From the Division of Molecular Neurobiology,
Department of Neuroscience, Karolinska Institute, S-17177 Stockholm,
Sweden and the ¶ Division of Cellular Biochemistry, The
Netherlands Cancer Institute, NL-1066 CX
Amsterdam, The Netherlands
Received for publication, June 15, 2000, and in revised form, November 13, 2000
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ABSTRACT |
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The signaling capabilities and
biological functions of activin receptor-like kinase 7 (ALK7), a type I
receptor serine/threonine kinase predominantly expressed in the nervous
system, are unknown. We have constructed a cell line derived from the
rat pheochromocytoma PC12 in which expression of a constitutively
active mutant of ALK7 (T194D) is under the control of a
tetracycline-inducible promoter. For comparison, another cell line was
engineered with tetracycline-regulated expression of a constitutively
active variant of the transforming growth factor- The transforming growth factor
(TGF)1 Activin receptor-like kinase 7 (ALK7) is an orphan member of the type I
subfamily of receptor serine/threonine kinases (9-11). It is
predominantly expressed in distinct subpopulations of central neurons
during postnatal development and adulthood (9, 12). Presently, not only
the ligand of ALK7 but also its corresponding type II receptor partner
are unknown. ALK7 is highly similar in its intracellular domain to ALK5
(78% identity) and ALK4 (77% identity), type I receptors for TGF- In the present work, we have investigated the signaling and biological
activities of ALK7 using a constitutively active form of this receptor
in an inducible expression system in PC12 cells. Taking advantage of
the ability of these cells to adopt a neuronal phenotype after
differentiation with nerve growth factor (NGF), we have also studied
the interactions between this well characterized cell system of
neuronal differentiation and ALK7 signaling.
Subcloning and Mutagenesis--
Single-stranded DNA from rat
ALK7 subcloned in pBS KS+ (Stratagene) was used for
oligonucleotide-based site-directed mutagenesis as described previously
(13).
Cell Culture and Tetracycline System--
The rat
pheochromocytoma cell line PC12 was cultured in Dulbecco's modified
Eagle's medium containing 10% horse serum, 5% fetal bovine serum, 2 mM L-glutamine, and 60 µg/ml gentamycin (Life
Technologies, Inc.) at 37 °C in a 5% CO2 humidified
atmosphere. All serum was screened for absence of tetracycline using
the CHO-AA8-Luc Tet-Off control cell line from
CLONTECH. Low serum experiments where done using
one-twentieth of the serum concentrations specified above. Perfect
Lipid 4 (Invitrogen) was used for cell transfections according to the
manufacturer's instructions. PC12 cells were transfected with an
expression construct of the reverse tetracycline transactivator
(pUHD172-1neo) and constitutively active His6-tagged T194D-ALK7 or hemagglutinin (HA)-tagged T204D-ALK5 under the control of
the tet-operon and a minimal cytomegalovirus promoter (pUHD10-3) (14).
Stably transfected cells were selected with 200 µg/ml geneticin
(G-418) and 1 µg/ml puromycin (Life Technologies, Inc. and Sigma,
respectively), expanded, and individually screened for low leakage and
high tetracycline inducibility by RNase protection assay (ALK7) or
Western blotting (ALK5). The tetracycline analogue doxycycline was used
in all inductions at 1 µg/ml or at the concentrations indicated.
RNase Protection Assay--
RNA isolation was done by
guanidinium isothiocyanate lysis and acid phenol extraction as
previously described (9). For RNase protection assays, a 310-bp-long
HincII-PvuII fragment of rat ALK7 cDNA was
subcloned into pBS KS+, linearized, and used as template for T7 RNA
polymerase. The rat c-fos probe has been previously
described (15). A rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
riboprobe was used for normalization. 10 µg of total RNA was
hybridized to [ Western Blotting, JNK in Vitro Kinase Assay, and PAI-1
Assay--
For protein analyses, cells were lysed in 1% Nonidet P-40
with 150 mM NaCl, 10 mM Tris-HCl (pH 7.5), 10 mM NaF, 2 mM Na3VO4, and Complete EDTA-free protease inhibitor mixture (Roche Molecular Biochemicals). The supernatant after mild centrifugation constituted the cytosolic fraction. The nuclear pellet was then further treated by
trituration through a 23-gauge needle and boiling in SDS to lyse the
nuclei and solubilize nuclear proteins. The fractions were then
separated by SDS-PAGE. Bands were electroblotted onto Amersham Hybond-P
polyvinylidene difluoride transfer membranes and further processed
using standard Western protocols. Protein bands were visualized using
enhanced chemifluorescence (Amersham Pharmacia Biotech) and a STORM840
fluorimaging system and quantification software as described above.
