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Action in Normal Mammary Epithelial Cells1
Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263
Address all correspondence and requests for reprints to: Dr. Margot M. Ip, Grace Cancer Drug Center, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York 14263. E-mail: mip{at}sc3101.med.buffalo.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF) can
regulate normal mammary epithelial cell (MEC) growth, morphogenesis,
and, under certain circumstances, functional differentiation in a
manner similar to epidermal growth factor (EGF). As TNF has been
shown to up-regulate EGF receptor (EGFR) expression and function in
other systems, the present studies were undertaken to determine whether
TNF action in MEC was indirect through stimulation of the EGFR. An
inhibitor of EGFR tyrosine kinase activity, PD158780, failed to block
proliferation induced by 40 ng/ml TNF and only partially inhibited
growth in response to 2 ng/ml TNF. PD158780 was also unable to
suppress the extensive morphological development induced by either
TNF concentration. In contrast, the effects of TNF and PD158780
on functional differentiation (i.e. casein accumulation)
were time dependent. When measured on day 7 after 48 h of
treatment, casein accumulation was unaffected by either concentration
of TNF or by PD158780. When assessed on day 21 after 16 days of
treatment, however, casein levels were decreased by 40 ng/ml TNF and
increased by PD158780. Significantly, this PD158780-induced increase in
casein was not observed in MEC that had been treated with both PD158780
and TNF. These results thus suggest that EGFR tyrosine kinase
activity is not necessary for TNF action in normal MEC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF) is a
multifunctional cytokine that affects the growth, differentiation,
and/or function of virtually all cell types (1, 2, 3, 4). These pleiotropic
effects are mediated in part through two distinct cell surface
receptors of molecular masses 55 and 75 kDa (p55 and p75,
respectively), which are expressed specifically and independently of
each other in varying proportions on virtually all cells (5, 6, 7).
Although the receptors have some sequence homology in their
extracellular domains, evidence suggests that the two actually have
unique postreceptor signaling pathways and ultimately induce different
responses. For example, a large majority of TNF activities,
including cytotoxicity (8, 9), antiviral activity (10), and stimulation
of fibroblast proliferation (11), are mediated by p55, whereas the
75-kDa TNF receptor transduces signals for thymocyte proliferation (8),
inhibition of hematopoiesis (12), and granulocyte-macrophage
colony-stimulating factor secretion (13).
In contrast to the well documented cytotoxic or cytostatic effects of
TNF in breast cancer cells (2, 14, 15), our laboratory has
demonstrated that TNF actually stimulates the growth and
morphological development and regulates the function of normal mammary
epithelial cells (MEC) in primary culture (16). These studies used a
primary culture system in which undifferentiated rat MEC, cultured in
the presence of a defined, serum-free medium, proliferate,
morphologically develop, and functionally differentiate to an extent
comparable to that of cells within the mammary gland of a rat during
lactation (17, 18, 19). Specifically, TNF was found to stimulate MEC
proliferation in both the presence and absence of epidermal growth
factor (EGF), a major growth and differentiation factor for the MEC in
this culture system, and we have recently determined that the p55 TNF
receptor is the primary mediator of this TNF-induced growth (20).
Both TNF and EGF also stimulated the morphological development of
the MEC organoids, inducing the formation of large, well differentiated
alveolar colonies that, in the case of TNF, had extensive ductal
branching (16). Lastly, TNF modulated the functional differentiation
(casein accumulation) of the MEC organoids in culture; however, these
effects were more complex than its effect on either growth or
morphogenesis. Higher concentrations of TNF inhibited casein
accumulation, but in the absence of EGF, lower concentrations of TNF
were found to stimulate casein accumulation at later times in culture,
albeit to a lesser extent than EGF (16). In addition, we have recently
determined that the two TNF receptors have opposing effects on MEC
functional differentiation, with the p55 receptor signaling inhibition
of casein accumulation, and p75 signaling stimulation (20).
Despite these recent discoveries, the postreceptor pathway(s) of TNF
action in normal MEC is still unknown. Indeed, it is not yet known
whether the actions of TNF are direct or indirect via the induction
of another cytokine or growth factor. One potential indirect signaling
pathway may involve the EGF receptor (EGFR). Previous studies have
indicated that EGFRs are present on normal MEC (21, 22, 23), and that MEC
can synthesize various EGFR ligands, including TGF (24, 25),
amphiregulin (26), and EGF (21, 27). In addition, numerous studies have
shown that TNF can affect the EGFR on various levels, including
messenger RNA (mRNA) expression, number and binding affinity of
receptors, phosphorylation status, and tyrosine kinase activity (26, 28, 29, 30, 31), and TNF can also induce the expression of various ligands
of the EGFR, including TGF and amphiregulin (26, 29, 32).
