(Received for publication, June 10, 1994; and in revised form, December 2, 1994)
From the
The glucocorticoid and transforming growth factor-
(TGF-
) regulation of growth and cell-cell contact was investigated
in the Con8 mammary epithelial tumor cell line derived from a
7,12-dimethylbenz(
)anthracene-induced rat mammary adenocarcinoma.
In Con8 cell monolayers cultured on permeable filter supports, the
synthetic glucocorticoid, dexamethasone, coordinately suppressed
[
H]thymidine incorporation, stimulated monolayer
transepithelial electrical resistance (TER), and decreased the
paracellular leakage of [
H]inulin or
[
C]mannitol across the monolayer. These
processes dose dependently correlated with glucocorticoid receptor
occupancy and function. Constitutive production of TGF-
in
transfected cells or exogenous treatment with TGF-
prevented the
glucocorticoid growth suppression response and disrupted tight junction
formation without affecting glucocorticoid responsiveness. Treatment
with hydroxyurea or araC demonstrated that de novo DNA
synthesis is not a requirement for the growth factor disruption of
tight junctions. Immunofluorescence analysis revealed that the ZO-1
tight junction protein is localized exclusively at the cell periphery
in dexamethasone-treated cells and that TGF-
caused ZO-1 to
relocalize from the cell periphery back to a cytoplasmic compartment.
Taken together, our results demonstrate that glucocorticoids can
coordinately regulate growth inhibition and cell-cell contact of
mammary tumor cells and that TGF-
, can override both effects of
glucocorticoids. These results have uncovered a novel functional
``cross-talk'' between glucocorticoids and TGF-
which
potentially regulates the proliferation and differentiation of mammary
epithelial cells.
The combined effects of systemic steroid and protein hormones as
well as locally acting growth factors control the normal development,
differentiation, and proliferation of the mammary
gland(1, 2, 3, 4, 5) .
Depending on their bioavailability, these extracellular signaling
molecules can interdependently, and in a temporally appropriate manner,
regulate the function of the three main differentiated cell types of
the mammary gland (i.e. stromal, myoepithelial, and epithelial
cells). The systemic humeral signals acting on mammary tissue include
the ovarian steroids and lactogenic hormones, such as prolactin and
glucocorticoids(6, 7, 8) . In addition, each
type of mammary cell can produce and/or respond to several classes of
growth factors (1, 2, 3, 4, 5) . Two
important mammary-derived mitogens are transforming growth factor-
(TGF-
) (
)and epidermal growth factor (EGF) which both
act through the tyrosine kinase EGF receptor (9, 10) .
TGF-
and EGF immunolocalize to morphologically distinct areas of
the mammary gland(11) , and the expression of these growth
factors is regulated at different stages of mammary gland growth and
development(1, 12, 13) . Both in vitro and in vivo studies have shown that TGF-
can have a
selective effect on the proliferation of normal mammary epithelial
cells(1, 5, 12, 14, 15, 16, 17) .
For example, exogenous administration of TGF-
stimulates ductal
and terminal end bud proliferation in the virgin mouse mammary
gland(11) , enhances lobuloalveolar development in
mitogenically primed mice(1, 14) , and stimulates
angiogenesis(15) .
Recent evidence suggests that TGF-
is involved in the early stages of breast tumorigenesis (1, 2, 5, 18) and acts as a potent
autocrine growth regulator of mammary tumor
cells(5, 12, 14, 19) . For example,
constitutive expression of TGF-
in a nontumorigenic mouse mammary
epithelial cell line induces anchorage-independent growth in
vitro(20) , while introduction of TGF-
cDNA into the
germ line of transgenic mice induces the appearance of
hormone-dependent mammary adenocarcinomas(21) . Many human and
rodent mammary tumors inappropriately express high levels of TGF-
in vivo(22) and in vitro(16) which
has been proposed to account, in part, for the cellular escape from
hormonal growth control of some transformed mammary epithelial cells.
Consistent with the proliferative advantage of mammary tumor cells that
produce this mitogen, TGF-
mRNA or protein expression has been
found in 40-70% of primary metastatic human breast
tumors(18, 23, 24) . Moreover, the expression
and secretion of TGF-
can be coordinately regulated with the
proliferative state of certain mammary tumors. In hormone-responsive
human mammary tumor cell lines, ovarian steroids stimulate in vitro proliferation with a concomitant increase in the production of
TGF-
(1, 5, 12, 14) , whereas
treatment of breast cancer patients with the anti-estrogen tamoxifen
often results in a significant decrease in the levels of TGF-
in
the tumor(25) .
