From the Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine 04074
Received for publication, January 15, 2003, and in revised form, February 10, 2003
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ABSTRACT |
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Aberrant activations of the Notch and fibroblast
growth factor receptor (FGFR) signaling pathways have been correlated
with neoplastic growth in humans and other mammals. Here we report that
the suppression of Notch signaling in NIH 3T3 cells by the expression
of either the extracellular domain of the Notch ligand Jagged1 or
dominant-negative forms of Notch1 and Notch2 results in the
appearance of an exaggerated fibroblast growth factor
(FGF)-dependent transformed phenotype characterized by
anchorage-independent growth in soft agar. Anchorage-independent growth
exhibited by Notch-repressed NIH 3T3 cells may result from prolonged
FGFR stimulation caused by both an increase in the expression of
prototypic and oncogenic FGF gene family members and the nonclassical
export of FGF1 into the extracellular compartment. Interestingly, FGF
exerts a negative effect on Notch by suppressing CSL
(CBF-1/RBP-Jk/KBF2 in mammals, Su(H) in
Drosophila and Xenopus, and Lag-2
in Caenorhabditis elegans)-dependent transcription, and the ectopic expression of constitutively active forms of Notch1 or Notch2 abrogates FGF1 release and the phenotypic effects of FGFR stimulation. These data suggest that
communication between the Notch and FGFR pathways may represent an
important reciprocal autoregulatory mechanism for the regulation of
normal cell growth.
Notch receptors and their ligands are components of an
evolutionarily conserved signaling pathway that regulate cell
proliferation, differentiation, and survival in a cell- and
tissue-specific manner (for reviews, see Refs. 1 and 2). Notch
receptors and their ligands are structurally conserved transmembrane
polypeptides, and four Notch receptors (Notch1-4) and six ligands
(Delta1-4, Jagged1, and Jagged2) have been identified in vertebrates
to date (3-8). Upon ligand binding, the intracellular domain of the
Notch receptor is released by proteolytic cleavage and becomes a
nuclear transcriptional regulator by interacting with members of the
CSL (CBF-1/RBP-Jk/KBF2 in mammals, Su(H) in
Drosophila and Xenopus, and Lag-2 in
Caenorhabditis elegans) family of transcription factors (9).
Notch receptors have also been reported to regulate cellular processes
through CSL-independent pathways that may involve interactions with
other signaling molecules such as nuclear factor We have reported previously that suppression of endogenous Notch
signaling mediated by the ectopic expression of either an extracellular
and soluble form of Jagged1
(sJ1)1 or dominant-negative
mutants of Notch1 (dnN1) or Notch2 (dnN2) induces dramatic changes in
the NIH 3T3 cellular phenotype in comparison with NIH 3T3 cells stably
transfected with the empty vector (vector control). These changes in
cellular phenotype include chord formation on collagen matrices;
increased Src activation and enhanced phosphorylation of the Src
substrate, cortactin; a decrease in the formation of actin filaments
and focal adhesion sites; impaired migratory ability; and increased
survival at high cell densities (11, 24, 25). In contrast, NIH 3T3
cells stably expressing either constitutively active mutants of Notch1 (caN1) or Notch2 (caN2) display a phenotype similar to that exhibited by the vector control cells (11).
Our initial interest in Notch originated from our observation that
Jagged1 was an FGF response gene in human endothelial cells undergoing
differentiation on fibrin clots (26). Because several of the phenotypic
characteristics displayed by Notch-repressed cells were dependent on
the activity of the FGFR effector molecule Src (11), we anticipated
that communication between Notch and FGFR signaling pathways may also
represent an important mechanism regulating cellular behavior in
fibroblasts. Whereas several studies report that the expression of
Notch and/or its ligands is correlated with activation of the FGFR
signaling pathway (27-29) and vice versa (30), little is
known about how interactions between these two important and ubiquitous
pathways influence cellular phenotype, including growth. To address
this question, we examined the effects of FGFR stimulation in
combination with Notch repression or activation in NIH 3T3 cells. We
decided to study these interactions in the NIH 3T3 cell because we had
already identified phenotypic characteristics associated with the
down-regulation of Notch signaling (11), and the NIH 3T3 cell
represents a relatively simple system because it primarily expresses
transcripts encoding Notch1 and Notch2, but not Notch3 and
Notch4.2 In addition,
aberrant activation of both Notch (21-23) and FGFR (31-33) signaling
pathways have been found to be associated with neoplastic growth, and
the NIH 3T3 cell is uniquely sensitive to oncogene-mediated
transformation (34).
We report that antagonistic interactions between the Notch and FGFR
signaling pathways regulate anchorage-independent growth in murine
fibroblasts. Stimulation of the FGFR pathway by exogenous FGF1 causes
Notch-repressed cells to grow as detached spheroids in tissue culture
and to aggressively form colonies in soft agar, phenotypic
characteristics associated with cellular transformation (reviewed in
Ref. 35). The transformed phenotype exhibited by Notch-repressed cells
may be attributed to their maintenance of FGFR-generated signals
generated by an increase in both the expression and release of FGF
family members. Furthermore, FGF1 has an inhibitory effect on
Notch/CSL-dependent transcription. In contrast, the expression of caN1 or caN2 protects the NIH 3T3 cell from FGF-induced anchorage-independent growth and suppresses the release of FGF1 under
normal growth conditions. These results suggest that cross-talk between
the Notch and FGFR signaling pathways may represent an important
autoregulatory mechanism that is involved in the regulation of cell growth.