Furthermore, the two TNF receptors have been shown to have diverse
effects on EGF ligand and receptor family members, with signaling via
p55 mediating the up-regulation of EGFR mRNA in epithelial cells and
that via p75 transducing the signal for the up-regulation of TGF
mRNA (29).
We therefore hypothesized that signals from the TNF receptors might be
up-regulating EGFR expression, ligand production, and/or tyrosine
kinase activity in MEC to indirectly stimulate MEC proliferation and/or
differentiation. To test this latter hypothesis, we used a specific
inhibitor of EGFR tyrosine kinase activity, PD158780 (compound 7f
of Table 1 in 33 in an attempt to block TNF action on normal
MEC in primary culture. This compound is a potent inhibitor of the
tyrosine kinase activity of members of the EGFR family, which includes
the EGFR itself as well as erbB2, -B3, and -B4, but has poor
inhibitory activity against the platelet-derived growth factor or
fibroblast growth factor receptor tyrosine kinases (Fry, D., personal
communication).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(2.5 x 106 U/mg) was a gift from Asahi Chemical
Industry Co. (Fuji, Shizuoka, Japan), and the inhibitor of EGFR
tyrosine kinase activity, PD158780, was provided by Dr. David Fry at
Parke-Davis (Ann Arbor, MI).
Animals
For the isolation of MEC for primary culture, virgin 50- to
55-day-old female Sprague-Dawley Crl:CD BR rats were purchased from
Charles River (Wilmington, MA) and used as the source of mammary
glands. Female CD2F1 mice, purchased from NCI-Frederick Cancer Research
Facility, Biological Testing Branch (Frederick, MD), were used to
passage the Engelbreth-Holm-Swarm (EHS) sarcoma, which was the source
of the reconstituted basement membrane (RBM) matrix for the primary
culture of the MEC organoids. Rats and mice were fed chow diets
(Teklad, Madison, WI) ad libitum and had free access to
water. Animal rooms were humidity controlled and air conditioned, with
14-h light, 10-h dark cycles (rats) or 12-h light, 12-h dark cycles
(mice). The care and use of the animals was in accordance with NIH
guidelines and Institute Animal Care and Use Committee regulations.
RBM matrix preparation
The RBM matrix used for the primary culture of the MEC organoids
was extracted from the EHS sarcoma as previously described (17).
MEC isolation and cell culture
For the primary culture of MEC, epithelial cell organoids were
isolated and cultured as previously described. Briefly, mammary glands
were excised from virgin 50- to 55-day old female rats (eight per
experiment), mechanically minced, suspended in 10 ml/g wet wt digestion
solution [phenol red-free RPMI 1640 containing 0.2% (wt/vol) dispase
II, 0.2% (wt/vol) collagenase type III, 5% (vol/vol) NCS, and 50
µg/ml gentamicin], and incubated at 37 C for approximately 13.5
h. The resultant epithelial cell organoids were pelleted by
centrifugation, washed twice with DMEM-F12, and resuspended in
DMEM-F12. The MEC suspension was then filtered through a 530-µm Nitex
filter (Tetko, Depew, NY) to remove any large aggregates of glandular
material and subsequently passed through a 60-µm Nitex filter, which
trapped the MEC organoids but allowed the passage (and removal) of
small cell clusters and single cells. The organoids were rinsed off the
Nitex filter with DMEM-F12 (phenol red-free) containing 5% (vol/vol)
NCS and 50 µg/ml gentamicin, refiltered through the 530-µm Nitex,
and incubated in a plastic tissue culture flask for 4 h at 37 C;
this step facilitated the attachment and removal of any remaining
stromal cells from the nonadherent mammary epithelial organoids. The
number of MEC within the organoids was then enumerated by counting of
nuclei as previously described (17). The nonadherent MEC organoids were
pelleted by centrifugation and resuspended in ice-cold RBM matrix at a
concentration of 3 x 105 cells/0.2 ml matrix. Two
hundred microliters of RBM matrix containing epithelial organoids was
layered on top of 200 µl pregelled, cell-free RBM matrix/well of a
24-well plate. After the matrix was allowed to gel for 3 h at 37
C, 1 ml medium was added to each well. The medium was changed on day 5
as indicated below; thereafter, the medium was changed twice per week.
