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J. Biol. Chem., Vol. 278, Issue 37, 35444-35450, September 12, 2003
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-mediated Epithelial Cell Growth Suppression*
From the Cell Biology Program and Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
Received for publication, February 10, 2003 , and in revised form, June 23, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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enforces homeostasis of epithelia
by activating processes such as cell cycle arrest and apoptosis. Id2
expression is often highest in proliferating epithelial cells and declines
during differentiation. Recently, Id2 expression has been found to
depend on Myc-Max transcriptional complexes. We observed that TGF-
signaling inhibits Id2 expression in human and mouse epithelial cell
lines from different tissue origins. Furthermore, the observed Id2
down-regulation by TGF- in mouse mammary epithelial cells occurs without
a concurrent drop in c-Myc levels. However, sustained Id2 repression
in these cells and in human keratinocytes coincides with induction of the Myc
antagonistic repressors Mad2 and Mad4, decreased formation of Myc-Max
heterodimers and the replacement of Myc-Max complexes with Mad-Max complexes
on the Id2 promoter. These results argue that induction of Mad
expression and Id2 down-regulation are important events during the TGF-
cytostatic program in epithelial cells. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(TGF-)1 plays a
central role in maintaining epithelial tissue homeostasis under normal
physiological conditions (1).
Through anti-proliferative or apoptotic mechanisms, TGF- can inhibit the
growth of virtually all non-transformed epithelial cell types
(2,
3). Disruption of
TGF--mediated growth control occurs in many carcinomas
(4). Furthermore, a selective
loss of the antimitogenic effect in the cancerous state can reprogram the
cellular response to TGF-, which then becomes a stimulus for tumor cell
invasion and metastasis (5).
Identifying the TGF- gene responses underlying the cytostatic effect in
epithelial cells is therefore of great interest.
TGF- signals through a ligand-activated serine/threonine kinase
complex that phosphorylates Smad transcription factors
(6,
7). Upon translocation into the
nucleus, the activated Smad proteins are rapidly directed to several hundred
target genes by a host of Smad-binding cofactors
(8,
9). In epithelial cells, a
small group of TGF- gene responses is devoted to arresting the cell
cycle. These include induction of the G1 cyclin-dependent kinase
(cdk) inhibitors, p15Ink4b, which directly inhibits cdk4 and cdk6
(10–12),
and p21Cip1, which inhibits cdk2
(11,
13,
14). As a result of these cdk
inhibitory events, cdk substrates such as pRb remain underphosphorylated and
block cell cycle progression
(15,
16).
Epithelial cells normally display another important set of TGF-
anti-proliferative gene responses involving repression of growth-promoting
transcription factors. This group is best represented by c-Myc, a
transcriptional regulator that can both activate or inhibit gene expression in
favor of cell proliferation
(17–19).
In association with the protein Max, c-Myc binds to cognate E-box DNA
sequences present in the promoter of many cell cycle-related genes
(18). The formation of Myc/Max
heterodimers is antagonized by members of the Mad family: Mad1, Mxi-1/Mad2,
Mad3, and Mad4. Mad proteins bind to Max in competition with Myc and repress
transcription through the same E-box motifs
(18,
20).
c-Myc is rapidly downregulated by TGF- in a variety of cell types
(2). A c-myc repressor
complex consisting of Smad3, Smad4, E2F4/5 and the transcriptional repressor
p107 mediates this effect
(21). c-Myc down-regulation by
TGF- not only deprives the cell of growth promoting functions but also
facilitates the induction of p15Ink4b and p21Cip1
(22–24).
TGF--activated Smad complexes induce transcription when bound to
specific sites within the p15Ink4b and p21Cip1 promoters,
while TGF--mediated c-Myc repression removes this inhibitory influence
from the same promoters (23).
Thus, c-Myc down-regulation plays an integrative role in the TGF-
cytostatic program. However, not all cell types that are growth inhibited by
TGF- undergo c-myc down-regulation
(25,
26), raising the question of
whether a different but functionally equivalent mechanism is at play.