It is tempting to consider that TGF-
mediates the dysregulation of mammary tumor cell responsiveness to
environmental cues by modulation or inhibition of the ability of
systemic factors, such as steroid hormones, to control mammary cell
differentiation and growth. To test this notion, we are utilizing Con8
mammary tumor cells, which are an epithelial cell line derived from a
7,12-dimethylbenz(
)anthracene-induced rat mammary
adenocarcinoma(4, 26, 27) . These rat mammary
tumor cells constitutively express cell surface glycoprotein antigens
related to the human PAS-O human milk fat globule protein demonstrating
that these cells retain differentiated characteristics of their mammary
epithelial origin(26) . We have shown that TGF-
and
glucocorticoid hormones have opposing actions on the proliferation of
these mammary tumor cells. Glucocorticoid hormones suppress the
growth(26, 27, 28) , induce c-jun expression(28) , and inhibit production of an
autocrine-acting TGF-
(29, 30) , whereas treatment
of steroid growth-suppressed Con8 cells with TGF-
restimulates DNA
synthesis (28, 29, 30) and induces expression
of c-myc and cyclin D1 transcripts(28) .
A key
issue is whether particular differentiated properties usually
associated with nontransformed mammary cells are also under reciprocal
control by glucocorticoids and TGF- in transformed Con8 cells. One
such differentiated property is the regulation of tight junction
permeability, since the onset of lactation in the normal mammary gland
coincides with an increase in the structural development of the tight
junction(31) . Thus, tight junction formation is important in
establishing cell-cell interactions by the formation of
``seals'' between laterally adjacent
cells(32, 33, 34) . We have recently shown in
a nontransformed mouse mammary epithelial cell line of ductal origin
that dexamethasone regulates tight junction
permeability(35, 36) . Our results suggest that a
similar ``normal-like'' differentiated property may be
conferred upon Con8 mammary tumor cells by glucocorticoids. In this
study, we show that glucocorticoids coordinately regulate tight
junction permeability and suppress the growth of Con8 mammary tumor
cells and that both steroid effects are reversed by exposure to
TGF-
.
CAT activity in the cell extracts containing 30-50 µg of
lysate protein was measured by the nonchromatographic assay of Neumann
and co-workers(38) . The enzyme assay was carried out in a
final reaction volume of 250 µl in the presence of 1 µCi of
[H]acetyl coenzyme A (specific activity 200
mCi/mmol, DuPont NEN), 25 µl of 1 M Tris-HCl, pH 7.8, and
chloramphenicol (50 µl of a 5 mM aqueous solution). The
reaction mixture was gently overlaid with 4 ml of a water-immiscible
scintillation fluor (Econofluor, DuPont NEN) and incubated at 37 °C
for 4 h. The CAT activity was monitored by direct measurement of
radioactivity by liquid scintillation counting. Measurements of CAT
activity were in the linear range of the assay as determined by a
standard curve using bacterial CAT enzyme. The enzyme activity was
expressed as a function of protein present in corresponding cell
lysates (
H-acetylated chloramphenicol produced, cpm/µg
of protein/4 h). Pure bacterial CAT enzyme (0.01 unit; Pharmacia,
Uppsala, Sweden) was utilized as a positive control for the CAT
enzymatic assays, while mock-transfected cells were used to establish
basal level activity. Each experiment was performed in triplicate and
was repeated two or more times.
Figure 1:
Dexamethasone stimulates
transepithelial electrical resistance and suppresses DNA synthesis of
Con8 mammary tumor cells. Upper panel, Con8 cells were
cultured on permeable supports in the presence (+DEX) or
absence (-DEX) of 1 µM dexamethasone. At
the indicated times, the monolayer transepithelial electrical
resistance was monitored, and the ohmscm
were
calculated as described in the text. Each assay was performed in
triplicate, and the results are an average from three separate
experiments. Lower panel, Con8 cells were cultured on
permeable supports in the presence (+DEX) or absence (-DEX) of 1 µM dexamethasone. The rate of
DNA synthesis was monitored at the indicated times during a 60-h time
course by determining the incorporation of
[
H]thymidine after a 1-h pulse label as described
in the text.
