Notch signaling is necessary for epithelial growth arrest by TGF-β
ABSTRACT Transforming growth factor beta (TGF-beta) and Notch act as tumor suppressors by inhibiting epithelial cell proliferation. TGF-beta additionally promotes tumor invasiveness and metastasis, whereas Notch supports oncogenic growth. We demonstrate that TGF-beta and ectopic Notch1 receptor cooperatively arrest epithelial growth, whereas endogenous Notch signaling was found to be required for TGF-beta to elicit cytostasis. Transcriptomic analysis after blocking endogenous Notch signaling uncovered several genes, including Notch pathway components and cell cycle and apoptosis factors, whose regulation by TGF-beta requires an active Notch pathway. A prominent gene coregulated by the two pathways is the cell cycle inhibitor p21. Both transcriptional induction of the Notch ligand Jagged1 by TGF-beta and endogenous levels of the Notch effector CSL contribute to p21 induction and epithelial cytostasis. Cooperative inhibition of cell proliferation by TGF-beta and Notch is lost in human mammary cells in which the p21 gene has been knocked out. We establish an intimate involvement of Notch signaling in the epithelial cytostatic response to TGF-beta.
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ABSTRACT: The contribution of epithelial-to-mesenchymal transitions (EMT) in both developmental and pathological conditions has been widely recognized and studied. In a parallel process, governed by a similar set of signaling and transcription factors, endothelial-to-mesenchymal transitions (EndoMT) contribute to heart valve formation and the generation of cancer-associated fibroblasts. During angiogenic sprouting, endothelial cells express many of the same genes and break down basement membrane; however, they retain intercellular junctions and migrate as a connected train of cells rather than as individual cells. This has been termed a partial endothelial-to-mesenchymal transition. A key regulatory check-point determines whether cells undergo a full or a partial epithelial-to-mesenchymal transitions/endothelial-to-mesenchymal transition; however, very little is known about how this switch is controlled. Here we discuss these developmental/pathological pathways, with a particular focus on their role in vascular biology. © 2014 American Heart Association, Inc.Arteriosclerosis Thrombosis and Vascular Biology 11/2014; · 5.53 Impact Factor
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ABSTRACT: Epithelial-mesenchymal transition (EMT) and cell transformation have been well-documented in multiple cancer cell models and are believed to be one of the earliest events in tumor progression. Genetic and epigenetic modifications shift cells toward either end of the EMT spectrum, and can be influenced by the microenvironment surrounding a tumor. EMT and mesenchymal-epithelial transition are critical to normal function and development and an intricate network of transcription factors and transcriptional regulators tightly regulates these processes. As evidenced in normal and transformed cell lines, many signaling pathways trigger EMT during development and differentiation. The signaling pathways include those triggered by different members of the transforming growth factor superfamily, epidermal growth factor, fibroblast growth factor, hepatocyte growth factor, hypoxia-inducible factor, Wnt, Notch, and many others. Functional redundancies allow cells to undergo EMT even if these key transcriptional regulators are lacking, but these same redundancies also make these pathways particularly susceptible to gain-of-function mutations or constitutive signal activation; the "forced" transition toward either a mesenchymal or epithelial phenotype.Frontiers in oncology. 01/2014; 4:358.
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ABSTRACT: Background Endothelial cells (ECs) are responsible for creating a tumor vascular niche as well as producing angiocrine factors. ECs demonstrate functional and phenotypic heterogeneity when located under different microenvironments. Here, we describe a tumor-stimulated mesenchymal phenotype in ECs and investigate its impact on tumor growth, stemness, and invasiveness.Methods Xenograft tumor assay in NOD/SCID mice and confocal imaging were conducted to show the acquisition of mesenchymal phenotype in tumor-associated ECs in vivo. Immunocytochemistry, qPCR and flow cytometry techniques showed the appearance of mesenchymal traits in ECs after contact with breast tumor cell lines MDA-MB231 or MCF-7. Cell proliferation, cell migration, and sphere formation assays were applied to display the functional advantages of mesenchymal ECs in tumor growth, invasiveness, and enrichment of tumor initiating cells. qPCR and western blotting were used to investigate the mechanisms underlying EC mesenchymal transition.ResultsOur results showed that co-injection of ECs and tumor cells in NOD/SCID mice significantly enhanced tumor growth in vivo with tumor-associated ECs expressing mesenchymal markers while maintaining their intrinsic endothelial trait. We also showed that a mesenchymal phenotype is possibly detectable in human neoplastic breast biopsies as well as ECs pre-exposed to tumor cells (ECsMes) in vitro. The ECsMes acquired prolonged survival, increased migratory behavior and enhanced angiogenic properties. In return, ECsMes were capable of enhancing tumor survival and invasiveness. The mesenchymal phenotypes in ECsMes were the result of a contact-dependent transient phenomenon and reversed upon removal of the neoplastic contexture. We showed a synergistic role for TGFß and notch pathways in this phenotypic change, as simultaneous inhibition of notch and TGFß down-regulated Smad1/5 phosphorylation and Jag1KD tumor cells were unable to initiate the process.Conclusions Overall, our data proposed a crosstalk mechanism between tumor and microenvironment where tumor-stimulated mesenchymal modulation of ECs enhanced the constitution of a transient mesenchymal/endothelial niche leading to significant increase in tumor proliferation, stemness, and invasiveness. The possible involvement of notch and TGFß pathways in the initiation of mesenchymal phenotype may propose new stromal targets.Journal of translational medicine. 01/2015; 13(1):27.
T H E J O U R N A L O F C E L L B I O L O G Y
© The Rockefeller University Press $15.00
The Journal of Cell Biology, Vol. 176, No. 5, February 26, 2007 695–707
TGF-β inhibits cell growth and acts as a tumor suppressor (Levy
and Hill, 2006). TGF-β signals via receptor serine/threonine ki-
nases that phosphorylate Smad proteins, which move to the nu-
cleus and regulate gene transcription (Massagué et al., 2005).
During epithelial cytostasis (growth arrest), Smads induce cell
cycle inhibitors p15 and p21 and repress c-Myc and inhibitors of
differentiation Id1, Id2, and Id3 (Pardali and Moustakas, 2007).
TGF-β up-regulates rapidly and maintains prolonged p21
mRNA and protein levels, which is critical for epithelial cyto-
stasis (Nicolas and Hill, 2003; Pardali et al., 2005). The me ch-
anism of sustained p21 maintenance is not clear, and we
hypothesized that it could be achieved by a secondary wave
of TGF-β signaling that activates new factors capable of main-
taining p21 levels. A candidate pathway for involvement in such
a scenario is Notch, a major regulator of cell fate (Lai, 2004).
Four distinct mammalian receptors (Notch1–4) interact
extracellularly with transmembrane ligands Jagged1, 2, and
Deltalike1–3 (DLL1–3), which are expressed by adjacent cells
(Lai, 2004). Such an interaction leads to the proteolytic cleav-
age of Notch by the γ-secretase activity of presenilin, thus re-
leasing the Notch intracellular domain, which enters the nucleus
and regulates transcription after binding to the transcription
factor CSL (Lai, 2004).
Retroviral insertions in mice and chromosomal transloca-
tions in human leukemias cause oncogenic truncations or fu-
sions of Notch (Radtke and Raj, 2003). The skin- or liver-specifi c
knockout of Notch1 leads to tumorigenesis, classifying Notch1
as a tumor suppressor (Nicolas et al., 2003; Croquelois et al.,
2005). Notch1 inhibits epidermal, endothelial, and hepatic cell
growth (Rangarajan et al., 2001; Qi et al., 2003; Noseda et al.,
2004). Notch arrests the keratinocyte cell cycle by transcrip-
tionally inducing p21 via CSL or calcineurin–nuclear factor of
activated T cells pathway activation (Rangarajan et al., 2001;
Mammucari et al., 2005).
Notch and TGF-β pathways cross talk, as TGF-β induces
Jagged1 expression, leading to epithelial-mesenchymal transi-
tion (Zavadil et al., 2004). During heart organogenesis, Notch
uses TGF-β signaling to cause the epithelial-mesenchymal tran-
sition (Timmerman et al., 2004). Alternatively, Notch induces
nodal, a TGF-β family regulator of embryogenesis (Raya et al.,
2003). The Notch intracellular domain directly binds to Smads,
leading to the coregulation of gene expression in neuronal and
endothelial cells (Blokzijl et al., 2003; Itoh et al., 2004).