The serum-free medium used for these studies consisted of 10 µg/ml
insulin, 1 µg/ml progesterone, 1 µg/ml hydrocortisone, 5 µg/ml
transferrin, 1 µg/ml PRL, 5 µM ascorbic acid, 1 mg/ml
fatty acid-free BSA, and 50 µg/ml gentamicin in phenol red-free
DMEM-F12. EGF (10 ng/ml; 1.6 nM) or TNF (2 or 40 ng/ml;
0.1 or 2.3 nM, respectively) was added where noted in the
text. PD158780 was dissolved in 0.1% DMSO and added to the medium
immediately before use.
[3H]Thymidine incorporation assay
The MEC organoids were cultured until day 5 in the EGF- and
TNF-free medium described above. On day 5, the medium was changed,
and EGF (10 ng/ml; 1.6 nM) or TNF (2 or 40 ng/ml; 0.1 or
2.3 nM, respectively) in either the presence or absence of
the EGFR tyrosine kinase inhibitor PD158780 (0.5 µM) was
added, and the cultures were incubated for either 48 h (until day
7) or 16 days (until day 21) at 37 C. As a control for these studies,
PD158780 alone was added to the EGF- and TNF-free culture medium.
After the cultures were pulse labeled with [3H]thymidine
(5 µCi/well) for the last 4 h of incubation on either day 7 or
day 21, the medium was removed, and the RBM matrix was digested away
using 5 caseinolytic units/ml liquid dispase for 2 h at 37 C. The
MEC organoids were washed with cold PBS, and the acid-insoluble
fraction was precipitated overnight at 4 C with 1 ml 5% (wt/vol)
trichloroacetic acid. The pellets were washed twice with cold 5%
trichloroacetic acid, solubilized in 1 ml 0.1 N NaOH
containing 0.1% (vol/vol) Triton X-100, neutralized with 100 µl 1
N HCl, and [3H]thymidine incorporation was
determined by liquid scintillation counting.
Quantitation of cell number (MTT assay)
Culture conditions and treatment were the same as those
described above for the [3H]thymidine incorporation
assay. Cell number was quantitated on days 7 and 21 using the MTT assay
that we have previously described (16). In this assay, viable cells
convert the soluble tetrazolium MTT dye into insoluble blue formazan
crystals, whereas nonviable cells are unable to metabolize the MTT
substrate and, thus, are not counted. The total viable cell number is
then determined by extrapolation of the absorbance intensity from a
standard curve that is constructed for each experiment. In brief, 200
µl MTT (5 mg/ml in PBS) were added per ml medium, and cultures were
incubated for 16 h at 37 C. After removal of the medium and
rinsing of each well with PBS, the RBM matrix was digested by adding 1
ml dispase (5 caseinolytic units/ml)/well and incubating for 2 h
at 37 C. The digested material was transferred to glass tubes, and each
well was rinsed with 1 ml PBS. The cells were then separated from the
digested matrix by centrifugation, and the pellet was dissolved in
isopropanol and recentrifuged. Absorbance at 570 nm was read in a
Bio-Tek model EL311 plate reader (Winooski, VT). Standard curves using
the newly isolated MEC were set up for each experiment. It was also
established that PD158780 did not interfere with the MTT assay, as
nuclei counts on day 21 did not differ significantly from cell counts
obtained using the MTT assay (data not shown).
Morphological analysis
Culture conditions and treatment were as described above for the
[3H]thymidine incorporation assay. The morphological
appearance of the MEC organoids was assessed and quantitated on days 7
and 21 of culture (during the last 4–6 h of treatment) by light
microscopic observation. Colonies were classified into four main
groups: end bud-like, alveolar, squamous, and atypical hybrid (19). The
end bud-like colonies are pale rust in color, have a more simplistic
lobular structure with fewer, smaller ductal projections, and are
primarily composed of immature epithelial cells that show little or no
evidence of functional differentiation. In contrast, the dark brown or
black alveolar colonies are larger, have a more complex, multilobular
structure with extensive ductal projections, and are composed of
morphologically and functionally differentiated MEC organized into a
classical alveolar arrangement. Squamous colonies contain
concentrically compacted acellular material in a keratotic whorl
pattern (16, 34), and atypical hybrid colonies are defined as hybrids
of alveolar and squamous colonies. Photographs of the organoids were
taken with a Nikon FX-35A camera mounted on an Olympus CK2 inverted
microscope (Melville, NY).