Id proteins function as negative regulators of basic helix-loop-helix
(bHLH) transcription factors critical for cell differentiation
(27,
28). Through interactions with
Rb, Id proteins can also actively promote cell proliferation
(29,
30) and recently, a role for
Id2 as a survival and differentiation factor in the mammary gland during
pregnancy has been demonstrated
(31–33).
TGF- inhibits Id1, Id2, and Id3 expression in several
cell types (34,
35). Transcriptomic analysis
of different human epithelial cell lines has revealed that repression of these
three Id genes, together with repression of c-myc and
induction of p15Ink4b and p21Cip1, constitute a TGF-
cytostatic program shared by human epithelial cell lines of different tissue
origins (36). Thus, in a
manner reminiscent of c-Myc, the Id proteins are emerging as important targets
of repression by TGF- in the cytostatic program.
A link between c-Myc and Id2 expression was recently established
by the demonstration that c-Myc binding to E-box motifs in the Id2
promoter supports Id2 expression
(34). Consequently, c-Myc
down-regulation by TGF- may secondarily lead to Id2 inhibition
(34). However, we observed a
poor correlation between c-Myc levels and Id2 repression by
TGF-, particularly in mouse mammary epithelial cells. Therefore, we
searched for a possible mechanism that might be functionally equivalent to
c-Myc down-regulation. Here we show that in mouse mammary epithelial cells and
human keratinocytes, sustained Id2 repression by TGF- is
paralleled by a strong induction of Mad4 and a replacement of Myc-Max
complexes with Mad-Max complexes on the Id2 promoter.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-actin riboprobe was generously provided by Mark Moasser
(Memorial Sloan-Kettering Cancer Center, New York), and contains nucleotides
1005–1155 of human -actin (GenBankTM accession
number M10277
[GenBank]
) inserted as an EcoRI/HindIII fragment into
pSP65. The human Id2 cDNA was inserted as a BamHI fragment
into pBluescript II SK for use as a template for riboprobe synthesis.
Cell Culture and Growth Inhibition Assays—HaCaT
keratinocytes were maintained in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum (FBS). NMuMG cells were maintained in
DMEM supplemented with 10% FBS, 2 mM glutamine, and 20
mM HEPES. Both HaCaT and NMuMG cells were obtained from the ATCC.
In all cases, the culture media contained 100 units/ml penicillin G, 100
µg/ml streptomycin, and 0.5 µg/ml fungizone. Cells were cultured with
100 pM TGF- for various lengths of time, ranging from
0–24 h. In some instances, NMuMG cells were preincubated with 10
µg/ml cycloheximide for 1 h prior to 6 h of TGF- stimulation.
125I-Deoxyuridine incorporation assays were performed in 10% FBS as
described previously (37).
RNase Protection and Northern Blot Analysis—RNA was isolated
using ULTRASPEC RNA isolation reagent (BIOTECX Laboratories, Inc.) following
the manufacturer's instructions. RNase protection assays were performed as
previously described (38). To
generate an antisense riboprobe specific for human Id2, the template
plasmid was linearized with XbaI and was used in an in vitro
transcription reaction with T7 RNA polymerase. To synthesize the human
-actin antisense riboprobe, the template plasmid was digested with
HindIII and transcribed with SP6 RNA polymerase.
Northern blot analysis was performed as previously described with the
exception that 20 µg of total RNA was used in each case
(39). Membranes were
prehybridized in QuikHyb (Stratagene) at 68 °C for several hours and
subsequently hybridized for 2 h at 68 °C with random-primed
[
-32P]dCTP radiolabeled probes. Membranes were washed in low
stringency wash buffer (2.0x SSC, 0.1% SDS) at room temperature followed
by a high stringency (0.2x SSC, 0.1% SDS) wash at 55 °C. Prior to
rehybridization, membranes were stripped by the addition of boiling 0.5% SDS
and allowed to cool to room temperature. Probes for northern blots were
synthesized by random priming (Prime-it II, Stratagene) following the
manufacturer's protocol. The fragment used for the mouse Id2 probe
corresponds to nucleotides 432–852 (GenBankTM accession number
M69293
[GenBank]
). A fragment encompassing nucleotides 2578–3294 of mouse
c-myc (GenBankTM accession number L00038
[GenBank]
) and the full-length
human c-myc cDNA were used to generate probes for northern blotting.
