(Received for publication, July 19, 1995)
From the
Transcription of tissue-specific genes in mammary gland requires
signals from both prolactin and basement membrane. Here we address the
mechanism by which this specialized extracellular matrix regulates
transcription. Using mammary cell cultures derived from transgenic mice
harboring the ovine -lactoglobulin gene, we show that either a
basement membrane extract, or purified laminin-1, induced high levels
of
-lactoglobulin synthesis. It is known that prolactin signals
through Stat5 (signal transducer and activator of transcription). This
transcription factor interacts with
-interferon activation
site-related motifs within the
-lactoglobulin promoter, which we
show are required for matrix dependence of
-lactoglobulin
expression. The DNA binding activity of Stat5 was present only in
extracts of mammary cells cultured on basement membrane, indicating
that the activation state of Stat5 is regulated by the type of
substratum the cell encounters. Thus, basement membrane controls
transcription of milk protein genes through the Stat5-mediated
prolactin signaling pathway, providing a molecular explanation for
previous studies implicating extracellular matrix in the control of
mammary differentiation.
Cell-matrix interactions are critical for regulating the
phenotype of many cells. In mammary gland, basement membrane is
necessary for the prolactin-mediated control of
lactation(1, 2, 3) . However, the mechanism
by which extracellular matrix (ECM) ()influences
differentiation in mammary epithelial cells has not been elucidated. We
have shown previously that functional
integrins are
required for mammary differentiation (4) and that the basement
membrane component laminin-1 directs milk protein gene transcription
coordinately with prolactin(5) .
The prolactin pathway is driven through the protein tyrosine kinase Jak2 (6, 7, 8, 9, 10) and one of its substrates, the transcription factor Stat5 (11, 12) . Stat factors associate with cytokine receptors following ligand binding and subsequently become phosphorylated by receptor-associated Jaks. They then dimerize and translocate to the nucleus where they bind specific DNA sequence motifs, thus contributing to transcriptional activation(13, 14, 15) .
In this paper we
examine whether an element of the prolactin signaling pathway is
modulated directly by cell-matrix interactions, thereby mediating the
ECM control of transcription. Using primary and first passage cultures
of mammary epithelial cells, we demonstrate that the activity of the
promoter for the milk protein -lactoglobulin (BLG) is dependent on
basement membrane and that Stat5 recognition sites within this promoter
are required for transcription. Moreover, we show that the ability of
Stat5 to bind its cognate DNA sequence within the BLG promoter requires
cell interactions with both basement membrane and prolactin. Thus,
matrix and cytokine signals converge on a single pathway.
Our data demonstrate, for the first time, that the activity of a Stat transcription factor is a target for regulation by ECM. This establishes a novel signaling route that is subject to control by cell-matrix interactions, in addition to those already described involving mitogen-activated protein kinases(16, 17) , insulin receptor substrate-1(18) , and Ras(17) .
Figure 1:
Structure of the BLG gene and
constructs used for generating transgenic mice. The structure of the
ovine BLG gene is shown at the top. BLG.SX/45 contains the
full gene including a 4.3-kb 5`-flanking sequence and 1.9-kb
3`-flanking sequence (20) . The 5`-promoter region was
truncated to 406 bp in BLGDp/46 and BLG
Dp/39(20) .
This region of the promoter contains three nucleotide sequences that
are bound by Stat5. Sites 1-3 are named, respectively, A1, A3S,
and STM, and the relative binding affinities of Stat5 to these sites
are 1:8:44(26) . Dinucleotides were mutated within each of
these factor-binding regions to generate BLG-SAA(22) . The wild
type 406-bp proximal promoter was also used to drive expression of CAT
in BC
Dp/188.
In order to dissect the mechanism of basement membrane control of milk protein expression, we used primary and first passage cultures of mammary epithelial cells. This enabled us to examine the signaling requirements for cells in culture that were as closely related as possible to those for cells in vivo. Initially we studied the activity of a transgenic ovine BLG promoter using a well established culture model for mammary differentiation(1, 2, 4, 5) .