Assessment of functional differentiation (measurement of casein
accumulation)
Culture conditions and treatment were as described above, and
samples were harvested on days 7 and 21 of culture as previously
described (16). Casein levels were assayed in three wells per treatment
group using the standard casein enzyme-linked immunosorbent assay
(ELISA) previously developed by our laboratory (18). In addition,
casein accumulation by MEC was determined using a modification of a
recently described Western blot procedure (20). In this case, samples
were mixtures of the triplicate wells for each treatment group. The
samples were normalized for loading on the basis of either equivalent
protein contents (after Bio-Rad protein analysis) or equivalent cell
number. In either case, extracts of MEC plus RBM matrix were diluted
with 4-fold concentrated Laemmli sample buffer (35), boiled for 4 min,
subjected to electrophoresis on a 12.5% polyacrylamide-SDS gel
according to the method of Laemmli (35), and transferred to
nitrocellulose. After the membranes were blocked overnight at 4 C with
5% (wt/vol) Blotto (nonfat dried milk) in TBS buffer (150
mM NaCl and 10 mM Tris, pH 7.4, at 25 C), they
were rinsed in TBS containing 0.5% (wt/vol) BSA (TBS/BSA) and
incubated for 2 h at room temperature with a rabbit polyclonal
antibody against the rat casein proteins (18) (1:3000 dilution) in
TBS/BSA. Blots were then washed in TBS/BSA, incubated with
peroxidase-conjugated donkey antirabbit antiserum (1:5000 dilution in
TBS/BSA) for 60 min at room temperature, washed in TBS/BSA containing
0.01% (vol/vol) Tween-20, and developed using the enhanced
chemiluminescence system (Amersham).
Statistics
Statistical significance was determined using a one-way ANOVA
with the Student-Newman-Keuls test for pairwise multiple comparisons.
P < 0.05 was judged to be statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced proliferation of normal
MEC on normal MEC growth and
differentiation.
First, the ability of PD158780 to block TNF-induced DNA synthesis in
normal MEC was investigated using a [3H]thymidine
incorporation assay. When assessed on day 7 of culture after 48 h
of treatment, the EGFR tyrosine kinase inhibitor only modestly
suppressed the increase in [3H]thymidine incorporation
stimulated by 40 ng/ml TNF (Fig. 1
, bars 5 and 6), but reduced DNA synthesis in response to 2 ng/ml TNF
by approximately 50% (Fig. 1, bars 7 and 8). In contrast, the
EGF-induced increase in DNA synthesis was completely blocked by
PD158780 (Fig. 1, bars 3 and 4). Moreover, the EGFR tyrosine kinase
inhibitor alone was found to cause 50% inhibition of
[3H]thymidine incorporation. As no EGF was present in the
culture medium, we believe that this latter inhibition was due to the
blockage of the activity of endogenous EGFR ligands either produced by
the MEC themselves or present in the RBM matrix.
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action changed as the MEC differentiated, the
cells were also treated until the end of the 21-day culture period (for
a total of 16 days). When measured on day 21,
[3H]thymidine incorporation by MEC treated with either
concentration of TNF was not increased relative to the control value
as it had been on day 7 (Figs. 1
and 2,
compare bars 5 and 7 to bar 1). However, it can be seen that PD158780
still had no effect on DNA synthesis in the presence of 40 ng/ml TNF
(Fig. 2, compare bars 6 and 5), but again caused a 50% inhibition at
the lower, 2 ng/ml concentration (Fig. 2, compare bar 8 with bar 7). Of
considerable interest was the marked difference in thymidine
incorporation between TNF- and EGF-treated MEC at this time. In
contrast to both TNF-treated groups and to day 7, thymidine
incorporation by MEC grown in the presence of EGF for 16 days was
dramatically decreased compared with the control group; moreover,
PD158780 had no further inhibitory effect on DNA synthesis in the
presence of EGF. Also noteworthy was the marked inhibitory effect of
PD158780 on thymidine incorporation in the absence of added growth
factor (Fig. 2, compare bars 2 and 1).