Nucleotides 1–630 of mouse mad4 (GenBankTM accession
number NM_010753
[GenBank]
) composed the probe used for Northern blotting. The
full-length rat GAPDH cDNA (GenBankTM accession number X02231
[GenBank]
)
was used to generate radiolabeled probe to control for RNA loading.
Immunoprecipitation and Immunoblotting—Cell lysis and
immunoblot analysis were performed following protocols that have been
described in detail elsewhere
(40). Immunoblots on total
protein were performed using 50 µg of protein lysate. Antibodies against
Id2 (C-20, sc-489), Max (C-17, sc-197), Mad1 (C-19, sc-222), Mad3 (H-206,
sc-770), and Mad4 (H-209, sc-771) were purchased from Santa Cruz
Biotechnology. The Mxi-1/Mad2 antibody (Cat. 610591) was obtained from BD
Transduction Laboratories, and the c-Myc antibody (C-8, AHO0072) was purchased
from BIOSOURCE. To control for protein loading, membranes were probed with
anti--tubulin antibodies (mouse ascites fluid, clone DM 1A, T9026)
obtained from Sigma. In most cases, horseradish peroxidase (HRP)-conjugated
anti-mouse, ant-rabbit, or anti-goat secondary antibodies (Amersham
Biosciences) were used and proteins were visualized by enhanced
chemiluminescence (ECL or ECL plus, Amersham Biosciences). In certain
instances, primary antibodies were detected with biotinylated secondary
antibodies followed by streptavidin-HRP (Jackson Laboratories) to enhance the
sensitivity of detection.
For Fig. 4, Max was
immunoprecipitated from 1 mg of NMuMG lysates stimulated with TGF- for
the indicated times using anti-Max antibodies (C-17, sc-197) and detected by
immunoblot analysis with Max antibodies (H-2, sc8011). For the
co-immunoprecipitation studies, HaCaT cells cultured in the absence or
presence of TGF- for 24 h were lysed in 250 mM NaCl; 50
mM HEPES, pH 7.0; 0.1% Nonidet P-40, and protease inhibitors.
Lysates were quantified by Bradford assay and 1 mg total protein was
immunoprecipitated on a rocking platform for 3 h with control normal rabbit
IgG (Upstate Biotechnology, Cat. 12-370) or Max antibodies (C-17, sc-197) and
protein G-Sepharose. Immunoprecipitates were washed three times in lysis
buffer and resuspended in SDS load buffer, resolved through SDS-polyacrylamide
gels, and transferred to polyvinylidene difluoride membranes (Immobilon P,
Millipore). The membrane was cut in two, the upper portion was probed with
c-Myc (9E10, sc-40) and the bottom with Max (H-2, sc-8011) antibodies.