Generation of Stable NIH 3T3 Transfectants--
Stable NIH 3T3
transfectants for vector control, sJ1, dnN1, dnN2, caN1, and caN2 were
obtained and screened for expression as described previously (11). The
FGF1 mutant containing the 3' signal peptide sequence of FGF4
(hst- Colony Formation in Soft Agar--
Clonal populations of NIH 3T3
cells stably transfected with vector control, sJ1, caN1, caN2, dnN1,
dnN2, hst- Analysis of FGF1-induced Spheroid Formation--
Clonal
populations of NIH 3T3 stable transfectants were resuspended and grown
in media containing 10% BCS, 10% BCS plus 1 µM
PD166866, 10% BCS plus 10 ng/ml recombinant human FGF1 and 10 units/ml
heparin, or 10% BCS plus 10 ng/ml recombinant human FGF1, 10 units/ml
heparin, and 1 µM PD166866 as indicated in the figure
legend. Approximately 2 × 105 cells were plated per
well (6-well dish), and 3 days after plating, phase-contrast
micrographs of the cells were taken.
Analysis of Spheroid Formation and Soft Agar Colony Growth in
hst- Analysis of the Expression of the fgf/fgfr Gene Family
Members--
Total RNA from vector control, sJ1, caN1, and dnN1
stable transfectants was isolated using Tri ReagentTM
(Sigma) according to the manufacturer's protocol. cDNA was
obtained from 5 µg of total RNA with SuperScriptTM
(Invitrogen) reverse transcriptase using an oligo(dT) primer (Invitrogen). The following specific primers were purchased from IDT and used for RT-PCR analysis (sense primers are indicated by
(s); antisense primers are indicated by (as)): FGF1(s),
5'-ATGGCTGAAGGGGAGATCACAACC-3'; FGF1(as), 5'-CGCGCTTACAGCTCCCGTTC-3';
FGF2(s), 5'-ATGGCTGCCAGCGGCATCAC-3'; FGF2(as),
5'-GAAGAAACAGTATGGCCTTCTGTCC-3'; FGF3(s), 5'-GCCTGATCTGGCTTCTGCTGC-3'; FGF3(as), 5'-GCAGCTGGGTGCTTGGAGGTGG-3'; FGF4(s),
5'-ACCACAGGGACGACTG-3'; FGF4(as), 5'-CATACCGGGGTACGCGTAGG-3'; FGF6(s),
5'-GGGCCATTAATTCTGACCACGTGCCTG-3'; FGF6(as),
5'-GGTCCTTATATCCTGGGGAGGAAGTGAGTG-3'; FGF7(s),
5'-CACGGATCCTGCCAACTCTGC-3'; FGF7(as), 5'-CCACAATTCCAACTGCCACGGTC-3';
FGF8(s), 5'-CTCTGCCTCCAAGCCAGGTAAG-3'; FGF8(as),
5'-GCTGATGCTGGCGCGTCTTGGAG-3'; FGF9(s), 5'-GGTGAAGTTGGGAGCTATTTCG-3'; FGF9(as), 5'-CATAGTATCTCCTTCCGGTGTCCAC-3'; FGF10(s),
5'-CACATTGTGCCTCAGCCTTTC-3'; FGF10(as), 5'-CCTCTATTCTCTCTTTCAGCTTAC-3';
FGFR1(s), 5'-AGGCCAGCCCCAACCTTG-3'; FGFR1(VT + as),
5'-GGAGTCAGCTGACACTGTTAC-3'; FGFR1(VT
PCR amplification was performed for 45 cycles as follows: 40 s at
94 °C, 40 s at 50 °C (for FGF2, FGF3, FGF4, and FGFR3) or at
55 °C (for FGF1, FGF6, FGF7, FGF8, FGF9, FGF10, FGFR1, and FGFR2),
and 1 min at 72 °C. For FGF5, RT-PCR analysis was performed as
described previously (37), and all amplified DNA was visualized with
ethidium bromide on 1.5% agarose gels.
Analysis of FGF1 Release in Stable NIH 3T3 Cell
Transfectants--
Adenovirus vector expressing lacZ, FGF1, caN1,
dnN1, caN2, or dnN2 was prepared as described previously (38) at a
titer of ~1012 viral particles/ml. For adenoviral
transduction, NIH 3T3 stable transfectants were incubated in serum-free
medium with ~103 viral particles/cell in the presence of
poly-D-lysine hydrobromide (Sigma) (5 × 103 molecules/viral particle) for 2 h at 37 °C,
after which the adenovirus-containing medium was removed and replaced
with serum-containing medium (10% BCS) for an additional 24 h.
The transduced cells were harvested by trypsin digestion and seeded for
the heat shock experiments as described previously (39). After heat
shock, the conditioned media from cells exposed to either 37 °C
(normal conditions) or 42 °C (heat shock conditions) were treated
with 0.1% dithiothreitol for 2 h at 37 °C, adsorbed to
heparin-Sepharose, and eluted from the column with 1.5 M
NaCl. The eluants were resolved by 15% (w/v) SDS-PAGE and evaluated by
FGF1 immunoblot analysis as described previously (39).
Transient Transfection Assays of CSL-regulated
Transcription--
NIH 3T3 cells were plated onto fibronectin-coated
(10 µg/cm2) 12-well tissue culture dishes and transiently
transfected at ~80% confluency with 500 ng of a luciferase construct
activated by four tandem copies of the CSL (CBF1) response element
(40), 100 ng of the TK Renilla (Promega) construct as
an internal control for transfection efficiency, and 500 ng of either
vector control, sJ1, or caN1 constructs using FuGENE 6 (Roche Molecular
Biochemicals) per the manufacturer's instructions. For analysis of
caN1 activity in the background of NIH 3T3 stable lines, vector
control, caN1, sJ1, and hst- Analysis of Jagged1 Expression--
Vector control, sJ1, caN1,
Jagged1, hst- Stimulation of the FGFR Pathway Potentiates a Transformed Phenotype
in Notch-repressed Fibroblasts--
In an effort to further our
understanding of how interactions between the Notch and FGFR signaling
pathways regulate cellular processes, we examined the response of
Notch-activated and Notch-repressed NIH 3T3 cells to the addition of
recombinant FGF1 to the growth media (Fig.
