ALK7 Induces Apoptosis through Activation of MAP Kinases ... file1 ALK7 Induces Apoptosis through...
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ALK7 Induces Apoptosis through Activation of MAP Kinases in a
Smad3-dependent Mechanism in Hepatoma Cells
Byung-Chul Kim,1,2 Howard van Gelder,1 Tae Aug Kim,1 Ho-Jae Lee,1 Kim G. Baik,1
Hyun Hye Chun,1 David A. Lee,1 Kyeong Sook Choi,3 and Seong-Jin Kim1#
1Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda,
MD 20892-5055, USA, 2College of Natural Sciences, Kangwon National University,
Chuncheon, Korea, and and 3Laboratory of Endocrinology, Institute for Medical
Sciences, Ajou University School of Medicine, Suwon, Korea
# To whom correspondence should be addressed. Tel: 301-496-8350; Fax: 301-496-8395;
E-mail: [email protected]
Running title: ALK7 induces apoptosis
Key words: ALK7, apoptosis, TGF-β, Smad3, hepatoma, JNK, p38, cytochrome c
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Abstract
ALK7 is a type I seine/threonine kinase receptor of the TGF-β family of proteins
that has similar properties to other type I receptors when activated. To see if ALK7
can induce apoptosis like some of the other ALK proteins, we infected the FaO rat
hepatoma cell line with adenovirus expressing a constitutively active form of the
ALK7. Cells infected with active ALK7 adenovirus showed an apoptotic positive
phenotype as opposed to those that were infected with a control protein. DNA
fragmentation assays and FACS analysis also indicated that ALK7 infection
induced apoptosis in FaO cells. We also confirmed this finding in Hep3B human
hepatoma cells by transfecting the constitutively active form of ALK7,
ALK7(T194D) transiently. Investigation into the downstream targets and
mechanisms involved in ALK7 induced apoptosis revealed that the TGF-β signaling
intermediates, Smads 2 and 3 were activated, as well as the activation of MAP
kinases JNK and p38. In addition, caspases 3 and 9 were also activated, and
cytochrome c release from the mitochondria was observed. siRNA-mediated
inhibition of Smad3 markedly suppressed ALK7-induced caspase 3 activation.
Treatment with protein synthesis inhibitors or the expression of dominant negative
form of the SEK1 abolished not only JNK activation, but apoptosis as well. Taken
together, these results suggests that ALK7 induces apoptosis through activation of
the traditional TGF-β pathway components, resulting in new gene transcription and
JNK and p38 activation that initiates cross talk with the cellular stress death
pathway, ultimately leading to apoptosis.
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Introduction The TGF-β superfamily of cytokines is responsible for regulating a wide range of
cellular responses in many different cell types, including differentiation, cell growth, and
apoptosis (1-5) TGF-βs signal through a serine/theorine kinase pathway that begins upon
ligand binding to a set of two transmembrane receptors, termed type I or type II, located
on the surface of the cell plasma membrane (1). The type II receptor is responsible for
initial ligand binding, which then acts to recruit and activate, via phosphorylation, the
type I receptor. Following activation, the type I receptor phosphorylates a set of proteins
named Smads that are specific for each kind of type I receptor. After the Smads are
activated, they interact with another protein, Smad4, which together translocate to the
nucleus to modify the cellular response through transcription of other gene products. To
date, the exact genes targeted by the Smad pathway have not been fully elucidated, with
less information still on the mechanisms by which these genes carry out their function.
ALK7 (Activin receptor-like kinase) is a serine/threonine kinase consistent with the
characteristics of a type-I receptor. Originally identified and cloned from Rat brain (6),
ALK7 mRNA is present throughout the digestive and central nervous system of rats.
The transmembrane receptor has a similar intracellular domain to TGF-β type I receptor
(TβR1) and ActRIB, but a different extracellular domain. The only reported interacting
ligand for ALK7 is mouse Nodal and xenopus related nodal (XnR1) (7). The function of
ALK7 as a type I receptor was confirmed with a constitutively active mutant form that
activated a TGF-β/Activin response reporter (6). ALK7 has also been found to activate
some components of the smad pathway such as Smad 2 and Smad 3 in fetal and adult rat
pancreas, (8). In the rat pheochromocytoma PC12 cell line, ALK7 not only activated
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both samd 2 and smad 3, but the map kinases of ERK and JNK, as well as inhibiting cell
proliferation (9). The human gene for ALK7 has been mapped to the genetic location of
2q24.1-q3, with most of the mRNA located in the brain, pancreas, and colon. (10)
Recently the human form of ALK7 has been identified along with three splice variants
that are expressed in the placenta throughout various stages of pregnancy (11).