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Chromatin Immunoprecipitation—HaCaT cells grown to
60–70% confluency, were cultured in the presence or absence of
TGF- for 24 h and subsequently cross-linked with 1% formaldehyde at room
temperature for 15 min. Chromatin immunoprecipitations were performed
essentially as described previously
(41). The antibodies used for
chromatin immunoprecipitations included normal rabbit (Upstate Biotechnology,
Cat. 12-370) and normal mouse (Upstate Biotechnology, Cat. 12-371) IgG,
acetylated histone H4 (Upstate Biotechnology, Cat. 06-866), Max (C-17,
sc-197), c-Myc (N262, sc-764), Mxi-1/Mad2 (BD Transduction, Cat. 610591), and
Mad4 (G-16, sc-1042). A 408-bp region of the Id2 promoter
(nucleotides 846–1254; GenBankTM accession number AF270493
[GenBank]
),
containing three E-boxes, was amplified with the following primers pairs:
5'-TCTGTTCCACTGTGGCACGTATG-3' (sense) and
5'-AAGCTCGATAATGGGGAAACAGTG-3' (antisense). As a negative control,
a 166-bp region of the -actin promoter (nucleotides 29–195;
GenBankTM accession number M10277
[GenBank]
) was amplified with the following
primers: 5'-AAACTCTCCCTCCTCCTCTTCC-3' (sense) and
5'-TCGAGCCATAAAAGGCAACTT-3' (antisense).
Retroviral Infection—Amphotropic and ecotropic Phoenix packaging cell lines were first transfected with MSCV-empty-IRES-GFP, MSCV-Mxi1/Mad2-IRES-GFP, or MSCV-Mad4-IRES-GFP retroviral constructs using LipofectAMINE following the manufacturer's instructions (Invitrogen). Viral supernatants were harvested 48 h post-transfection, filtered, and used for overnight infections of NMuMG or HaCaT cells in the presence of 5 µg/ml polybrene. GFP-positive cells were sorted 48 h following infection on a Vantage cell sorter.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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without
Concurrent c-Myc Down-regulation—We observed Id2
down-regulation within 3 h of TGF- addition to epithelial cell lines
from different tissue origins including NMuMG mouse mammary epithelial cells,
HaCaT human skin keratinocytes, HPL1 human lung epithelial cells, MCF10A human
mammary epithelial cells, and A549 human lung carcinoma cells
(Fig. 1). To define the
mechanism mediating this response we focused on NMuMG and HaCaT cells, which
were efficiently growth arrested by TGF- in a dose-dependent manner
(Fig. 2A). We compared
the kinetics of Id2 down-regulation in these cells with the kinetics
of another important growth inhibitory gene response, namely, c-myc
down-regulation (Fig.
2B). TGF- caused a decrease in Id2
message, which dropped to undetectable levels by 6 h and remaining low at 24 h
after TGF- addition (Fig.
2B). Although two transcripts were evident by
Id2 Northern blot analysis in HaCaT cells
(Fig. 1, second panel;
Fig. 2B, right
panel), subsequent RNase protection analysis with an Id2
specific ribroprobe revealed a rapid and sustained down-regulation of
Id2 mRNA levels (Fig.
2C). This suggests that the lower transcript represents
Id2 (Figs. 1 and
2B). The upper
transcript has been detected by others
(42), but its relationship to
Id2 is unclear. Id2 protein levels were also markedly reduced in
NMuMG cells over a similar time course, as determined by immunoblot analysis
(Fig. 2D).
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A sharp decline in c-myc message was observed at early time points
in these cell lines (Fig.
2B). In NMuMG cells, this decrease was followed by a
recovery of c-myc transcript levels that was noticeable by 3 h and
complete by 12 h after TGF- stimulation
(Fig. 2B).
c-myc down-regulation was more sustained in HaCaT cells, which showed
only a partial recovery of c-myc transcript levels by 24 h
(Fig. 2B). The
transient decrease in c-Myc expression in NMuMG cells was verified at the
protein level by immunoblot analysis (Fig.
2D). The drop in c-Myc levels in these cells was more
limited and transient than the drop in c-myc message, suggesting an
additional attenuation of this response at the c-Myc translation and/or
protein stability levels. This was in marked contrast to the effect of
TGF- on Id2 mRNA and protein levels, which remained low out to 24 h
post-stimulation (Fig. 2,
B–D).
It has been suggested that TGF--mediated suppression of Id2
expression is downstream of c-Myc down-regulation by TGF- given that
c-Myc binding to E-box motifs in the Id2 promoter supports
Id2 expression (34).
