From the Division of Cellular Biochemistry, The
Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The
Netherlands and § Ludwig Institute for Cancer Research, Box
595, S-751 24, Uppsala, Sweden
Received for publication, August 13, 2002, and in revised form, November 22, 2002
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ABSTRACT |
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Transforming growth factor- Transforming growth factor- TGF- Members of the small GTP-binding proteins, including Rac, Rho, and
Cdc42, have been reported to become activated by TGF- The MAP kinase pathways have also been shown to modulate the Smad
pathway. Overexpression of dominant negative members of the JNK pathway
inhibited TGF- To investigate the pathways that are activated in the absence of Smad
activation we have generated a TGF- Expression Plasmids--
All of L45 mutants in human ALK5 were
made by the QuikChange site-directed mutagenesis kit (Stratagene) using
pCDNA3-ALK5/HA and pCDNA3-caALK5/HA as templates. pMEP-ALK5/HA,
pMEP-ALK5(D266A)/HA, and pMEP-ALK5(3A)/HA were constructed by ligation
of the insert from pCDNA3-ALK5/HA or its mutants with pMEP4
(Invitrogen). The chimeric constructs between GM-CSF Adenovirus Constructs--
To create adenoviruses for ALK5/HA or
ALK5(3A)/HA, the insert of pCDNA3-ALK5/HA or
pCDNA3- ALK5(3A)/HA was ligated into pAdTrack-CMV (53). After
recombination of pAdTrack-CMV-ALK5/HA or pAdTrack-CMV-ALK5(3A)/HA with
pAdEasy-1, the obtained plasmids were transfected into 293T cells, then
adenoviruses were amplified.
Cell Culture--
293T and Phoenix-A cells were cultured in
Dulbecco's modified Eagle's medium (DMEM, Invitrogen) containing
10% fetal calf serum (FCS, Invitrogen) and 1×MEM nonessential amino
acids (Invitrogen). Mv1Lu, R4-2, MDA-MB-468, and COS7 cells were
maintained in DMEM containing 10% FCS. Mouse NMuMG breast epithelial
cells were grown in DMEM containing 10% FCS, 1× nonessential amino
acids, and 10 µg/ml insulin. For selection of stable transformants
with pMEP4 in R4-2 and retroviral vectors in NMuMG, the cells were
cultured in the presence of 400 units/ml hygromycin B (Calbiochem) and 5 µg/ml puromycin (Sigma), respectively.
Retroviral Infection--
Phoenix-A cells (4 × 106/10 cm dish) were transfected with 20 µg of retroviral
expression plasmid with FuGENE 6 (Roche Molecular Biochemicals). The
next day the cells were reseeded in a 175-cm2 flask and
subsequently cultured in the presence of 2 µg/ml puromycin for 1 week. After confluence, the cells were grown in 15 ml of DMEM
containing 10% FCS without drug selection for 16 h. This culture
medium was used as a source for retrovirus after filtration. For
retroviral infection, NMuMG cells (2 × 106/10 cm
dish) were infected with 5 ml of cultured medium containing pBabe- Transcriptional Reporter Assays--
One day before transfection
Mv1Lu or R4-2 cells were seeded at 4.0 × 105
cells/well in 12-well plates. The cells were transfected using FuGENE
6. Where indicated, TGF- Immunoprecipitation and Western Blotting--
To detect Smad2
phosphorylation by constitutively active (ca) ALK5 or its mutant
derivatives in COS7 cells, 3 µg of pDEF3-FLAG-Smad2 and 3 µg of
caALK5 or its mutant derivatives were transfected in COS7 cells at
1.5 × 106 cells/10-cm dish using FuGENE 6. Forty
hours after transfection, the cells were lysed in 1 ml of lysis buffer
(20 mM Tris (pH 7.4), 150 mM NaCl, 10%
glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 2.5 µg/ml aprotinin, 2 mM
sodium vanadate, 40 mM NaF, and 20 mM
Receptor Autophosphorylation Assay--
One day before
transfection, COS7 cells were seeded at 2.0 × 105
cells/3.5-cm dish. The cells were transfected with 1.5 µg of pCDNA3-caALK5/HA or its mutant derivatives by FuGENE 6. Forty hours
later, the cells were lysed with lysis buffer, precleared with protein
G-Sepharose beads, and incubated with anti-HA12CA5 antibody for 2 h at 4 °C. Protein G-Sepharose beads were then added to the reaction
mixture, and incubation was continued another 30 min at 4 °C. The
immunoprecipitates were washed with lysis buffer three times and with
the kinase reaction buffer (10 mM Tris (pH 7.4), 10 mM MgCl2, and 2 mM
MnCl2) twice and then incubated with kinase reaction buffer
containing 14.8 kBq/ml [ Growth Inhibition Assay--
R4-2 transformants were seeded at
1 × 104 cells/well in 24-well plates. Before the
addition of TGF- Extracellular Matrix Formation Assay--
Cells were seeded in
6-well plates at a density of 2 × 106 cells/well in
medium containing 100 µM ZnCl2. After 18 h, the medium was changed to cysteine/methionine-free medium
(Invitrogen) with 100 µM ZnCl2 in the
presence of 10 ng/ml TGF- In Vitro JNK Assay--
JNK activation was determined by an
immune complex kinase assay using GST-c-Jun 1-135 as a substrate (56).
293T cells were seeded at 6.0 × 106/10 cm dish 1 day
before transfection. The cells were transfected with 3 µg of
FLAG-JNK1 Staining of Actin--
NMuMG cells or its stable transformants
were seeded at 1 × 104 cells/well in 8-well glass
slides (LAB-TEK) coated with 0.1% gelatin. Eighteen hours later, the
cells were starved in DMEM containing 0.3% FCS for 4 h and then
stimulated with 10 ng/ml TGF- Reverse Transcription-PCR Analysis--
One day before the
infection, MDA-MB-468 cells were plated in 15-cm dishes. The infection
with adenoviruses expressing GFP or wild type human Smad4 was carried
out as previously described (57). After 24 h, the cells were
treated with vehicle or 5 ng/ml TGF- Northern Blot Analysis--
NMuMG transformants were cultured in
DMEM, 0.3% FCS 12 h before stimulation. Total RNAs from NMuMG
transformants stimulated with either 10 ng/ml TGF- Generation of ALK5 L45 Mutants Defective in Smad
Activation--
To study ALK5-initiated signaling responses that are
Smad-independent, we mutated the L45 loop in ALK5 to selectively
perturb the ability of ALK5 to bind and activate Smad2 and Smad3. We
mutated the aspartic acid residue that is conserved in all type I
receptors and the four amino acid residues that we and others
previously demonstrated to be important determinants for binding
specificity for Smad isoforms between ALK5 and ALK6 (7, 8) (Fig.
1, A and
B). We generated single mutants, i.e.
ALK5(D266A), ALK5(N267A), ALK5(D269A), ALK5(N270A), and
ALK5(T272A) and a triple mutant, i.e.