1). Surprisingly, NIH 3T3 cells in which
endogenous Notch signaling was repressed (sJ1 and dnN1) formed
multicellular, spheroid-like structures similar to those observed in
NIH 3T3 cells stably expressing an oncogenic mutant of FGF1 engineered with the FGF4 signal peptide sequence (hst-
Cellular proliferation despite detachment from the extracellular matrix
is indicative of anchorage-independent growth in most cell types and is
an in vitro signature of the NIH 3T3 cell transformed phenotype (34). Therefore, we examined our Notch-activated and Notch-repressed cell lines for anchorage-independent growth in soft
agar in both the presence and absence of exogenously added recombinant
FGF1 (Fig. 2, A and
B). Although we have previously reported (25) that sJ1 NIH
3T3 stable transfectants do not form colonies in soft agar when plated
at low seed densities (100 cells/6-cm dish) in growth media containing
10% BCS, we have found during the course of these studies that sJ1and
dnN1 transfectants do form small, pinpoint-sized colonies in soft agar
when plated at seed densities greater than 1,500 cells/6-cm dish. The
addition of FGF1 to the growth media greatly exaggerated the size of
the colonies formed by sJ1 and dnN1 transfectants so that they were clearly visible to the eye, although the number of colonies did not
significantly increase. Indeed, the intensity of the transformed phenotype induced by FGF1 in the Notch-repressed NIH 3T3 cells resembled that exhibited by NIH 3T3 cells stably expressing either an
oncogenic Ras construct (caRas) or an oncogenic mutant of FGF1 (hst- Activation of Notch Inhibits FGFR-mediated Transformation--
To
further explore the possibility that Notch signaling may protect the
NIH 3T3 cell from FGF-mediated anchorage-independent growth, we assayed
the ability of sJ1 NIH 3T3 stable transfectants cotransfected with caN1
to form colonies in both the presence and absence of FGF1. Expression
of caN1 in the sJ1 NIH 3T3 background dramatically inhibited, in terms
of both number and size, colony formation that occurred in the presence
or absence of FGF1. Indeed, the number of colonies formed by the
sJ1:caN1 cotransfectants was similar to that observed in vector control
and caN1 stable lines (Fig. 2, A and B). Unlike
sJ1 single transfectants, sJ1:caN1 cotransfectants did not form
spheroids in the presence of FGF1. However, these cells were less
adherent to the tissue culture dish than the vector control or caN1
cells (Fig. 1). Although analysis of several different clonal cell
lines of the various stable transfectants used in these studies yielded
similar results (data not shown), we wanted to confirm that
constitutively active Notch could repress FGFR-mediated transformation
by transducing hst- Notch Signaling Regulates the Expression of the fgf Gene
Family--
Prior to this study, we reported (11) that Notch-repressed
cells exhibit a pattern of tyrosine phosphorylation similar to that
observed in NIH 3T3 cells stimulated by FGF1. In this study, we found
that sJ1 and dnN1 cells form small, PD166866-sensitive colonies in soft
agar even in the absence of exogenous FGF1. Taken together, these
observations suggested that Notch-repressed transfectants were
releasing FGF into the extracellular compartment. Therefore, we
examined vector control and sJ1 cells for expression of the first 10 of
the 23 known members of the fgf gene family, including the
prototypes fgf1 and fgf2, by using RT-PCR (Fig.
4A). Whereas transcripts for
fgf2, fgf7, and fgf10 are expressed in
all of the cell lines examined, expression of mRNAs encoding
fgf1, fgf3, fgf4, and fgf5
is limited to the sJ1 stable transfectants. In contrast, the pattern of
fgf gene family expression in caN1 transfectants is the same
as that seen in the vector control lines (data not shown). Transcripts
encoding fgf6, fgf8, and fgf9 were not
found in any of the cell lines examined by RT-PCR (data not shown). No
differences were found in the expression pattern of the
fgfrs because all cell lines examined expressed both the 2 and 3-Ig loop isoforms of fgfr1, fgfr2, and
fgfr3 as well as the VT Repression of Notch Signaling Induces FGF1 Release--
Although
most of the FGF family members contain signal peptides that facilitate
their secretion through the classical ER-Golgi exocytosis pathway, it
is well established that the prototype members of the FGF gene family
(FGF1 and FGF2) do not contain a signal peptide and are instead
released by nonclassical mechanisms. Whereas the pathway utilized by
FGF2 to gain access to the extracellular compartment is not known, FGF1
is released in response to environmental stress as a component of a
copper-dependent, multiprotein aggregate that includes the
p40 extravesicular domain of p65 synaptotagmin-1 and S100A13 (42-45).