Monoclonal anti-HA (12CA5) and anti-His6 were from BAbCo;
anti-P-ERK, anti-ERK, and anti-JNK antibodies were from New England
Biolabs; polyclonal antibodies against ALK7 (RQC), Smad1,
phospho-Smad1, Smad2, phospho-Smad2, Smad3, and Smad7 have been
previously described (9, 16-18).
For JNK in vitro kinase assays, total cell lysates were
incubated with glutathione-Sepharose beads that had been previously loaded with a purified c-Jun-glutathione S-transferase
fusion protein (Upstate Biotechnology). After three washings in lysis buffer, the beads were further washed once in 0.1 M Tris,
pH 7.5, 5 mM LiCl and once in kinase buffer (25 mM HEPES, pH 7.0, 20 mM MgCl2, 1 mM Na3VO4, 20 mM
Levels of PAI-1 were determined by SDS-PAGE of extracellular matrix
proteins of metabolically labeled cells as previously described (19,
20). Nearly confluent 35-mm wells were stimulated with doxycycline at 1 µg/ml in full serum Dulbecco's modified Eagle's medium. After
24 h the cells were washed once in phosphate-buffered saline
(PBS), and the medium was replaced with serum-free Dulbecco's modified
Eagle's medium without methionine and cysteine. After 2 h,
35S-labeled methionine and cysteine (Amersham Pharmacia
Biotech; PRO-MIX, SJQ0079) were added at 40 µCi/ml, and the cells
were incubated for an additional 2 h. The cells were then removed
by washing on ice once in PBS, three times in 10 mM
Tris-HCl, pH 8.0, 0.5% sodium deoxycholate, 1 mM
phenylmethylsulfonyl fluoride, two times in 2 mM Tris-HCl,
pH 8.0, and once in PBS. SDS samble buffer containing
Luciferase Reporter Assays--
Transcriptional response assays
with luciferase reporters were analyzed using the Dual-Luciferase
Reporter Assay System kit from Promega. Briefly, 6-well dishes of cells
at 50% confluence were transfected as indicated above with 2 µg of
the appropriate firefly luciferase reporter construct. For internal
control of cell number and transfection efficiency, 50 ng of
Renilla luciferase under a minimal cytomegalovirus promoter
(pRL-CMV, Promega) was included in the transfection. After
transfection, full medium was added with or without doxycycline at 1 µg/ml, and the cultures were incubated for an additional 48 h
before cell lysis as indicated by the manufacturer. Firefly luciferase
activity was normalized to the Renilla luciferase activity,
and fold induction was calculated relative to the luciferase activity
in the absence of doxycycline for each reporter construct and cell
line. Luciferase expression was quantified on a 1450 MicrobetaJet
luminescence counter (Wallac).
Thymidine Incorporation Assay--
Proliferation assays were
done on cells cultured on poly-D-lysine-coated 12-well
plates at an density of 10 × 103 cells/well. For cell
viability assays, 24 h after plating, cells were pulsed for 4 h with 2.0 µCi/ml [methyl-3H]thymidine (Amersham
Pharmacia Biotech), washed, and then incubated for an additional
48 h with or without doxycycline at 1 µg/ml. For DNA synthesis
assays, the 4 h pulse with [methyl-3H]thymidine was
done after the 48-h treatment with doxycycline. At the end of either
type of experiment, the medium was replaced by 0.25% trypsin and 10 mM EDTA in PBS and, after a 30-min incubation at 37 °C,
the cells were harvested onto filters mats using a cell harvester
(Skatron Instruments). [3H]Thymidine incorporation was
quantified on a 1450 Microbeta liquid scintillation counter (Wallac).
Filamentous Actin Staining and Morphological
Studies--
F-actin was stained using a fluorescent phallotoxin from
Molecular Probes (Alexa 488 phalloidin, A-12379) according to
manufacturer's instructions. Briefly, cells were plated on
poly-D-lysine-coated chamber slides (Lab-Tek) and left to attach
overnight. The medium was then changed according to the experimental
parameters, and the cells were incubated for additional 4.5 days before
washing twice with PBS and fixing in 3.7% formaldehyde solution in PBS for 10 min at room temperature. After two additional washes in PBS,
each slide was stained at room temperature for 20 min with one unit of
Alexa 488 phalloidin in PBS. Following the staining, the slides were
washed twice in PBS and mounted under a coverslip in a 1:1 solution of
PBS and glycerol and examined by fluorescence microscopy. Morphological
studies were performed in PDL-coated 6-well tissue culture plates in a
similar fashion. Cells were always washed with PBS in between changes
of medium to avoid carry over. For neuronal differentiation, NGF
(Promega) was used at 50 ng/ml in low serum medium (i.e.