We studied the effects of infecting the FaO rat hepatoma cell line and Hep3B human
hepatoma cell line with a genetically modified adenovirus expressing HA-tagged ALK7
in order to determine whether or not ALK7 could induce apoptosis in these cells. The
FaO cell line has proven to be a useful model system to study apoptosis, especially for
TGF-β1, which induces cell death in liver cells both in vitro and in vivo. Apoptosis
normally occurs in cells by one of two pathways: cellular stress or death ligand (12). In
both pathways, the final step involves activation of the effector caspase proteins from
their inactive forms via large multi-protein complexes. Once activated, the caspases act
as proteases that cleave various substrates that lead to the death of the cell. In FaO cells,
the inhibition of caspases has been shown to prevent TGF-β1 induced apoptosis (13-15).
The release of cytochrome c from the mitochondrian is an important step in the cellular
stress apoptotic pathway and also in TGF-β1 sensitive cells, where inhibition of
cytochrome c release can completely abolishes TGF-β1 induced apoptosis (16). Use of
protein synthesis inhibitors suggests that new protein synthesis is required for TGF-β1
mediated apoptosis in FaO cells. Recently, microarray data of TGF-β1 treated FaO cells
indicated a number of anti-oxidant genes that are down regulated as well as many
reactive oxygen species that are up regulated (17). These genes may be the intended
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targets of the TGF-β pathway and could have a direct impact on the two major apoptotic
pathways.
Infection with ALK7 did in fact cause apoptosis in FaO cells. More specifically,
ALK7 infection activated a number of the same proteins and mechanisms necessary for
TGF-β1 induced apoptosis, including both Smad and caspase proteins, and also triggered
cytochrome c release from the mitochondrion. In addition, ALK7 was found to activate
JNK and p38, MAP kinases known to be involved in cell cycle regulation. Coupled to
the fact that dominant negative form of the upstream SEK1 blocked ALK7 induced
apoptosis, it is likely that JNK and p38 and the caspase proteins are downstream targets
of Smad pathway transcription products. In support of the idea of cross talk between the
Smad and apoptotic pathways, protein synthesis inhibitors blocked ALK7 induced
apoptosis. Thus ALK7 mediated apoptosis appears to employ some of the same proteins
used in cross talk used by TGF-β1-induced apoptosis.
Materials and Methods
Cell Culture, Ttransfection, and Treatment-FaO rat hepatoma cells and Hep3B human
hepatoma cells were maintained at 37 0C in DMEM (Dulbecco’s Modified Eagle Medium
supplemented with 10% heat-inactivated fetal bovine serum [FBS]). The 293-derived
PHOENIX E (kind gift of Lisa Choy, University of California) or GP-293 packaging
cells were maintained in DMEM supplemented with 10% heat-inactivated FBS. FaO
stable cells were transfected in six-well plates using Lipofectin (Life Technology,
Rockville, MD) according to the manufacturer’s instructions. For TGF-β1 treatment,
cells were incubated with 5 ng/ml TGF-β1 for 24 hrs in media. For treatment with
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protein synthesis inhibitors, cells were incubated with puromycin and cycolhamide for 24
hours in media.
Plasmids and Adenoviral Infections-Recombinant adenoviruses expressing LacZ, HA-
tagged active ALK5, and HA-tagged active ALK7 was used at a multiplicity of infection
(m.o.i.) ranging form 0 to 250 with single viruses as described by Fujii et al., (18). High-
titered stocks of recombinant adenoviruses were grown in 293 cells and purified.
Infection of recombinant adenoviruses was performed at a multiplicity of infection
(m.o.i.) of less than 8 x 102 (pfu/cell). FaO cells were seeded at 0.3 x 106 cells per well in
six-well dishes and cultured in 2 ml of DMEM medium supplemented with 10% FBS.
Twenty-four hours later, the medium was replaced with fresh medium and adenovirus
vectors (m.o.i.=100~250) were added. The cells were incubated for 8 h for infection and
24 h after infection, the cells were harvested for gene expression.
DNA fragmentation assay-FaO cells were treated with lysis buffer (10 mM Tris-Cl, pH
7.4, 10 mM NaCl, 10 mM EDTA, and 0.5 % SDS, and 0.1 mg/ml proteinase K) and were
incubated at 50 0C for 2 hr. The lysate was extracted with phenol, phenol/chloroform (1:1)
and chloroform, precipitated with 2.5 volume of ice-cold ethanol. The DNA was
resuspended in Tris-EDTA buffer supplemented with 100 µg/ml RNase A. DNA samples
were electrophoretically separated on 2% agarose gel for 1 hour at 120V.
Flow Cytometry Analysis-For flow cytometry assay, FaO cells were grown in six-well
plates and incubated for 24 h at 370 C and then infected with adenoviruses carrying
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ALK7. After 36 h, cells were harvested, and washed twice with PBS buffer (pH 7.4).
After fixing in 80% ethanol for 30 min, cells were washed twice, and resuspended in PBS
(pH 7.4) containing 0.1% Triton X-100, 5 µg/ml propidium iodide (PI) and 50 µg/ml
ribonuclease A for DNA staining. Cells were then analyzed by a FACScan cytometer
(program CELLQUEST, Becton Dickinson), Red fluorescence due to PI staining of DNA
was expressed on a logarithmic scale simultaneously to the forward scatter of the
particles. Four thousand events were counted on the scatter gate. The number of apoptotic
nuclei was expressed as a percentage of the total number of events.