ALK5(D269A,N270A,T272A), called ALK5(3A).
First we tested the in vitro kinase activity of caALK5
mutants and compared them with caALK5. The constitutively active
variants were made by converting threonine residue 204 into aspartic
acid (60). caALK5(D266A), caALK5(N267A), and caALK5(3A) but not
caALK5(269A), caALK5(N270A), and caALK5(T272A) were found to have a
similar enhanced in vitro kinase activity as caALK5 (Fig.
1C, lanes 1-8). We also saw a comparable kinase
activity among ALK5, ALK5(D266A) and ALK5(3A) (Fig. 1C,
lanes 9, 10, and 12). All ALK5 mutants were also found to form heteromeric complexes with the TGF-
To examine whether ALK5(D266A) and ALK5(3A) can activate
Smad-dependent reporter activity, we first transfected
caALK5 or its mutants in Mv1Lu cells. As expected, caALK5 dramatically
induced (>70-fold) Smad-dependent luciferase activity.
However, caALK5(D266A) and caALK5(3A) exhibited only 4.1- and 2.3-fold
activation, respectively (Fig. 1E). ALK5 is known to be
present in cells as a dimer in the absence of ligand (61). It is
possible that the exogenous caALK5 L45 mutants can make complexes with
endogenous ALK5, which can explain the low residual activity of caALK5
L45 mutants. Thus, introduction of ALK5 L45 mutant in the cells, which
possess endogenous functional ALK5, may not lead to a complete
Smad-independent signaling. Therefore, we transfected ALK5 L45 mutants
in R4-2 cells that lack functional ALK5. As seen in Fig. 1F,
neither ALK5(D266A) nor ALK5(3A) activated (CAGA)12-Luc
(i.e. a readout for activation of the Smad3/Smad4 pathway)
or 2×ARE-luc reporter (i.e. a read-out for the Smad2/Smad4
pathway). These results were fully consistent with the inability of
ALK5(D266A) and ALK5(3A) to induce Smad2 phosphorylation. We therefore
used ALK5(D266A) and ALK5(3A) in the following experiments to examine
Smad- (in)dependent signaling.
ALK5 L45 Mutants Do Not Rescue TGF- ALK5 L45 Mutant Interacts with I-Smads but Not XIAP or
Dab-2--
I-Smads bind to activated type I receptors and can compete
with R-Smads for receptor binding (62-64). However, the domain(s) in
the type I receptor responsible for I-Smad binding have not been
characterized. To test the interaction of Smad6 or Smad7 with ALK5(3A)
mutant, I-Smads and receptors were transfected into COS7 cells and
subjected to immunoprecipitation followed by Western blotting. The
ALK5(3A) mutant was found to interact with Smad6 or Smad7 as
efficiently as wild type ALK5 (Fig.
3A). This suggests that the
L45 loop is not important for interaction with I-Smads. However, when
we analyzed the interaction of two other components known to bind type
I receptors, i.e. XIAP (47) (Fig. 3B) and Dab-2
(10) (Fig. 3C), we found that none of them interacted with
the ALK5(3A) mutant using a similar strategy as above. The L45 loop
region may, thus, not only be important for Smad binding but also for
interaction with other signaling components.
ALK5 L45 Mutant Activates JNK MAP Kinase--
TGF- Characterization of Chimeric GM-CSF/TGF- GM-CSF/ALK5 L45 Mutants Can Modulate Gene Expression but
Fail to Induce Stress Fibers in NMuMG Cells--
Transcriptional
analysis on
NMuMG cells transdifferentiate from an epithelial phenotype to a
spindle-shaped morphology in response to TGF- Smads are pivotal intracellular mediators of TGF- Consistent with previous data that implicate Smads in TGF- The ALK5(3A) mutant was found to interact with I-Smads (Fig. 3).
I-Smads have been shown to compete with R-Smads for interaction with
activated type I receptors (62-64). Taken together, these results
suggest that R-Smads and I-Smads interact also with a region in the
receptor, such as glycine-serine-rich domain (74). The weak binding of
XIAP and Dab-2 was found to be dependent on the intact L45 loop in
ALK5. Thus, the L45 loop region may be important for interaction not
only R-Smads but also with other signaling components. Therefore, to
complement our studies on Smad-independent signaling using ALK5 L45
mutant-expressing cells, we are currently examining the responses on
TGF- Activation of JNK was shown to be enhanced by the caALK5(3A) mutant
(Fig. 4). Thus, this response is independent of the Smad pathway. The
observation that ALK5(3A) is weaker than wild type ALK5 in activating
c-Jun-based transcriptional reporters suggests that Smad signaling
contributes to the activation of this reporter, as previously shown
(32-37). XIAP has been implicated as a link between the type I
receptor and the JNK pathway (26). However, ALK5(3A), which is not
capable of binding to XIAP, can induce moderate JNK-mediated reporter
activities (Fig. 4B). This suggests that XIAP is dispensable
for JNK activation mediated by TGF- The TGF- In conclusion, using Smad-activation-defective L45 loop mutants of
ALK5, we have shown that ALK5-mediated JNK activation, and certain gene
responses can occur independent of the Smad pathway. The ALK5 L45
mutants will be important tools to examine the requirement of Smads,
and other signaling components that bind to the L45 loop for the
various effects by TGF- (TGF-
) elicits
cellular effects by activating specific Smad proteins that control the
transcription of target genes. Whereas there is growing evidence that
there are TGF-
type I receptor-initiated intracellular pathways that are distinct from the pivotal Smad pathway, their physiological importance in TGF-
signaling is not well understood. Therefore, we
generated TGF-
type I receptors (also termed ALK5s) with mutations in the L45 loop of the kinase domain, termed ALK5(D266A) and ALK5(3A). These mutants showed retained kinase activity but were unable to
activate Smads. Characterization of their signaling properties revealed
that the two L45 loop mutants did not mediate
Smad-dependent transcriptional responses, TGF-
-induced
growth inhibition, and fibronectin and plasminogen activator-1
production in R4-2 mink lung epithelial cells lacking functional ALK5
protein. Mutation in the L45 loop region did not affect the binding of
inhibitory Smads but did abrogate the weak binding of X-linked
inhibitor of apoptosis protein and Disabled-2 to ALK5. This suggests
that the L45 loop in the kinase domain is important for docking of other binding proteins. Interestingly, JNK MAP kinase activity was
found to be activated by the ALK5(3A) mutant in various cell types. In
addition, TGF-
-induced inhibition of cyclin D1 expression and
stimulation of PMEPA1 (androgen-regulated prostatic mRNA) expression were found to occur, albeit weakly, in an Smad-independent manner in normal murine mammary gland cells. However, the
TGF-
-induced epithelial to mesenchymal transdifferentiation was
found to require an intact L45 loop and is likely to be dependent on
the Smad pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-
)1 belongs to a
family of cytokines that regulate cell proliferation and
differentiation of many different cell types (1). TGF-
family
members, which include TGF-
s, activins, and bone morphogenetic
proteins, were found to possess critical roles during embryogenesis and
in maintaining tissue homeostasis during adult life. Deregulated
TGF-
family signaling has been implicated in multiple developmental
disorders and in various human diseases, including cancer, fibrosis,
and auto-immune diseases (2).