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and to determine whether this balance
was affected by inhibition of EGFR tyrosine kinase activity. In
contrast to the stimulatory effect on thymidine incorporation, neither
concentration of TNF, in either the presence or absence of PD158780,
had any effect on viable cell number after 48 h (day 7). Cell
number was increased in the EGF-treated group, however, and this
increase was completely suppressed by PD158780, whereas cell number was
unaffected by the 48-h treatment with PD158780 alone (Fig. 3).
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-treated groups was increased compared with that
on day 7 and was significantly higher than the cell number in the
EGF-treated culture (Fig. 4, compare bars
5 and 7 to bar 3). More significantly, PD158780 was unable to block the
increase in viable cell number induced by 40 ng/ml TNF (Fig. 4, compare bars 5 and 6), but partially suppressed the increase in cell
number in response to 2 ng/ml TNF. Finally, the EGF-induced increase
in viable cell number was blocked by PD158780 (Fig. 4) as it had been
on day 7, whereas the EGFR tyrosine kinase inhibitor alone had no
effect (compare bars 1 and 2). Thus, when taken together, these data
suggest that EGFR tyrosine kinase activity is not required for TNF
to induce the proliferation of normal MEC in culture, although the
pathway(s) activated by both the TNF and EGF receptors may actin
concert to stimulate MEC growth under certain circumstances.
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-induced morphological
differentiation and EGF can stimulate
the ductal and alveolar morphogenesis of MEC in culture while
suppressing the outgrowth of colonies with an atypical or squamous
epithelial morphology (16, 34). To determine whether the EGFR was
involved in the pathway by which TNF stimulated the morphological
development of the MEC, the ability of PD158780 to inhibit
TNF-induced morphogenesis was assessed. Morphological changes after
48 h of treatment were subtle (data not shown). In both TNF-
and EGF-treated groups, the percentage of the less differentiated, end
bud-like colonies was slightly decreased, whereas the percentage of the
more differentiated, alveolar colonies was increased; the percentage of
squamous and atypical hybrid colonies in all groups was negligible at
this time. The EGFR tyrosine kinase inhibitor had no significant effect
on colony morphology in either the presence or absence of TNF or
EGF.
In contrast, TNF induced significant changes in both colony type and
size after 16 days of treatment. Specifically, 40 ng/ml TNF
stimulated the formation of complex, lobulo-alveolar colonies that were
significantly larger than those induced by EGF (Fig. 5, compare j with f and g) and which were
interconnected by extensive ductal branching (Figs. 5j and 6).
Furthermore, TNF also inhibited the development of both squamous and
atypical colonies (Fig. 6). More
importantly, the EGFR tyrosine kinase inhibitor was unable to inhibit
this expansive TNF-induced morphogenesis, as colonies that developed
in the presence of TNF plus PD158780 were as large, viable, and
morphologically developed as those that developed in the presence of
TNF alone (Fig. 5, j and k, and Fig. 6). The 2 ng/ml concentration
of TNF also stimulated alveolar and ductal morphogenesis, although
the effects were less pronounced than those of 40 ng/ml TNF, and
PD158780 was still unable to block this TNF-induced morphological
differentiation (16) (data not shown).
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, the EGFR tyrosine kinase inhibitor had several
noteworthy effects on MEC morphogenesis when added alone or in
combination with EGF. Unexpectedly, PD158780 alone mimicked the ability
of EGF to both suppress squamous and atypical colony formation and to
stimulate the formation of the more differentiated, lobular-alveolar
colonies (Fig. 6). These latter colonies, however, tended to be quite
small compared to TNF- or EGF-treated colonies (Fig. 5d), and any
larger alveolar colonies that developed in the presence of PD158780
alone appeared to be highly disrupted and were surrounded by small
cellular bodies that had apparently broken off of the periphery of the
alveolar organoids (Fig. 5e). Furthermore, organoids treated with both
EGF and PD158780 were not as large as colonies treated with EGF alone
(Fig. 5, compare h and i to g), and many of the alveolar colonies in
this group also appeared to be highly disrupted (Fig. 5i).