Transient Transfection of ALK7 vectors and Assessment of Cell Survival - For transient
expression of constitutively active ALK7 (ALK7-TD: T194D)) or kinase-inactive ALK7
(ALK7-KR: K222R), Hep3B cells were co-transfected with pEGFP or ALK7-KA,
ALK7-KD, and pcDNA vectors. 48 h after transfection, the apoptotic enhanced green
fluorescent protein (EGFP)-positive cells in the same field were assessed. The percentage
of apoptotic cells was calculated relative to the numbers present in the control (pcDNA3)
wells. The number of transfected cells counted was at least 200. The values are the means
of counts on three wells ± SEM. Similar results were obtained in three additional
independent experiments.
Immunoblot Analysis-Whole-cell extracts were obtained in a 1% Triton X-100 lysis
buffer (50 mM Tris-Cl, pH 8.0, 150 mM sodium chloride, 1 mM EDTA, 1 mM EGTA,
2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM β–
glycerophosphate, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Western
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blotting was performed using anti-phospho-JNK (Cell Signaling Technology Inc.,
Beverly, MA), anti-phospho-P38(Cell Signaling Technology Inc., Beverly, MA), anti
phsopho-p42 (Cell Signaling Technology Inc., Beverly, MA) anti-caspase-8 (P-20, Santa
Cruz Biotechnology, Inc., Santa Cruz, Calif.), anti-phospho-Smad2 (Zymed Laboratories
Inc., South Sanfrancisco, Calif.), anti-phospho-Smad3 (Santa Cruz Biotechnology, Inc. ),
anti-cytochrome c (7H8.2C12; PharMingen, San Diego, CA), and anti-HA(Y-11; Santa
Cruz Biotechnology, Inc.) antibodies. Protein samples were heated at 95 0C for 5 min and
analyzed by sodium dodecyl sulfate (SDS)-16% polyacrylamide gel electrophoresis
(PAGE).
Analysis of Cytochrome c Release-For mitochondria cytochrome c release assay, FaO
cells were scraped off in isotonic isolation buffer (10 mM HEPES, 1 mM EDTA, 250
mM Sucrose, pH 7.6), collected by centrifugation at 2,500 g for 5 min at 4 0C and
resuspended in hypotonic isolaton buffer (10 mM HEPES, 1 mM EDTA, 50 mM
Sucrose, pH 7.6). Cells were disrupted by passing through a 27 gauge needle 5-10 times
and checked for cracked cells by trypan blue staining. Hypertonic isolation buffer (10
mM HEPES, 1 mM EDTA, 450 mM Sucrose, pH 7.6) was added to balance the buffer’s
tonicity. Samples were centrifuged at 1,000 x g (2,100 rpm) at 4 0C for 10 min.
Supernatants were recovered and centrifuged again at 100,000 x g. The mitochondria
pellet proteins were extracted in isotonic isolation buffer and supernatant contained the
cytosolic protein extract. Protein concentration of lysates was determined using the Bio-
Rad protein assay kit (Bio-Rad, Hercules, CA) according to the manufacturer’s
instructions. After electrophoresis separation of 50 µg protein/condition in sodium
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dodecyl sulfate (SDS)-16% polyacrylamide, gels were transferred by semidry transfer
(Bio-Rad Labs, Richmond, CA) to nitrocellulose membranes. Immunoblots were blocked
in TBS-T (10 mM Tris/HCl, 150 mM NaCl, pH 7.5, 0.05% Tween 20) containing 5%
non-fat dried milk and incubated overnight with the primary antibody (monoclonal anti-
cytochrome c diluted 1:1000 in TBS-T 5% BSA). After washing, membranes were
incubated with peroxide-conjugated anti-mouse immunoglobulin (1:3000 in TBS-T 0.5%
non-fat dried milk) for 1 h and the blot was developed with the ECL kit (Pierce Chemical
Co., Rockford, IL).
siRNA Methods- We used the siRNA desigh tool (Dharmacon Inc., Lafayette, CO) to
identify target siRNAs. The Smad3-specific sequence was 5’-
UCCGCAUGAGCUUCGUCAAAdTdT-3’ (Smad3 nucleotides 1181 to 1200;
GenBank Accession number U68019). Hep3B cells were seeded at 30% density the
day before transfection. Transfections were performed by using TransIT-TKO reagent
(Mirus, Madison, WI) according to the manufacturer’s instructions with 200 pmole of
siRNA and 10 µl of transfection reagent/ 10 cm dish for Hep3B cells. 24 hr after
transfection, cells were infected with adenovirus expressing LacZ or active ALK7.
Results Constitutive active ALK7 in Fao Cells results in Apoptosis
In order to investigate the possible role ALK7 plays in apoptosis, a constitutive active
mutant form bearing a HA-tag was inserted into an adenovirus expression system and
used for infection into cultures of FaO cells. Two other adenovirus constructs expressing
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either HA-tagged ALK5 or LacZ were infected into separate sets of FaO cells. To
confirm the expression of the virally infected proteins, the cells were harvested for their
protein and detected using an antibody against HA. Both the HA-tagged proteins were
successfully expressed (Fig. 1a) whereas the LacZ protein could not be detected. After
sufficient time had elapsed, the phenotype of the cells was observed and compared to the
known apoptotic positive control phenotype exhibited by cells treated with TGF-β1.