family members transduce their signals across the plasma
membrane by inducing the formation of heteromeric complexes of specific
type I and type II serine/threonine kinase receptors (3). The type I
receptor is phosphorylated and activated by the type II receptor (4)
and initiates intracellular signaling through activation of downstream
signaling components, including the phosphorylation of
receptor-regulated (R)-Smad proteins at their extreme C-terminal serine
residues. Using chimeric TGF-
family receptors, the exposed L45
loop, a nine-amino acid sequence between kinase subdomains IV and V,
was found to be important in determining the signaling specificity of
type I receptors (5, 6) by specifying which Smad isoform is activated
(7, 8). Accordingly, Smad2 and Smad3 act downstream of TGF-
and
activin type I receptors, whereas Smad1, Smad5, and Smad8 are
phosphorylated by bone morphogenetic protein type I receptors. A number
of proteins with anchoring, scaffolding, and/or chaperone activity have
been identified that regulate the recruitment of Smads to the type I-type II receptor complex, including Smad anchor for receptor activation (SARA) (9) and Disabled-2 (Dab-2) (10). Whether these
components or other identified TGF-
receptor-binding proteins may
play a role in presentation of other substrates besides Smads remains
to be explored. Activated R-Smads form heteromeric complexes with
common partner (Co)-Smad, i.e. Smad4, which accumulates in the nucleus, where they control gene expression of genes involved in
e.g. growth inhibition, apoptosis, migration, and
extracellular matrix production (11). Whereas Smads have been
identified as pivotal intracellular effectors for TGF-
, there is
growing evidence that additional pathways are activated downstream of
TGF-
receptors (11). However, the molecular mechanisms by which
these responses are transduced and their physiological significance in
TGF-
signaling have remained poorly characterized.
and to
potentiate TGF-
-induced transcriptional activation (12, 13) and
induce mobilization of the actin cytoskeleton (14). Three distinct MAP
kinases, i.e. extracellular-regulated kinase, c-Jun
N-terminal kinase (JNK), and p38, have been shown to be activated by
TGF-
and other family members (15-22). Both rapid (within 10-30
min) as well as delayed (after several hours) activation of MAP kinase
have been reported, of which the delayed activation has been shown to
depend on Smad pathways (19). The mitogen-activated protein kinase
TGF-
-activated kinase 1 (TAK1) has been shown to be phosphorylated
upon TGF-
stimulation and can lead to the activation of JNK and p38
MAP kinase pathways (17, 23, 24). TAK1-binding protein (TAB1) was
identified as an activator of TAK1 (25). Hematopoietic progenitor
kinase-1 (HPK1) and X-chromosome-linked inhibitor of apoptosis (XIAP)
may provide a direct link between TAK1-binding protein (TAB1) and the
type I receptor (26-28). Interestingly, the TGF-
-induced expression
of fibronectin and inhibition of insulin-like growth factor-binding
protein-5 or NOV, a secreted glycoprotein, were shown to require JNK
activation but to be independent of Smad pathways (29-31).
/Smad-mediated responses (19, 29). Smad3 and AP-1
family members have been shown to cooperate with each other in
transcriptional responses by forming direct protein interactions
(32-37). In addition, TGF-
-induced activation of p38 MAP kinase was
shown to induce ATF-2 phosphorylation, which acted synergistically with
Smads in transcriptional activation (38). JNK was found to be rapidly
activated by TGF-
in mink lung epithelial cells (Mv1Lu) and to
facilitate TGF-
-induced Smad activation (19). In contrast,
activation of extracellular-regulated kinase MAP kinase has also been
shown to induce the phosphorylation in the linker region of R-Smads
and, thereby, to inhibit the ligand-induced nuclear accumulation of
R-Smads (39-41).
type I receptor (ALK5) with
mutations in the L45 loop of the kinase domain that are defective in
Smad activation and characterized the signaling properties of these
mutated type I receptors.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
R and ALK5
(pNA
I) and between GM-CSF
R and T
R-II (pHa-
II) were
provided by Dr. E. B. Leof (42). To create chimeric constructs
between human GM-CSF
R and human ALK5/HA, the intracellular domain
of ALK5 from pNA
I was substituted into the corresponding domain of
ALK5 from pCDNA3-ALK5/HA or its mutants. Subsequently, the inserts
of chimeric constructs were ligated into the LZRS-pBMN-ms(-IRES)-eGFP
retroviral vector constructed from LZRS-pBMN-LacZ, which was obtained
from Dr. G. Nolan. pBabe-
RII, a retroviral expression vector of the
chimeric construct between human GM-CSF
R and human T
R-II, was
constructed by ligating the insert from pHa-
II to pBabe (43). To
generate pDEF3-FLAG-Smad2, the insert from pCDNA3-FLAG-Smad2 (44),
digested with KpnI and XbaI, was subcloned into
the KpnI-XbaI site of pDEF3 (45). Human FAST-1
(46), GST-XIAP (47), FLAG-Dab-2 (10), and GST-c-Jun 1-135 (49)
were kindly provided by Drs. B. Vogelstein, C. H. Duckett, P. H. Howe, and G. A. Ruiter, respectively. The constructs for
pCDNA3-ALK5/HA, pCDNA3-caALK5/HA, FLAG-Smad4,
(CAGA)12-luc, 2×ARE-luc, 6×Myc-Smad6, and 6×Myc-Smad7
have been described previously (44, 50-52). HA-p38 and FLAG-JNK1
were obtained from Drs. E. Nishida and R. Davis, respectively.
pFA2-cJun, pFA-CHOP, pAP-1-Luc, and pFR-Luc were purchased from Stratagene.
RII retrovirus in the presence of 10 µg/ml DOTAP
(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium salts; Roche Molecular Biochemicals). After selection with 5 µg/ml puromycin,
RII-positive cells were sorted by fluorescence-activated cell sorter using anti-human GM-CSF
R monoclonal antibody (Santa Cruz) and R-phycoerythrin goat anti-mouse IgG (H+L) antibody (Molecular Probes). Then,
RII-positive cells were collected and cultured. Subsequently,
RII-expressing cells were infected with
LZRS-pBMN-ms(-IRES)-eGFP-
ALK5/HA, LZRS-pBMN-ms(-IRES)-eGFP-
ALK5(D266A)/HA,
LZRS-pBMN-ms(-IRES)-eGFP-
ALK5(3A)/HA, or LZRS-pBMN-ms(-IRES)-eGFP by
the above procedure. Because the expression of
ALK5 or its mutants
is linked to that of GFP, both GFP- and
RII-positive cells were
collected by fluorescence-activated cell sorter to get double-positive cells.