PC12 Cell Lines with Inducible Expression of Constitutively Active
ALK7 and ALK5 Receptors--
In the absence of the ALK7 ligand and its
corresponding type II receptor partner, an alternative system was
developed to study the downstream signaling mechanism and potential
biological activities of the ALK7 receptor. Type I receptor
serine/threonine kinases can be activated in a ligand- and type II
receptor-independent way by replacing an acidic residue for a specific
threonine within the juxtamembrane region of the intracellular domain,
a segment known to be involved in kinase regulation (21). An equivalent mutation in the ALK7 receptor, corresponding to replacement of Asp for
Thr194 (T194D), also results in constitutive activation of
ALK7 as evaluated using TGF-
Under basal conditions, ALK7-PC12 or ALK5-PC12 cells did not express
any detectable levels of the introduced ALK7 or ALK5 receptors,
respectively (Fig. 1). However, treatment
with the tetracycline analogue doxycycline resulted in a marked
increase in the expression of the respective receptors (Fig. 1).
Induction of ALK7 mRNA expression was dose-dependent
over a wide range of doxycycline concentrations (Fig. 1A),
with a peak 8 h after doxycycline treatment followed by a slow
decline (Fig. 1B). Expresion of ALK7 and ALK5 protein could
be first detected 4 h after doxycycline stimulation and was
maximal at 24 h (Fig. 1C). The delay observed between
the time course of induction of protein and mRNA expression following Dox treatment is unclear and could be due to differences in
the sensitivity of the two methods. As shown below, several ALK7-induced signaling events can be detected as early as 3 and 6 h following Dox treatment, indicating that levels of ALK7 protein must
in fact be present at these early time points. Expression of the
exogenous receptors declined 5 days after uninterrupted doxycycline
treatment, perhaps because of autoinhibition following continuous
signaling (data not shown).
Activation of Smad Proteins and MAP Kinases in Response to ALK7
Signaling in PC12 Cells--
A universal signal transduction event
following activation of ALK receptors is the phosphorylation and
nuclear translocation of members of the Smad protein family.
Phosphorylation and nuclear accumulation of receptor specific Smads
after ALK7 activation was investigated in nuclear fractions of ALK7-
and ALK5-PC12 cells treated with doxycycline using specific antibodies
for Smad1, Smad2, and Smad3. Activation of ALK7 resulted in
phosphorylation and nuclear translocation of Smad2 but not Smad1 (Fig.
2A). Smad3 was also found to
accumulate in the nuclei of ALK7-PC12 cells treated with doxycycline
(Fig. 2A). Similar results were obtained in ALK5-PC12 cells
in agreement with previous observations. In agreement with the kinetics
of ALK receptor expression, activation of Smad2 and Smad3 could first
be detected 3 h after doxycycline stimulation and was maximal at
24 h (Fig. 2A).
We also looked for activation of the MAP kinases ERK and JNK in ALK7-
and ALK5-PC12 cells after doxycycline treatment. Increased phosphorylation of ERK1 and ERK2 could be detected 6 and 12 h following doxycycline stimulation in both ALK7- and ALK5-expressing cells (Fig. 2B). Using a c-Jun-glutathione
S-transferase fusion protein as substrate, we assayed the
activity of JNK in cell lysates of ALK7- and ALK5-PC12 cells after
doxycycline treatment. JNK activity was significantly elevated in both
cell lines after 12 h of doxycycline treatment (Fig.
2B).
Thus, in agreement with the structural similarity between the kinase
domain of ALK7 and those of the TGF- Activation of TGF- Regulation of Endogenous Gene Expression by ALK7 Signaling in PC12
Cells--
We also investigated whether ALK7 is able to regulate
endogenous genes known to be targets of TGF- Anti-proliferative Effect of ALK7 Activation--
Thymidine
incorporation experiments were performed to investigate the effects of
ALK7 signaling on cell survival and proliferation. In the first set of
experiments, the cells were pulsed at the beginning with
[3H]thymidine for 4 h and then cultured for another
48 h in the presence or absence of doxycycline. Under these
conditions, there was no change at the end of the experiment between
parental PC12, ALK7-PC12, or ALK5-PC12 cells (Fig.
5A), indicating that neither ALK7 nor ALK5 signaling affected the viability of PC12 cells. In the
second experiment, the cells were first incubated for 48 h in the
presence or absence of doxycycline and then pulsed for 4 h with
[3H]thymidine prior to harvesting. In this experiment,
however, both ALK7 and ALK5 signaling produced a pronounced decrease in thymidine incorporation in a dose-dependent manner (Fig.
5B), indicating that activation of ALK7, like the TGF- Effects of ALK7 Signaling on PC12 Cell Morphology, Actin
Cytoskeleton, and Neuronal Differentiation--
During the course of
the studies described above, it became evident that ALK7 signaling had
very characteristic effects on cell morphology. Under control
conditions, ALK7-PC12 cells were indistinguishable from parental cells.