Both the active ALK5 and ALK7 infected cells showed positive apoptotic phenotypes,
whereas the cells infected with LacZ did not (Fig. 1b-d). The ALK7 also induced
apoptosis in the untransformed hepatocyte cell line AML12 (data not shown).
To further analyze if ALK7 activation could cause apoptosis, cells were infected with
increasing amounts of the active mutant form along with cells that were treated with
TGF-β1, serum starved, unaltered, or infected with LacZ and run in a DNA laddering
assay. When cells undergo apoptosis, a series of small DNA fragments separated by a
hundred or so base pairs in length are generated, creating a characteristic “ladder”
appearance (19). DNA in cells treated with ALK7 resembled the DNA ladder seen in the
TGF-β1 apoptotic positive control, whereas the other DNA did not (Fig. 1c). As a final
test to see if ALK7 induces apoptosis in FaO cells, a FACS analysis was conducted of
cells that had been infected with LacZ, or the active forms of either ALK5 or ALK7. The
resulting percentage of cells that appeared in the population of cells in sub-G1 phase for
ALK7 infected cells was similar to the apoptotic positive control ALK5 infected cells and
significantly higher than LacZ (Fig. 1d).
We next examined the effect of ALK7 on apoptosis in Hep3B human hepatoma cells. We
transiently transfected the constitutively active form of ALK7 (ALK7-TD) and kinase-
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inactive form of ALK7 (ALK7-KR) which acts as a dominant-negative receptor.
Comparable expression levels of the ALK7 mutant proteins were obtained when
immunoblotting was performed using the anti-HA antibody (Fig. 2a). By 48 h after
transfection, the phenotype of the cells was observed and compared to the known
apoptotic positive control phenotype exhibited by cells treated with TGF-β1. Hep3B cells
transfected with ALK7-TD showed positive apoptotic phenotypes, whereas the cells
transfected with ALK7-KR did not (Fig. 2a-c). These results taken together indicate that
activated ALK7 induces apoptosis in hepatoma cells.
ALK7 induced apoptosis in FaO cells activates JNK and requires new protein synthesis-
To better understand the mechanisms and pathways involved in ALK7 induced apoptosis,
we examined the possible activation of MAP kinases known to be important in other
cytokiene mediated cell death. The MAP kinases play crucial roles in relaying
extracellular signals regarding cell cycle regulation and cell proliferation (20). To study
the possible role of MAP kinase involvement in ALK7 induced apoptosis, increasing
amounts of active ALK7 were introduced into FaO cells and the activity of three MAP
kinases measured. The amount of active form of the three MAP kinases (JNK, p38, and
ERK) was measured using antibodies specific to the phosphorylated forms of each
kinase. Only active JNK (P-JNK) showed increased levels of expression with increasing
amounts of virus (Fig. 3).
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Dominant Negative SEK1 blocks ALK7 induced apoptosis by preventing caspase
activation and Cytochrome c release- The Map Kinase JNK can be activated by upstream
kinases, including SEK1 (21). To determine whether ALK7 induced apoptosis activates
JNK through SEK1, the dominant negative form of SEK1 (dnSEK1) was created and
introduced into FaO cells (22) that were infected with increasing amounts of ALK7. JNK
activation is almost completely abolished in cells expressing dnSEK1 when compared to
the cells with the control vector (Fig. 4a). To see whether the loss of activated JNK could
halt apoptosis, Lac Z or ALK7 infected cells carrying the dnSEK1 expressing vector or
the control vector were subjected to FACS analysis. As expected, the dnSEK1
containing cells had a similar number of cells in sub-G1 phase when compared to LacZ,
even in the presence of ALK7 (Fig. 4b). This result confirms that dnSEK1, through the
inhibition of JNK activation, prevents apoptosis. It also suggests that SEK1 acts
upstream of JNK in the ALK-7 apoptotic pathway.
Caspase proteins are known to be important in most apoptotic pathways. To see if
caspase activation plays a role in ALK7 induced apoptosis, increasing amounts of active
ALK7 protein were infected in FaO cells and caspase activation measured. Using
antibodies that were specific to the active, inactive, or both forms of caspase 3, 7, 8 and 9
it was discovered that only caspase 3 and 9 are activated by ALK-7. (Fig. 5a) To
determine whether caspase 9 activation is upstream or downstream of SEK1, FaO cells
expressing dnSEK1 or vector control were infected with either LacZ or ALK7
adenovirus. ALK7 activation of caspase 9 is almost completely abolished in cells that
also express dnSEK1 (Fig. 5b), indicating that caspase 9 activation occurs downstream of
SEK1. To confirm that caspase activation is necessary for apoptosis, the polycaspase
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inhibitor Z-VAD-FMK was added to FaO cells infected with ALK7 or LacZ, and
compared to cells treated with DMSO in a FACS analysis. The number of cells in sub-
G1 phase in the cells treated with inhibitor resembles the number seen in the control cells,
which was significantly lower than the number seen in ALK7 infected cells without
inhibitor (Fig. 5c). The data confirm that caspase activation is necessary for ALK7
induced apoptosis in FaO cells.