3 was added into the dishes 24 h after
transfection. Subsequently, the cells were cultured in DMEM containing
0.3% FCS for 18 h. In all experiments,
-galactosidase (pCH110,
Amersham Biosciences) activity was measured to normalize for
transfection efficiency. Each transfection was carried out in
triplicate and repeated at least twice.
-glycerophosphate). The cell lysates were precleared with protein
G-Sepharose beads (Amersham Biosciences) and incubated with FLAG M5
antibody (Sigma) for 2 h at 4 °C. Subsequently, protein
G-Sepharose beads were added to the reaction mixture and incubated for
30 min at 4 °C. After washing the immunoprecipitates with lysis
buffer three times, the proteins in immunoprecipitates and aliquots of
total cell lysates were separated by SDS-PAGE and transferred to a
Hybond-C Extra membrane (Amersham Biosciences). The membrane was
subsequently probed with phosphorylated Smad2-specific antibodies (pS2)
(8) or FLAG M5 antibody. Primary antibodies were detected with
horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse
antibody (Amersham Biosciences) and chemiluminescent substrate. The
expression of ALK5 receptors was determined by immunoprecipitation of
the [35S]cysteine/methionine-labeled cells (44). The
detection of the interactions between caALK5 and I-Smads, between
caALK5 and XIAP, or between ALK5 and Dab-2 was performed by
immunoprecipitation followed by Western blotting according to the above
method, except that cells were lysed in TNE buffer (10 mM
Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1%
Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml
leupeptin, 2.5 µg/ml aprotinin, 2 mM sodium vanadate, 40 mM NaF, and 20 mM
-glycerophosphate). The
expression of proteins in R4-2 or NMuMG stable transformants was
detected by Western blotting of total cell lysates in TNE buffer. pS2
antibody for phosphorylated Smad2, anti-Smad2 antibody (Transduction
Laboratories) for total Smad2, anti-HA12CA5 antibody (Roche Molecular
Biochemicals) for ALK5 or
ALK5 chimera, and anti-GM-CSF
R
antibody for
RII chimera were used as primary antibodies.
-32P]ATP (Amersham
Biosciences) for 30 min at 25 °C. Immunoprecipitates were separated
by SDS-PAGE. The expression of receptor proteins was determined by
35S-metabolic labeling and immunoprecipitation (44).
3, the cells were simultaneously treated (or not
treated) with 100 µM ZnCl2 to induce ALK5
expression. Two hours before harvest, the cells were pulsed with 18.5 kBq of [methyl-3H]thymidine (Amersham
Biosciences) in 0.5 ml of culture medium. The cells were fixed with
ice-cold 5% trichloroacetic acid for more than 20 min and washed twice
with 5% trichloroacetic acid and once with water. Solubilization of
the cells was done in 400 µl of 0.1 M NaOH for 20 min at
room temperature. The 3H radioactivity incorporated into
DNA was determined by liquid scintillation counting.
3, and the incubation was prolonged for
another 4 h. During the last 2 h, cells were incubated with
148 kBq/ml 35S-labeling mixture ProMix (Amersham
Biosciences). The cells were removed by washing on ice, once in
phosphate-buffered saline (PBS), 3 times in 10 mM Tris (pH
8.0), 0.5% sodium deoxycholate, and 1 mM
phenylmethylsulfonyl fluoride, twice in 2 mM Tris (pH 8.0), and once in PBS. Extracellular matrix proteins were scraped off, extracted into SDS sample buffer, and analyzed by SDS-PAGE. Plasminogen activator-1 (PAI-1) was identified as a 45-kDa protein in the extracellular matrix fraction (54). For the detection of fibronectin, R4-2 transformants were seeded at 1.0 × 106
cells/6-well plate. After 18 h, the cells were cultured in the presence of 100 µM ZnCl2 for 5 h.
Subsequently, the culture medium was changed to DMEM containing 0.3%
FCS and 100 µM ZnCl2 with or without 10 ng/ml
TGF-
3. After 16 h, the medium was changed to 1 ml of
cysteine/methionine-free medium containing 100 µM
ZnCl2 and 74 kBq/ml 35S-labeling mixture ProMix
with or without 10 ng/ml TGF-
3. Two hours later, 0.5 ml of culture
medium was collected into the tube and incubated with 100 µl of
gelatin-Sepharose (50% slurry in 50 mM Tris (pH 7.4) and
150 mM NaCl; Amersham Biosciences) and 30 µl of 10%
Triton X-100 for 16 h at 4 °C. Fibronectin is known to bind
strongly to gelatin (55). The beads were successively washed once with
solution A (50 mM Tris (pH 7.4) and 150 mM
NaCl), once with solution B (50 mM Tris (pH7.4) and 500 mM NaCl), and once again with solution A. Gelatin-bound
proteins were analyzed by SDS-PAGE.
or 3 µg of caALK5/HA or its mutant derivatives by
FuGENE 6. After 40 h, cells were lysed in 1 ml of TNE buffer. The
cell lysates were precleared with protein G-Sepharose beads and
incubated with FLAG M2 (Sigma) antibody for 2 h at 4 °C.
Subsequently, protein G-Sepharose beads were added to the reaction
mixture, and incubation was prolonged another 30 min at 4 °C. The
immunoprecipitates were washed 3 times with TNE buffer, twice with LiCl
buffer (500 mM LiCl, 100 mM Tris (pH 7.6),
0.1% Triton X-100, and 1 mM dithiothreitol), and 3 times
with 2× assay buffer (40 mM MOPS (pH 7.2), 4 mM EGTA, 20 mM MgCl2, 2 mM dithiothreitol, and 0.2% Triton X-100). Then the
immunoprecipitates were incubated in the reaction buffer (20 mM MOPS (pH 7.2), 2 mM EGTA, 15 mM
MgCl2, 1 mM dithiothreitol, 0.1% Triton X-100,
25 µM ATP, and 74 kBq/µl [
-32P]ATP)
containing 0.14 µg/µl GST-c-Jun-(1-135) at 30 °C for 20 min.
Simultaneously, the expression of FLAG-JNK1
and caALK5/HA or its
mutant derivatives was detected by Western blotting using total
lysates. To detect the JNK activity in R4-2 cells infected with
adenoviral ALK5 or its mutant, R4-2 cells were seeded at 1.0 × 106/10-cm dish 1 day before the infection. After infection
with adenoviruses (multiplicity of infection 100), the cells were
cultured in DMEM, 10% FCS for 24 h. Subsequently, the cells were
starved in DMEM, 0.3% FCS 12 h before TGF-
3 was added in the
dish. After cell lysis with TNE buffer, the kinase assay was carried
out as the above except that
JNK1 antibody (C-17; Santa Cruz) was
used for immunoprecipitations. The anti-HA and pS2 antibodies were used for detection of ALK5 and phosphorylated Smad2, respectively.