Conceivably, dexamethasone may be
stimulating monolayer tight junction-like properties by causing an
overgrowth of Con8 mammary tumors on the filters. However, consistent
with our previous observations using a plastic
substratum(25, 26, 28) , dexamethasone
significantly suppressed [H]thymidine
incorporation of Con8 mammary tumor cells grown on permeable supports
within 36 h of steroid treatment (Fig. 1, lower panel).
The suppression of [
H]thymidine incorporation
using cells cultured on permeable supports is within the time frame
observed for glucocorticoids to induce an early G
block in
cell cycle progression of cells cultured on plastic
substratum(28) . Thus, in the absence of glucocorticoids, Con8
mammary tumor cells rapidly proliferate and fail to produce a tight
monolayer, whereas stimulation of tight junction formation was
coincident with the dexamethasone inhibition of cell growth.
Figure 2:
Dexamethasone dose-response of
transepithelial electrical resistance, DNA synthesis, and receptor
function. Upper panel, Con8 mammary tumor cells were cultured
on permeable supports for 2 days in the presence of the indicated
concentrations of dexamethasone. The monolayer transepithelial
electrical resistance was determined, and the ohmscm
were calculated as described in the text. The apical to
basolateral leakage of [
C]mannitol (M
= 182) was monitored as described in the
text. The results are an average of triplicate samples. Lower
panel, Con8 cells were cultured on permeable supports and analyzed
for DNA synthesis by incorporation of
[
H]thymidine. A parallel set of cultures were
transfected with the GRE-CAT chimeric reporter gene by electroporation
and assayed for dexamethasone-stimulated CAT activity as described in
the text. The results are an average of triplicate
samples.
The
dexamethasone stimulation in TER also dose-dependently correlated with
glucocorticoid receptor transcriptional activation of the chimeric
GRE-CAT reporter gene as well as with an inhibition of DNA synthesis (Fig. 2, upper versus lower panels). Steroids which are
either neutral with respect to their glucocorticoid activity (such as
-estradiol or testosterone) or which show glucocorticoid
antagonist activity (RU38486, progesterone) failed to induce Con8
monolayer TER (data not shown). Thus, the dexamethasone stimulation of
Con8 monolayer TER dose-dependently correlated with glucocorticoid
receptor occupancy and function which suggests that tight junction
permeability is regulated in Con8 cells by a glucocorticoid
receptor-mediated process, rather than as a result of an aberrant
interaction between the steroid and the plasma membrane. Furthermore,
consistent with our previous results with nontransformed mammary cells (35) , the glucocorticoid-induced formation of tight junctions
in Con8 mammary tumor cells required de novo protein synthesis
and normal levels of extracellular calcium (data not shown).
Figure 3:
Effects of dexamethasone and TGF- on
cell cycle phase distribution of Con8 mammary tumor cells. Con8 cells
were treated with the indicated combinations of 1 µM dexamethasone (DEX) and 10 ng/ml human recombinant
TGF-
, cell extracts were stained with propidium iodide, and
nuclei were analyzed for DNA content by flow cytometry with a Coulter
Elite Laser. Approximately 10,000 cells were analyzed from each sample.
The percentages of cells within the G
, S, and
G
/M phases of the cell cycle were determined with the
Multicycle computer program as described under ``Experimental
Procedures.''
To determine whether TGF- reverses the
glucocorticoid-stimulated formation of tight junctions under the
conditions that promote cell cycle progression, confluent mammary tumor
cell monolayers grown on permeable supports were treated with 1
µM dexamethasone for 24 h to induce tight junction
formation and then incubated in the presence or absence of growth
factor. As shown in Fig. 4, the monolayer transepithelial
electrical resistance was reduced to basal levels within 18 h of
TGF-
treatment. We have previously shown that this time course is
sufficient for TGF-
to stimulate the cells to move through G
and S phases(28) . Thus, under conditions in which
TGF-
restimulates proliferation of glucocorticoid suppressed
cells, the function of pre-formed tight junctions is coordinately
disrupted.
Figure 4:
TGF- overrides the
dexamethasone-stimulated monolayer transepithelial electrical
resistance. Con8 mammary tumor cells were cultured on permeable
supports in the presence (+DEX) of 1 µM dexamethasone, and one control culture was incubated in the
absence of steroid (-DEX). After 24 h in dexamethasone,
cells were treated in the presence (+DEX/+TGF
)
or absence (+DEX) of 10 ng/ml of human recombinant
TGF-
. Throughout the 42-h time course, the monolayer
transepithelial electrical resistance was determined at the indicated
times, and the ohms
cm
were calculated. The results
are an average from triplicate samples.
