal at 50
ng/ml pIFN, and maximum signal between 0.5 and 5 µg/ml (not
shown). Furthermore, the two DNA binding activities induced by PV were
inhibited by the staurosporine pretreatment in the same manner as MGF
and PME
GAF (Fig. 4C, lanes3, 5, and 7). Interestingly, IFN enhanced both MGF
and PME
GAF complexes when added to PV-treated cells (lane8), suggesting that common protein kinases may be
involved in MGF and PMEGAF activation. Additionally, another
tyrosine phosphatase inhibitor, phenylarsine oxide, failed to induce
MGF DNA-binding factors and even blocked PRL induction of MGF complex
(data not shown). These data demonstrate that PV is able to stimulate
the activation of two DNA-binding complexes that appear to be MGF and
GAF-related, and suggest that tyrosine phosphatase in addition to a
tyrosine kinase is involved in the activation of casein gene
expression.MGF Is Rapidly Activated in the Cytoplasm by PRL and GH
prior to Nuclear Translocation
While rapid activation of nuclear
STAT5 was described elsewhere and confirmed in the present work,
nothing is known about the possible cytoplasmic activation of this
factor. To address this question, cytoplasmic and nuclear extracts were
prepared and tested by EMSA using the rabbit -CAS-MGF probe. To
further characterize complex hormonal regulation of mammary epithelial
cells, we examined the ability of other lactogenic hormones, human and
bovine GH, to stimulate MGF in mammary epithelial cells. Human GH and
oPRL bind the rabbit PRL-R with high affinity and exhibit a potent
lactogenic activity in these cells. However, bovine GH is considered as
totally devoid of PRL-like activity in explants from bovine mammary
gland and has a very low affinity for recombinant rabbit
PRL-R(54, 55) . (
)Cells were treated by
oPRL, pIFN, hGH, and bGH for 15 min. MGF activity was detected in
the cytoplasm within 5 min after PRL stimulation, with comparable
intensity at the 15-min time point (data not shown). As expected, hGH
markedly induced a DNA binding activity that comigrated with MGF both
in cytoplasm and nuclear extracts (Fig. 5, lanes2, 4, 7, and 9). bGH was only
able to induce a weak complex that comigrated with MGF (Fig. 5, lanes5 and 10). Interestingly, pIFN
induced two complexes in the cytoplasm, but only one of them was
translocated to nuclei. The nature of the slower complex, which
comigrates with MGF, but fails to translocate to the nucleus is
unclear.
-CAS GAS binding activities by PRL, hGH, bGH, and pIFN.
Mammary epithelial cells were induced in the synthetic medium for 15
min by oPRL (lanes2 and 7), pIFN
(lanes3 and 8), hGH (lanes4 and 9), and bGH (lanes5 and 10) at 500 ng/ml each. The cytoplasmic (lanes1-5) and nuclear (lanes 6-10)
extracts were prepared and analyzed by EMSA with the rabbit
-CAS
GAS probe as described in Fig. 1.
CAS GAS binding activities all
competed well with the rabbit -CAS GAS (MGFw) and IFP GAS (whose
consensus is identical to those of -CAS GAS), less effectively
with the Ly6 GAS (whose sequence lacks G residue, critical for MGF
binding), and not at all by a mutated -CAS GAS (MGFm) or a
nonspecific heterologous oligonucleotide, NTAT (Fig. 6). The
competition profile of MGF from lactating mammary gland was identical
to MGF from primary mammary cells (data not shown). PMEGAF DNA
binding was also well competed by MGFw IFP and Ly6 GAS but not with
MGFm or heterologous NTAT. Competition experiments with PV-induced
complexes indicate that they had the same profile as MGF and
PMEGAF. These studies further support the notion that PRL, hGH,
and bGH activate the same MGF complex. In addition, PV appears to
induce simultaneously MGF and another factor different from MGF.
-CAS
GAS nuclear binding factors with oligonucleotides containing various
GAS elements. To analyze DNA-binding specificities of different factors
induced in mammary epithelial cells, 4 µg of nuclear extracts from
these cells treated with oPRL, hGH, bGH, and pIFN, as described in
the legend to Fig. 5, or with PV at 10 µM for 1 h
were subjected to gel retardation assay with the rabbit
-CAS GAS
probe. Different treatment agents are indicated on the left of
the panel. As competitors, a 50 M excess of each of the
following unlabeled double-stranded oligonucleotides was added to the
binding reaction: homologous rabbit -CAS GAS MGF site (MGFw, lane2), mutated -CAS GAS MGF
site (MGFm, lane3), human IFP 53-IFN
response element (IFP, lane4), human
Ly6E/A-IFN
response element (Ly6, lane5), and an heterologous oligonucleotide rich in A and T
as control (NTAT, lane6).