3 or 50 ng/ml human GM-CSF for 20 h. After treatment, the slides were washed once with PBS, fixed for 10 min with 3.7% paraformaldehyde, washed 3 times with PBS, subsequently
permeabilized with 0.1% Triton X-100 in PBS for 2 min, and washed
again 3 times with PBS. Slides were blocked with 5% normal swine serum
(DAKO) in PBS at 37 °C for 1 h, and incubated with 5% normal
swine serum (in PBS) containing rhodamine-conjugated phalloidin
(diluted 1:200) (Molecular Probes). The slides were then washed five
times with PBS. To visualize the fluorescence, a confocal
laser-scanning microscope (Leica) was used.
1 for 2 or 12 h. At the end
of each incubation time total cellular RNA was extracted using RNeasy
(Qiagen). Then total RNA (1 µg) was digested with RQ1 RNase-free
DNase (Promega) and included in cDNA synthesis reaction using
Superscript II reverse transcriptase (Invitrogen) with conditions as
previously described (58). PCR for fibronectin was performed with the
forward primer 5'-TGGAACTTCTACCAGTGCGAC-3' and the reverse primer
5'-TGTCTTCCCATCATCGTAACAC-3'. PCR conditions were as follows: 95 °C
for 5 min followed by 33 cycles of 95 °C for 30 s, 59 °C for
1 min, and 72 °C for 1 min, with a final incubation at 72 °C for
10 min. PCR for glyceraldehyde-3-phosphate dehydrogenase was carried
out with the forward primer 5'-ATCACTGCCACCCAGAAGAC-3' and the reverse
primer 5'-ATGAGGTCCACCACCCTGTT-3'. PCR conditions were 95 °C for 5 min followed by 29 cycles of 95 °C for 30 s, 57 °C for 1 min, and 72 °C for 1 min, with a final incubation at 72 °C for 10 min. PCR products were analyzed by 2% agarose electrophoresis and
ethidium bromide staining.
3 or 50 ng/ml
GM-CSF were prepared with RNeasy. Fifteen micrograms of total RNA were
loaded on the denatured1.5% agarose gel. Blotting and hybridization
were performed as previously described (59). The partial cDNAs for
mouse PMEPA1 (GenBankTM accession number BG075859)
and cyclin D1 (GenBankTM accession number BG083088) were
obtained from The Wellcome Trust Sanger Institute (Cambridge, UK), and
their sequences were verified.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Identification of ALK5 L45 mutants that are
defective in Smad activation, but with retained kinase activity.
A, schematic representation of ALK5 and location of L45 loop
in the kinase domain. TM, transmembrane domain.
GS, glycine-serine rich domain. BMPR, bone
morphogenetic protein receptor. B, comparison of amino acid
sequences in L45 loop region among different human ALKs. The amino acid
residues that were selected for mutation into alanine residues are
indicated with numbers, referring to their position in the
ALK5 sequence. The two -strands (
4 and
5) that flank the L45
loop are shown as arrows. C, in
vitro autophosphorylation activity of caALK5 and wild type ALK5
and various L45 mutant derivatives. ALK5 constructs were transfected
into COS7 cells, immunoprecipitated (IP) with anti-HA
antibody, and subjected to an autophosphorylation reaction. Expression controls for ALK5
are shown below by immunoprecipitation of 35S-labeled cell
lysates from parallel transfected COS7 cells with anti-HA antibody.
D, effect of caALK5 and L45 mutant derivatives on Smad2
phosphorylation. caALK5 or L45 mutant derivatives were transiently
co-transfected with FLAG-Smad2 into COS7 cells. The level of C-terminal
Smad2 phosphorylation was determined by Western blotting
(WB) with anti-phospho-Smad2 (pS2) antibody.
Expression controls for ALK5 and Smad2 are shown below. E,
caALK5 L45 mutants activate Smad-dependent luciferase
reporter weakly in Mv1Lu cells. caALK5 and its derivatives were
transiently transfected with (CAGA)12-Luc into Mv1Lu cells.
Luciferase values were normalized for transfection efficiency. Part of
the original figure is expanded in the inset to compare the
values of caALK5 L45 mutants with those of mock transfection. All
values represent the mean ± S.D. Significantly different from the
mock: *, p < 0.05; **, p < 0.005. F, effect of ALK5 and L45 mutant derivatives on
TGF-
-induced transcriptional responses. ALK5 and L45 mutant
derivatives were transiently transfected with (CAGA)12-Luc
(left panel) or ARE-Luc (right panel) into R4-2
cells that lack functional ALK5 and treated with TGF-
, and
transcriptional response was determined by measuring luciferase
activity. Luciferase values were normalized for transfection
efficiency. All values represent the mean ± S.D.
type II
receptor (T
R-II) and to bind TGF-
with equal efficiency as compared with wild type ALK5 (data not shown). Next, we tested the
ability of ALK5 mutants to phosphorylate Smad2 in transfected COS7
cells. Among the three mutants with equivalent kinase activity compared
with caALK5, caALK5(D266A) and caALK5(3A) did not phosphorylate Smad2
(Fig. 1D). In addition, we could not see any enhancement of
Smad1 phosphorylation by caALK5 or caALK5 mutants (data not shown).
-induced Growth Inhibition,
Fibronectin, and PAI-1 Production in Mink Cells That Are Deficient in
ALK5--
Mv1Lu cells are potently inhibited in their growth and
produce high levels of fibronectin and PAI-1 upon TGF-
stimulation. To elucidate the abilities of ALK5(D266A) and ALK5(3A) to mediate these
responses, we stably transfected these receptors in R4-2 cells that are
deficient in functional ALK5. All receptor constructs were placed under
the transcriptional control of the metallothionein promoter, which can
be induced by ZnCl2. Expression analysis of type I
receptors in R4-2 stable transformants revealed
ZnCl2-inducible expression of wild type ALK5, ALK5(D266A),
and ALK5(3A) (Fig. 2A).
Consistent with the experiment in COS7 cells, Smad2 phosphorylation by
TGF-
was observed only in wild type ALK5-expressing R4-2 cells after
the addition of ZnCl2 (data not shown). We then
investigated whether ALK5(D266A) and ALK5(3A) mutants could be
substituted for wild type ALK5 with respect to TGF-
-induced growth
inhibition (Fig. 2B), fibronectin, and PAI-1 protein
production (Fig. 2C). However, ALK5(D266A) and ALK5(3A)
mutants were not able to mediate these responses. Thus, they appear to
depend on an intact L45 loop and are likely to be
Smad-dependent responses. Consistent with the results
obtained from R4-2 cells that express ALK5 L45 mutants, TGF-
did not
induce fibronectin mRNA levels in MDA-MB-468 cells that are
deficient in Smad4. This TGF-
-induced response with delayed kinetics
(induction after 12 h, but not 2 h) could be rescued after
infection of MDA-MB 468 cells with Smad4 adenovirus (Fig.