Another known indicator of apoptosis in many cell types is the release of cytochrome c
from the mitochondrian during the cell stress pathway. It has been reported that TGF-β1
induced apoptosis can also stimulate release of cytochrome c in FaO Cells (13,16). To
see if ALK7 also has cross talk with the cellular stress pathway, FaO cells were infected
with LacZ, ALK-5, ALK7, or treated with TGF-β1 and then prepared for cytochrome c
measurement (see material and methods). Like ALK5 and TGF-β1, ALK7 also induced
cytochrome c release when compared with the control (Fig. 6a). To see if cytochrome c
release occurs downstream of SEK1, FaO cells were infected with LacZ, ALK5, ALK7
or treated with TGF-β1 and transfected with dnSEK1. Cytochrome c release was reduced
in all instances when compared to cells not treated with dnSEK1. (Fig. 6b)
ALK7 activates Smad2 and Smad3 that in turn, can activate JNK and p38 MAP kinases-
ALK7 has been shown to activate both the receptor Smad 2 and Smad 3 protein in a
variety of cell types, (8,9) which is consistent with ALK-7’s function as a type I receptor.
To investigate whether ALK7 can activate Smads in both FaO and Hep3B cells,
increasing amounts of active ALK7 were infected into cells and analyzed for Smad
activation using antibodies specific to the phosphorylated forms of Smad2 or Smad3.
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Both Smad2 and Smad3 were activated in the presence of significant expression ALK7 in
FaO cells (Fig. 7a) and Hep3B cells (Fig. 7c). To see whether over-expression of Smad
proteins is sufficient to stimulate activation of JNK and p38 MAP kinases in the presence
of ALK7, FaO cells were infected with active ALK7 and transfected with Flag-tagged
Smad 2, Smad 3, Smad 4, or a control vector and analyzed for JNK activation. Smad
expression was confirmed with an antibody specific for the Flag-tag. Though the three
Smads tested all activated JNK to some degree, only the receptor activated Smads,
Smad2 and Smad3, showed significant activation in FaO cells (Fig. 7b). By contrast,
overexpression of the co-Smad 4 protein did not result in similar levels of activation, but
was comparable to the control vectors in overall activation. We also examined activation
of JNK and p38 MAP kinases in the presence of ALK7 in Hep3B cells (Fig. 7d). ALK7
induced activation of JNK and p38, as well as caspase 3 activation.
Downregulation of endogenous Smad3 markedly decreases ALK7-induced caspase 3
cleavage- In the previous study (13), we have demonstrated that overexpression of
Smad4 did not enhance the level of apoptosis induced by TGF-β1 and that Smad2
overexpression slightly enhanced TGF-β1-induced apoptosis. However, Smad3
overexpression significantly enhanced apoptosis induced by TGF-β1, suggesting that
Smad3 activation may mediate apoptosis in hepatoma cells. To confirm the critical role
of Smad3 in the ALK7-induced caspase 3 cleavage, we performed the loss of function
studies using Smad3-specific siRNA to evaluate the specific role of Smad3 on ALK7-
induced apoptosis. We tried to reduce endogenous Smad3 expression through RNA-
mediated (RNAi) interference using Smad3-specific siRNA. Transfection of the Smad3
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siRNA (100~200 nM) resulted in over 70-90% decreases in Smad3 protein levels (Fig.
8). When transfected with Smad3-specific siRNA, caspase 3 cleavage by the ALK7 was
markedly reduced (Fig. 8).
Discussion
In this study, we have demonstrated the first reported data that ALK7 can induce
apoptosis. By using a modified form of adenovirus expressing a constitutively active
ALK7 protein, we have demonstrated that ALK7 creates the same apoptotic positive
phenotype that treatment of TGF-β causes in FaO cells. We have also shown that ALK7
infection generates the breakup of DNA into smaller segments or “ladder” that is
consistent with cells that are undergoing apoptosis and not another form of cell death.
Aside from confirming apoptosis in FaO cells, we have shown some of the possible
pathway components and mechanisms that could be responsible for ALK7-induced
apoptosis. These include the activation of the Samd, caspase, and JNK and p38MAP
kinases, as well as the release of cytochrome c from the mitochondrion. We have also
shown that new protein synthesis is required for both JNK activation and cell cycle arrest.
Finally, we have demonstrated that dominant negative SEK1 blocks ALK7-induced
apoptosis, suggesting SEK1 is a crucial intermediate protein.
The data are highly similar to data reported for TGF-β1 or active ALK5 pathways in
general. This is not surprising, as ALK7 has a very similar intracellular domain to ALK5
and is known to activate some of the same receptor smads and reporter constructs (9).