Effect of PD158780 on TNF-modulated MEC functional
differentiation
Previous studies by our laboratory determined that TNF had a
complex, biphasic effect on casein production by MEC; a higher
concentration of TNF (40 ng/ml) inhibited functional
differentiation, whereas in the absence of EGF, a lower concentration
of TNF (2 ng/ml) enhanced casein accumulation (16). To determine
whether these effects of TNF might be mediated through the EGFR, the
ability of PD158780 to interfere with the effects of TNF on casein
accumulation by MEC was measured using both Western blot analysis and
an ELISA (18). When examined on day 7, casein accumulation was
unaffected by the 48-h treatment with either 2 or 40 ng/ml TNF (Fig. 7, lane 5, and data not shown), and
casein levels were also unchanged in MEC that had been treated with
both TNF and PD158780 (Fig. 7, lane 6, and data not shown). The EGFR
tyrosine kinase inhibitor completely blocked the increased accumulation
of all casein isoforms in response to EGF (Fig. 7, compare lanes 3 and
4), whereas PD158780 alone had no significant effect on casein levels
(Fig. 7, compare lanes 2 and 1). Casein levels in all but the
EGF-treated group were below the limits of detectability by ELISA (data
not shown).
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, and the EGFR
tyrosine kinase inhibitor had no effect on this TNF-induced
inhibition (Fig. 8A, compare lanes 1, 5,
and 6, and Fig. 8B). Despite its ability to significantly stimulate
both MEC growth and morphogenesis, 2 ng/ml TNF had no effect on
casein levels after 16 days of treatment, even though this TNF
concentration had previously been shown to increase casein accumulation
by MEC at later times in culture (Fig. 8A, lanes 1 and 7, and Fig. 8B).
Casein levels were also unchanged in MEC that had been treated with
both 2 ng/ml TNF and PD158780.
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, A and B). This latter effect was in direct contrast to the
inhibitory effect of PD158780 on EGF-induced casein accumulation after
48 h. Lastly, it should also be noted that this PD158780-induced
increase in casein accumulation was not observed in the presence of
either concentration of TNF (Fig. 8, A and B). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced
MEC proliferation and EGF on normal MEC were less than
additive (16). This suggested that TNF and EGF might be activating a
common growth stimulatory pathway, and that the mitogenic effect of
TNF might be mediated, at least in part, via activation of the EGFR
in response to signals from the p55 TNF receptor. On the other hand,
this earlier data also suggested that the mitogenic signaling pathway
triggered by the p55 TNF receptor may have an EGFR-independent
component, as TNF was shown to increase total cell number to a
higher level than that observed with optimal levels of EGF (16, 17, 34). The studies presented herein suggest that EGFR tyrosine kinase
activity is not required for TNF to stimulate MEC proliferation.
PD158780 was unable to inhibit proliferation in response to 40 ng/ml
TNF and only partially suppressed the growth-promoting effects of 2
ng/ml TNF. Although we cannot rule out the possibility that PD158780
may be inhibiting an, as yet undiscovered, tyrosine kinase that is
activated by 2 ng/ml TNF, this latter observation suggests that
signaling pathway(s) activated by endogenous EGFR ligands may act in
concert with pathways activated by the lower concentration of TNF to
stimulate growth. In support of this theory, EGF and TNF have been
shown to preferentially activate different members of the
mitogen-activated protein kinase family in other cell types. EGF
strongly stimulates ERK1 and ERK2 activity, whereas TNF induces a
more pronounced activation of the c-Jun N-terminal kinases (JNKs)
(36, 37, 38). Thus, when EGFR activation is blocked by PD158780, such that
ERK1 and/or ERK2 may no longer be activated by this receptor, growth in
the presence of 2 ng/ml TNF may be partially, but not completely,
suppressed.
In contrast, the higher (40 ng/ml) concentration of TNF may be able
to directly activate ERK1 and/or ERK2, such that the blockage of
endogenous EGFR ligand-induced action by PD158780 has no effect on DNA
synthesis in response to this TNF concentration. Even though TNF
is not as powerful a stimulus for ERK1 and ERK2 as EGF, numerous
studies have demonstrated that TNF can activate these enzymes
(39, 40, 41, 42), so it could be proposed that the higher concentration of
TNF may activate both the JNK and ERK enzymatic signaling cascades
to stimulate MEC growth. Furthermore, a study by Sluss et
al. (43) has demonstrated that there are two JNK protein kinase
isoforms, JNK1 and JNK2, and that the activation of these protein
kinases by TNF is concentration dependent. Therefore, it could be
postulated that 40 ng/ml TNF activates both JNK1 and JNK2, whereas
the lower TNF concentration only triggers the activation of one JNK
isoform. The higher TNF concentration may also preferentially
activate JNK2, which has been shown to be more potent in inducing the
phosphorylation of c-jun than JNK1 (43). The specific roles
of both of the ERK and JNK mitogen-activated protein kinase isoforms in
stimulating MEC growth in response to both TNF and EGF are currently
under investigation.