Treatment of Nb2 Cells by Lactogenic Hormones Induces
GAF-related Factor
We were interested in comparing the above
described PRL actions to those that have been extensively characterized
in Nb2 cells, which were used as a model to study the
prolactin-like activity of many hormones. These cells express
PRL-R and are PRL-dependent for their growth. They also proliferate in
response to hGH, but not of bGH. Cytoplasmic and nuclear extracts were
prepared from Nb2 cells after 15 min of oPRL, hGH, bGH, or pIFN
stimulation and compared with the complexes stimulated in mammary
epithelial cells. No complexes were detected after bGH or pIFN
treatment (Fig. 7A and data not shown). Two GAS-binding
complexes were detected in cytoplasmic and nuclear fractions of PRL-
and hGH-treated cells (Fig. 7A). None of the two
complexes comigrated with MGF. The faster complex comigrated with
PME
GAF and other GAFs, and the slower migrating complex had the
similar mobility to APRF/STAT3 complex from IL-6-stimulated HepG2 cells (Fig. 7, A and B).
,
activate rat
-CAS GAS-binding factors in Nb2 cells. A,
PRL and hGH rapidly activate rat -CAS GAS-binding factor in Nb2
cells to translocate from the cytoplasm to the nucleus. Cytoplasmic (lanes 7-10) or nuclear (lanes 11-14)
extracts prepared from Nb2 cells treated with oPRL (lanes8 and 12), bGH (lanes9 and 13), or hGH (lanes10 and 14) for
15 min at 500 ng/ml each were assayed by EMSA with rat -CAS GAS
probe in 4.5% polyacrylamide gel. Lanes7 and 11, untreated Nb2 cells; lanes1-5,
nuclear extracts from mammary epithelial cells (PME) untreated (lane1) or treated with oPRL (lane2), pIFN (lane3), hGH (lane4), or bGH (lane5). Lane6, whole cellular extracts from IL-6-treated HepG2 cells.
The position of migration is shown for MGF from mammary epithelial
cells extract and for faster migrating complex (designated as FMC), for Nb2 extracts. B, analysis of the
electrophoretic mobility of STF-PRL from Nb2 cells with different GAFs
by EMSA. Nuclear extracts from human U937 cells treated by IFN
(lane1), rat Nb2 cells treated by oPRL (lane2), rabbit mammary epithelial cells treated by pIFN
(lane3), murine M12 cells treated by IFN
(lane4), and IL-6-treated HepG2 cells were prepared
as described under ``Materials and Methods'' and assayed by
EMSA in 5% polyacrylamide gel using the rat IRF-1 GAS
probe.
, induce a STAT1-related factor. This is consistent with
reports indicating that STAT1 is activated in Nb2 cells (24) .
PRL- and GH-activated DNA-binding Proteins Are
Antigenically Similar and Recognized by MGF-specific Antiserum;
Anti-p91 Antibodies Recognize PME
We wanted
to further confirm whether MGF complexes induced by PRL, GH, and PV are
identical, by testing each DNA-binding complex for reactivity to a
panel of antisera generated against various conserved domains of STAT1,
as well as naturally occurring MGF-specific antiserum (NIS93). To
confirm the antibody nature of supershifted complexes, the IgG fraction
from NIS93 and from a control nonimmune rabbit (SCL) was prepared by
ammonium sulfate precipitation(56) . Nuclear or cytoplasmic
extracts were incubated with antibodies and then analyzed by EMSA.
These experiments revealed a dose-dependent supershift of the MGF
complex by the IgG fraction of NIS93 (Fig. 8A).