Figure 5:
TGF- disrupts the
glucocorticoid-stimulated formation of tight junctions in the presence
of DNA synthesis inhibitors. Con8 mammary tumor cells were cultured on
permeable supports in the presence of 1 µM dexamethasone (DEX), while one set of control cultures were not treated with
hormone (No Additions). After a 48-h steroid treatment (arrow), dexamethasone-treated cells were incubated with the
indicated combinations of 10 ng/ml TGF-
(TGF-
) or either 10
µM cytosine
-D-arabinofuranoside (top
panels: araC) or 1 mM hydroxyurea (lower panels:
HU). At the indicated times, the monolayer transepithelial
electrical resistance was determined, and the ohms
cm
were calculated (left panels). At the final time point,
DNA synthesis was monitored by the incorporation of
[
H]thymidine as described in the text (right
panels). The results are an average from triplicate
samples.
Figure 6:
TGF- does not inhibit glucocorticoid
receptor function. Con8 mammary tumor cells were transfected with the
GRE-CAT chimeric reporter gene by electroporation and then cultured on
permeable supports for 48 h with the indicated combinations and time of
incubation with 1 µM dexamethasone (DEX) and 10
ng/ml TGF-
(TGF-
). Cell extracts were assayed for
CAT specific activity as described in the text. The results are an
average of two independent sets of triplicate
samples.
Figure 7:
Constitutive expression of transforming
growth factor- blocks the glucocorticoid stimulation of monolayer
transepithelial electrical resistance. Con8 mammary tumor cells and
CT93 cells, which constitutively express TGF-
, were cultured on
permeable supports in the presence (+DEX) or absence (-DEX) of 1 µM dexamethasone. The
transepithelial electrical resistance was monitored over a 48-h time
course, and the ohms
cm
were determined as described
in the text.
Figure 8:
Effects of glucocorticoids and
constitutive expression of transforming growth factor- on
paracellular transport of [
H]inulin. Con8 mammary
tumor cells and CT93 cells, which constitutively express TGF-
,
were cultured on permeable supports in the presence or absence of 1
µM dexamethasone for 3 days.
[
H]Inulin (M
= 5000)
was added apically and assayed in the basolateral media after 4 h. The
paracellular transport was calculated as the amount of radiolabeled
[
H]inulin detected in the basolateral media,
divided by the total amount of [
H]inulin added to
the apical media compartment. The baseline used to determine the -fold
induction is defined by the amount of [
H]inulin
which diffused through the support membrane of cell-free
filters.
To confirm that constitutive
expression of TGF- overrides the glucocorticoid-mediated growth
suppression response, nontransfected Con8 cells and
TGF-
-transfected CT93 cells were cultured on permeable supports,
and DNA synthesis was examined in cells treated with or without
dexamethasone. Dexamethasone inhibited
[
H]thymidine incorporation in nontransfected Con8
cells but not in the CT93 cells (Fig. 9). Thus, constitutive
expression of TGF-
prevents glucocorticoids from suppressing the
growth and regulating tight junction permeability. Conceivably, the
failure of glucocorticoids to stimulate TER and reduce paracellular
transport in CT93 cells could be due to clonal variation of transfected
cells and not due to the effects of TGF-
per se. Two
lines of evidence argue against this possibility. First, several
independently isolated subclones of transfected Con8 cells which
constitutively express TGF-
(30) show the same phenotype
as CT93 cells (data not shown). Secondly, the direct addition of
TGF-
to dexamethasone-treated Con8 cells reduced the monolayer TER
back to basal levels within 24 h of growth factor treatment,
concomitantly with a stimulation in [
H]thymidine
incorporation (Fig. 9). TGF-
addition to transfected CT93
cells did not further reduce the TER or stimulate DNA synthesis (Fig. 9).
Figure 9:
Effects of glucocorticoids and
transforming growth factor- on DNA synthesis and transepithelial
electrical resistance of mammary tumor cells. Con8 mammary tumor cells
and CT93 cells, which constitutively express TGF-
, were cultured
on permeable supports in the presence or absence of 1 µM dexamethasone for 3 days. Cells which had been treated with 1
µM dexamethasone for 48 h were then exposed to medium
containing dexamethasone and 10 ng/ml human recombinant TGF-
. The
cells were then incubated with both factors for 2 days. The
incorporation of [
H]thymidine and transepithelial
electrical resistance were monitored as described in the
text.