This could suggest that ALK7 may stimulate transcription of the same genes that are
activated by Smads in the ALK5 or TGF-β signaling or apoptotic pathways.
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Some similarities to TGF-β1-induced apoptosis and ALK7-induced apoptosis in FaO
cells are present in this study, including the release of cyctochrome c and JNK activation.
JNK activation, coincidently, has been implicated as a possible downstream target of the
Rho family of proteins, which have been shown to be involved in cross talk during TGF-
β signaling. This is in agreement with our results, which suggests cross talk between the
Smad and other pathways, such as the cellular stress death pathways, must be occurring.
Evidence of this cross talk is found in our data with the activation of caspase 9, which is
activated upon interaction with Apaf1, a component of the apoptosome. However, Apaf1
requires ctyochrome c release in order to self aggregate and interact with the inactive
form of caspase 9 (12), strongly supporting the theory that ALK7-induced apoptosis
occurs via cross talk with the cellular stress pathway. Importantly, caspase 8 was not
activated, suggesting that the death ligand response does not play a role in ALK7 induced
apoptosis.
Experiments involving a tetracycline inducible active form of ALK7 have been shown
to create morphological changes and arrest proliferation in the rat pheochromocytoma
PC12 cell line (9). In addition, ALK7 activated Smad2 and Smad 3 as well as stimulated
transcription from Smad binding elements in these cells, including genes often activated
by TGF-β. An analysis of microarray data of gene transcription upon TGF-β treatment
of FaO cells found up-regulation of many pro-apoptotic genes, such as CTGF, which
promotes fibroblast proliferation, and down regulation of many anti-oxidant genes, such
as GLCLC, which helps synthesize glutathione (17). Based on the high similarity of the
downstream targets studied so far, it’s likely that ALK7 activation stimulates these same
kind of pro and anti-apoptotic genes in FaO cells. Further work is also required to
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identify the ligand responsible for signaling apoptosis in these cells, though Nodal is a
possible candidate (7). Experiments that find ALK7 induction of apoptosis in human
cells will also provide some valuable information, notably in brain development, where it
is thought that high levels of ALK7 mRNA are located.
In summery, ALK7 is a type I serine/threonine kinase of the TGF-β signaling cytokines
that when transiently expressed in hepatoma cells causes apoptosis. The pathway by
which ALK7 carries out apoptosis is similar to other ALKs, in that it begins with Smad
signaling that results in transcription of various gene products. These newly synthesized
proteins are necessary to complete the final stages of apoptosis, which involve cross-talk
with other cell pathways that eventually culminate in cytochrome c release and caspase 9
activation.
Acknowledgements- We thanks K. Miyazono, T. Imamura, and M. Fujii for ALK7
constructs.
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Figure Legends
Fig. 1. ALK7 Induces Apoptosis in FaO Cells. FaO cells were treated with differing
amounts of modified adenovirus expressing LacZ, HA-tagged active ALK5, or HA-
tagged active ALK7 proteins for studies in apoptosis. (a) Western blot analysis with
primary antibody against HA-tag showing successful expression of ALK5 and ALK7
proteins in infected cell extracts when compared with negative control LacZ. (b) Effect
of ALK7 on cellular morphology. Differing multiplicity of infections (m.o.i) of viral
construct ALK7 were administered to cultures of FaO cells (bottom panel). In association
with the significant decrease in cell count, a dramatic change in cellular morphology
suggestive of cell death appeared after infection with adenovirus carrying a constitutively
active form of ALK7. The top panel shows apoptotic positive (ALK5 infection and TGF-
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β treatment) and negative (LacZ infection) controls. (c) Cellular fragmentation after
infection with adenovirus carrying a constitutively active form of ALK7. Cells were
treated with increasing amounts of adenovirus expressing active ALK7 or with LacZ for
24 h. Total DNA was isolated and run on a 1.5% agarose gel for comparison with DNA
fragmentation positive (TGF-β treated cells) and negative (FaO cells in 10% serum
containing media) controls. (d) A representative illustration is shown of PI incorporation
measured in FaO cells infected with adenovirus vectors carrying constitutively active
ALK7 or active ALK5 by flow cytometry analysis. Adenovirus carrying β-galactosidase
(m.o.i. of 250) was used as a control. The number of apoptotic nuclei is expressed as a
percentage of the total number of events. Similar results were achieved in three separate
experiments with comparable outcomes.
Fig. 2. ALK7 Induces Apoptosis in Hep3B Cells. Hep3B cells were transfected with
vectors expressing HA-tagged active ALK7 (ALK-KR), or HA-tagged inactive ALK7
(ALK7-TD) together with pEGFP vector for studies in apoptosis. (a) Western blot
analysis with primary antibody against HA-tag showing successful expression of ALK7-
KR and ALK7-TD proteins in transfected cell extracts when compared with negative
control pcDNA3. Caspase 3 activation was examined by Western blot analysis using
antibodies specific to the cleaved forms of caspase 3. As a control, activation of caspase
3 by TGF-β1 (5 ng/ml) was also examined 24 h after TGF-β1 treatment in Hep3B cells.