Moreover, ovine anti-rabbit IgG (SMAL) gave an additional supershift,
further indicating an IgG-dependent MGF supershift with the NIS93 (Fig. 8B). Interestingly, the two p91-specific
antisera, one of which (Ab 3) recognizes APRF/STAT3, failed to
recognize MGF, as judged by the results obtained with the rabbit
GAF but Not MGFCAS GAS probe (Fig. 8B). In more detailed studies
with the rat CAS GAS probe (Fig. 8C), we used Ab
1-3 (which recognize different conserved domains of STAT1), Ab 4
(which recognizes only the denatured form of STAT1), or NIS93. As
expected, MGF was recognized by NIS93 but not with Ab 1-4 (Fig. 8C). NIS93 was not able to recognize STAT1
induced by IFN in U937 cells or STAT3 induced by IL-6 in HepG2
cells (not shown), suggesting that this antibody is directed against a
domain of STAT5, not conserved in other STAT factors. Moreover, NIS93
recognized hGH- and bGH-induced complexes (Fig. 9A), a
fact also consistent with their presumed identity to the PRL-induced
complex. Furthermore, Ab 3 supershifted PME
GAF complexes from
either IFN- and PV-treated mammary epithelial cells (Fig. 9, A and B). These data support the idea
that this shifted complex is related to STAT1. Our study indicates that
PRL, GH, and PV activate STAT5. In addition, it confirms that PV
induces in mammary epithelial cells another factor, apparently
identical to IFN
-induced complex and related antigenically to
STAT1, but different from MGF.
-CAS GAS probe was then added for additional 15 min prior to the
electrophoresis in nondenaturing 5.0% polyacrylamide gel. The volume
(in µl) of antiserum added to the reactions is indicated on the top
of the panel. SCL: control rabbit serum (lane2). B, NE from PRL-induced mammary epithelial cells were incubated
for 15 min with different antibodies: STAT1-specific Ab 3 (lane2) and Ab 1 (lane3), MGF-recognizing
IgG fraction of NIS93 (0.5 µl) (lanes 4-6), ovine
antiserum raised against rabbit IgG, SMAL (lane7),
and rabbit control serum, SCL (lane8). Additionally,
SMAL and SCL antisera were also added in the two reactions with NIS93
antiserum 7 min later (lanes5 and 6). Gel
retardation analysis was performed after additional incubation for 15
min with the labeled rabbit -CAS GAS probe followed by the
electrophoresis in nondenaturing 5.0% polyacrylamide gel. C,
MGF binds to the IRF-1 GAS element but does not cross-react with
anti-p91 antibodies. Supershift reactions were performed with anti-p91
Ab 1-4 (lanes3-6), NIS93 (lane2), and nonimmun serum (lane7)
preincubated with nuclear extract of PRL-treated mammary epithelial
cells (PME/PRL) before adding IRF-1 GAS probe and analyzed in the 5%
polyacrylamide gel.
, and PV in mammary epithelial cells.
Ab interference mobility shift assay was performed as described in Fig. 7A using the rabbit
-CAS GAS probe. A, mammary epithelial cells were treated as described in
legend to Fig. 5. Nuclear extracts from cells stimulated with
pIFN (lanes 1-5), hGH (lanes6-10), and bGH (lanes 11-15) were
incubated with control serum (lanes2, 7, and 12), Ab raised against STAT1 (lanes3, 4, 8, 9, 13, and 14) or with NIS93 for 10 min followed by a 15-min incubation
with the labeled probe before submitting the reaction mixture to the
gel electrophoresis. B, NE from mammary epithelial cells
treated with PV (10 µM) for 1 h was incubated as in A with control serum (lane2), Ab raised against
STAT1 (lanes 3 and 4) or with NIS93 (lane5) and analyzed by supershift antibody assay with the
rabbit
-CAS GAS probe as described in A.
-casein gene(49) . This enhancer is able to respond to PRL
in the absence of ECM. The presence of the MGF consensus sequence
within the BCE-1 enhancer suggests that MGF is involved in its
activation by PRL, but its contribution to ECM response is not clear.
However, our results, demonstrating the activation of MGF in secondary
cultures where ECM proteins are not detectable (
)(under
conditions where only limited casein synthesis occurs), suggest that
ECM exerts its effect on casein genes expression independently of MGF
activation. Furthermore, our results suggest that ECM signaling is
distinct from that of PRL(71) .-casein expression(1, 71, 72) . These
observations are consistent with very low PKC activity in mammary cells
during late pregnancy and lactation, periods closely associated with
the expression of milk protein genes(58) .
Our studies demonstrate
for the first time that two different GAS-binding complexes are
simultaneously detected in the NE of cells treated with sodium
pervanadate. We used a unique properties of rabbit mammary cells which
respond to PRL as well to IFN to characterize these complexes.
PME
GAF, the factor induced by IFN in mammary epithelial
cells, is different from MGF, based on their distinct mobilities in
EMSA and their different reactivity to a panel of antisera.