Figure 10:
Effects of glucocorticoids and
transforming growth factor- on ZO-1 localization. Con8 mammary
tumor cells were treated with no hormones (panel A: Con8 - DEX), 1 µM dexamethasone (panel B: Con8 + DEX), or dexamethasone and 10 ng/ml human recombinant TGF-
(panel C: Con 8 + DEX/+ TGF
) for 2 days. CT93
cells, which constitutively express TGF-
, were treated with 1
µM dexamethasone (CT93 + DEX) for 2 days.
The cells were fixed and analyzed for ZO-1 localization by indirect
immunofluorescence as described in the text. Cell pictures were
originally photographed at
430
magnification.
Our results with Con8 mammary tumor cells have demonstrated
that glucocorticoids can coordinately suppress cell proliferation and
stimulate tight junction formation and thereby confer to this
transformed cell type normal-like growth and differentiation
characteristics. Exposure of glucocorticoid growth-suppressed cells to
the mammary mitogen TGF- rapidly stimulated cell proliferation and
caused the dysregulation of tight junction permeability, resulting in a
loss of monolayer tightness. Moreover, constitutive expression of
TGF-
precluded glucocorticoids from mediating either the growth
suppression or tight junction responses. The TGF-
disruption of
tight junctions is based on the observed reduction in monolayer
transepithelial electrical resistance, stimulation of paracellular
transport, and redistribution of the ZO-1 tight junction protein.
Malignantly transformed mammary cells can often display a loss of
responsiveness to particular sets of extracellular signals. One
mechanism of dysregulation is the inappropriate production of growth
factors and/or function of their cognate receptors, which alter
proliferative and/or differentiated
properties(1, 2, 3, 4, 5) .
It is therefore tempting to consider that TGF-
may exert many of
its tumorigenic effects on mammary epithelial cells by not only
providing a proliferative advantage to transformed cells expressing EGF
receptors, but also by altering the way in which the cells respond to
steroid-induced signals, which are normally responsible for maintaining
critical cell-cell interactions at junctional complexes.
The
glucocorticoid stimulation of transepithelial electrical resistance is
a receptor-dependent process which occurs under conditions in which the
mammary tumor cells are growth-suppressed, whereas TGF-
simultaneously stimulates DNA synthesis and reverses the steroid
effects on tight junction permeability. This inverse relationship
between cell proliferation and regulation of cell-cell interactions may
have important implications for understanding mechanisms of
invasiveness and metastasis of mammary tumors. The unrestricted growth
of tumors is dependent upon vascularization of the tumor(42) .
It is conceivable that autocrine or paracrine growth factors with
angiogenic activities may regulate this process, in part, by causing
the dissolution of epithelial cell tight junctions to allow invasion of
new blood vessels. A number of other studies have shown that other
types of cell-cell interactions are altered in transformed cells. For
example, the formation of desmosomes, which are patches of
intercellular contacts(43) , is inversely related to the stage
of lung cancers and their ability to metastasize(44) .
Similarly, it was found that certain connexins, which are gap junction
proteins that regulate the formation of intercellular
channels(45, 46) , are transcriptionally
down-regulated in human mammary tumor cell lines but not in primary
normal or nontransformed cells(47) . It has been proposed that
the connexin-mediated channels help to transmit growth-controlling
signals between cells(47, 48) . Several connexins have
been shown to be selectively produced in nontumorigenic cells and when
overexpressed slow the growth of transformed
cells(48, 49) . Thus, an alteration in cell-cell
communication may protect transformed cells from particular types of
growth inhibition, or alternatively, growth-inhibited cells may be more
capable of forming particular intercellular junctional complexes. In
this regard, we have previously shown that glucocorticoids induce a
G
block in cell cycle progression of Con8 mammary tumor
cells(28) , suggesting that this growth suppression is a
prerequisite for the assembly of functional tight junctions.
The
TGF- disruption of tight junction formation did not require the
mammary tumor cells to be actively cycling since the growth
factor-mediated reduction in monolayer TER occurs in the presence of
two different inhibitors of DNA synthesis. Both araC and hydroxyurea
block cell cycle progression at the G
/S
boundary(39, 40) , whereas, as discussed above,
TGF-
overrides the glucocorticoid-mediated block in cell cycle
progression early in the G
phase(28) . These
results suggest that the regulation of tight junction functionality may
either be directly linked to the control of the G
phase of
the cell cycle up to the S phase boundary or that tight junction
formation may not be a cell cycle-regulated process per se.