Given that FGF1 but not FGF2 is differentially expressed in the sJ1
stable transfectants (Fig. 4A), we examined these cells for
FGF1 release. Because expression of the FGF1 steady-state translation
product is undetectable in the NIH 3T3 cell (39) and very low in sJ1
transfectants (data not shown), we examined vector control, sJ1, caN1,
dnN1, caN2, and dnN2 transfectants for the release of adenovirally
transduced FGF1 under normal (37 °C) and heat shock (42 °C)
conditions (Fig. 5). Whereas vector control, sJ1, and sJ1:caN1 transfectants were able to export FGF1 at
similar levels into the extracellular compartment in response to
temperature stress, a significant release of FGF1 at 37 °C was
observed only in the sJ1 cells. Expression of caN1 into the sJ1
background (Fig. 5a, sJ1:caN1) significantly reduced FGF1 release at 37 °C, but not at 42 °C, indicating that activation of
Notch attenuates FGF1 release under normal but not heat shock conditions. A role for Notch as a regulator of FGF export was further
substantiated by the reverse experiment in which NIH 3T3 cells stably
transfected with FGF1 were transduced with either the lacZ-, caN1-,
dnN1-, caN2-, or dnN2-expressing adenovirus (Fig. 5b). Under
these conditions, FGF1 was also released into the extracellular
compartment in response to heat shock in all cell lines examined, but
its release at 37 °C was limited to those cells expressing either
dnN1 or dnN2.
Exogenous FGF1 Represses CSL-dependent
Signaling--
We have previously observed (11) that the ability of
caN1 to up-regulate a CSL-luciferase reporter construct is
significantly diminished in sJ1 stable transfectants in comparison with
vector control cells (Fig. 6). Because
FGF release appears to be up-regulated in Notch-repressed cells, we
wanted to determine whether caN1/CSL-mediated transcription was
negatively correlated with activation of the FGFR signaling pathway.
The ability of caN1 to stimulate transcription of a CSL-luciferase
reporter in hst- FGF1 Up-regulates the Expression of Jagged1--
Because we have
previously reported that Jagged1 was an FGF response gene in
human endothelial cells, we examined the NIH 3T3 stable transfectants
used in this study for expression of Jagged1 by immunoblot analysis
using an antibody directed against an epitope located within the
intracellular C-terminal domain of full-length Jagged1 (Fig.
7). We found that Jagged1 expression was
more pronounced in sJ1 and hst- Whereas both the Notch and FGFR signaling pathways have long been
recognized as important regulators of cell fate determination events in
a variety of cell types, little is known about how interactions between
these two major signaling pathways impact cellular processes. In this
report, we present evidence that supports the existence of an important
cellular mechanism that may be involved in balancing the signals
generated by these signaling pathways. Interestingly, whereas the
stimulation of the FGFR by FGF1 exerts a negative regulatory effect on
Notch signaling by repressing Notch-mediated CSL transcription, Notch,
in turn, tempers FGF-generated signals by regulating the extracellular
appearance of fgf gene family members. In addition, whereas
activated Notch also represses the effects of FGFR stimulation through
an as yet unidentified mechanism other than FGF export, the
perturbation of endogenous Jagged1/Notch signaling disrupts the
equilibrium between the Notch and FGFR pathways and leads to the
manifestation of a transformed phenotype.
Our results indicate that endogenous Jagged1/Notch signaling may
regulate the FGFR signaling pathway by controlling the availability of
FGF in the extracellular compartment. Indeed, the detection of mRNA
encoding FGF1 and the proto-oncoproteins FGF3, FGF4, and FGF5 in sJ1
but not vector control or caN1 stable NIH 3T3 transfectants indicates
that interference with endogenous Jagged1/Notch signaling alters the
expression pattern of FGF gene family members. Whereas a
Notch-responsive element, such as a CSL-binding site, has yet to be
identified in any of the fgf gene family members, it is known that during the development of the Drosophila tracheal
system, conventional Notch signaling negatively regulates the
transcription of the fgf homologue, branchless,
resulting in an attenuation in the activity of breathless,
the fgfr homologue (30). Therefore, it is not unreasonable
to suggest the presence of a similar regulatory mechanism in vertebrates.
In addition to regulating the expression of fgf gene family
members, our data also suggest that Notch may act as a gatekeeper that
represses the nonclassical release of FGF1 under normal growth conditions. We have previously found (42-45) that the export of FGF1
only occurs in the NIH 3T3 cell under conditions of cellular stress
such as heat shock or hypoxia through a copper-dependent mechanism that requires the formation of a multiprotein complex consisting of FGF1, S100A13, and the p40 extravesicular domain of
synaptotagmin-1. Therefore, we were quite surprised to find that NIH
3T3 cells with suppressed Notch signaling released FGF1 not only during
heat shock, but also under normal growth conditions. Although we cannot
dismiss the possibility that caN1 inhibits FGF1 release under normal
growth conditions by interacting directly with the FGF1 secretory
complex, the observation that caN1 and caN2 stable transfectants
continue to release FGF1 under heat shock conditions to the same extent
as vector control transfectants argues against this possibility.
Instead, it is more likely that repression of Notch in the sJ1, dnN1,
and dnN2 stable transfectants induces a stress/survival response that
enables FGF1 export.
Because expression of caN1 or caN2 strongly inhibited FGFR-mediated
anchorage-independent growth in hst- The unanticipated observation that FGF1 suppresses Notch1/CSL-mediated
transcription suggests that Notch may also protect the NIH 3T3 cell
from abnormal growth through the transcriptional regulation of
Notch/CSL-responsive genes. FGF export would then reinforce the
inhibition of Notch/CSL activation already present in Notch-repressed
cells. This is consistent with the requirement for the
Notch/CSL-dependent induction of p21waf/cip for the
stimulation of keratinocyte differentiation by the regulation of growth
arrest (keratinocytes require exogenous FGF for cell division) (50). However, this regulatory mechanism may also contain a cell- and tissue-specific as well as an age-dependent component
because in the developing tooth bud, FGF10 is able to induce the
Notch/CSL-dependent transcription of hes1 (51),
and this may be complicated by an additional level of specificity for
some but not all of the 23 members of the fgf gene family.