In
transgenic mice, the BLG promoter sequence is strongly active in
mammary gland during pregnancy and lactation(20) . To assess
whether the activity of this promoter is regulated by cell-matrix
interactions, mammary epithelial cells from a series of transgenic mice
expressing BLG constructs (Fig. 1) were plated on plastic dishes
or on a reconstituted basement membrane extract from the EHS tumor and
cultured under differentiation conditions with lactogenic hormones.
After 3 days, the cultures were pulse-labeled with
[S]methionine, and cell lysates were prepared by
detergent extraction(5, 23) . Total lysates of mammary
epithelial cells isolated from pregnant BLG
Dp/39 mice showed that
although BLG was synthesized by cells cultured both on plastic and EHS
matrix, its expression was up-regulated dramatically by basement
membrane (Fig. 2A). Quantitative measurements indicated
a 3.7-fold increase of BLG expression on EHS matrix over plastic.
Immune precipitation experiments showed that endogenous mouse milk
protein synthesis was also up-regulated by this matrix (Fig. 2B), as expected from earlier
studies(1, 2, 4, 5) . Very similar
results were obtained from cultures of BLG.SX/45 mice, which contain
4.3 kb of the BLG promoter sequence rather than just the 406 bp of
BLG
Dp/39. Thus, the expression of a transgenic ovine BLG gene in
mammary epithelial cells is under the control of ECM.
Figure 2:
Basement membrane- and laminin-dependent
control of BLG promoter activity. Mammary glands from midpregnant
BLGDp/39 or BC
Dp/188 mice were digested and the epithelial
cells established in culture. A and B show that
expression of BLG was dependent on the type of substratum that the
cells encounter. First passage BLG
Dp/39 cells were plated on
plastic (P) or on a reconstituted basement membrane from the
EHS tumor (E) and cultured for 3 days with the lactogenic
hormones, prolactin, hydrocortisone, and insulin. At the end of the
culture period, the cells were pulse-labeled with
[
S]methionine for 27 min, the medium was
collected, and the cells were extracted with radioimmune precipitation
buffer. Aliquots of cell lysate representing equal amounts of newly
synthesized protein were (A) separated by 15% reducing
SDS-PAGE and (B) immune precipitated using an antibody
specific for mouse milk proteins, but not ovine BLG, before separation
by SDS-PAGE. The band designated BLG was confirmed by immune
precipitation with a BLG-specific antibody (not shown). No proteins of
this size were detected in radiolabeled whole cell extracts from
non-transgenic mouse cultures. Similar results to those shown were
obtained from BLG
Dp/46 and BLG.SX/45 mice. C shows that
purified laminin-1 induced expression of BLG. For this assay, first
passage BLG
Dp/39 cells were seeded on coverslips and then treated
with 200 µg/ml soluble ECM proteins diluted into the culture medium
for 4 days in the presence of lactogenic hormones(5) . Controls
received hormones but no ECM protein. Alternatively, cells were plated
on top of EHS gels. At the end of the culture period, cells were
pulse-labeled with [
S]methionine for 75 min and
extracted, and equal aliquots of newly synthesized total cell protein
were separated by SDS-PAGE. Shown is the region of the gel containing
BLG. D shows that the 406-bp promoter of the BLG gene itself
contains matrix-dependent control elements. Mammary epithelial cells
from pregnant BC
Dp/188 mice were either plated directly on plastic
or EHS matrix (P and E; leftpanel)
or were cultured on collagen before being replated as first passage
cultures on plastic, collagen I, or EHS matrix (P, C, E; rightpanel). After 4 days in the
presence of lactogenic hormones, cells were lysed and the activity of
CAT enzyme was measured. CAT enzyme activity in the EHS cultures was
increased 3.4-fold over plastic in primary cells and 2.9-fold over
plastic in first passage cells.