PME
GAF interacts with the MGF site of the rabbit -casein
promoter. However, IFN alone is not able to induce caseins.
Furthermore, our preliminary results indicate that treatment of mammary
epithelial cells with pIFN
shows a dose-dependent inhibitory
effect on PRL induced casein synthesis (results not shown). This
suggests that PME
GAF might have an inhibitory effect on casein
gene expression. The mechanism that would allow the -casein
promoter to bind yet discriminate between STF-IFN and STF-PRL is
unknown but could be related to the presence of a YY1 site, which is
juxtaposed to the
-CAS GAS. It has been proposed that YY1 is
involved in the repression of casein gene
transcription(61, 62) .-inducible factor. Their activation is prevented by the protein
kinase inhibitor staurosporine, indicating that tyrosine kinases are
involved in the PV mediated activation of GAS-binding complexes.
Although the exact mechanism of activation by PV remains unclear,
studies on the receptors for IL-3, granulocyte-macrophage
colony-stimulating factor, erythropoietin, and ciliary neurotrophic
factor
IL-6 suggest that a receptor-associated tyrosine
phosphatases SH-PTP1 may play an essential role in signal
down-regulation (see (59) , and references therein).GAF could
exert its inhibitory effect on mRNA casein synthesis by competing with
MGF when both factors are induced by PV. Furthermore, it is not clear
if possible post-translational modifications, other than tyrosine
phosphorylations, are necessary to confer the transactivation potential
to STAT5 by PRL action. If it is the case, then PV treatment could only
induce MGF DNA binding but not its transcriptional activity.-induced factor in rabbit mammary cells and
other GAFs, and is antigenically related to STF-IFN
. This
indicates that in these cells, PRL signaling could use a STAT1-related
factor. The characterization of a second complex will indicate if rat
STAT5 is also induced with PRL and hGH. Data from another group
indicate that PRL can activate factor immunologically related to STAT5
in Nb2 cells. (
)Our observations together with data from
other groups raise the question of weather some STAT proteins may be
involved in the proliferative response of immunocompetent cells to
prolactin and growth factors, while another set of specific STFs are
involved in the functioning of nonproliferating highly differentiated
mammary epithelial cells. The molecular mechanisms of such specificity
of response to the cytokines by cognate receptors and transcription
factors activation remains an area of intense investigation. Recent
publications of Heim et al.(64) and Stahl et al.(65) give insight in the understanding of molecular
mechanisms of ligand-dependent specificity activation of STFs. They
show that tyrosine-containing motifs in cytokine receptors and STAT SH2
groups may play a crucial role in determining the specificity of their
interactions. Proposed models involve ligand-driven receptor
dimerization and activation of associated kinases, which in turn
activate STATs. The use of chemically defined medium clearly
demonstrate in the present work that pervanadate alone is able to
induce the formation of two distant DNA-binding complexes. This first
example of simultaneous activation of different STFs in the absence of
ligands for IFN-R and PRL-R, which suppose the presence of only
monomeric forms of cognate receptors, raises the question how
pervanandate could activate signal transducing factors with different
specificity. Moreover, our data indicate that the binding activity of
MGF does not strictly reflect its ability to induce casein gene
expression. These data and our other results (71) suggest that
a tyrosine phosphatase is also involved in triggering the induction of
casein gene expression.
)-activated factor; PME
GAF, primary mammary epithelial
cells IFN-induced complex; GAS,
activation site; APRF,
acute-phase response factor; GH, growth hormone; bGH, bovine GH; hGH,
human GH; IL-2-7, interleukins 2-7; EGF, epidermal growth
factor; PKC, protein kinase C; NE, nuclear extract; ECM, extracellular
matrix; EMSA, electromobility shift assay; CHX, cycloheximide; PV,
sodium pervanadate; TP, tyrphostin 25; Ab, antibody; MOPS,
4-morpholinepropanesulfonic acid.
)))))
We thank Drs. C. La Bonnardière,
J. Djiane, G. Kann, A. Gertler, and G. Kirilowsky for providing us,
respectively, with: pIFN, hGH, bGH and SMAL antiserum, Nb2 cells,
and GF 109203X.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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M. Wartmann, N. Cella, P. Hofer, B. Groner, X. Liu, L. Hennighausen, and N. E. Hynes 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., December 13, 1996; 271(50): 31863 - 31868. [Abstract] [Full Text] [PDF]
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