Notch signaling is necessary for epithelial growth
arrest by TGF-β
Hideki Niimi, Katerina Pardali, Michael Vanlandewijck, Carl-Henrik Heldin, and Aristidis Moustakas
Ludwig Institute for Cancer Research, Biomedical Center, Uppsala University, SE-751 24 Uppsala, Sweden
invasiveness and metastasis, whereas Notch supports onco-
genic growth. We demonstrate that TGF-β and ectopic
Notch1 receptor cooperatively arrest epithelial growth,
whereas endogenous Notch signaling was found to be re-
quired for TGF-β to elicit cytostasis. Transcriptomic analysis
after blocking endogenous Notch signaling uncovered
several genes, including Notch pathway components and
cell cycle and apoptosis factors, whose regulation by TGF-β
ransforming growth factor β (TGF-β) and Notch act
as tumor suppressors by inhibiting epithelial cell
proliferation. TGF-β additionally promotes tumor
requires an active Notch pathway. A prominent gene co-
regulated by the two pathways is the cell cycle inhibitor
p21. Both transcriptional induction of the Notch ligand
Jagged1 by TGF-β and endogenous levels of the Notch
effector CSL contribute to p21 induction and epithelial
cytostasis. Cooperative inhibition of cell proliferation by
TGF-β and Notch is lost in human mammary cells in which
the p21 gene has been knocked out. We establish an inti-
mate involvement of Notch signaling in the epithelial cyto-
static response to TGF-β.
H. Niimi and K. Pardali contributed equally to this paper.
Correspondence to Aristidis Moustakas: firstname.lastname@example.org
H. Niimi’s present address is Toyama University Faculty of Medicine, Dept. of
Clinical and Molecular Pathology, Toyama City 930-0194, Japan.
K. Pardali’s present address is Molecular Medicine, Dept. of Genetics and
Pathology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden.
Abbreviations used in this paper: GAPDH, glyceraldehyde-3′-phosphate
dehydrogenase; GSI, γ-secretase inhibitor; HMEC, human mammary epithelial
cell; MOI, multiplicity of infection; N1ICD, Notch1 intracellular domain.
JCB • VOLUME 176 • NUMBER 5 • 2007 696
Based on these facts, we investigated cross talk between TGF-β
and Notch during epithelial cytostasis. We demonstrate that the
TGF-β cytostatic response at least partly requires Notch signaling.
A novel mechanism based on transcriptional induction of the
Notch ligand Jagged1, involvement of the Notch effector CSL,
and sustained p21 induction explains the interdependent roles
of TGF-β and Notch during cytostasis.
Notch and TGF-훃 cooperatively arrest
epithelial cell growth
To study cross talk between Notch and TGF-β, we ectopically
expressed the human Notch1 intracellular domain (N1ICD;
Rangarajan et al., 2001). Usually, 70–80% of cells expressed
N1ICD at roughly endogenous levels, which induced a classic
target of this pathway (transcription factor Hes1; unpublished
data). In mock-infected (Ad-GFP) mouse mammary epithelial
NMuMG cells, TGF-β1 suppressed S-phase entry by 60–70%
(Fig. 1 A). Ectopic N1ICD did not have much effect on its
own, but N1ICD plus TGF-β1 suppressed S-phase entry by
80–95% (Fig. 1 A). This effect was dependent on TGF-β1 dose
(Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb
.200612129/DC1) and was also confi rmed in human mammary
MCF-10A cells (see Fig. 8 A).
TGF-β1 stimulation in the presence of a γ-secretase
inhibitor (GSI), which blocks endogenous Notch signaling
(Brunkan and Goate, 2005), led to a substantial but not complete
restoration of S-phase entry (Fig. 1 B), which was confi rmed in the
mammary epithelial MCF-10A cells (see Fig. 8 C) and in immor-
talized human mammary epithelial cells (HMECs; Fig. S1 B).
In contrast, in mink lung epithelial cells, ectopic N1ICD inhib-
ited the suppressive effect of TGF-β1 (Fig. S1 C). This high-
lights the cell context dependency of the cytostatic response
and confi rms a recent study that shows c-Myc up- regulation
by Notch signaling, which counteracts cytostasis by TGF-β
(Rao and Kadesch, 2003).
In human HaCaT keratinocytes, Ad-N1ICD alone sup-
pressed S-phase entry almost to the same extent as 2 ng/ml
TGF-β1 (Fig. 1 C). Ad-N1ICD combined with TGF-β1 led to
>95% growth suppression, and up to 80% of the cells were
arrested in G1 phase of the cell cycle (Fig. 1 E). This showed
strong Notch1–TGF-β1 cooperativity that was blocked by
TGF-β receptor kinase inhibitors (Fig. S1 D), suggesting the
interdependence of the two pathways. GSI also blocked cyto-
stasis by TGF-β1 in HaCaT cells (Figs. 1 D and S1 E) and
shifted the cell cycle profi le to that of mock-treated cells (Fig. 1 F).
We conclude that Notch and TGF-β cooperatively induce
growth arrest in human and mouse epithelial cells of mammary
and skin origin. Endogenous Notch signaling is partly neces-
sary for growth arrest by TGF-β.
Notch signaling is required for the
regulation of many genes by TGF-훃
To further understand the Notch–TGF-β cross talk, we per-
formed a transcriptomic screen in HaCaT cells stimulated with
TGF-β1 in the absence or presence of GSI. We measured gene
expression after cycloheximide pretreatment after 2, 6, and 48 h
of TGF-β1 stimulation, aiming at immediate/early, intermediate,
and sustained gene responses. Several hundred TGF-β–responsive
genes were measured (Fig. 2 A and Table S1, available at http://
www.jcb.org/cgi/content/full/jcb.200612129/DC1), which is in
accordance with previous microarray analyses in the same cell
line (Akiyoshi et al., 2001; Zavadil et al., 2001; Kang et al.,
2003). GSI decreased the number of TGF-β–regulated genes
by 36% (Fig. 2 A and Table S1). At 2 h, only immediate/early
TGF-β gene targets were measured, and GSI had no effect. At
6 h, we observed 85% inhibition. At 48 h, we did not observe
dramatic effects on total gene numbers, but effects were seen on
individual gene profi les.
A comparison of the two gene lists (minus and plus GSI)
showed 198 genes whose response to TGF-β was unaffected by
GSI (Fig. 2, B and C). Examples are TIEG (TGF-β–inducible early
growth response protein 1), a zinc fi nger transcription and pro-
apoptotic factor; TNFSF10 (TNF ligand superfamily member 10),
Figure 1. Notch and TGF-훃 cooperate during epithelial growth arrest.
(A–D) Thymidine incorporation assays in NMuMG (A and B) or HaCaT
(C and D) cells infected with Ad-GFP or Ad-N1ICD (multiplicity of infection
[MOI] of 50) and stimulated with vehicle (−) or 2 ng/ml TGF-β1 for 60 h
(A and C) or stimulated with TGF-β1 in the presence or absence of 4 μM
GSI (B and D). In D, 0.5–5 ng/ml TGF-β1 was used. (E and F) HaCaT cell
cycle analysis under conditions as in C and D. The percentage of cells per
cell cycle phase is plotted in bar graphs. Error bars represent SD.
NOTCH AND TGF-β IN EPITHELIAL CYTOSTASIS • NIIMI ET AL.697
a proapoptotic secreted protein; NF2 (neurofi bromin 2), a cyto-
skeletal regulator; and IFITM (interferon-induced trans-
membrane protein 1), a cell surface antigen. The expression of
394 TGF-β1–responsive genes (roughly 50% of the regulated
genes) was neutralized by GSI, demonstrating a strong depen-
dency on Notch (Fig. 2, B and C). Examples are STRAP (serine-
threonine kinase receptor-associated protein), an adaptor that
binds to TGF-β receptor and inhibitory Smad7 to mediate the
termination of TGF-β signaling; SMURF1 (Smad ubiquity-
lation regulatory factor 1), an E3 ubiquitin ligase that causes
TGF-β receptor and Smad degradation; S100A11, a calcium-
binding protein that mediates epithelial cytostasis by TGF-β as
it transcriptionally induces the cell cycle inhibitor p21; and IVL
(involucrin), a keratinocyte differentiation marker that cross-
links to the keratin cytoskeleton. Finally, 179 genes were not
previously recognized as TGF-β targets, as their regulation is
revealed only after GSI treatment (Fig. 2 B), suggesting that
Notch signaling may repress genes in a manner that prohibits
responses to TGF-β. This experimental design did not test for
adverse effects of GSI on gene expression in general, which is
formally possible. However, GSI both inhibited and induced
specifi c gene expression when combined with TGF-β, and we
never observed adverse effects of GSI in the absence of TGF-β
in RT-PCR assays.
For the fi rst time, we uncovered large gene sets that are co-
regulated by TGF-β and Notch positively or negatively (Fig. 2
and Table S1). Notch seemed to counteract the regulation of
many genes by TGF-β1. This suggests that to a large extent, the
transcriptomic response to TGF-β incorporates regulation by
TGF-훃1 induces the expression
of Notch ligands and modulates
the Notch receptor profi le
Among the genes identifi ed, two were members of the Notch
pathway: TGF-β1 induced JAGGED1 (JAG1) and repressed
NOTCH1 (Fig. 3 A). We examined whether TGF-β1 regu-
lates the expression of all Notch ligands and receptors in
HaCaT (Fig. 3, B and C) and NMuMG cells (Fig. S2, available
TGF-β1 considerably induced JAG1 mRNA and protein and
DLL4 mRNA, weakly induced DLL3 mRNA at 24 h, and did
not appreciably affect JAG2 or DLL1 mRNA in HaCaT cells. In
NMuMG cells, TGF-β1 induced Jag1 mRNA and protein and
Dll1 mRNA but did not appreciably regulate Jag2 mRNA
levels (Fig. S2, A and B). On the other hand, TGF-β1 repressed
NOTCH1 mRNA and protein in HaCaT cells (Fig. 3, B and C).