Although our present observations do not exclude a link between c-myc
and Id2 down-regulation during the initial phase of the TGF-
response, they suggest that additional mechanisms must be involved in
Id2 repression by prolonged TGF- signaling. In support of this
possibility, we observed that the sustained repression of Id2 by
TGF- requires ongoing protein synthesis as the protein synthesis
inhibitor cycloheximide blocked TGF--mediated Id2
down-regulation in NMuMG cells (Fig.
3). Thus, long-term down-regulation of Id2 depends on a
TGF--induced inhibitory activity.
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Prolonged TGF- Signaling Induces Max, Mad2, and Mad4
Expression in NMuMG and HaCaT Cells—c-Myc has been shown to mediate
induction of Id2 expression through E-box motifs located within the
Id2 promoter (34).
Using luciferase reporter assays we confirmed that the same E-box motifs were
involved in TGF--controlled suppression of transcription from the
Id2 promoter (data not shown). Therefore, we investigated the
hypothesis that a TGF- inducible transcriptional repressor factor(s)
capable of binding to these E-box elements may mediate repression of
Id2 transcription.
We focused our attention on the Mad family of transcription factors, which
bind and inhibit transcription from E-box motifs by heterodimerizing with Max,
thereby excluding c-Myc from these promoter elements
(18,
20). TGF- stimulation
over 24 h induced a pronounced and sustained increase in Max and Mad4 protein
levels, and to a lesser extent Mxi1/Mad2, which was initiated 6 h
post-cytokine addition in both NMuMG and HaCaT cells
(Fig. 4A). Of the
remaining members of the Mad family, Mad1 was transiently up-regulated in
HaCaT cells at early time points (Fig.
4A), in agreement with a recent report
(43). However, Mad1 was not
detected in NMuMG cells and Mad3 was absent from both cell types
(Fig. 4A). The
observed increase in Mad4 protein results from elevated mad4 mRNA
levels in both NMuMG and HaCaT cells (Fig.
4B). These data argue that Mad4, and to a lesser extent
Mxi-1/Mad2, may function to inhibit Id2 expression in response to long term
TGF- treatment of several epithelial cell lineages.
Reduced Myc/Max Heterodimer Formation in Response to
TGF- Stimulation—The elevated levels of Mad2 and
Mad4 observed in response to TGF- suggests that the Mad proteins may
antagonize c-Myc function by binding to Max and displacing c-Myc from the
E-box element. To determine whether TGF- stimulation can inhibit
c-Myc/Max complex formation in the presence of appreciable c-Myc expression,
we investigated the effect of TGF- on the level of this complex.
Immunoprecipitation experiments revealed a significant and specific increase
in endogenous Max levels following TGF- stimulation
(Fig. 5). In the absence of
TGF-, c-Myc was efficiently associated with Max. However, treatment with
TGF- for 24 h dramatically reduced the amount of Myc that
heterodimerized with Max despite the observed increase in endogenous Max
levels (Fig. 5). This suggests
that the formation of Myc/Max heterodimers is specifically reduced in response
to TGF-. The observed increase in Max levels therefore suggests that a
significant amount of unbound Max is present to heterodimerize with members of
the Mad family.
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Reduced c-Myc and Enhanced Mad Binding to the Id2 Promoter in Response
to Sustained TGF- Stimulation—In order to directly
determine the impact of these TGF--induced events on the protein
complexes bound to the Id2 promoter, we decided to examine the levels
of Max, c-Myc, Mxi-1/Mad2, and Mad4 that were bound to the Id2
promoter in intact cells, in the absence or presence of prolonged TGF-
treatment. Chromatin immunoprecipitation assays were performed using a region
of the Id2 promoter containing the E-box elements. Consistent with
the decreased formation of Myc/Max heterodimers, TGF- stimulation caused
a reduction in the level of c-Myc that was bound to the Id2 promoter
(Fig. 6A). In
contrast, a constant amount of Max was bound to the same region of the
Id2 promoter in the absence or presence of TGF-
(Fig. 6A). The
observed decreases in c-Myc, but not Max, binding to the Id2 promoter
suggests that a shift from Myc/Max to Mad/Max complexes has occurred on the
Id2 promoter in TGF--treated cells. Indeed, TGF-
increased the amount of endogenous Mad4, and to a lesser extent Mxi-1/Mad2,
that was bound to the Id2 promoter
(Fig. 6B). This
suggests that the TGF--induced accumulation of Max-Mad complexes results
in the binding of these transcriptional repressors to the Id2
promoter, contributing to the sustained repression of Id2.