The up-regulation of Jagged1 by FGF in the NIH 3T3 cell agrees with our
previous results, in which Jagged1 expression was up-regulated in human
endothelial cells undergoing FGF-induced but not VEGF-induced tube
formation (26). These results are also consistent with those of
Matsumoto et al. (29), who found Jagged1 to be up-regulated
by FGF2 in bovine capillary endothelial cells. In addition, several
other studies have also correlated the expression of polypeptide
components of the Notch signaling pathway with FGFR stimulation. For
example, FGF2 increases the expression of Notch1 in immortalized mouse
oligodendrocytes (27), as do both FGF prototypes in murine
neuroepithelial precursor cells (28). Furthermore, FGF10 also induces
the Notch signal modifier Lunatic Fringe in the murine
developing tooth bud (52), suggesting a role for the cooperativity
between FGFR and Notch signaling in the development of cell fate by
lateral specification, and this is consistent with the report by Ikeya
and Hayashi (30) that the interplay between Notch and FGFR
(breathless) signaling regulates cell fate in
vivo. Thus, the up-regulation of Notch signaling components
including Jagged1 may represent a negative autoregulatory mechanism to
balance the FGFR mitogenic response in the NIH 3T3 cell.
The aggressive transformed phenotype displayed by Notch-repressed cells
in the presence of exogenous FGF1 was unexpected. Indeed, only those
FGF family members that contain a classical signal peptide and are
secreted through the traditional ER-Golgi apparatus have been
considered oncogenes, indicating that FGF must be continuously released
by a cell to become oncogenic (31, 53). Removal of the signal peptide
from FGF4 decreases its transforming potential (54), whereas
constitutive secretion of an oncogenic mutant of FGF1 engineered with
the FGF4 signal peptide sequence (hst- Numerous studies have demonstrated that the activity of Notch is highly
dependent on cell type and environmental context, and this is
particularly true for the activity of soluble ligands and for Notch
regulation of cell growth. In the NIH 3T3 cell, we have suggested that
the soluble, non-transmembrane form of Jagged1 acts to inhibit Notch
signaling, possibly by interfering with endogenous transmembrane ligand
interaction with Notch receptors. Naturally occurring soluble forms of
the Notch ligands arising from proteolytic cleavage (55) or perhaps
differential mRNA processing (19, 26) have been identified, yet the
functional activities of these modified ligands are not clear. The
observation that soluble forms of Notch ligands have been demonstrated
to be both agonists (19, 56, 57) and antagonists of Notch signaling (58-61) may be due to factors including oligomerization or
immobilization of ligands (62-64). Although it is possible that
proteolytic cleavage of Notch ligands generates nonfunctional soluble
fragments that reduce endogenous ligand availability (65), our data
support a model where soluble ligands have significant activity in
regulating Notch signaling. In addition, a preponderance of the human
jagged1 mutations found in individuals afflicted with
Alagille syndrome results in the production of soluble ligands (66),
suggesting a functional consequence in human disease. We predict that
the generation of soluble ligands represents an immediate cellular response to down-regulate Notch signaling events.
Although aberrant activation, not inhibition, of Notch signaling
pathways has been reported to be associated with neoplastic growth in
mammals (4, 23, 67, 68), it is clear that the influence of Notch on
cellular decisions including growth is dependent on cell type and
environmental context (50, 57, 69, 70). Indeed, Notch has also been
reported by several groups to be a suppressor of cellular growth. For
example, the activation of Notch1 causes the arrest of cell cycle
progression in the chicken B-cell line DT40 (71) as well as in small
cell lung cancer cells (72), and prevents myeloid cell but not
erythroid cell proliferation in the absence of polypeptide mitogens
(73). Furthermore, down-regulation, not up-regulation, of Notch1
signaling is required for progression into the late stages of human
papillomavirus-induced cervical carcinogenesis (49). Because both Notch
and FGF are important regulators of many common physiological
processes, including neurogenesis and angiogenesis, further elucidation
of the cellular mechanisms mediating communication between the Notch
and FGFR pathways will be important for our understanding of
pathological conditions mediated by these signaling pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B and Src (10-12).
Phenotypic analysis of mice null for Notch receptors or their ligands
emphasizes the requirement for proper Notch signaling not only during
development but also in the adult (13-17). Indeed, aberrant Notch
signaling has been implicated in several human pathological conditions
including the development of the CADASIL (18) and Alagille
syndromes (19, 20) and the formation of neoplasias in mice and humans
(21-23).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(FGF4):FGF1) and FGF1 (pXZ38) stable transfectants were
obtained and screened for expression as described in Ref. 31. Stably
transfected constitutively active Ras (caRas) clonal populations were
obtained by previously described methods (11) using the activated H-Ras
pUSEamp plasmid (Upstate Biotechnology).
(FGF4):FGF1, or caRas were plated on 6-cm tissue culture
dishes with 0.5% agar in an overlay containing Dulbecco's modified
Eagle's medium (Invitrogen), 10% bovine calf serum (BCS; Hyclone),
and 0.33% agar at 1.5 × 103 cells/dish. As
indicated, some dishes were also treated with 1 µM of the
FGFR1-specific inhibitor, PD166866 (Ref. 36; a generous gift from
R. L. Panek, Park-Davis), and/or 10 ng/ml recombinant human
FGF1 and 10 units/ml heparin (Sigma). Cells were fed with 0.5 ml of
media with or without FGF1 and/or the FGFR1-specific inhibitor every 3 days as indicated. Twenty days after plating, colonies were stained
with p-iodonitrotetrazolium violet (Sigma) for
visualization. Quantitation of colony formation was achieved by
counting all p-iodonitrotetrazolium violet-stained colonies consisting of more than 4 cells under a Zeiss Stemi SVII Apo dissecting microscope.