We have shown
that the basement membrane ligand responsible for triggering
-casein transcription in mammary epithelial cells is
laminin-1(5) . We therefore examined whether laminin-1 could
also activate BLG transcription in mammary cultures derived from
BLG
Dp/39 transgenic mice. Using an assay that we developed
previously(5) , first passage cells were plated on coverslips
and treated for 4 days with 200 µg/ml purified laminin-1 (19) or complete basement membrane matrix diluted into the
culture medium, together with lactogenic hormones. The rate of protein
synthesis was examined by pulse-labeling the cultures, followed by
detergent extraction and analysis by SDS-PAGE. As above, the cells
cultured on EHS matrix expressed high levels of transgenic BLG protein (Fig. 2C). We also found that the cells treated with
laminin-1 expressed high levels of BLG, whereas control cultures
treated with no exogenous ECM proteins expressed barely detectable
amounts of BLG. Similar levels of BLG synthesis to those in the
laminin-treated cells were seen in cultures exposed to the complete
basement membrane matrix diluted into the medium. These results
indicate that an individual, highly purified basement membrane
component can induce transcriptional activation from the BLG promoter.
To demonstrate that the matrix control elements are contained within
the proximal 406-bp promoter region rather than in sequences either
further upstream or in the 3`-flanking region of the BLG gene or in the
BLG coding region, mammary cultures were prepared from BCDp/188
transgenic mice
expressing the 406-bp promoter linked to
the chloramphenicol acetyltransferase reporter gene (Fig. 1).
Primary and first passage cells were cultured on tissue culture
plastic, a thin layer of collagen I, or EHS matrix in the presence of
lactogenic hormones (Fig. 2D). When cell extracts were
analyzed using a CAT assay, we found that CAT enzyme activity was
increased by 2.9-3.4-fold in cells cultured on the basement
membrane, a similar increase to that of BLG protein in cells from
BLG
Dp/39 mice.
Together, these experiments show that positively acting, basement membrane-dependent transcription control elements are contained within the proximal 406 bp of the BLG promoter and that these elements can be activated in cells treated with purified laminin-1.
The BLG promoter contains three DNA recognition sequences for the
transcription factor, Stat5 (also known as mammary gland factor (11, 12) or milk protein binding factor(26) ).
Stat5 is expressed in pregnant and lactating mammary gland (28) and binds -interferon activation site (GAS)-related
DNA sequence elements(29) . Since the GAS motifs bound by Stat5
are required for maximum BLG promoter function in
vivo(22) , we asked whether they were also required for
its activity in culture. Dinucleotide sequences within the three GAS
motifs in the proximal 406 bp of the BLG promoter were mutated to
abolish Stat5 binding activity, and mice that contained multiple copies
of this transgene were produced. In mammary cultures prepared from the
resulting BLG-SAA transgenic mice (Fig. 1), low levels of BLG
were transcribed from the mutant promoter, but there was no increased
synthesis of BLG in cells cultured on EHS matrix (Fig. 3, A and B). This result contrasted with that for endogenous
-casein, which was induced to a similar extent by the EHS matrix,
both in cultures from BLG
Dp/39 and BLG-SAA transgenic mice
harboring the wild type or mutant BLG promoter and in cultures from
BC
Dp/188 mice expressing the CAT reporter gene (Fig. 3, A and C). These results confirm previous conclusions
that Stat5 binding sites within the BLG promoter are required for a
high level of activity(22, 29) . Our observation that
BLG expression was not completely abrogated in the BLG-SAA cultures
indicates that additional matrix-independent factors have a role in
transcription from this promoter. However, since there was no increased
expression of BLG in cells cultured on the basement membrane matrix,
our results suggested strongly that Stat5 itself was necessary for the
matrix-dependent control of expression.
Figure 3:
Requirement of Stat5 binding sites for
activation of the BLG promoter by basement membrane. First passage
cultures were prepared from BLGDp/46 and BLG-SAA transgenic mice
and analyzed as described in Fig. 2. A shows a
gel of total lysates from cells cultured for 4 days with lactogenic
hormones on plastic (P), collagen I (C), or EHS
matrix (E) and then pulse labeled for 45 min with
[
S]methionine. A similar gel was exposed to a
storage phosphor imaging plate, and the radioactivity in the BLG and
-casein bands was determined. After normalizing to the total
radioactivity in each lane, the levels of BLG (B) and
-casein (C) were determined. EHS matrix increased the
activity of the wild type (BLG
Dp/46) promoter by 2.6-fold over
plastic but had virtually no effect on the activity of the mutant
(BLG-SAA) promoter. For the quantitative analysis, we also included
data from cell extracts of BC
Dp/188 cultures prepared at the same
time, where no proteins corresponding to BLG were detected. In contrast
to the results for BLG, the increase in endogenous
-casein
expression was similar for each of the three transgenic
lines.