The differential effects of TNF and EGF on MEC proliferation
The distinct effects of TNF and EGF on MEC
proliferation after different times in culture were intriguing. When
assessed on day 7, thymidine incorporation, but not cell number, was
increased in response to TNF, whereas both DNA synthesis and cell
number were increased by EGF. This observation further supports our
contention that the pathways of TNF- and EGF-induced growth in
normal MEC are different. In addition, this difference in cell number
between the TNF- and EGF-treated groups may be due to the ability of
TNF to increase the overall rate of cell turnover at this time. As
several studies have demonstrated that TNF can be either cytotoxic
or mitogenic to certain cell types (44, 45), it is possible that in
addition to an overall stimulation of MEC proliferation, TNF may be
cytotoxic to a specific subset of MEC within the cultures, such that
there is no initial net increase in cell number. However, once the MEC
to which TNF is cytotoxic have been eliminated, the proliferative
effects of TNF may become apparent. EGF, in contrast, may have no
effect on the rate of cell turnover at this time; thus, its growth
stimulatory effects are immediately evident.
In contrast to day 7, the total cell number on day 21 in the
groups treated with either 2 or 40 ng/ml TNF was significantly
higher than that in the EGF-treated cultures. Of further interest,
however, was the observation that MEC cultured in the presence of
either concentration of TNF were still actively synthesizing DNA at
this time, albeit at a reduced level compared with that on day 7,
whereas DNA synthesis by EGF-treated MEC was significantly lower than
the level in TNF-treated cells. The level of
[3H]thymidine incorporation by the EGF-treated cells on
day 21 was also significantly lower than the level on day 7 and was
actually 3.5-fold lower than thymidine incorporation by MEC cultured in
the absence of EGF or TNF. This latter observation suggests that EGF
may be acting to inhibit cell growth at this time; however, the fact
that PD158780 did not reverse this inhibition argues against this
supposition. Rather, the data suggest a loss of responsiveness of the
EGF-treated cells to EGF, perhaps by down-regulation of EGFR activity
in response to the 16-day EGF treatment. Alternatively, or perhaps in
addition, several groups have reported that EGFR levels decrease during
pregnancy and lactation (22, 23) (Darcy, K. M., manuscript in
preparation), and we have recently determined that EGFR levels decline
in conjunction with the morphological and functional differentiation of
MEC in primary culture (Darcy, K. M., manuscript in
preparation).
In any case, the different effects of TNF and EGF on DNA synthesis
and cell number at different times in culture strongly suggest that the
mitogenic actions of TNF and EGF are mediated through independent
pathways, even though there may be cooperativity between these
pathways under some circumstances. TNF may also be acting on a cell
population that is unresponsive to EGF. For example, both TNF and
EGF may be able to regulate the growth and differentiation of immature
MEC, whereas TNF may also be able to stimulate the proliferation of
a putative stem cell population and/or effect the death of a small
subset of cells early in culture.
The EGFR tyrosine kinase inhibitor did not block TNF-induced
morphological development of normal MEC
The extensive branching alveolar morphogenesis that was
stimulated by both concentrations of TNF was completely unaffected
by PD158780, suggesting that the pathway of TNF-induced
morphological development is not dependent on EGFR tyrosine kinase
activity. This conclusion is further supported by the observation that
TNF actually induced the formation of larger colonies with more
expansive ductal branching than EGF. This difference between TNF-
and EGF-stimulated morphogenesis may be due to the differential ability
of TNF and EGF to modulate the production and/or activity of matrix
metalloproteinases (MMPs), which could then affect the remodeling of
the ECM. Previous studies in our laboratory have shown that MMP-9 (the
92-kDa type IV collagenase) activity in conditioned medium from MEC was
increased by TNF, but decreased by EGF (46, and our unpublished
observations). Therefore, the TNF-induced remodeling of the ECM may
permit the pronounced ductal branching and alveolar morphogenesis that
occurs in response to this cytokine, whereas the inability of EGF to
increase MMP-9 activity may explain why EGF does not stimulate
branching morphogenesis to the same extent as TNF.