(FGF4):FGF1 Transfectants after Adenoviral
Transduction--
hst-
(FGF4):FGF1 stable transfectants were
transduced with either adenovirus expressing lacZ, dominant negative
FGFR, caN1, or caN2 as described below. After 24 h, the cells were
plated on 6-well dishes at a concentration of 105
cells/well, and 3 days after plating, phase-contrast micrographs of the
cells were taken. For colony formation in the soft agar assay, the
hst-
(FGF4):FGF1 transfectants were transduced with the indicated
recombinant adenoviral vectors and plated into 0.33% agar 24 h
after the transduction. Two weeks after plating, colonies were
visualized by staining with p-iodonitrotetrazolium violet.
as),
5'-CACTGGAGTCAGCTGACACC-3'; FGFR2(s), 5'-TCCTTCAGTTTAGTTGAGGATAC-3';
FGFR2(as), 5'-GCAGCTTTCAGAACCTTGAGG-3'; FGFR3(s),
5'-CAAGTGCTAAATGCCTCCCAC-3'; and FGFR3(as),
5'-GCAGAGTATCACAGCTGC-3'.
(FGF4):FGF1 stable transfectants were
plated onto fibronectin-coated (10 µg/cm2) 12-well tissue
culture dishes and transiently transfected with 500 ng of the
CSL-luciferase construct, 100 ng of the TK Renilla (Promega)
construct as an internal control for transfection efficiency, and 200 ng of caN1. For all experiments, the medium was replaced 24 h
after transfection with fresh Dulbecco's modified Eagle's medium
(Invitrogen) supplemented with 10% BCS or with 10% BCS containing 1, 2.5, 5, or 10 ng/ml recombinant human FGF1 and 10 units/ml heparin as
indicated in the figure legend. The cells were harvested 48 h
after the media change, and luciferase/Renilla activity was
measured using Promega's Dual-Luciferase Reporter Assay System. The
efficiency of transcription was measured and normalized in relationship
to the activity of pRL-TK Renilla, and the activity is
reported as the ratio of luciferase/Renilla activity. Each
experiment was done in triplicate, and error bars represent the
S.E.
(FGF4):FGF1, and sJ1:caN1 stable NIH 3T3 cell
transfectants were plated on tissue culture dishes in either normal
growth media (Dulbecco's modified Eagle's medium; Invitrogen), 10%
BCS (Hyclone), or normal growth media containing 10 ng/ml recombinant
FGF1 and 10 units/ml heparin. Cells were harvested at confluence (based
on the status of vector control cells grown in normal growth media).
Because hst-
(FGF4):FGF1 stable transfectants and the sJ1
transfectants grown in FGF1-containing media form spheroids, all cells
were harvested by scraping in the presence of the growth media, and
then the cells/growth media were collected into sterile 15-ml conical
tubes. Cells were pelleted by centrifugation at 800 × g for 5 min. After centrifugation, the supernatant
was removed, and the cell pellets were washed three times with 1×
phosphate-buffered saline. Cells were lysed in 500 µl of 20 mM Tris, pH 7.5, containing 300 mM sucrose, 60 mM KCl, 15 mM NaCl, 0.5 mM EDTA,
0.5% (v/v) Triton X-100, 1 mM phenylmethylsulfonyl
fluoride, 0.1% SDS, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM sodium orthovanadate for 15 min on ice. Cell lysates
were pelleted, and the supernatant containing soluble proteins was
removed. Samples were normalized for protein concentration using the
BCA Protein Assay Kit (Pierce). Equal protein loads were resolved by
8% acrylamide (w/v) SDS-PAGE, transferred to Hybond C (Amersham
Biosciences), and immunoblotted with the Jagged1 antibody (Santa Cruz
Biotechnology). Jagged1 was visualized using a horseradish
peroxidase-conjugated antibody against goat IgG (Sigma) and the ECL
detection system (Amersham Biosciences).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(FGF4):FGF1) to force constitutive secretion of FGF1 through the conventional ER-Golgi pathway (31). The cells contained within the spheroid structures were
viable because they continued to proliferate over time and also grew as
a monolayer when replated onto fresh tissue culture dishes in the
absence of recombinant FGF1 in the growth media (data not shown). In
contrast, vector control and caN1 NIH 3T3 cell stable transfectants did
not form spheroids but instead continued to grow as a monolayer in
the presence of recombinant FGF1. Spheroid formation was a
specific response to FGF1 because treatment with the FGFR1-specific
inhibitor PD166866 (36) completely abolished FGF1-induced spheroid
formation in sJ1, dnN1, and hst-
(FGF4):FGF1 stable
transfectants.
View larger version (129K):
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Fig. 1.
Spheroid formation is regulated by FGF1 and
the Notch pathway. NIH 3T3 cell transfectants were grown in media
containing either 10% BCS or 10% BCS with the addition of FGF1 (10 ng/ml) and heparin (10 units/ml), or 1 µM PD166866 as
indicated. Phase-contrast photomicrographs (original magnification,
×40) of the cells 3 days after plating are shown.