We therefore examined the activity of Stat5 directly, in cells cultured on the different substrata in the presence or absence of prolactin. Cells from non-transgenic mice were cultured on plastic, collagen I, or EHS matrix and then harvested, and nuclear extracts were prepared(26) . DNA electrophoretic mobility shift assays were performed using the oligodeoxynucleotide sequence corresponding to the highest affinity Stat5 binding site in the BLG promoter(26) . Stat5 DNA binding activity was strongly induced in cells cultured on basement membrane matrix providing prolactin was present (Fig. 4A), and a similar DNA binding activity was present in extracts of whole mammary gland tissue (Fig. 4B). However, prolactin was unable to induce Stat5 activity in the absence of basement membrane.
Figure 4: Matrix dependence of transcription factor activity. A, primary mammary epithelial cells from non-transgenic midpregnant mice were cultured on plastic (P), collagen I (C), or EHS matrix (E) for 2 days in serum-containing medium. The cultures were washed, and the medium was changed to differentiation medium containing lactogenic hormones with or without prolactin for 0, 2, or 3 days. The cells were then harvested and equal quantities of cell protein were used for electrophoretic mobility shift assays. The DNA oligodeoxynucleotides used were the recognition motifs for Stat5, NF-I, and SP1. In the control lanes (Co), an additional extract from mammary gland was included for the Stat5 assay, and partially purified NF-I protein was included for the NF-I assay. B, equal quantities of cell protein from nuclear extracts of mammary gland or from the EHS sample prepared above (2 days in differentiation medium) were used for electromobility shift assays with the Stat5 probe. Antibodies, as indicated, were incubated with nuclear extract before addition of the probe. PY, phosphotyrosine.
To confirm that gel retardation was due to binding of Stat5 rather than other Stat factors, competition experiments were carried out in the presence of function-blocking anti-Stat antibodies. Complex formation was abolished by an anti-Stat5 antibody but not by antibodies specific for Stat1 (Fig. 4B) or for Stat2, -3, -4, and -6 (not shown). In addition, Stat5 gel shifts of mammary cell and tissue lysates were substantially blocked by anti-phosphotyrosine antibodies (Fig. 4B), confirming previous studies showing that phosphorylated tyrosine residues on Stat5 are required for its DNA binding activity(12) .
As controls, the DNA binding activity of Sp1 and NF-I transcription factors in these nuclear extracts were also determined. Sp1 was more active in cells cultured on plastic or collagen I substrata, whereas NF-I was more active in the EHS cultures, but this was not dependent on prolactin (Fig. 4A).
Together, our data show that the activities of transcription factors in mammary epithelial cells are regulated differentially by the type of substratum the cell encounters and that Stat5 only binds its cognate DNA sequence in cells that are cultured on a basement membrane.
It has been known for some time that expression of tissue-specific milk protein genes in mouse mammary gland requires cell interactions with basement membrane(1, 2, 3) . The milk-producing cells of alveoli interact with a laminin-rich basement membrane in vivo. Culture studies have demonstrated that this association with basement membrane provides signals for milk protein synthesis that are mediated through integrins (4) and derived from the ECM component laminin(5) . These locally acting signals operate together with lactogenic hormones, resulting in transcriptional activation of milk protein promoters (24, 30) via a mechanism that has not yet been elucidated.
In this work we have taken the problem one step back from the level of the promoter and shown that the DNA binding activity of a Stat transcription factor essential for milk protein transcription is controlled by cell-matrix interactions. This conclusion is based on a combination of studies with (i) cultures from transgenic mice, which showed that the BLG promoter was responsive to basement membrane signals but only if Stat5 DNA recognition motifs were intact and (ii) nuclear extracts of cultured cells, showing that the DNA binding activity of Stat5 required signals from an appropriate ECM. The type of matrix able to activate Stat5 was specific since cells cultured on collagen I had low levels of BLG promoter function and no detectable Stat5 DNA binding activity. Instead, the activation signal was contained within a basement membrane matrix and acted coordinately with prolactin.