In addition, it should be noted that PD158780 alone had several
interesting morphological effects. Earlier studies in our laboratory
have shown that in the absence of EGF, branching alveolar morphogenesis
was decreased, and the formation of both atypical hybrid and squamous
colonies was enhanced (16, 34). In the current studies, however,
treatment of the MEC with an inhibitor of EGFR tyrosine kinase activity
actually permitted alveolar morphogenesis, although the
PD158780-treated colonies were smaller than those treated with either
EGF or TNF. As PD158780 was not present for the first 5 days of
culture, endogenous EGFR ligands produced by the MEC or within the RBM
may have initiated the process of morphological differentiation such
that the subsequent inhibition of EGFR tyrosine kinase activity was
unable to inhibit morphogenesis once it had begun. This hypothesis
concurs with previous studies in our laboratory in which alveolar
morphogenesis proceeded even when EGF was removed from the culture
medium after the first few days of culture (34). However, PD158780 also
induced the apparent disintegration of the alveolar colonies when added
alone or in combination with EGF (but not in the presence of TNF),
so even though PD158780 did not inhibit alveolar morphogenesis,
inhibition of the EGFR tyrosine kinase activity resulted in the
apoptotic death of the lobulo-alveolar organoids (47). When taken
together with the aforementioned data on the effects of EGF on DNA
synthesis at different times as well as with earlier studies by both
our laboratory (34) and the Hynes group (48), it thus appears that EGF
is needed at early times in culture (e.g. day 7) for
proliferation as well as for rendering MEC competent to respond to
lactogenic hormones. After differentiation, however, EGF may become a
survival factor that serves to prevent apoptosis and maintain the
existing state of the gland rather than stimulate further growth and/or
development.
The EGFR tyrosine kinase inhibitor did not block the effects of
TNF on MEC functional differentiation
In accordance with our previous studies, casein accumulation
by MEC was inhibited by a 16-day treatment with 40 ng/ml TNF. As
PD158780 was unable to alter this effect, it appears that the EGFR is
not involved in the pathway by which 40 ng/ml TNF regulates casein
accumulation. In contrast to our previous studies, casein accumulation
was not increased after long term treatment with 2 ng/ml TNF. This
apparent discrepancy may be due to the fact that TNF was not added
until day 5 in the current study, whereas TNF had been present from
days 0–21 of culture in the former studies. In addition, the
stimulatory effect of 2 ng/ml TNF on casein may have been masked
because casein levels in the control group were unusually high in this
series of experiments; this phenomenon may be due to the activity of
endogenous EGFR ligands either produced by the MEC themselves or
present in the RBM matrix.
Surprisingly, casein accumulation was increased in MEC that had been
treated for 16 days with PD158780 alone or in combination with EGF. As
studies by our laboratory have shown that casein accumulation by MEC is
significantly decreased in the absence of EGF, this observation was
somewhat unexpected. It could be argued that this PD158780-induced
increase in casein may be due in part to the 2-fold increase in the
percentage of the alveolar casein-producing colonies in this group;
however, several factors argue against this explanation. First, in both
EGF- and EGF- plus PD158780-treated cultures, the percentage of
alveolar colonies was increased to the same extent compared with the
control group, yet casein levels were higher in MEC treated with both
EGF and PD158780 than in MEC treated with EGF alone. In addition,
TNF increased the percentage of alveolar colonies, but casein
accumulation by TNF-treated MEC was either decreased or unaffected
depending on the TNF concentration. Therefore, the increased
percentage of alveolar colonies cannot completely account for the
increase in casein levels in the PD158780-treated cultures.
In summary, the studies presented herein suggest that the
modulation of MEC growth and development by TNF does not require
EGFR tyrosine kinase activity. Specifically, EGFR tyrosine kinase
activity is not necessary for TNF to stimulate the proliferation of
normal MEC in culture; however, low concentrations of TNF may act in
concert with EGF to stimulate MEC growth. In addition, the EGFR
tyrosine kinase inhibitor had no effect on the ability of TNF to
either stimulate MEC morphogenesis or to regulate casein accumulation
by MEC. Finally, the data suggest that the function of the EGFR changes
during MEC development in vitro, possibly as the result of a
decrease in EGFR expression. Further analysis of both the pathway of
TNF action and the role of EGF are currently underway to determine
their respective contributions to the regulation of mammary gland
development.
|
Acknowledgments |
|---|
. We are also indebted
to Ms. W. Shea, M. Jane Ehrke, and Drs. Molly Kulesz-Martin and Daniel
Medina for helpful discussions. |
Footnotes |
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Received April 3, 1997.
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