The lower blots in each panel show reprobing of the same filters with anti-β-actin
antibodies and demonstrate comparable amounts of protein in all the lanes. (b) Effect of
transient transfection of ALK7-KR and ALK-TD on apoptosis. Hep3B cells were
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transiently transfected with the expression vector encoding ALK7-KR, ALK7-TD or
pcDNA3 as a control, together with pEGFP-Vector as described in Materials and
Methods. Representative photomicrographs of fluorescence images are shown. The
arrows indicate fluorescence of the GFP and those of the nuclei stained by DAPI of
Hep3B cells transfected with pEGFP-Vector (original magnification X 400). (c) The
percentage of apoptotic cells of Hep3B cells transiently transfected with pcDNA3,
ALK7-KR, or ALK-TD.
Fig. 3. Activation of MAP kinases during ALK7-induced apoptosis. FaO cells were
infected with increasing amounts of adenovirus vectors carrying constitutively active
ALK7. 24 h after infection, the cells were harvested for protein, which was then
subjected to Western blot analysis. Western blot analysis using respective phospho-
specific antibodies to detect their activated forms of MAP kinases was performed.
Immunoactive JNK1/2, and p38 were probed by using anti-JNK1/2 and p38 antibody.
Similar results were achieved in three separate experiments with comparable outcomes.
An increase in activation with increased infection of ALK7 is seen only for JNK. An
antibody specific to HA-tag was used to confirm increased expression of HA-ALK7 with
increasing ALK7 m.o.i.
Fig. 4. Dominant-negative SEK1 blocks ALK7 induced apoptosis and JNK
activation. FaO cells expressing either the dominant negative form of the protein SEK1
(dnSEK1) or the control vectors (pcDNA3) were described previously (22). These cells
were infected with increasing amounts of adenovirus vectors carrying constitutively
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active ALK7. (a) After sufficient time was allowed for infection, the cells were
harvested for protein, which was then subjected to western blot analysis. Antibodies
specific to the phosphorylated form of JNK (P-JNK) were used to measure JNK
activation and antibodies specific to β-actin were used to confirm sample normalization.
(b) FACS analysis showing the percentage of cells counted in sub-G1 phase when FaO
cells were infected with LacZ or ALK7 (m.o.i. of 250) in cells expressing either dnSEK1
or pcDNA3 vectors.
Fig. 5. ALK7 induces caspase 3 and caspase 9 activation. (a) FaO cells were infected
with increasing amounts of adenovirus vectors carrying constitutively active ALK7. The
cells were harvested for protein 24 h after infection, which was then subjected to Western
blot analysis. Antibodies specific to the pro and/or cleaved forms of caspases, 3, 7, 8 and
9 were used to detect caspase activation. (b) FaO cells were transfected with a vector
expressing either dnSEK1 or the control vector pcDNA3 and then were infected with
either ALK7 or LacZ expressing adenovirus (m.o.i.of 250). The cells were harvested for
protein 24 h after infection, which was then subjected to western blot analysis with
antibodies sepcifc for caspase 9 activation. Antibodies specific for β-actin were used to
confirm sample normalization. (c) FACS analysis showing the percentage of cells
counted in sub-G1 phase when FaO cells were infected with LacZ or ALK7 (m.o.i. of
250) in cells treated with the broad-range caspase inhibitor Z-VAD-FMK or DMSO.
Fig. 6. ALK7 induces cytochrome c release. (a) FaO cells expressing control vector
pcDNA3 were infected with LacZ, active ALK-5, active ALK7 expressing adenovirus
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(m.o.i. of 250), or treated with TGF-β1 (5 ng/ml). After 24 h after infection, the
mitochondrion was harvested (see material and methods) and the contents were subjected
to Western blot analysis. (b) The same conditions as found in (a) FaO cell expressing
dnSEK1.
Fig. 7. ALK7 activates Smad2 and Smad 3 in both FaO and Hep3B cells. (a) FaO
cells were infected with increasing amounts of adenovirus vectors carrying constitutively
active ALK7. FaO cells were harvested for protein 24 h after infection, which was then
subjected to Western blot analysis. Antibodies specific for the phosporylated forms of
Smad2, 3, and 4 were used to detect the level of Smad activation. Antibodies specific for
HA-tag were used to confirm ALK7 expression and antibodies specific for β-actin were
used to confirm sample normalization. (b) FaO cells infected with adenovirus vectors
carrying constitutively active ALK7 (m.o.i. of 100) were transfected with Flag-tagged
Smad2, Smad3, Smad4 proteins, or control vectors (designated as “c”). The cells were
harvested for protein 24 h after transfection, which was then subjected to Western blot
analysis. Antibodies specific for the phosphorylated form of JNK (P-JNK) were used to
detect the level of JNK activation. Antibodies for Flag were used to confirm expression
of Smad proteins. (c) Hep3B cells were infected with increasing amounts of adenovirus
vectors carrying constitutively active ALK7 for 24 h. After infection, the cells were
harvested and cell lysates were subjected to Western blot analysis for activated Smad2
and Smad3. (d) Hep3B cells were infected with increasing amounts of adenovirus vectors
carrying constitutively active ALK7 for 24 h. After infection, the cells were harvested
and cell lysates were subjected to Western blot analysis. Western blot analysis using the
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respective phosphor-specific antibodies to detect their activated forms of MAP kinases
was performed. Immunoreactive JNK, p38, and cleaved caspase 3 were probed by using
anti-JNK, p38 and cleaved caspase 3 specific antibody.