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We next tested whether forced expression of Mxi-1/Mad2 or Mad4 was
sufficient to repress Id2 expression in the absence of TGF-
stimulation. HaCaT and NMuMG cells were infected with retroviruses expressing
either Mxi-1/Mad2-IRES-GFP, Mad4-IRES-GFP, or control vector and RNA was
isolated from GFP positive cells recovered by cell sorting. Northern blot
analyses indicated that expression of either Mxi-1/Mad2 or Mad4 was not
sufficient to repress Id2 expression in HaCaT or NMuMG cells
(Fig. 6C). Given that
TGF- signaling reproducibly causes Id2 repression in these
cells (Figs. 1 and
2), our results argue that
additional TGF- induced events, in concert with c-Myc down-regulation
and Mad induction, are necessary for sustained Id2 repression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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mediates the cytostatic response in epithelial cell lineages. While c-Myc and
Id2 are similarly downregulated during the acute phase of the TGF-
response in NMuMG and HaCaT cells, the sustained repression of Id2
may additionally involve c-Myc independent mechanisms since c-Myc levels may
return to the basal state in the face of prolonged TGF- signaling. This
phenomenon is particularly evident in NMuMG cells, among the epithelial cell
lines that we have investigated. Moreover, chronic Id2 repression is
coincident with increased expression of Mxi-1/Mad2 and Mad4. We propose that
TGF- induced expression of Mxi-1/Mad2 and Mad4 causes a shift from
Myc/Max to Mad/Max complexes that bind to the Id2 promoter,
contributing to Id2 transcriptional repression
(Fig. 7). The delayed kinetics
of Mad induction by TGF- suggest that Mxi-1/Mad2 and Mad4 function to
help sustain, but not initiate, Id2 repression in response to TGF-. The
inability of Mxi-1/Mad2 or Mad4, when expressed exogenously, to repress
Id2 expression indicates that additional TGF- signals
participate in this gene response (Fig.
7).
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Given the observation that c-Myc can induce Id2
(34), and TGF-
suppresses c-Myc expression (1,
2), the ability of TGF-
to downregulate Id2 may represent a secondary event down-stream of
c-Myc repression. Indeed, the down-regulation of c-Myc has been shown to be an
obligate event for Id2 repression in a mink lung epithelial cell line
(34). However, our data from
immortalized mouse mammary epithelial cells suggest that Id2 down-regulation
can occur in the face of sustained c-Myc expression. The decrease in c-Myc
expression observed at early time points in NMuMG cells following TGF-
addition is subsequently erased by the re-emergence of c-Myc expression, while
Id2 levels still remain low. Rather, our observations suggest a different
mechanism that relies on the antagonism of c-Myc function. We show that
TGF- induces the expression of Mad family members, including a small
increase in Mxi-1/ Mad2 and a strong increase in Mad4 levels in NMuMG and
HaCaT cells. Recently, it has been shown that c-Myc can repress Mad4
transcription through Miz-1 in undifferentiated mouse erythroleukemia cells,
and that c-Myc down-regulation can lead to Mad4 induction upon
differentiation of these cells
(44). In the case of
TGF--stimulated NMuMG cells, Mad4 expression is induced at time points
when c-Myc levels are beginning to re-emerge, suggesting that c-Myc does not
repress Mad4 in this context. We demonstrate that elevated levels of Mad
proteins can influence the composition of transcriptional complexes on the
Id2 promoter, resulting in a shift from Myc/Max complexes that occupy
the E-box region in the absence of TGF-, to Mad/Max complexes following
TGF- stimulation. Interestingly, individually expressing either
Mxi-1/Mad2 or Mad4 was not sufficient to repress Id2 expression in
NMuMG and HaCaT in the absence of TGF- treatment. While these data do
not exclude a role for Mxi-1/Mad2 or Mad4 in the down-regulation of
Id2 expression, they indicate that additional TGF- signals
assist in mediating this gene response. Therefore, based on previous work, a
diminished recruitment of Myc-associated TRRAP/GCN5 complexes
(20) and enhanced binding of
Mad-associated corepressor and histone deacetylase complexes
(45), is predicted to
contribute to the maintenance of Id2 repression in epithelial
cells.