The initial response
to a large class of ligands including prolactin is the rapid activation
of gene transcription mediated by Stat factors. Engagement of receptor
triggers a cascade of tyrosine phosphorylation events resulting in
transcriptional activation by
Stats(13, 14, 15) . In COS and Chinese
hamster ovary cells, which do not normally synthesize either an
endogenous prolactin receptor or Stat5, prolactin can induce de
novo transcription from a transfected casein promoter once its
receptor and Stat5 are ectopically expressed(11, 12) .
However, these minimal requirements for casein promoter activity are
not sufficient in mammary gland since prolactin does not induce
transcriptional activation of the -casein gene or high levels of
BLG expression in mammary cells cultured on plastic or collagen I. This
implies that tissue-specific gene expression in the natural context is
under complex, multifactorial control.
Two lines of evidence show
that the appropriate signaling components are present in mammary
epithelial cells cultured in the absence of suitable ECM cues. First,
cells plated on plastic synthesize functional prolactin receptors,
since the mRNA for interferon-regulated factor-1 is induced under this
culture condition in response to prolactin. ()Second, both
Jak2 and Stat5 proteins are present in mammary cells cultured on
plastic or collagen I, and their levels are comparable with those in
cells cultured on basement membrane matrix. (
)The lack of
Stat5 activity may therefore indicate the existence of additional
regulatory factors in differentiating mammary cells. For example,
serine phosphorylation is required for the formation of active Stat3
homodimers(32) ; in addition to the Jak2 phosphorylation site
on residue 694, Stat5 also contains a consensus site for
mitogen-activated protein kinase. This enzyme is activated through
cooperative signaling between insulin and ECM(18) , although it
is known that insulin signals themselves are not required for
prolactin-mediated activation of Stat5 (33) . Alternatively,
tyrosine-specific protein phosphatases, which are known to modulate the
activity of the erythropoietin receptor(34) , may have a role
in controlling some downstream effects of the prolactin receptor.
Our experiments additionally suggest that there are at least two
tiers of matrix-dependent regulation at the transcription factor level.
First, increased NF-I and decreased Sp1 activity in cells cultured on a
basement membrane may reflect a hormone-independent induction of the
differentiated state involving cell interactions with the type of ECM
that mammary cells encounter in vivo. This control might be
mediated via mechanochemical signals from cell-matrix
interactions(35, 36, 37) , or alternatively,
it could be transmitted through conventional signaling pathways
initiated by membrane-associated kinases such as pp125(38) and/or GTPases such as Rho and Rac(39) . A
second level of regulation occurs through a coordinate basement
membrane- and hormone-dependent triggering of Stat5 function, which
directly controls transcription from milk protein promoters.
Several
milk protein gene control regions, including the 406-bp BLG
promoter(26) , the -casein proximal promoter and its
enhancer element BCE1(30) , and the whey acidic protein
promotor(31) , contain potential recognition sequences for
Stat5. Since all of these genes are controlled by ECM, we propose that
the matrix-dependent activation of Stat5 provides an essential
contribution to the mammary differentiation program.
In conclusion,
this study suggests one signaling mechanism to explain previous
observations in the mammary system that implicate ECM in the control of
differentiation(1, 2, 3, 4, 5) .
We have demonstrated that transcription from the BLG promoter requires
mammary cells to interact with a basement membrane and that ECM and
lactogenic hormones cooperate in the regulation of Stat5 transcription
factor activity. To our knowledge, this is the first demonstration that
the function of a Stat family member is dependent on signals other than
cytokines/growth factors. In the light of recent data showing that
cell-matrix interactions also control mitogen-activated protein kinase
and insulin receptor substrate-1 activity, our findings now reveal a
related mechanism by which extracellular matrix regulates cell
phenotype. It is known that proliferation of normal mammalian cells
requires not just growth factors but also ECM
proteins(40, 41) , and recent studies have suggested
that other fundamental processes such as cell survival are dependent on
matrix(21, 42) . ()Thus, together these
studies point to a general control principle involving cross-talk
between cytokine- and matrix-mediated signaling pathways.