2D). Thus, our results indicate that the expression of
fibronectin is regulated by the TGF-
/Smad pathway.
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Fig. 2.
R4-2 Mink cells expressing ALK5 L45 mutants
do not mediate TGF- -induced growth inhibition
or production of fibronectin and PAI-1. A, ALK5 or
mutant derivatives were subcloned into pMEP4, in which transcription
can be induced by ZnCl2. Expression of ALK5, ALK5(D266A),
and ALK5(3A) is shown by Western blot analysis of total cell lysates
with anti-HA antibody. Cells were treated with ZnCl2 before
lysis. B, ectopic expression of wild type ALK5, but not
ALK5(D266A) or ALK5(3A) mutants, rescue TGF-
-induced growth
inhibition in R4-2 mink cells. Cells were stimulated with
ZnCl2 where indicated. The relative growth compared with
non-treated cells is plotted against the concentration of TGF-
. All
values represent the mean ± S.D. C, ectopic expression
of wild type ALK5, but not ALK5 (D266A) or ALK5(3A) mutants, rescue
TGF-
-induced fibronectin and PAI-1 production. Fibronectin and PAI-1
protein levels were analyzed by SDS-PAGE followed by PhosphorImager
analysis (Fuji). The asterisk shows a nonspecific band.
D, regulation of the fibronectin gene by TGF-
is
Smad4-dependent. MDA-MB-468 cells were infected with the
indicated recombinant adenoviruses and treated with TGF-
for the
indicated times. Ad-GFP, GFP expressing adenovirus;
Ad-Smad4, Smad4 expressing adenovirus. Reverse
transcription-PCR reactions with specific primers for fibronectin and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a
internal control were performed, and products were analyzed by ethidium
bromide staining.
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Fig. 3.
ALK5(3A) mutant interacts with I-Smads but
not with XIAP nor Dab-2. A, caALK5 or caALK5(3A) mutant
was cotransfected with 6×Myc-Smad6 or 6×Myc-Smad7. To show
interaction between ALK5 and I-Smads, the cell lysates were subjected
to immunoprecipitation (IP) with anti-HA antibody followed
by Western blotting (WB) with anti-Myc antibody. Expression
controls for ALK5 or I-Smads are shown below by Western blotting on
total cell lysates. B, caALK5 without or with 3A mutation of
the L45 loop was cotransfected with expression constructs for GST-XIAP.
To show interaction between components, cell lysates were first
subjected to immunoprecipitation with anti-HA antibody followed by
Western blotting with anti-GST antibody. Expression controls are shown
below by Western blot analysis of total cell lysates. C,
ALK5 or ALK5(3A) was cotransfected with FLAG-Dab-2 in COS7 cells. The
cell lysates were immunoprecipitated with anti-HA antibody followed by
Western blotting with FLAG M5 antibody. The expression of ALK5 and
Dab-2 is shown below by Western blotting of total lysates.
has been
shown to activate JNK MAP kinase in certain cell types (18, 19, 21,
27-30). We investigated the ability of caALK5 or caALK5(3A) to induce
phosphorylation of c-Jun in 293T cells. 293T cells were chosen because
they have very little endogenous ALK5 and can be efficiently
transfected. Expression constructs for HA-tagged caALK5 or caALK5(3A)
was co-transfected with an expression construct for JNK in 293T cells,
and cell lysates were subjected to immunoprecipitation with anti-FLAG
M5 antibody followed by in vitro kinase reaction using
GST-c-Jun 1-135 as a substrate. Like caALK5, caALK5(3A) was found to
potently activate JNK (Fig.
4A). In contrast, the
phosphorylation of Smad2 in 293T cells was not enhanced by caALK5(3A)
but was dramatically induced by caALK5 (data not shown). Consistent
with these results, TGF-
induced a significant JNK activation in
R4-2 cells that were infected with wild type ALK5 or ALK5(3A) mutant
adenoviruses. Peak levels of JNK activation were reached 10 min after
TGF-
challenge (Fig. 4B). TGF-
induced Smad2
phosphorylation in R4-2 cells expressing wild type ALK5 but not in
cells expressing ALK5(3A) mutant (Fig. 4B). Thus,
TGF-
-induced JNK activation occurs in a Smad-independent manner.
Further support for ALK5(3A)-induced activation of JNK was obtained by
analyzing the activation of Gal4-c-Jun in R4-2 cells. caALK5(3A)
significantly induced the Gal4-cJun transcriptional reporter, albeit
weaker than caALK5 (Fig. 4C, upper panel).
Consistent with the Gal4-cJun assay, the activation of pAP-1-Luc, which
can be activated by JNK, was also weakly induced by caALK5(3A) compared with that of caALK5 (Fig. 4C, middle panel).
Transfection of ALK5(3A) in R4-2 cells mediated a 2-fold activation of
pAP-1-Luc reporter in response to TGF-
(Fig. 4D). In
contrast, the Gal4-CHOP assay, which exhibits a readout of the
activated p38 pathway (65), was not significantly affected by
caALK5(3A) but was influenced by caALK5 (Fig. 4C,
lower panel).
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Fig. 4.
The ALK5(3A) mutant activates JNK MAP
kinase. A, to test the effect of ALK5 and the ALK5(3A)
mutant on JNK activation, HA-tagged expression constructs encoding
these receptors were cotransfected with FLAG-tagged JNK into 293T
cells. Cell lysates were immunoprecipitated with anti-FLAG M2 antibody
and subjected to an in vitro phosphorylation assay using
GST-c-Jun 1-135 as a substrate (upper panel). Expression
controls for ALK5 (middle panel) and JNK (lower
panel) are shown by Western blots (WB) on total cell
lysates with anti-HA and anti-FLAG M2 antibodies, respectively. Kinase
activity was calculated relative to the value in the absence of ALK5.
B, R4-2 mink cells were infected with ALK5 adenoviruses.
Infected cells were stimulated with 10 ng/ml TGF- 3 for the indicated
times. Cell lysates were immunoprecipitated with anti-JNK1 antibody and
subjected to an in vitro phosphorylation assay using
GST-c-Jun 1-135 as a substrate (upper panel). Expression
for ALK5 (middle panel) and phosphorylated Smad2
(lower panel) are shown by Western blots on total cell
lysates with anti-HA and anti-pS2 antibodies, respectively. Relative
kinase activity was calculated with each value of non-stimulated cells.
C, R4-2 mink cells were transfected with Gal4-cJun, pFR-Luc
reporter, and JNK together with constructs for caALK5 or caALK5(3A)
(top panel), AP-1-Luc reporter and JNK together with
expression constructs for caALK5 or caALK5(3A) (middle
panel), and Gal4-CHOP, pFA-CHOP, and HA-p38 together with
expression constructs for caALK5 or caALK5(3A) (bottom
panel). Luciferase values normalized for transfection efficiency
are shown. All values represent the mean ± S.D. Significantly
different from the mock: *, p < 0.05; **,
p < 0.005. D, R4-2 mink cells were
transfected with AP-1-Luc reporter and JNK together with expression
constructs for ALK5(3A). Twenty-four hours after transfection, the
cells were stimulated with 10 ng/ml TGF-
3 for 18 h. Luciferase
values normalized for transfection efficiency are shown. All values
represent the mean ± S.D. Significantly different from the mock
in the absence of TGF-
3: *, p < 0.05.