Fig. 8. siRNA-mediated inhibition of Smad3 suppresses ALK7-induced caspase 3
cleavage. Smad3 siRNA efficiently blocks expression of endogenous Smad3. Hep3B
cells were transfected with either control (scrambled) siRNA or increasing concentration
of Smad3 siRNA, and 24 hr after transfection, cells were infected with adenovirus
expressing LacZ or active ALK7 (m.o.i. of 100). Hep3B cells were harvested 24 h after
infection. Cell lysates were immunoblotted with antibodies against Smad3, and β-actin
(loading control). Suppression of endogenous Smad3 expression by Smad3 siRNA in
Hep3B cells results in an increase of ALK7-induced caspase 3 cleavage.
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Fig. 1
Cont
rol
Lac
Z 25
0 m
.o.i
TGF-β1
ALK7
50
m.o
.iAL
K7 2
50 m
.o.i
c100 m.o.i.50 m.o.i. 250 m.o.i.
LacZ TGF-βALK5
ALK7
bAnti-HA
LacZ
HA-ALK7
HA-ALK5
a
β-actin
M1
M2
M3
Lac Z
4.6%M1
M3
M2
ALK 5
27% M3
M2
M1
ALK 7
20%5.9%M1
M2
M3
Control
5.9%
d
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TGF-β1 - +
Caspase3
β-actin
pcD
NA
3
ALK
7-K
R
ALK
7-TD
HA
Caspase3
β-actin
0
5
10
15
20
25
pcDNA3ALK7-KR
ALK7-TD
Apo
ptot
ic c
ell (
%)
ALK7-TD
pEGFP DAPI
pcDNA3
Fig. 2
a
b c
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Fig. 3
p-JNK/SAPK
p-p38 MAPK
p-p42/44 MAPK
HA-ALK7
0 100 250 500 (m.o.i.)
ALK7
β-actin
0 250
LacZ
p42/44 MAPK
JNK/SPAK
p38 MAPK
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M1
M2
M32.1%M1
M2
M33.1%
M1
M2
M32.6%
LacZ
M1
M2
M339.4%
ALK7
pcDNA3
dnSEK1
b
Fig. 4
FaO-pcDNA3 FaO-dnSEK1
p-JNK/SAPK
β-actin
a
FaO-dnSEK10 100 250 0 100 250 0 100 250
LacZ ALK7
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Caspase 3
Caspase 7
Caspase 8
Caspase 9
0 25 100 250 (m.o.i.)
ALK7a
M1
M2
M32.0%M1
M2
M310.2%
M1
M2
M32.6%
LacZ
M1
M2
M339.4%
ALK7
DMSO
Z-VAD-FMK
c
ALK7LacZ
pcDNA3 dnSEK1
Caspase 9
-+ -- -
++ +
b
β-actin
Fig. 5
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Fig. 6
Cyt c
LacZ ALK7 ALK5 TGF-β
14 kDa
pcDNA3a
Cyt c14 kDa
LacZ ALK7 ALK5 TGF-β
dnSEK1b
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Fig. 7
p-Smad2
p-Smad 3
Smad 3
Con
trol
Lac
Z
ALK
7 (5
0 m
.o.i.
)
ALK
7 (1
00 m
.o.i.
)
Smad2
HA-ALK7
c
Smad2
Smad3
a ALK7
HA-ALK7
p-Smad2
0 10 25 100 (m.o.i.)
p-Smad3
β-actin
0 100
LacZ
p-JNK/SAPK
FLAG-Smads
S2 S3 S4C C
ALK7b
JNK/SAPK
p-38
p-JNK
JNK
Con
trol
Lac
Z
ALK
7 (5
0 m
.o.i)
ALK
7 (1
00 m
.o.i)
p38
HA-ALK7
d
β-actin
19 kDa17 kDa
CleavedCaspase-3
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+++ALK7-adeno
+++LacZ-adeno
++
++ +++Smad3 Si
+Scramble Si
Smad3
Cleaved Caspase-3
β-actin
Fig. 8
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Chun, David A. Lee, Kyeong Sook Choi and Seong-Jin KimByung-Chul Kim, Howard van Gelder, Tae Aug Kim, Ho-Jae Lee, Kim G. Baik, Hyun Hye
mechanism in hepatoma cellsALK7 Induces apoptosis through activation of MAP kinases in a Smad3-dependent
published online April 23, 2004J. Biol. Chem.
10.1074/jbc.M313277200Access the most updated version of this article at doi:
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