(FGF4):FGF1). Interestingly, the size of the colonies also increased at seed densities of 5,000-10,000 cells/6-cm dish, even in
the absence of exogenously added recombinant FGF1 (data not shown). In
contrast, the addition of FGF1 had either no effect or only resulted in
the formation of sparse and very small colonies in the caN1 and vector
control transfectants. These transfectants also do not form colonies,
regardless of plating concentration, in the absence of FGF1. NIH 3T3
cells stably expressing dnN2 but not caN2 also formed small colonies
whose size was dramatically increased in the presence of FGF1 (data not
shown). Similar to spheroid formation, FGF potentiation of soft agar
growth in NIH 3T3 cells was a specific response to FGFR
stimulation because the addition of the FGFR1 inhibitor PD166866
substantially reduced FGF1-mediated colony formation in sJ1, dnN1, and
the hst-
(FGF4):FGF1 transfectants but had no effect on colony
formation in the caRas cells. Treatment with PD166866 also inhibited
small colony growth exhibited by sJ1 and dnN1 in the absence of FGF1.
These data suggest that repression of endogenous Notch signaling
sensitizes the NIH 3T3 cell to FGFR-mediated cellular transformation
and that activation of the Notch signaling pathway may protect the NIH
3T3 cell from abnormal growth.
View larger version (28K):
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Fig. 2.
Anchorage-independent growth requires FGF1
signaling and Notch repression. As indicated in the figure, cells
were grown in soft agar as described under "Experimental
Procedures," with some dishes treated with 1 µM of the
FGFR1-specific inhibitor, PD166866, and/or 10 ng/ml recombinant human
FGF1 and 10 units/ml heparin as indicated. Cells were fed every 3 days,
and colony number was quantified after 20 days. A,
representative wells for each growth condition are shown (original
magnification, ×4). B, the number of colonies was
quantified by counting all stained colonies from two plates for each
experimental condition. Graphed values are the means ± S.E.
Error bars represent the S.E., and the data reflect a
representation of one of several soft agar experiments conducted.
(FGF4):FGF1 stable transfectants with adenovirus
expressing either caN1 or caN2. Adenoviral expression of either caN1 or
caN2, but not a lacZ control, reduced the number of colonies formed in
soft agar to a level similar to that observed in the hst-
(FGF4):FGF1 NIH 3T3 cell transfectants transduced with a dominant negative FGFR1
construct (Fig. 3A) and
completely abolished spheroid formation (Fig. 3B). These
data support our observation that activation of the Notch
pathway protects the NIH 3T3 cell from FGFR-mediated transformation.
View larger version (52K):
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Fig. 3.
The expression of activated Notch suppresses
spheroid formation and colony growth in soft agar in
hst- (FGF4):FGF1 NIH 3T3 cell transfectants.
A, colony formation in soft agar exhibited by
hst-
(FGF4):FGF1 NIH 3T3 cell transfectants adenovirally transduced
with either lacZ, dominant negative FGFR1, caN1, or caN2. Positive
colonies were visualized by staining with
p-iodonitrotetrazolium violet 2 weeks after plating.
B, phase-contrast photomicrographs (original magnification,
×40) of hst-
(FGF4):FGF1 NIH 3T3 cell transfectants adenovirally
transduced with either lacZ, dominant-negative FGFR1, caN1, or
caN2.
and VT+ isoforms (41) of
fgfr1 (Fig. 4B). These data indicate that sJ1-mediated repression of endogenous Notch signaling regulates the
FGFR signaling pathway by a mechanism that includes changes in
fgf but not fgfr mRNA expression.
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[in a new window]
Fig. 4.
Regulation of FGF/FGFR gene
expression by soluble Jagged1. A, the expression of
fgf family members in vector control and sJ1 stable NIH 3T3
transfectants as determined by RT-PCR using primers and conditions
described under "Experimental Procedures." RT-PCR using primers
specific for murine GAPDH was performed as a control for
cDNA synthesis. B, the expression of fgfr
family members in vector control and sJ1 NIH 3T3 stable transfectants
as determined by RT-PCR using primers and conditions as described under
"Experimental Procedures." Each primer set was designed to detect
both the 2- and 3-Ig loop forms for fgfr1, fgfr2,
and fgfr3 as well as the VT and VT+ isoforms of
fgfr1.
View larger version (24K):
[in a new window]
Fig. 5.
Inhibition of Notch induces secretion of FGF1
under normal growth conditions. a, immunoblot analysis
of FGF1 export into the extracellular compartment in pMEXneo, sJ1, and
sJ1:caN1 stable transfectants transduced with FGF1 adenovirus and
subsequently subjected to heat shock (42 °C, 2 h) or maintained
under normal growth conditions (37 °C, 2 h). b,
immunoblot analysis of FGF1 export into media conditioned by stable
FGF1 NIH 3T3 cell transfectants adenovirally transduced with either
LacZ, caN1, dnN1, caN2, or dnN2 and maintained under normal growth
conditions (37 °C, 2 h) or subjected to heat shock conditions
(42 °C, 2 h).
(FGF4):FGF1 transfectants was significantly reduced
in comparison with its activity in vector control and caN1 stable
transfectants (Fig. 6C). In addition, exogenous FGF1
also repressed caN1/CSL transcription in a dose-dependent fashion in caN1 transfectants (Fig. 6A). The low levels of
endogenous CSL-dependent transcription in vector control and sJ1 NIH
transfectants also displayed a dose response to exogenous FGF1,
although the magnitude of the FGF1 effect was less than that achieved
in the caN1 transfectants (Fig. 6, A and B).
However, whereas caN1/CSL-dependent transcriptional
activation is significantly repressed by FGF1, this repression is not
complete. These results are interesting because the vector control
cells (Fig. 2, A and B) still do not proliferate
in response to exogenous FGF1 under conditions of soft agar growth.
Therefore, it is possible that this relatively low level of
conventional CSL-dependent activity is sufficient to
repress the ability of the NIH 3T3 cell to respond to exogenous FGF1 as
an agent of cell transformation.