Receptors--
GM-CSF is known to be a species-specific ligand.
Chimeric receptors between GM-CSF and TGF-
receptors have shown that
TGF-
signaling can be reconstituted in a system independent of
TGF-
ligand (42, 66). We generated a chimeric receptor between the
extracellular domain of GM-CSF
R and the intracellular domain of
ALK5(3A) or ALK5(D266A), which allowed us to study TGF-
signaling in
a cell upon stimulation with GM-CSF when co-transfected with GM-CSF
R/T
R-II chimera (Fig.
5A) . Both chimeric receptor
chains were subcloned in retroviral expression vectors. After infection of NMuMG cells, chimeric receptor-expressing clones were sorted by
fluorescence-activated cell sorter analysis. We analyzed TGF-
- or
GM-CSF-induced Smad2 phosphorylation in wild type NMuMG cells and in
cells expressing GM-CSF
R/T
R-II chimera alone (termed
II) or
together with GM-CSF
R/ALK5 (termed
wt/
II), GM-CSF
R/ALK5(D266A) (termed
(D266A)/
II), or GM-CSF
R/ALK5(3A)
(termed
(3A)/
II) (Fig. 5A). As expected, TGF-
induced Smad2 phosphorylation in all cells, whereas GM-CSF only induced
Smad2 phosphorylation in cells expressing
wt/
II but not in other
cell clones (Fig. 5B). Thus, consistent with previous
results, the intracellular domain of neither ALK5(D266A) nor ALK5(3A)
induced Smad2 phosphorylation when the chimeric receptors were
activated. In addition, GM-CSF stimulated a significant increase in
AP1-Luc activity in
(3A)/
II cells. Interestingly, the luciferase
activity was higher after 6 h compared with 18 h of GM-CSF
stimulation (Fig. 5C).
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Fig. 5.
Characterization of chimeric
GM-CSF/TGF- receptors. A,
schematic representation of chimeric GM-CSF·TGF-
receptor
constructs. B, chimeric GM-CSF·TGF-
receptors were
stably expressed in NMuMG cells. The Western blotting (WB)
of cell lysates by pS2, anti-Smad2, anti-HA, and anti-GM-CSF
R
antibodies revealed expression of phosphorylated Smad2, total Smad2,
GM-CSF
R/ALK5 chimera, and GM-CSF
R/T
R-II, respectively, in
different transformants. G, 50 ng/ml GM-CSF stimulation for
1 h; T, 10 ng/ml TGF-
stimulation for 1 h. C,
NMuMG transformants expressing
II or
(3A)/
II were transfected
with pAP-1-Luc reporter and JNK. Twenty-four hours after transfection,
the cells were stimulated with 50 ng/ml GM-CSF for 6 h (left
panel) or 18 h (right panel). Luciferase values
normalized for transfection efficiency are shown. All values represent
the mean ± S.D. *, p < 0.05, significantly
different from the data in the absence of GM-CSF.
wt/
II- and
(3A)/
II-expressing NMuMG cells upon
stimulation with GM-CSF indicated that the genomic response is much
stronger in the presence than the absence of Smad
signaling.2 Among the limited
group of genes that were found to be up-regulated and down-regulated by
GM-CSF (and TGF-
) in
wt/
II- and
(3A)/
II-expressing cells
are PMEPA1 and cyclin D1, respectively (Fig.
6, A and B). Wild
type ALK5 was found to mediate a stronger signal than ALK5(3A) mutant;
this suggests that both Smad-dependent and Smad-independent signaling are needed to efficiently regulate these genes by TGF-
. PMEPA1 was initially identified as an androgen-regulated prostatic mRNA (67) without functional annotation. Cyclin D1 was previously shown to be a target of TGF-
(68) and has been implicated in TGF-
-induced growth arrest.
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Fig. 6.
GM-CSF/ALK5 L45 mutants can mediate effects
on gene expression of specific genes but fail to induce stress fibers
in NMuMG cells. The expression of PMEPA1 and cyclin D1 were
enhanced and decreased in the chimeric GM-CSF/ALK5 L45 mutant
stimulated with GM-CSF, respectively. A, Northern blot using
total RNA from each transformant stimulated with 10 ng/ml TGF- 3
(T) or 50 ng/ml GM-CSF (G) for 1 and 6 h was
performed using PMEPA1 as a probe (upper panel). Relative
expression levels (normalized using 28 S) compared with non-stimulated
cells are indicated. Equal loading of RNA samples is shown by ethidium
bromide stain of gel before Northern blotting (lower panel).
B, cyclin D1 mRNA in NMuMG transformants stimulated with
10 ng/ml TGF-
3 (T) or 50 ng/ml GM-CSF (G) for
the indicated times was detected by Northern blot analysis (upper
panel). Relative expression levels (normalized using 28 S)
compared with non-stimulated cells are indicated. Equal loading of RNA
samples is shown by ethidium bromide stain of gel before Northern
blotting (lower panel). C, cells stably
expressing
II in the absence or presence of
wt and
(3A) or
nontransfected cells treated without or with TGF-
3 or GM-CSF were
stained with phalloidin for polymeric actin.
as can be demonstrated
by a reorganization of the actin cytoskeleton (69, 70). We therefore
analyzed
II,
wt/
II, and
(3A)/
II for TGF-
- and
GM-CSF-induced stress fiber formation. As expected, we found that
TGF-
induced stress fiber formation in all cell lines (Fig. 6C), whereas GM-CSF induced stress-fiber formation in
wt/
II cells (Fig. 6C) but not in
(3A)/
II cells
(Fig. 6C),
(D266A)/
II cells (data not shown), and
II cells (Fig. 6C). In addition, we also observed stress
fiber formation by GM-CSF in an
wt/
II-expressing Swiss3T3
transformant but not in
(D266A)/
II- or
(3A)/
II-expressing Swiss3T3 transformants (data not shown). Thus, an intact L45 loop is
critical for ALK5-mediated induction of stress fibers.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-induced
responses by relaying the signal from the activated TGF-
receptor to
the nucleus, where they affect the transcription of target genes.