The mammary epithelium is a major physiological target of TGF-
(46) and its growth inhibitory
action continually enforces homeostasis except during pregnancy
(47–49).
Id2 function has also been shown to be important for lobulo-alveolar
development during pregnancy
(31). Recently, studies have
argued that Id2 protein is induced late during pregnancy
(32) and functions as a
differentiation factor in the mammary gland
(33). Interestingly, enforced
TGF- signaling during late pregnancy in transgenic mice induces
apoptosis that occurs at the same time that mammary gland apoptosis is
observed in Id2-null mice (31,
50).2
Therefore, it is possible that loss of TGF- signaling during mid to late
pregnancy allows Id2 expression and mammary gland differentiation.
Expression of the mad gene family has often been correlated with differentiation, although certain members are easily detected in proliferating cells (18). However, Mad overexpression has not been shown to be an efficient inducer of differentiation in all cell lineages (51, 52). Instead, ectopic expression of Mad family members has been shown to impair proliferation in both non-transformed and tumor-derived cell lines, and can block transformation by multiple oncogenes (18). Although the loss of Mad1 or Mad3, by gene-targeting approaches, does not lead to developmental defects nor to tumor susceptibility (53, 54), hyperplasia in multiple tissues are observed in Mxi-1/Mad2-null animals (55). Together, these observations indicate that Mad proteins may function primarily to inhibit proliferation rather than promote differentiation.
The ability of TGF- to induce Mad expression may antagonize c-Myc
function in cell types that lack profound and sustained c-Myc down-regulation
in response to this cytokine. Increased Mad levels, in concert with Max,
compete with Myc/Max heterodimers for E-boxes within the Id2
promoter, providing a basis for sustained Id2 repression by
TGF-. In this regard we have identified a mechanism for Id2
down-regulation in response to chronic TGF- signaling, that is distinct
from rapid Id2 repression by this cytokine. This mechanism may contribute to
the observed effects of extended TGF- signaling in mammary epithelial
cells. Given the tumor suppressive effects of TGF- during mammary
tumorigenesis (56), and the
anti-proliferative effects ascribed to Mad family members
(18), it will be interesting
to determine if the tumor suppressive effects of TGF- signaling in
epithelial cell lineages relies on the induction of Mad expression.
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FOOTNOTES |
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Supported by a fellowship from the Cancer Research Fund of the Damon
Runyon-Walter Winchell Foundation (DRG-1532).
An Investigator of the Howard Hughes Medical Institute. To whom correspondence
should be addressed: Cell Biology Program, Box 116, Memorial Sloan-Kettering
Cancer Center, 1275 York Ave., New York, NY 10021. Tel.: 212-639-8975; Fax:
212-717-3298; E-mail:
j-massague{at}ski.mskcc.org.
1 The abbreviations used are: TGF-, transforming growth factor ;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; cdk, cyclin-dependent
kinase.
2 Siegel, P. M., Shu, W., Cardiff, R. D., Muller, W. J., and Massagué,
J. (2003) Proc. Natl. Acad. Sci. U. S. A. 100,
8430–8435.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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