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Fig. 6.
FGF1 suppresses caN1/CSL-activated
transcription. A, dose-response curve of endogenous
CSL-activated transcription in the presence of increasing
concentrations of recombinant human FGF1 in vector control, caN1, and
sJ1 transfectants. Reporter activity is represented as the ratio of
luciferase/Renilla activity. B, inset
highlighting the FGF1 dose-response curve of CSL-activated
transcription in vector control and sJ1 NIH 3T3 cell transfectants
shown in A. C, CSL-activated transcription in
vector control, hst- (FGF4):FGF1, caN1, and sJ1 NIH 3T3 stable
transfectants transiently transfected with caN1. Reporter activity is
represented as the ratio of luciferase/Renilla
activity.
(FGF4):FGF1 transfectants than in
vector control and caN1 transfectants grown in normal growth medium
(Fig. 7, 10% Serum). The presence of FGF1 in the growth
media increased Jagged1 expression in all stable lines examined, and
caN1 still displayed the lowest level of Jagged1 expression. We have
not found that NIH 3T3 cells express other Notch ligands including
Jagged2, Delta1, Delta3, or Delta4 in either the absence or presence of
FGF1 in the growth media (data not shown). In addition, we have also
observed that steady-state levels of mRNA encoding Notch1 and
Notch2 appear to be unchanged as determined by RT-PCR (data not shown),
although slight differences in the expression of these transcripts
detectable by more sensitive assays cannot be ruled out.
View larger version (21K):
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Fig. 7.
FGF1 increases the steady-state level of
Jagged1 protein expression. The expression of endogenous Jagged1
was determined by immunoblot analysis as described under
"Experimental Procedures" in vector control, sJ1, caN1, and
sJ1:caN1 stable transfectants plated in normal growth media (10%
Serum) or in normal growth media containing 10 ng/ml FGF1 and 10 units/ml heparin (10% Serum + FGF1). As a control for the
Jagged1 immunoblot, cell lysates obtained from NIH 3T3 cells stably
transfected with a full-length, human Jagged1 construct were also
included in the immunoblot analysis. The expression of Jagged1 was
determined in hst- (FGF4):FGF1 stable transfectants plated in only
normal growth media.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(FGF4):FGF1 transfectants, it is
likely that activated Notch1/Notch2 interferes with FGFR-mediated cellular transformation at a level other than its regulation of FGF
release. Although aberrant expression of FGFRs and their isoforms has
also been associated with cellular transformation (33, 46, 47), this is
probably not a contributing factor in our system because the expression
of fgfr mRNA, including those transcripts that represent
splice variants, is similar in all of the NIH 3T3 stable lines examined
in this study. Although it is currently unknown which other signaling
pathways are involved in facilitating cross-talk between Notch and
FGFR, it is possible that suppression of FGF-mediated
anchorage-independent growth by caN1 and caN2 may occur through its
regulation of activator protein-1-dependent transcription.
In fibroblasts, continuous exposure to FGF1 increases the transcription
of fos (44), a polypeptide component of the activator
protein-1 complex. Notch activation has been reported to inhibit
activator protein-1-mediated transcription in HeLa and human
erythroleukemia cell line K562 (48), and caN1 suppression of activator
protein-1 may be the underlying mechanism behind its inhibition of
human papillomavirus-induced transformation in cervical carcinoma cells
(49).
(FGF4):FGF1) induces a
transformed phenotype in NIH 3T3 cells in vitro (Ref. 31;
Figs. 1A and 2, A and B) and produces aggressive, metastatic tumors in vivo (31). It is possible
that continuous exposure to low levels of endogenous FGF may
"prime" the Notch-repressed cells such that any additional FGFR
stimulation results in aggressive and uncontrolled growth. The
proto-oncoprotein Src is an FGFR downstream effector molecule whose
activity is increased in Notch-repressed cells (11), and it is possible that further potentiation of Src signaling by FGFR stimulation may be
the primary mechanism driving FGF-mediated anchorage-independent growth
in these cells. However, we have been unable to block FGF-mediated soft
agar colony growth or spheroid formation through the expression of a
dominant negative mutant of Src (data not shown), indicating that
another, as yet unidentified pathway may be a contributing factor to the exaggerated transformed phenotype mediated by exogenous FGF1 in Notch-repressed cells.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. T. Kadesch for the Notch1 and CSL-reporter constructs, Dr. S. Artavanis-Tsakonas for the Notch2 constructs, Dr. R. Friesel for the dominant-negative FGFR1 construct, Dr. R. L. Panek for the FGFR1 inhibitor PD166866, and Norma Albrecht and Gloria Ledoux for expert administrative assistance.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants CA92255 (to D. S.), HL32348 and HL35627 (to T. M.), and RR15555 (to T. M. and L. L.) and by American Cancer Society Grant RPG97-093-03 (to L. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to this work.
§ Present address: Dept. of Animal, Nutritional and Medical Laboratory Sciences, College of Life Sciences and Agriculture, University of New Hampshire, Durham, NH 03824.
¶ To whom correspondence should be addressed: Center for Molecular Medicine, Maine Medical Center Research Institute, 81 Research Dr., Scarborough, ME 04074. Tel.: 207-885-8200; Fax: 207-885-8179; E-mail: maciat@mmc.org.
Published, JBC Papers in Press, February 21, 2003, DOI 10.1074/jbc.M300464200
2 D. Small, R. Trifonova, and T. Maciag, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: sJ1, soluble Jagged1; BCS, bovine calf serum; caN, constitutively active Notch; dnN, dominant-negative Notch; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; RT-PCR, reverse transcription-PCR.
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