Even more dramatic was NOTCH3 repression by TGF-β1 in the
same cells (Fig. 3, B and C). NOTCH2 and NOTCH4 expres-
sion was not appreciably affected by TGF-β1 in HaCaT cells
(Fig. 3 B). Although similar HaCaT expression profi les were
measured for the Notch1 receptor in NMuMG cells respond-
ing to TGF-β1, Notch4 mRNA and protein were considerably
induced in NMuMG cells (Fig. S2, C and D).
In HaCaT cells, GSI primarily perturbed the expres-
sion profi le of JAG1 and, to a lesser extent, that of NOTCH3
(Fig. 3, B and C), weakly induced DLL1 at 24 h, and repressed
the weak induction of DLL3 at 24 h but did not affect the
other regulated ligands or receptors. This agrees with the effect
of cycloheximide that blocks the induction of JAG1, DLL1,
and DLL3 by TGF-β1 (unpublished data), which represents
indirect Notch-mediated responses to TGF-β. The lack of
effect of GSI on Notch receptor profi les is also seen at the
protein levels of TβRI, which is slowly down-regulated dur-
ing the time course but is not affected by GSI (Fig. 3 C). We
conclude that TGF-β1 induces the expression of endogenous
Notch ligands in keratinocytes and mammary epithelial cells,
whereas the regulation of Notch receptors is complex and tis-
sue type dependent. Between the two regulated ligands JAG1
and DLL4, we could only verify the regulation of JAG1 pro-
tein (Fig. 3 C), as our DLL4 antibody showed poor effi cacy
(unpublished data). Thus, during the stimulation of epithelial
cells with TGF-β, the initial induction of various Notch ligands
may activate this pathway, whereas the delayed repression of
Notch receptors may refl ect a negative loop of Notch receptor
Figure 2. Transcriptomic analysis of the dependence of TGF-훃1 on Notch
signaling. (A and B) Cumulative gene expression data from HaCaT cells (A)
and Venn diagrams (B) that cluster genes to each category of cell treatment.
The total (Tot) gene numbers indicate the number of annotated (a) and
non annotated (na) genes. Gray table cells indicate signifi cant deviations
(P < 0.01) upon GSI treatment relative to the control. In B, up- and down-
regulated (arrows) gene numbers are shown within each Venn diagram.
(C) Kinetic graphs of eight representative genes with expression values
(arbitrary units [au]) calculated from the microarray data. Error bars
JCB • VOLUME 176 • NUMBER 5 • 2007 698
TGF-훃 target genes that regulate the cell
cycle or induce apoptosis
11 genes with known links to the cytostatic and apoptotic pro-
grams of TGF-β were identifi ed in the transcriptomic screen: the
cell cycle inhibitors p21 (CDKN1A) and p15 (CDKN2B), cyclins
B2 (CCNB2), D1 (CCND1), and D2 (CCND2), the transcrip-
tional regulators c-Myc (MYC) and Id2 (IDB2), the signal trans-
ducer S100A11 (S100A11), and the apoptotic/survival regulators
GADD45β (GADD45B), GADD45γ (GADD45G), and TIEG
(TIEG-1/KLF10; Fig. 4 A). Prolonged up- or down-regulation
of many of these genes was neutralized by GSI (Fig. 4 A) as
verifi ed by quantitative RT-PCR analysis (Fig. 4 B).
The c-MYC gene, a well studied transcriptional target of
TGF-β/Smad signaling that plays major regulatory roles in
the epithelial cytostatic program of TGF-β (Chen et al., 2002),
exhibited its characteristic repression phase followed by the re-
covery of basal mRNA levels after 24 h of TGF-β stimulation
(Fig. 4 B). GSI did not affect the c-MYC expression profi le, sug-
gesting that endogenous Notch signaling is not involved in the
c-MYC response of keratinocytes, which is in contrast to what
was previously reported for mink lung epithelial cells that over-
expressed N1ICD (Rao and Kadesch, 2003). Similar to c-MYC,
cyclin B2 (CCNB2) also exhibited relative insensitivity to GSI
throughout the time course. Among the 11 genes of the cyto-
static/apoptotic program, GSI most prominently affected p21,
p15, IDB2, S100A11, and GADD45B expression profi les from
6 h onwards (Fig. 4, A and B; p21, p15, and GADD45B). The
immediate/early response of all the genes measured after 2 h of
stimulation with TGF-β1 in the presence of cycloheximide was
not substantially affected by GSI (Fig. 4, A and B).
GSI quantitatively reduced the amplitude of the mRNA
profi les of the aforementioned genes (Fig. 4 B, GADD45B and
CDKN2B) but preserved the dynamic changes in the overall
profi le of mRNA expression. In the case of the p21 cell cycle
inhibitor, GSI not only reduced the amplitude of the response
but also distorted the expression profi le beyond 2 h dramatically
(Fig. 4 B). Although the immediate/early response of p21 to
TGF-β1 was unaffected by GSI, long-term p21 mRNA induc-
tion was substantially blocked by GSI, suggesting that Notch
signaling was critical for this response, acting as a secondary
signal to the primary TGF-β stimulus. The effect of GSI was also
considerable at the protein level because sustained (6–24 h) p21
protein induction by TGF-β1 was converted to an early response
(1.5–3 h) in the presence of GSI (Fig. 4 C). This prompted us to
further analyze the profi le of p21 expression and also test the func-
tional relevance of this profi le. The present data demonstrate that
although endogenous Notch signaling contributes to regulation of
a substantial subset of the TGF-β cytostatic gene program, Notch
is not involved in the regulation of every gene in this program.
Induction of Jagged1 by TGF-훃
contributes to p21 gene regulation
and epithelial cytostasis
The evidence so far has led to a working model in which TGF-β
signaling induces Jagged1 production, which then leads to
Notch receptor activation and further signaling via CSL, leading
to regulation of the cell cycle inhibitors p15 and p21 and, thus,
mediating epithelial cell cycle arrest (Fig. 5 A). To examine the
functional relevance of JAG1 induction by TGF-β during
cytostasis, we depleted endogenous JAG1 by siRNA (Fig. 5 B).
The three- to fourfold induction of JAG1 mRNA throughout the
24-h time course in response to TGF-β was reduced to a mere
1.3–1.6-fold induction in the presence of siRNA. Under the
same conditions of endogenous JAG1 depletion, JAG1 protein
accumulation in the 6–24-h interval of the time course was se-
verely lost to essentially undetectable levels (Fig. 5 C). The
specifi city of JAG1 siRNA–mediated depletion was verifi ed by
demonstrating that three unrelated proteins, Smad2, Smad3, and
α-tubulin, were not affected by the same siRNA. In addition to
the total Smad2 and Smad3 levels, TGF-β–inducible phospho-
Smad2 and -Smad3 levels were not appreciably affected by
JAG1 siRNA during the 24-h time course (Fig. 5 C). Notably, the
Figure 3. TGF-훃1 regulates the expression of Notch ligands and receptors.
(A) Kinetic expression profi les of JAG1 and NOTCH1 measured via micro-
array analysis (expressed in arbitrary units [au]). (B) Quantitative RT-PCR of
JAG1,2, DLL1,2,3, and NOTCH1,2,3,4 mRNAs in HaCaT cells stimulated
with 2 ng/ml TGF-β1 for 2, 6, 24, and 48 h in the absence (DMSO, −;
gray lines) or presence of 4 μM GSI (+; black lines). Relative gene expres-
sion values (fold change) after normalization to GAPDH gene expression
are shown. (C) Immunoblot analysis of JAG1, NOTCH1, NOTCH3, TβRI,
and control β-tubulin protein levels from HaCaT cells treated with TGF-β1
and GSI as indicated. Immunoblots of total cell lysates (IB) or immunoblots
after immunoprecipitation (IP/IB) are shown. Error bars represent SD.
NOTCH AND TGF-β IN EPITHELIAL CYTOSTASIS • NIIMI ET AL.699
knockdown of JAG1 mRNA and protein resulted in a concomi-
tant decrease in the TGF-β–inducible levels of p21 mRNA and
protein (Fig. 5, D and E). This decrease was evident throughout
the time course and was more robust during the 6–24-h interval
when endogenous JAG1 protein accumulated at maximal levels.
