alk7 induces apoptosis through activation of map kinases ... file1 alk7 induces apoptosis through...

1

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

by guest on July 2, 2019http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

2

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.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

3

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

4

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

5

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

6

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

7

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

9

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

10

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-


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

11

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).


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

12

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

13

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.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

14

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

15

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.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

16

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

17

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.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

18

References

1. Massagué, J. and Wotton, D. (2000) EMBO J. 19, 1745–1754

2. Attisano, L. and Wrana, J. L. (2000) Curr. Opin. Cell Biol. 12, 235–243

3. Hsing, A. Y., Kadomatsu, K., Bonham, M. J., and Danielpour, D. (1996) Cancer

Res. 56, 5146–5149

4. Choi, K. S., Lim, I. K., Brady, J. N., and Kim, S.-J. (1998) Hepatology 27, 415-

421.

5. Choi, K. S., Eom, Y. W., Kang, Y., Ha, M. J., Rhee, H., Yoon, J. W., and Kim,

S.-J. (1999) J. Biol. Chem. 274, 31775-31783.

6. Tsuchida K, Sawchenko PE, Nishikawa S, Vale WW. (1996) Mol Cell Neurosci.

7(6):467-78.

7. Reissmann E, Jornvall H, Blokzijl A, Andersson O, Chang C, Minchiotti G,

Persico MG, Ibanez CF, Brivanlou AH. (2001), Genes Dev. 15(15), 2010-22

8. Watanabe R, Yamada Y, Ihara Y, Someya Y, Kubota A, Kagimoto S, Kuroe A,

Iwakura T, Shen ZP, Inada A, Adachi T, Ban N, Miyawaki K, Sunaga Y, Tsuda

K, Seino Y. (1999), Biochem Biophys Res Commun. 254(3):707-12.

9. Jornvall H, Blokzijl A, ten Dijke P, Ibanez CF. (2001), J Biol Chem. 276(7),

5140-6.

10. Bondestam J, Huotari MA, Moren A, Ustinov J, Kaivo-Oja N, Kallio J, Horelli-

Kuitunen N, Aaltonen J, Fujii M, Moustakas A, Ten Dijke P, Otonkoski T, Ritvos

O. (2001), Cytogenet Cell Genet. 95(3-4), 157-62

11. Roberts HJ, Hu S, Qiu Q, Leung PC, Cannigia I, Gruslin A, Tsang B, Peng C.

(2002), Biol Reprod 68(5):1719-26

12. Green, DR. (1998) Cell. 18;94(6):695-8.

13. Kim, B. C., Mamura, M., Choi, K. S., Calabretta, B., and Kim, S.-J. (2002) Mol.

Cell. Biol. 22, 1369-1378.

14. Cain, K., Inayat-Hussain, S.H., Couet, C., Cohen, G. M.. (1996) Biochem. J. 314,

27-32

15. Inayat-Hussain, S.H., Couet, C., Cohen, G.M., and Cain, K. (1997) Hepatalogy

25, 1516-1526.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

19

16. Herrera, B., Alvarez, A. M., Sanchez, A., Fernandez, M., Roncero, C., Benito, M.,

and Fabregat, I. (2001) FASEB J. 15, 741-751

17. Coyle B, Freathy C, Gant TW, Roberts RA, Cain K. (2003), J Biol Chem.

278(8):5920-8.

18. Fujii M, K. Takeda, T. Imamura, H. Aoki, T. K. Sampath, S. Enomoto, M.

Kawabata, M. Kato, H. Ichijo, and K. Miyazono. 1999. Mol. Biol. Cell. 10:3801-

3813.

19. Duvall, E., Wyllie, A.H. (1986) Immunol. Today. 7, 115.

20. Gallo, KA, Johnson, GL. (2002) Nat Rev Mol Cell Biol. (9):663-72.

21. Kishimoto H, Nakagawa K, Watanabe T, Kitagawa D, Momose H, Seo J, Nishitai

G, Shimizu N, Ohata S, Tanemura S, Asaka S, Goto T, Fukushi H, Yoshida H,

Suzuki A, Sasaki T, Wada T, Penninger JM, Nishina H, Katada T. (2003) J Biol

Chem. 278(19):16595-601

22. Park, H. J., Kim, B. C., Kim, S.-J., and Choi, K. S. (2002) Hepatology. 35, 1360-

1371

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-


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

20

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

21

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

22

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

23

(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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

24

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.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

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

by guest on July 2, 2019 http://www.jbc.org/ Downloaded from

http://www.jbc.org/

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


http://www.jbc.org/

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


http://www.jbc.org/

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


http://www.jbc.org/

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


http://www.jbc.org/

Fig. 6

Cyt c

LacZ ALK7 ALK5 TGF-β

14 kDa

pcDNA3a

Cyt c14 kDa

LacZ ALK7 ALK5 TGF-β

dnSEK1b


http://www.jbc.org/

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


http://www.jbc.org/

+++ALK7-adeno

+++LacZ-adeno

++

++ +++Smad3 Si

+Scramble Si

Smad3

Cleaved Caspase-3

β-actin

Fig. 8


http://www.jbc.org/

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:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M313277200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M313277200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2004/04/23/jbc.M313277200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2004/04/23/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2004/04/23/jbc.M313277200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

alk7 induces apoptosis through activation of map kinases ... file1 alk7 induces apoptosis through...

Documents