Regardless of the precise connection between cell cycle control and
tight junction formation, the disruption of monolayer TER by TGF-
in the presence of DNA synthesis inhibitors implicates the tight
junction machinery as a selective target of EGF receptor signaling and
not just an indirect consequence of cell cycle progression after growth
factor treatment.
Components of intercellular junctional complexes
have recently been implicated in growth regulation, such as the genes
which encode certain tight junction-associated proteins and adhesion
molecules(50) . One such gene product is the tight
junction-associated ZO-1 protein which is homologous to a class of
tumor suppressor genes. The amino-terminal half of ZO-1 displays
significant sequence homology to the product of the lethal discs large (dlg) gene of Drosophila(50, 51) .
The dlg gene product is localized in the undercoat of the
septate junction in Drosophila which is considered to be
analogous to the tight junction of vertebrate epithelial cells.
Mutations in dlg result in a loss of apical-basolateral
epithelial cell polarity and in neoplastic growth which implicates this
gene as a tumor suppressor gene(50) . It is conceivable that
junctional plaque proteins play a role in suppression of the malignant
phenotype by orchestrating the interactions of junctional adhesion
receptors and cytoplasmic signal transducers which are involved in the
negative regulation of cell growth. Consistent with this idea, we have
shown that under conditions in which TGF- mediates a dysregulation
of Con8 cell growth and tight junction permeability, ZO-1 is
redistributed from a pericellular location to a more cytoplasmic
compartment. The regions of ZO-1 most homologous to the tumor
suppressor genes are the filamentous domain, an SH3 domain, and a
guanylate kinase domain(50, 51) . These domains
represent interesting potential targets for steroid or growth factor
control of the localization and/or function of this tight
junction-associated protein, since similar structural features are
fundamentally involved in receptor-mediated signal transduction (52) .
The maintenance of tight junction function is a
normal-like differentiated property which prevents the mixing of
molecules from the apical and basolateral membranes and which precludes
paracellular
permeability(34, 53, 54, 55) . Tight
junction permeability of mammary epithelia is regulated during the
onset of lactation in which milk components are strictly secreted into
the lumen of the ducts via apical-directed secretory
pathways(31) . Lactogenic steroid and protein hormones and a
variety of growth factors are known to be involved in regulating the
temporal and tissue-specific development of the lactogenic state. Our in vitro work with nontransformed mammary epithelial cells (35, 36) suggests that glucocorticoids are likely to
be the lactogenic hormones responsible for regulation of tight junction
permeability. Glucocorticoids can exert their effects on gene
expression by specific binding of the steroid receptor complex to DNA
transcriptional enhancer elements which are present in promoters of
steroid controlled genes, or by interfering with the action of other
transcription factors, such as the JunFos AP-1 transcription
complex, via protein-protein
interactions(56, 57, 58, 59) . Given
this mechanism of glucocorticoid hormone action, is it likely that
dexamethasone regulates the transcription of key genes encoding protein
components or regulatory factors which modulates tight junction
formation. The timing of glucocorticoid-induced gene expression is
critical in that it may initiate a transcriptional cascade in which
early regulated gene products initiate the growth suppression response,
whereas later-acting response genes may maintain the growth-inhibited
state and regulate tight junction permeability.
TGF- overrides
glucocorticoid growth suppression and coordinately causes a
dysregulation of tight junction permeability and ZO-1 localization
which suggests a novel role for TGF-
in disrupting the
differentiated function of mammary tumor cells. The degree of
permeability of the tight junctions is known to be regulated by
intracellular signals initiated by protein kinase C, phospholipase C,
adenylate cyclase, and GTP-binding proteins, as well as calcium (60, 61, 62, 63, 64) . Any
of these signaling components may be downstream targets of
TGF-
-mediated cascades initiated by activation of the EGF receptor
tyrosine kinase. We are currently attempting to elucidate the
transcriptional and secondary signaling events underlying TGF-
control that operate in mammary tumor cells and allow this growth
factor to override the effects of glucocorticoids. Conceivably, such
pathways represent an important ``cross-talk'' between growth
factor and steroid receptor signal transduction cascades that are
necessary to guide the functional relationships between particular sets
of environmental cues acting on mammary epithelial cells.