Finally, we demonstrated that JAG1 knockdown reverted the
70% growth inhibition by TGF-β1 to a mere 25% inhibition
(Fig. 5 F), suggesting that endogenous JAG1 participates in the
TGF-β cytostatic response. These data strongly suggest that
transcriptional induction of the JAG1 gene by TGF-β is inti-
mately linked to the robust transcriptional induction of the p21
cell cycle inhibitor and to the growth inhibitory response of
HaCaT keratinocytes (Fig. 5 A).
Importance of CSL during p21 induction
and epithelial growth inhibition by TGF-훃
Knockdown of endogenous Notch1 via siRNA was effective but
failed to inactivate Notch signaling (unpublished data), as epithe-
lial cells express other Notch receptors whose expression is regu-
lated by TGF-β (Figs. 3 and S2). Therefore, we depleted CSL,
which is the only known common mediator of all Notch sig-
naling pathways. siRNA reduced CSL mRNA expression by
85% (Fig. 6 A) and reduced protein to undetectable levels (Fig. 6 F),
whereas mock siRNA had no effect in HaCaT cells. The CSL
knockdown was specifi c as verifi ed by demonstrating that three
unrelated proteins (Smad2, Smad3, and α-tubulin) were not af-
fected by the same siRNA. In addition, the TGF-β–inducible
phospho-Smad2 and -Smad3 levels were not appreciably affected
by the CSL siRNA during the 48-h time course (Fig. 6 B). A com-
parable 65–75% knockdown of endogenous CSL was achieved
when HaCaT cells were simultaneously infected with mock
(Ad-GFP) or specifi c (Ad-N1ICD) adenoviruses (Fig. 6 C). During
the concomitant stimulation of HaCaT cells with TGF-β1, we
observed a minor trend for the induction of endogenous CSL
mRNA levels (Fig. 6, A and C), which we could not reproduce at
the protein level (Fig. 6 F). As an additional confi rmation of the
specifi city of CSL siRNA, ectopic N1ICD mRNA levels obtained
after adenoviral infection of HaCaT cells were not affected by
knocking down endogenous CSL (Fig. 6 D). Notably, under the
same conditions of the combined knockdown of endogenous
CSL and ectopic N1ICD expression, endogenous p21 mRNA in-
duction was dramatically reduced (Fig. 6 E). Under mock infec-
tion conditions, the 2.5–3-fold induction of endogenous p21
mRNA by TGF-β1 was reduced to a weak 1.3-fold induction
(Fig. 6 E), which was correspondingly refl ected at the p21 pro-
tein level (Fig. 6 F). Furthermore, the synergistic p21 induction
by TGF-β1 and N1ICD also depended on proper endogenous
CSL levels because knockdown of the latter considerably reduced
the inducible p21 mRNA levels (Fig. 6 E) and even more dramat-
ically reduced the corresponding p21 protein levels (Fig. 6 F).
Finally, CSL knockdown substantially reverted cytostasis
by TGF-β1; in mock-transfected cells, TGF-β1 stimulation
caused a suppression of thymidine incorporation to 35% of
unstimulated cells, whereas after CSL knockdown, the sup-
pression was only to 64% of unstimulated cells (Fig. 6 G). In
addition, siCSL reverted the cytostatic effect of N1ICD alone to
control levels and strongly blocked synergistic cytostasis by
TGF-β1 plus N1ICD (Fig. 6 G). These experiments with CSL
knockdown demonstrate a similar phenotype to JAG1 knock-
down (Fig. 5) or the inhibition of γ-secretase activity by GSI
(Figs. 1 and 4) and collectively prove that endogenous Notch/
CSL signaling is critical, at least in part, for the antiproliferative
response of HaCaT cells to TGF-β.
Partial dependence of TGF-훃 receptor
signaling on 후-secretase activity
The γ-secretase activity of presenilin regulates Notch, Wnt/
β-catenin, CD44, ErbB signaling, and β-amyloid processing and
Figure 4. TGF-훃 target genes of the cytostatic
and apoptotic program and their dependence
on Notch signaling. (A) Table listing 11 regu-
lated genes with links to cell cycle and apop-
tosis and statistically signifi cant (black;
P < 0.05) or nonsignifi cant (gray; P > 0.05)
expression values. Gray cells indicate genes
for which GSI treatment had a clear impact.
(B) Quantitative RT-PCR of c-Myc, CCNB2,
GADD45B, CDKN2B, and CDKN1A mRNAs in
HaCaT cells treated as in Fig. 3 B. (C) Immuno-
blot of endogenous p21 and control β-tubulin
in HaCaT cells stimulated with 2 ng/ml TGF-β1
for 0–24 h in the absence (−, DMSO) or pres-
ence (+) of 4 μM GSI.
JCB • VOLUME 176 • NUMBER 5 • 2007 700
deposition in Alzheimer’s disease (Brunkan and Goate, 2005).
TGF-β did not appreciably affect the expression or activation
of CD44 and ErbB2 in our cell models nor did ligands for these
receptors show cooperation with TGF-β–induced cytostasis
(unpublished data). More convincingly, the similarity of cel-
lular phenotypes with respect to p21 gene regulation and kera-
tinocyte proliferation arrest obtained after the use of GSI and
knockdown of endogenous JAG1 and CSL after siRNA transfec-
tion strongly enforces the model that Notch signaling operates
downstream of TGF-β during epithelial cell growth inhibition
(Fig. 5 A). During the course of all of the previous experiments,
we also monitored the infl uence of Notch pathway inhibition on
the primary activation step of Smad signaling, namely the TGF-β
receptor–mediated phosphorylation of Smad2 and Smad3. As
previously presented, the knockdown of JAG1 or CSL did not
appreciably perturb the normal fl ow of TGF-β receptor signaling
as monitored by phospho-Smad protein levels in extensive time
course experiments (Figs. 5 B and 6 B).
In contrast, when the same experiment was repeated after
the stimulation of HaCaT cells with TGF-β1 in the presence of
GSI, we could observe a partial but considerable inhibition of
both phospho-Smad2 and -Smad3 levels (Fig. 7 A). The nega-
tive and adverse effects of GSI on phospho-Smad levels was
evident throughout extensive time course experiments and was
more prominent after 2 h of stimulation with TGF-β1. To test
whether activated Notch signaling led to the opposite effect,
namely the induction of phospho-Smad levels in HaCaT cells,
we infected cells with mock (Ad-GFP) or specifi c (Ad-N1ICD)
adenoviruses and measured phospho-Smad2/3 (Fig. 7 B).
Although TGF-β1 induced robust phospho-Smad2 and -Smad3
levels in HaCaT cells, in the presence of control (Ad-GFP) or
Ad-N1ICD adenovirus, N1ICD by itself failed to induce phospho-
Smad levels. Furthermore, TGF-β1 stimulation of cells ex-
pressing ectopic N1ICD did not lead to any further increase
of phospho-Smad levels compared with TGF-β1 stimulation
alone (Fig. 7 B). Therefore, we conclude that Notch signaling
does not seem to contribute to R-Smad phosphorylation by
TGF-β receptors in HaCaT cells. However, the use of GSI
demonstrates that γ-secretase activity is linked to the process
of R-Smad phosphorylation by TGF-β receptors via as yet
The aforementioned result on a potential role of γ-secretase
during R-Smad activation obliged us to test even more rigor-
ously the specifi city of the observed effects of GSI on p21
gene induction and epithelial cytostasis downstream of TGF-β.
If the GSI effect was primarily caused by the reduction on
phospho-Smad levels, Notch should not be able to rescue such
effects when provided ectopically. Upon control Ad-GFP infec-
tion, TGF-β1 induced endogenous p21 protein levels, and GSI
partially blocked this response (Fig. 7 C) as described for un-
infected cells (Fig. 4 C). Under such conditions, we also veri-
fi ed that p21 protein induction by TGF-β1 could be enhanced
by ectopic N1ICD (Fig. 7 C), confi rming a cooperative role
of TGF-β1 and Notch1 signaling in maintaining high p21 pro-
tein levels. Furthermore, the rescue of p21 expression could
be achieved by ectopic N1ICD in a dose-dependent manner
(Fig. 7 C). The 3.8-fold inhibition elicited by GSI became 1.3-fold
when GSI was combined with a high dose of N1ICD, confi rming
that GSI primarily blocks endogenous Notch signaling during
Figure 5. Jagged1 is a TGF-훃 target that reg-
ulates p21 induction and epithelial cytostasis.
(A) Diagram of the signaling pathway estab-
lished in this paper (black arrows). Gray
arrows point to previously established regula-
tory connections between components of the
pathway. Inhibitory connections with compounds
and siRNAs at the bottom illustrate the experi-
mental means used during this study. TβR,
TGF-β receptor; LY, TGF-β receptor type I inhib-
itor LY580276; KO, knockout. (B) Quantitative
RT-PCR analysis of endogenous JAGGED1
(JAG1) mRNA levels normalized over endog-
enous GAPDH from HaCaT cells transiently
transfected with siLuc (black lines) or siJAG1
(gray lines) and subsequently stimulated with
2 ng/ml TGF-β1 for 0, 2, 6, 12, and 24 h.