TGF-
-induced responses that are independent of Smads have been
reported (11). However, their physiological importance is not well
understood. Therefore, we investigated the pathways that are activated
in mutant TGF-
type I receptors (ALK5s) defective in Smad activation
but with retained kinase activity. Most of the TGF-
-induced
responses in a variety of cell lines that we examined, including growth
inhibition, fibronectin and PAI-1 protein production, and stress fiber
formation, were dependent on having an ALK5 with intact L45 loop and
are likely to be Smad-dependent. ALK5-mediated activation
of JNK was found to be independent of Smads. In addition, T
R-I was
found to weakly regulate the expression of specific genes in a
Smad-independent manner. Taken together these results indicate that
different independent signaling pathways are initiated from the
activated type I receptor. Although our results show that
Smad-independent signaling through TGF-
receptor is not sufficient
to mediate many of the regulatory effects of TGF-
on cell
proliferation and differentiation, further studies are under way to
investigate the effects of the mutants in more short term responses,
like mobilization of the actin cytoskeleton. In addition, our data
certainly do not rule out an important role for TGF-
-induced
Smad-independent signaling in either promoting, inhibiting, or
redirecting the Smad pathway or an important role for Smad-independent
signaling in TGF-
-induced responses that are induced via
Smad-mediated changes in expression of genes, e.g. growth
factors, their receptors, and AP1 family members.
-induced
growth arrest (71, 72), we found that ALK5 L45 mutants defective in
Smad activation were unable to mediate growth arrest despite being able
to down-regulate cyclin D1 (Fig. 6B). Cyclin D1 has been
implicated in TGF-
-induced growth arrest. However, either
TGF-
-induced down-regulation of cyclin D1 in Smad-independent manner
is not efficient enough or Smad-dependent regulation of other cell cycle regulators, e.g. up-regulation of CDK
inhibitors p15 and p21 and down-regulation of c-Myc, are needed for
anti-proliferative action of TGF-
. In EpH4 polarized mammary
epithelial cells ALK5-initiated signaling to growth arrest has been
shown to occur in part independently of the Smad pathway via
phosphatase 2A-mediated inactivation of p70S6K (73). Whether our ALK5
L45 mutants can mediate (partial) growth arrest in EpH4 cells remains
to be investigated. The dependence for intact L45 loop for the observed
ALK5-induced PAI-1 and fibronectin protein production in R4-2 cells
(Fig. 2C) is consistent with the inability of ALK5 L45
mutants to activate the PAI-1 promoter based reporter
(CAGA)12-Luc assay (Fig. 1F) and the lack of
TGF-
-induced fibronectin mRNA levels in Smad4 deficient
MDA-MB-468 cells (Fig. 2D). The observed TGF-
-induced
fibronectin has been previously shown to be dependent on JNK activation
(29). However, the ability of the ALK5 L45 mutant to activate JNK
without inducing fibronectin levels indicates that JNK activation alone
by TGF-
is not sufficient for the induction of fibronectin
production. TGF-
has been reported to induce fibronectin protein
levels independent of Smad4 (29). However, we found that
TGF-
-induced fibronectin mRNA levels require Smad4 (Fig.
2D). The reason for this discrepancy remains to be elucidated.
in cells that lack certain R-Smads or Smad4.
in R4-2 cells. Although the
TGF-
receptor-interacting protein Daxx has been known to mediate JNK
activity (21), we were unable to show enhancement of JNK activity in
the presence of caALK5 or caALK5(3A) in 293T cells (data not shown).
Receptor-mediated activation of Smads may occur at early endosomes as
SARA (Smad anchor for receptor activation), the molecule presenting
R-Smads to the type I receptor, is exclusively located in this
organelle (75-77). It will be of interest to examine whether
TGF-
-induced JNK activation is initiated at the plasma membrane or
at early endosomes.
-induced change of epithelial cells into
fibroblastoid-shaped cells and formation of actin stress fibers were
found to require an intact L45 loop. This result is consistent with previous data that show that Smads alone can weakly induce stress fiber
formation and cooperate with activated ALK5 for full
epithelial-to-mesenchymal transition (70). While our manuscript was in
preparation, Yu and co-workers reported on the characterization of a
similar ALK5 L45 mutant capable of inducing apoptosis but not
epithelial-to-mesenchymal transition via a p38 pathway in NMuMG cells
(48). We observed a weak but not consistent p38 phosphorylation in
response to activation of mutant chimeric GM-CSF·TGF-
R complex.
The differences between our observations and those of Yu et
al. (48) may be because of the use of constitutively active ALK5
L45 mutant by Yu et al. (48) compared with ligand-mediated
activation of chimeric GM-CSF/TGF-
receptor complex by us. An
advantage of the use of constitutively active receptor is that the
signal is built-up in the cell, allowing easier detection. However, we
found that caALK5 L45 mutants, when expressed in Mv1Lu cells expressing
endogenous ALK5, very weakly activated a Smad-dependent
reporter (Fig. 1E). In addition, caALK5 L45 mutant receptors will
induce a sustained response, which does not occur when ALK5 L45 mutant
receptors are activated by ligand.
.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. M. Laiho, K. Iwata, E. B. Leof, G. Nolan, B. Vogelstein, C. H. Duckett, P. H. Howe, G. A. Ruiter, E. Nishida, and R. Davis for reagents. We are grateful to Dr. L. Oomen, to L. Brocks for expert assistance with confocal microscopy, to A. Pfauth for cell sorting with fluorescence-activated cell sorter, and to the Sanger Centre microarray consortium for cDNA microarrays.
![]() |
FOOTNOTES |
---|
* This research was supported by Dutch Cancer Society Grant NKI 2000-2217 and NKI 2001-2481).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.
¶ To whom correspondence should be addressed. Tel.: 31-20-5121979; Fax: 31-20-5121989; E-mail: p.t.dijke@nki.nl.
Published, JBC Papers in Press, November 22, 2002, DOI 10.1074/jbc.M208258200
2 S. Itoh, M. Thorikay, M. Kowanetz, A. Moustakas, F. Itoh, C.-H. Heldin, and P. ten Dijke, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
TGF-, transforming growth factor-
;
ALK, activin receptor-like kinase;
AP, activating protein;
Dab-2, Disabled-2;
DMEM, Dulbecco's modified
Eagle's medium;
FAST, forkhead activin signal transducer;
FCS, fetal
calf serum;
GFP, green fluorescent protein;
GM-CSF, granulocyte/macrophage colony-stimulating factor;
GST, glutathione
S-transferase;
IRES, intra-ribosomal entry site;
JNK, c-Jun
N-terminal kinase;
MAP, mitogen protein kinase;
Mv1Lu, mink lung
epithelial cells;
NMuMG cells, normal murine mammary gland cells;
PAI-1, plasminogen activator inhibitor-1;
PMEPA1, androgen-regulated
prostatic mRNA;
PBS, phosphate-buffered saline;
pS2, phospho-Smad2
antibody;
Smad, Sma- and Mad-related protein;
TAK1, TGF-
-activating
kinase 1;
T
R, TGF-
receptor;
XIAP, X-chromosome-linked
inhibitor of apoptosis;
HA, hemagglutinin;
CMV, cytomegalovirus;
MOPS, 4-morpholinepropanesulfonic acid.
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REFERENCES |
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