(C) Immunoblot of endogenous JAG1, phospho-
Smad2, phospho-Smad3, total Smad2 and
Smad3, and α-tubulin control from HaCaT cells
transfected as in B and stimulated with 2 ng/ml
TGF-β1 for the indicated time points. (D) Quan-
titative RT-PCR analysis of endogenous p21
(CDKN1A) mRNA levels normalized over en-
dogenous GAPDH from HaCaT cells trans-
fected and stimulated as in B. (E) Immunoblot
of endogenous p21 and β-tubulin control from
HaCaT cells transfected as in B and stimulated
with 2 ng/ml TGF-β1 for the indicated time
points. (F) Thymidine incorporation assay in
HaCaT cells transfected as in B and stimulated
with vehicle (gray bars) or 2 ng/ml TGF-β1
(black bars) for 60 h. Error bars represent SD.
NOTCH AND TGF-β IN EPITHELIAL CYTOSTASIS • NIIMI ET AL.701
Similar to these rescue experiments of p21 induction,
thymidine incorporation assays confi rmed our conclusion about
a major role of GSI as a Notch pathway inhibitor (Fig. 7 D).
Accordingly, upon control Ad-GFP infection, TGF-β1 inhib-
ited S-phase entry, and GSI reversed this effect (Fig. 7 D).
Dose-dependent Ad-N1ICD infection reduced cell growth and,
combined with TGF-β1, suppressed growth by 98% (Fig. 7 D).
Under these conditions, GSI could weakly restore cell growth,
and the higher the N1ICD dose, the less effective GSI was.
Thus, N1ICD can antagonize GSI, suggesting that the Notch
pathway is a primary target of GSI in the cell model used.
It follows from the model we present in Fig. 5 A that if
p21 induction and epithelial cytostasis by TGF-β requires
downstream activation of Notch signaling and because JAG1
protein levels accumulate after 6 h of stimulation with TGF-
β1 (Fig. 5 B), the addition of GSI in HaCaT cells that are pre-
stimulated with TGF-β1 should effectively block the cytostatic
response. In all previous experiments, GSI was added 0.5–1 h
before TGF-β1 (Fig. 1). However, also when GSI was added
12–18 h after TGF-β1 stimulation, it was effective in blocking
the cytostatic response of TGF-β1 weakly by 20–30%, whereas
addition between 24 and 48 h gradually enhanced the potency
of TGF-β1 in causing cytostasis (Fig. 7 E). Thus, a time win-
dow for TGF-β1 cytostasis spans the fi rst 24 h. On the other
hand, the addition of GSI 12–48 h after TGF-β1 could not con-
siderably block p21 protein induction (Fig. 7 F). Thus, robust
levels of p21 correspond to the even weak (20%) suppression of
thymidine incorporation observed when GSI is added 12 h after
TGF-β1 (Fig. 7, E and F; GSI 48 h). This implies that the early
period of 0–12 h of TGF-β stimulation represents a critical
window during which both p21 induction and suppression of
S-phase entry is sensitive to GSI. The small difference observed
between the effect of GSI on p21 expression (Fig. 7 F) and thy-
midine incorporation (Fig. 7 E) when added after TGF-β stimu-
lation emphasizes the role of additional cytostatic regulators
such as p15, S100A11, and Id2, which are coregulated by TGF-β
and Notch, or c-Myc, which is not regulated by Notch (Fig. 4).
Thus, we conclude that GSI primarily acts as an inhibitor of the
Notch pathway, and its adverse effect on the accumulation of
phosphorylated R- Smads cannot fully explain the cellular pheno-
types under investigation.
p21 expression is a critical factor during
epithelial cytostasis by TGF-훃–Notch
The data support a model whereby TGF-β induces Notch lig-
ands that activate signaling. This supports the duration of TGF-β
signaling at suffi ciently high and long levels for cell cycle ar-
rest to occur (Fig. 5 A). The induction of JAG1 (and possibly
Figure 6. CSL signaling is critical for p21 induction and epithelial growth arrest by TGF-훃. (A) Quantitative RT-PCR of CSL mRNA levels in HaCaT cells trans-
fected with control siLuc or specifi c siCSL siRNAs and stimulated or unstimulated with 2 ng/ml TGF-β1 for 16 h. (B) Immunoblot of endogenous phospho-
Smad2, phospho-Smad3, total Smad2 and Smad3, and endogenous control α-tubulin levels from HaCaT cells transiently transfected with siCSL or siLuc
before stimulation with 2 ng/ml TGF-β1 for the indicated time points. (C–E) Quantitative RT-PCR analysis of CSL, ectopic Ad-N1ICD, and p21 (CDKN1A)
mRNA normalized over GAPDH in HaCaT cells transfected with siLuc or siCSL and subsequently infected with Ad-GFP or Ad-N1ICD (MOI of 50) before
stimulation with 2 ng/ml TGF-β1 for 24 h. (F) Immunoblot of endogenous p21 and CSL, ectopic Ad-N1ICD, and endogenous control β-tubulin levels from
HaCaT cells transiently transfected with siCSL or siLuc and subsequently infected with Ad-GFP or Ad-N1ICD (MOI of 50) before stimulation with 2 ng/ml
TGF-β1 for 24 h. The conditions are identical to those in C–E. (G) Thymidine incorporation assay in HaCaT cells transfected with siRNAs as in C, which
were subsequently coinfected with the indicated adenoviruses (MOI of 50) and were stimulated or unstimulated with 2 ng/ml TGF-β1 for 60 h. Error bars
JCB • VOLUME 176 • NUMBER 5 • 2007 702
DLL4) by TGF-β leads to the activation of endogenous Notch/
CSL signaling, which is required for sustained p21 induction and
is important for epithelial cytostasis by TGF-β. The latter con-
clusion was verifi ed after ectopic p21 expression (Fig. S3, avail-
able at http://www.jcb.org/cgi/content/full/jcb.200612129/DC1),
which antagonized the reversion in TGF-β1–mediated growth
arrest elicited by GSI and led to robust cytostasis (Fig. S3 A).
Interestingly, very high levels of ectopic p21 protein (6–10-fold
relative to the endogenous TGF-β–induced p21 level) were
required to bypass the neutralizing effect of GSI (Fig. S3 B).
This suggests that TGF-β in the presence of GSI might induce
target genes that permit sustained cell proliferation even in the
presence of high levels of potent cell cycle inhibitors such as
p21. This fi nding plus the previous result on p21 induction by
TGF-β1 in the presence of GSI that was added several hours
after TGF-β1 (Fig. 7 F) raised the possibility that although p21
clearly is a responsive gene to the TGF-β–Notch pathways, the
physiological relevance of p21 to epithelial cytostasis induced
by the same pathways remains to be determined.
To rigorously test the role of p21 on epithelial cytostasis
downstream of TGF-β–Notch signaling, we attempted to knock-
down p21 expression in HaCaT cells after siRNA transfection.
Such attempts always led to a partial reduction of p21 mRNA
and protein levels by 60–70%, which correlated with a partial
defect in the cytostatic response to TGF-β (unpublished data).
To obtain defi nitive evidence for a role of p21 in the TGF-β
cytostatic program, we made use of two individual cell clones
of human mammary epithelial MCF-10A cells, whose endoge-
nous p21 gene was deleted after homologous recombination
(Fig. 8 and Fig. S4, available at http://www.jcb.org/cgi/content/
full/jcb.200612129/DC1; Bachman et al., 2004). Similar to the
effect on HaCaT, NMuMG, and HMEC cells (Figs. 1 and S1),
TGF-β1 suppressed thymidine incorporation in control un-
infected MCF-10A cells (unpublished data) or in MCF-10A cells
transiently infected with control Ad-GFP (Fig. 8 A). Ad-N1ICD
infection led to a substantial suppression of S-phase entry,
which was comparable with that obtained by 2 ng/ml TGF-β1
(Fig. 8 A). The combination of TGF-β1 and N1ICD led to a
Figure 7. Role of 후-secretase on the accumulation of phospho-Smad levels. (A) Immunoblot of endogenous phospho-Smad2 and -Smad3 and corresponding
total Smad2 and Smad3 levels in HaCaT cells stimulated with 2 ng/ml TGF-β1 for the indicated time points in the presence of DMSO (−) or 4 μM GSI (+).
(B) Immunoblot of endogenous phospho-Smad2 and -Smad3 and corresponding total Smad2 and Smad3 levels in HaCaT cells transiently infected with Ad-
GFP or Ad-N1ICD (MOI of 50 each) before stimulation with 2 ng/ml TGF-β1 for the indicated time points. (C) Immunoblot of p21, ectopic N1ICD, and
control Smad2/Smad3 and β-tubulin from HaCaT cells infected with Ad-GFP (MOI of 50) or Ad-N1ICD (MOI of 10, 25, and 50) before stimulation with
2 ng/ml TGF-β1 for 24 h in the absence (−, DMSO) or presence (+) of 4 μM GSI. Densitometric values of p21 protein bands normalized over β-tubulin
are shown between the immunoblots. The 0-h TGF-β1 without GSI condition is normalized to 1.0, and all other values are expressed relatively. In the right
panel, the denominator represents the fold decrease in inducible p21 caused by GSI. (D) Thymidine incorporation assays in HaCaT cells infected with
Ad-GFP or Ad-N1ICD (MOI of 10, 25, and 50) and stimulated with vehicle (−) or 2 ng/ml TGF-β1 (+) for 60 h in the absence (DMSO) or presence of
4 μM GSI. (E) Thymidine incorporation assay in HaCaT cells stimulated with 2 ng/ml TGF-β1 for 60 h in the absence or presence of 4 μM GSI, which was
added after the onset of TGF-β1 stimulation and was present in the cell culture for the indicated time points. The top horizontal line indicates the level of thy-
midine incorporation that corresponds to 80% of the control level in the presence of GSI (third bar), and the bottom horizontal line corresponds to the level
of thymidine incorporation that shows a statistically signifi cant (P < 0.05) difference from the level of thymidine incorporation in the presence of TGF-β1 in
the control condition (second bar). All values below this line are not signifi cantly different from this reference point (P < 0.05) except for the last condition,
which is signifi cantly lower. (F) Immunoblot of endogenous p21 and α-tubulin control from HaCaT cells stimulated with 2 ng/ml TGF-β1 for 60 h in the ab-
sence or presence of 4 μM GSI that was added after TGF-β1 stimulation and stayed in the culture for the indicated time points. The conditions are identical
to those in E. Error bars represent SD.
NOTCH AND TGF-β IN EPITHELIAL CYTOSTASIS • NIIMI ET AL. 703
very strong cytostatic response (Fig. 8 A). In contrast, infection
of the p21 knockout clones of MCF-10A cells and stimulation
with TGF-β1 failed to show any measurable suppression of
thymidine incorporation (Figs. 8 B and S4 A). The reciprocal
experiment using GSI as a means of blocking endogenous
Notch signaling corroborated the results with ectopic N1ICD
expression. Thus, GSI effectively blocked the suppression of
thymidine incorporation by TGF-β1 in wild-type MCF-10A
(Fig. 8 C), whereas in the p21 knockout clones, thymidine levels
remained high in the absence or presence of GSI (Figs. 8 D and
S4 B). It is worth noting that the two p21 knockout clones incor-
porated substantially higher levels of thymidine compared with
wild-type cells (Figs. 8 and S4). This correlated well with the
absence and presence of endogenous p21 protein expression,
respectively (Fig. 8, bottom; and Fig. S4 C). Therefore, these
experiments strongly implicate a functional role of p21 in the
cytostatic response of epithelial cells downstream of TGF-β and
The impetus for this study was the realization that TGF-β and
Notch pathways act as tumor suppressor and prometastatic or
oncogenic pathways during carcinogenesis (Radtke and Raj,
2003; Pardali and Moustakas, 2007). We establish that Notch
and TGF-β cooperatively suppress epithelial cell growth when
both pathways are simultaneously activated. On the other hand,
TGF-β induces Jagged1 ligand synthesis, which then activates
Notch signaling in the same cell population, thus rendering
TGF-β partially dependent on Notch signaling during the es-
tablishment of cytostasis (Figs. 1, 3, and 5). We observed the
same type of interdependent relationship between the two path-
ways when large-scale gene expression analysis was performed
(Figs. 2 and 4). Additionally, however, we measured many
genes that were uniquely regulated by the combined input of
TGF-β1 and Notch1 both positively and negatively. Finally, we
demonstrate that TGF-β–induced cytostasis requires the durable
expression of factors such as p21, which is achieved by an ini-
tial TGF-β input followed by a secondary but indispensable
Notch input (Figs. 5 and 6). While p21 is not the only gene
of the cytostatic program of TGF-β that is affected by Notch
signaling inhibition, evidence derived from p21 knockout epi-
thelial cells strongly links this cell cycle regulator to the cyto-
static response of the cells. Analyzing in detail the functional
roles of other genes uncovered in this study and the detailed
mechanisms of their regulation by TGF-β and Notch may shed
light on even more novel facets of the binary roles these two
pathways play during the control of epithelial proliferation and
In analyzing the transcriptomic response of HaCaT keratino-
cytes to TGF-β in the presence of GSI (Figs. 2 and 4), we un-
covered an extensive dependence of gene expression regulation
on endogenous Notch pathway activation. Based on careful
control experiments in which we examined the role of GSI on
phospho-Smad accumulation in response to TGF-β (Fig. 7 A),
two possible working models can explain the transcriptomic re-
sults. First, an adverse negative effect of GSI on phospho-Smad
accumulation may be the main reason behind the substantial de-
crease in the number of TGF-β–responsive genes we measured,
especially at the 6-h time point (Fig. 2 A). In simple terms, GSI
lowers the active levels of Smads in the epithelial cell, thus re-
ducing the downstream output of these signal transducers as
measured by gene expression readouts. However, the majority
of the evidence presented here argues against this model. Two
examples are illustrative: (1) a large number of new target genes
of the TGF-β pathway were uncovered in our screen whose
expression is regulated only when cells are treated with GSI (Fig.
2 B and Table S1). This suggests that the inhibition of γ-secretase
activity in the cell redirects the specifi city of gene expression
regulation by TGF-β toward new targets. This phenomenon is
hard to reconcile based on a model in which Smad activation is
gradually diminishing as a result of GSI. (2) Specifi c gene targets
of TGF-β/Smad signaling such as TIEG (Fig. 2 C) or c-Myc
(Fig. 4 B) are not affected at all by the presence of GSI. If the inhibitor
Figure 8. The cell cycle inhibitor p21 is re-
quired for mammary epithelial cytostasis by
TGF-훃 and Notch. (A–D) Thymidine incorpora-
tion assays in MCF-10A wild-type (WT) cells
(A and C) or MCF-10A p21−/− homo zygous
knockout clone 2 cells (B and D) infected with
Ad-GFP or Ad-N1ICD (MOI of 50) and either
stimulated with vehicle (−) or 2 ng/ml TGF-β1
for 60 h (A and B) or stimulated with TGF-β1 in
the presence or absence of 4 μM GSI (C and D).
Immunoblots from the same cells for ectopic
N1ICD, endogenous p21, and control endog-
enous α-tubulin are shown below the bar
graphs. Error bars represent SD.
JCB • VOLUME 176 • NUMBER 5 • 2007 704
were simply reducing phospho-Smad levels, these two and other
genes should have shown new expression profi les in the presence
of GSI, which was never observed.
The second working model, which is corroborated by the
majority of the data presented here, is outlined in Fig. 5 A and
essentially favors a sequential mode of signaling starting with
TGF-β and later followed by Notch. This pathway targets
critical mediators of the cytostatic response of epithelial cells,
namely the cell cycle inhibitors p15 and p21. Depletion of en-
dogenous Jagged1 and CSL proteins supports this model, and no
evidence for a contribution of these classic Notch pathway com-
ponents to the process of Smad activation could be gained (Figs.
5 and 6). This model of sequential signaling suggests that gene
targets like p15 and p21 are directly regulated by the incoming
Smad pathway as previously established (Feng et al., 2000;
Pardali et al., 2000, 2005; Seoane et al., 2001, 2004; Gomis et al.,
2006b), and subsequent onset of Notch signaling after the accu-
mulation of ligands of this pathway, such as Jagged1, contributes
to a sustained and robust transcriptional induction of the same
genes. From this perspective, it would be interesting to examine
in deeper detail the transcriptional mechanisms that mediate the
regulation of p15 and p21 gene expression by the combined
TGF-β/Smad and Notch/CSL signaling inputs. In this respect, it
is interesting that Jagged1 clusters together with p15 and p21 as
genes of the same synexpression group downstream of TGF-β, as
all of these genes seem to require the activity of Smad signaling
and the cooperation of transcription factors of the FoxO family
(Gomis et al., 2006a). The mechanistic details of how Smads,
FoxO members, and additional cofactors orchestrate the time-
dependent induction of Jagged1 remain to be elucidated.
An interesting question remaining open at this stage
is the mechanism by which the inhibition of γ-secretase af-
fects the accumulation of phosphorylated R-Smads downstream
of the TGF-β receptor. Presently, we examine three alter-
native possibilities: (1) γ-secretase may be involved in the ac-
tivation process of the TGF-β receptor, thus playing a critical
role in the phosphorylation of R-Smads by the type I receptor;
(2) γ-secretase positively contributes to the stability of phos-
phorylated R-Smads, possibly by down-regulating an ubiquitin
ligase involved in phosphorylated R-Smad turnover; or (3)
γ-secretase negatively regulates the phosphatases that remove
the C-terminal phosphates from phospho–R-Smads. Ongoing
work aims at addressing these alternative mechanisms.
Among all components of the Notch pathway whose ex-
pression is regulated by TGF-β signaling, our evidence favors
more prominent roles for the ligands of these pathways such as
Jagged1 and DLL4 in keratinocytes (Fig. 3 B) or Jagged1 and
DLL1 in mammary epithelial cells (Fig. S2). The observed regu-
lation of receptors of the Notch pathway appeared to be indirect
(unpublished data) and possibly the result of an autogenous
negative feedback pathway whereby the activation of Notch sig-
naling itself leads to the down-regulation of its receptor genes.
Although our evidence favors this model, TGF-β was found
to up-regulate the expression of Notch4 concomitantly to the
down-regulation of Notch1, at least in mammary epithelial cells
(Fig. S2). The functional relevance of such a reciprocal regula-
tion of Notch receptors during cytostasis of mammary epithelial
cells remains unknown. Alternatively, TGF-β may instruct for
this switch of Notch receptor expression as it promotes epithelial-
mesenchymal transition of the mammary cells, a physiological
response in which the cross talk between TGF-β and Notch sig-
naling has already been established at least in keratinocytes
(Zavadil et al., 2004).
In establishing the sequential signaling pathway of TGF-β
followed by Notch as a critical regulator of epithelial cytostasis
(Fig. 5 A), we primarily focused on regulation of the cell cycle
inhibitor p21. This was prompted by the characteristic expres-
sion profi le measured for p21 during our experiments (Fig. 4 B).
However, regulation of additional factors such as p15, Id2, or
S100A11 seems to also be integrated in the same physiological
response. Thus, in emphasizing a role of p21 as a major target
gene of the sequential signaling cascade outlined here, one
should strongly consider the legitimate and equipotent con-
tribution of the other regulators of this multigenic response to
TGF-β. This point is underscored by the experiments using p21
knockout MCF-10A cells (Figs. 8 and S4). Our evidence fully
recapitulates the original fi ndings of Bachman et al. (2004) and
further demonstrates the role of p21 downstream of Notch sig-
naling in mammary epithelial cells. However, it should be kept
in mind that MCF-10A cells represent relatively normal immor-
talized human epithelial cells that have spontaneously lost the
expression of their endogenous p15 cell cycle inhibitor gene
(Chen et al., 2001). Thus, the p21 knockout MCF-10A clones
represent a double knockout for p15 and p21 expression, and
this is the main reason why TGF-β completely fails to elicit pro-
liferation arrest in these cell clones. Our attempts to deplete p15
or p21 individually from HaCaT or other epithelial cell models
in which TGF-β–mediated cytostasis is well understood always
led to partial and relatively weak phenotypes, presumably be-
cause of the compensation provided by the other genes of the
cytostatic program that remained intact (unpublished data).
In summary, this study establishes a relay mechanism of sig-
nal transduction that plays critical roles for the establishment of
epithelial cell cycle arrest. This mechanism fi ts well with the es-
tablished tumor suppressor roles of TGF-β and Notch signaling.
Additionally, this mechanism opens the exciting possibility whereby
the two signaling pathways may be misregulated in an inter-
dependent manner during human tumor progression, thus offering
a promising territory for future studies in cancer cell biology.
Materials and methods
Cells and reagents
Human HaCaT keratinocytes, human MCF-10A mammary epithelial cells,
human embryonic kidney 293 cells, mouse NMuMG mammary epithelial
cells, and their derivative clone NMe have been described previously
(Valcourt et al., 2005). Mink lung epithelial cells (Mv1Lu) were purchased
from the American Type Culture Collection, and HMECs were obtained
from R.A. Weinberg (Whitehead Institute for Biomedical Research/Massa-
chusetts Institute of Technology, Cambridge, MA). MCF-10A clones 1 and
2 defi cient in the endogenous p21 gene were obtained from B.H. Park
(The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins
University, Baltimore, MD; Bachman et al., 2004). Recombinant mature
TGF-β1 was purchased from PeproTech. The TGF-β type I receptor kinase
inhibitor LY580276 and TGF-β types I and II receptor kinase dual inhibitor
LY364947 were obtained from J.M. Yingling (Eli Lilly, Inc., Indianapolis,
IN; Peng et al., 2005). The inhibitor X against γ-secretase activity (GSI)
was purchased from Merck Biosciences/Calbiochem.
NOTCH AND TGF-β IN EPITHELIAL CYTOSTASIS • NIIMI ET AL. 705
Transient adenoviral infections and siRNA transfections
Adenoviruses expressing GFP were based on the bicistronic Adeasy vec-
tor obtained from B. Vogelstein (The Johns Hopkins Medical Institutions,
Baltimore, MD). Adenoviruses expressing N1ICD were based on
Adeasy, which was obtained from G.P. Dotto (Harvard Medical School,
Boston, MA) and F. Radtke (Ludwig Institute for Cancer Research [LICR],
Lausanne, Switzerland). Adenoviruses expressing wild-type human p21
were obtained from K. Walsh (Boston University School of Medicine,
Boston, MA). Adenoviruses were amplifi ed and titrated in human embry-
onic kidney 293 cells, and transient infections were performed as de-
scribed previously (Valcourt et al., 2005). Under standardized conditions,
epithelial cells were infected at a rate of 75–85% without any signs of
cytotox icity as assessed by live GFP autofl uorescence and immunofl uores-
The human CSL-specifi c (GenBank/EMBL/DDBJ accession no.
NM_005348; reagent number M-007772; human RBPSUH) and human
JAG1-specifi c (GenBank/EMBL/DDBJ accession no. NM_000214; reagent
number L-011060; human Jag1) siRNAs were pools of four RNA oligo-
nucleotides termed On-Target Plus SMARTpools that minimize off-target
effects; siRNA against the luciferase reporter vector pGL2 (GenBank/
EMBL/DDBJ accession no. X65324) served as a control. All siRNAs were
purchased from Dharmacon. HaCaT cells were transiently transfected
with 20 nM siRNA using siLentFect (Bio-Rad Laboratories) according
to the manufacturer’s protocol. Cells were transfected 1 d after seeding,
remained with transfection cocktail for 24 h, were switched to fresh
medium plus TGF-β1, and were retransfected with siRNA for another 24 h
before cell analysis.
Total proteins from NMuMG or HaCaT cells were extracted, subjected to
SDS-PAGE, and analyzed by Western blotting as described previously
(Valcourt et al., 2005). Mouse monoclonal anti–β-tubulin (T8535) antibody
was obtained from Sigma-Aldrich; mouse monoclonal anti-Cip1/WAF1
(clone 70) was purchased from BD Transduction Laboratories; rabbit
polyclonal anti-Notch1 (ab8925) was purchased from Abcam; mouse
monoclonal anti-Smad1/2/3 (H2), rabbit polyclonal anti-Notch4 (H-225),
rabbit polyclonal anti-Notch3 (M-134), rabbit polyclonal anti-Jagged1
(H-66), rabbit polyclonal anti-TGFβRI (V-22), rabbit anti-CSL/RBP-Jκ, rabbit
anti-DLL4/Delta-4 (H-70), and mouse anti–α-tubulin (TU-02) were obtained
from Santa Cruz Biotechnology, Inc. Secondary anti–mouse IgG and
anti–rabbit IgG coupled to HRP were obtained from GE Healthcare. The
ECL detection system was prepared in house, and immunoblots were
scanned on a CCD camera (LAS-1000; Fuji). Densitometry was performed
using the AIDA program of the scanner.
Thymidine incorporation, cell counting, and FACS assays
Cells were cultured, stimulated with growth factors, and labeled meta-
bolically with [3H]thymidine as described previously (Valcourt et al.,
2005). The data are plotted as mean values with SEMs of triplicate
repeats per independent experiment. Each independent experiment was
repeated at least three times. Cell monolayers were washed with PBS,
trypsinized, and stained with trypan blue (Sigma-Aldrich), and viable
cell numbers were calculated using a counter (Z1; Beckman Coulter).
Cell numbers are plotted as means from triplicate determinations with
SEMs per experiment.
For FACS analysis of cell cycle distribution, cells were trypsinized
and resuspended in ice-cold PBS/10 mM EDTA, centrifuged for 5 min at
2,500 rpm and 4°C, resuspended in PBS/0.1% wt/vol glucose, fi xed in
−20°C-cold 70% vol/vol ethanol, washed in PBS/10 mM EDTA twice,
and stained with 0.1 mg/ml propidium iodine by coincubation with
RNAase. Stained cells were analyzed in a Guava EasyCyte Mini System
(Guava Technologies, Inc.) according to the manufacturer’s protocol and
built-in software. Mean cell number per cell cycle phase was estimated
based on measurements from 5,000 cells per single reading. The data
are plotted in bar graphs representing percentiles of the total
Statistical analysis of thymidine incorporation assays was performed by two-
tailed paired t tests. Signifi cance was considered at a p-value of <0.05.
cDNA microarray analysis
HaCaT cells were cultured in the presence of 3% FBS and stimulated with
2 ng/ml TGF-β1 for 2, 6, and 48 h in the absence or presence of 4 μM
GSI. Cells for the 2-h time point were also incubated with 10 μg/ml
cycloheximide to block protein synthesis. Total RNA extraction and cDNA
probe labeling was performed as described previously (Valcourt et al.,
2005). Equal amounts of labeled cDNA probes per pair were hybridized
to cDNA microarray chips (Hver2.1.1) from the Sanger/LICR/Cancer
Research UK Consortium (see http://www.sanger.ac.uk/Projects/Micro-
arrays/ for details and hybridization protocols). The glass chips contained
14,633 single-stranded cDNA elements of 1.5-kb mean length, which
represent 10,252 unique human genes. The human IMAGE cDNA clone
collection was obtained from the Medical Research Council Human Ge-
nome Microarray Platform Resource Centre. cDNA clone resequencing
was performed by Team 56 at the Sanger Institute. Hybridizations were
performed in triplicate using RNAs from three independent cultures and
including the dye swap control. Microarray scanning, image analysis, and
primary spot intensity statistical analysis were performed as described
previously (Valcourt et al., 2005). Regulated genes were selected based
on the mean ratio value of ≥1.7 for up-regulated genes and ≤0.55 for
down-regulated genes. In addition, regulated genes had to be expressed
on three arrays out of three and with a t test value for the ratios within
replicates corresponding to a probability of <0.05. Statistically signifi cant
genes (P < 0.05) were clustered based on their expression values using the
K-means statistical algorithm that is incorporated into GeneSpring 7.2 data
mining software (Silicon Genetics/Agilent Technologies). For all time
points, we considered as a reference a duplicate cell culture in which
TGF-β1 was replaced by vehicle. Functional classifi cation of regulated
genes was performed manually based on exhaustive PubMed searches
Semiquantitative RT-PCR and quantitative real-time RT-PCR
Total RNA from NMuMG or HaCaT cells was analyzed by semiquantitative
RT-PCR as described previously (Valcourt et al., 2005) using specifi c
primers (Table I). Primers for mouse glyceraldehyde-3′-phosphate dehydro-
genase (Gapdh) were used to ascertain that an equivalent amount of
cDNA was synthesized. Specifi city controls included reactions in which
reverse transcrip tase was omitted (−RT) and in which cDNAs were re-
placed with water.
DNase RQI–digested RNA from NMuMG and HaCaT cells was an-
alyzed by quantitative real-time RT-PCR as described previously (Valcourt
et al., 2005). Primers (Table I) were designed with Primer Express
(Applied Biosystems). Reactions were performed in a sequence detector
(ABI-Prism 7000; Applied Biosystems) in triplicate, and, for each condition,
the ground condition (minus TGF-β1 and/or mock infected with Ad-GFP)
was set as 1; expression data are presented as bar graphs of mean
values plus SD.
Online supplemental material
Fig. S1 shows thymidine incorporation and cell counting assays in various
epithelial cell types. Fig. S2 shows semiquantitative RT-PCR assays and
corresponding immunoblot assays for Notch family member expression
in NMuMG cells. Fig. S3 shows thymidine incorporation and immunoblot
assays in HaCaT cells expressing ectopic p21. Fig. S4 shows thymidine
incorporation and immunoblot data from clone 1 of the p21 knockout MCF-
10A cells. Table S1 provides information about transcriptomic analysis of
the TGF-β1 response after Notch inhibition.Online supplemental material is
available at http://www.jcb.org/cgi/content/full/jcb.200612129/DC1.
We thank G.P. Dotto, B.H. Park, F. Radtke, B. Vogelstein, K. Walsh, R.A.
Weinberg, and J.M. Yingling for reagents and M. Kowanetz and members of
our group for help and suggestions. We thank the staff of the Sanger Institute
Microarray Facility for supplying arrays, laboratory protocols, and technical
advice (David Vetrie, Cordelia Langford, Adam Whittaker, and Neil Sutton) as
well as Quantarray/GeneSpring datafi les and databases relating to array
elements (Kate Rice, Rob Andrews, Adam Butler, and Harish Chudasama).
This work was supported by LICR, the Swedish Cancer Society (project
number 4855-B03-01XAC), the Natural Sciences Foundation of Sweden
(project number K2004-32XD-14936-01A), and Marie Curie Research
Training Network EpiPlastCarcinoma under the European Union FP6 program.
K. Pardali was supported by the X-109/2001-02 scholarship of the Alexander
S. Onassis Public Benefi t Foundation (Greece). The microarray consortium is
funded by the Wellcome Trust, Cancer Research UK, and LICR.
Submitted: 22 December 2006
Accepted: 26 January 2007
JCB • VOLUME 176 • NUMBER 5 • 2007 706
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Table I. Oligonucleotide primers used for RT-PCR analyses
Gene Primer sequence (strand)Product sizeTemperature PCR cycleAccession no.
5′-C A G A A C T T A C A G C T C C A G C C -3′ (+)
5′-C C T C T G G A A T G T G G G T G A T C -3′ (−)
5′-C A A G T T G C C T G G G G T C T T C C -3′ (+)
5′-G G C A A G G A G T C A T C A G C T G G -3′ (−)
5′-C A G T G G C T T G G G T C T G T T G C -3′ (+)
5′-C C T T C T C C T C T C T G T C T A C C -3′ (−)
5′-G A G T T C C A G T G T G A C G C C T A -3′ (+)
5′-G G G C C T C G T G A A T A T G A C C A -3′ (−)
5′-C A T C A T T G G G G C T A C C C A G A -3′ (+)
5′-C C T G A A C C T G G T T C T C A G C A -3′ (−)
5′-A T C A C T G C C A C C C A G A A G A C -3′ (+)
5′-A T G A G G T C C A C C A C C C T G T T -3′ (−)
5′-C C C A C C A A A A C A A C A A A T G T C A -3′ (+)
5′-C A T C A G A A A A A G C T T G G C A G A G A -3′ (−)
5′-C T G C C C A A G C T C T A C C T T C C -3′ (+)
5′-C A G G T C C A C A T G G T C T T C C T -3′ (−)
5′-T G G A C C T G G T G G C T A C G A A T -3′ (+)
5′-A G G G C C T A A G T T G T G G G T T C A -3′ (−)
5′-A G G T A A T T T C A T G C C A G T T C A C A -3′ (+)
5′-C A T G C C A G T A A C T G A G C A C A C A -3′ (−)
5′-T G A G G T G T A A A A T G G A A G T G A G A T G -3′ (+)
5′-A G A A C C T G C T C G G T C T G A A C T C -3′ (−)
5′-C C C C G G A C C G T C A G T -3′ (+)
5′-G A T G G A A G G A G C A G A T A T G A C A T A A A T -3′ (−)
5′-C T T G T G A A T G T C C C C C C A A C T -3′ (+)
5′-C A G T A G G T G C C C G T G A A T C C -3′ (−)
5′-G G G A A G G T T T T G G G C T C T C T -3′ (+)
5′-C G G T C A C C G T C C G C A T C T T -3′ (−)
5′-G A T G A T G G G A A C C C G A T C A A -3′ (+)
5′-G C A A G G G A A C A A G G A A A T C T G T -3′ (−)
5′-G A C T G C C G C A T C A A C A T G -3′ (+)
5′-C A C C A C A C C T T G C T G C A G T C -3′ (−)
5′-C G G G T C C A C C A G T T T G A A T G -3′ (+)
5′-G T T G T A T T G G T T C G G C A C C A T -3′ (−)
5′-A A A C A A G T G A A A G C A T A T G G G T -3′ (+)
5′-C C T G A A A C A A A G A T T C A T G A T T -3′ (−)
5′-T G T C T T C C A G A T T C T G A T T C G C -3′ (+)
5′-G G T G T C T C T T C C T T G T T G T C C -3′ (−)
5′-T G A T C G G C T C G G T A G T A A T G C -3′ (+)
5′-G A C A A C G C T C C C A G G T A G T C A -3′ (−)
5′-G G A G A G G G T A A A A A A T G A A A G A A T A C A T G -3′ (+)
5′-G G C A T A C A T T C A T T T T A G G A G A G A A A C -3′ (−)
5′-A G G G T C A A G T G G G A C A G T G T C A -3′ (+)
5′-A G C T C C G T T T T A G C T C G T T C C T -3′ (−)
5′-G G A G T C A A C G G A T T T G G T C G T A -3′ (+)
5′-G G C A A C A A T A T C C A C T T T A C C A G A G T -3′ (−)
458 6032 NM_010929
457 5832 NM_010588
482 58 36 NM_007865
443 5726 Valcourt et al., 2005
Pardali et al., 2005
151 60qPCR ENSG00000198719
151 60qPCR ENSG00000090932
232 60qPCR ENSG00000184916
76 60 qPCRENSG00000148400
154 60qPCR ENSG00000112049
15060 qPCR ENSG00000136997
Lowercase gene names refer to mouse sequences, and capitalized gene names refer to human sequences. When quantitative PCR (qPCR) assays are performed,
the PCR cycle number is not applicable.
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