However, within 12 h of doxycycline treatment, ALK7-PC12 cells
flattened out and began extending blunt, short cell processes. Four
days after doxycycline stimulation, ALK7-PC12 cells were unequivocally
different from parental cells (Fig.
6A). Conditioned medium from
doxycycline-treated ALK7-PC12 cells had no biochemical or morphological
effects on naïve PC12 cells (not shown), indicating that the
changes induced in ALK7-PC12 cells were produced by a direct, cell
autonomous mechanism and not via the production of a secondary signal.
The effects of ALK7 on PC12 cell morphology were most evident in the presence of serum. Although cultures grown in low serum did show some
changes, these were not as pronounced and mostly restricted to a few
cells (not shown). Intriguingly, the morphological changes induced by
ALK7 activation were still evident several days after removal of
doxycycline, suggesting a long lasting alteration. The effects of ALK7
activation on PC12 cell morphology were also reflected in the
distribution of actin filaments, as revealed by phalloidin staining
(Fig. 6A). ALK7 signaling resulted in an overall reduction
in actin staining, indicating actin filament depolimerization (Fig.
6A). Loss of cortical actin staining, characteristic of
round, phase bright parental PC12 cells, was also seen in several of
the most affected cells (Fig. 6A). Similar effects in cell morphology could also be observed in an independently isolated PC12
cell clone overexpressing constitutively active ALK7 (data not shown).
Surprisingly, ALK5 activation had no effect on the morphology or actin
filament distribution of PC12 cells (Fig. 6B), suggesting
that, despite their apparently similar signaling mechanisms, ALK5 and
ALK7 have the capacity to transmit distinct biological responses.
Although the effects of ALK7 signaling on cell morphology were clearly
different from those induced by NGF, both processes shared several
features, including a reduction in proliferation rate and the
elaboration of cell processes, albeit short in the case of ALK7. In the
absence of doxycycline, ALK7-PC12 cells responded to NGF just like
parental cells, differentiating at the same speed and to the same
extent after comparable treatments (data not shown). On the other hand,
cultures that were pretreated with doxycycline showed an accelerated
differentiation in response to NGF, already evident 12 h after the
onset of NGF stimulation (Fig. 7). At
this time, more than 50% of the ALK7-PC12 cells stimulated with
doxycycline and NGF showed signs of undergoing neuronal
differentiation, whereas only a smaller proportion (10%) did in the
cultures that received only NGF (Fig. 7). Two weeks into the
differentiation process, ALK7-PC12 cells treated with doxycycline
showed in addition a more robust phenotype with longer neurites and a
greater percentage of differentiated cells (Fig. 7). A similar, albeit
less pronounced effect was obtained if the cultures were simultaneously
exposed to both doxycycline and NGF (data not shown). Again, no
accelerated neuronal differentiation in response to NGF could be seen
in ALK5-PC12 cells that had been stimulated with doxycycline (data not
shown).
We have devised a system to study the function of type I
serine/threonine kinase receptors that bypasses the ligand and the type
II receptor. We have used this system to obtain insights into the
signaling capabilities and biological activities of the orphan type I
receptor ALK7.
The high similarity in the primary sequence of the kinase domain of
ALK7 with type I receptors for activin and TGF- In agreement with its ability to induce the promoters of
cycline-dependent kinase inhibitors p15INK4B
and p21, ALK7 activation had an anti-proliferative effect on PC12
cells. In this regard, ALK7 shares with other type I receptors of the
TGF- In line with a role in neuronal differentiation, ALK7 activation in
PC12 cells induced a marked and long lasting morphological change,
characterized by cell flattening, reduction of actin polymerization, loss of phase brightness and cortical actin filaments, and elaboration of short, blunt cell processes. In particular, the effects of ALK7
activity on the actin cytoskeleton suggest cross-talk with signaling
components that regulate actin reorganization, such as the small G
proteins of the Rho subfamily. Interestingly, JNK, a downstream
effector of the Rho family proteins Rac and cdc42, was also activated
by ALK7. Of note, TGF- The morphological changes induced by ALK7 activity were quite different
from those elicited by NGF, which does not induce cell flattening but
stimulates the growth of round, phase bright cell bodies and long, thin
neuritic processes. Nevertheless, ALK7 collaborated with NGF in
promoting neuronal maturation, as demonstrated by an acceleration of
the differentiation process in cells exposed to both signals. In this
regard, the effects of ALK7 activation in PC12 cells resemble the
"priming" effect of a transient exposure to NGF, which also results
in accelerated differentiation during a subsequent encounter with this
factor (30). Although a direct extrapolation of the effects of ALK7 on
PC12 cells to other cell types expressing endogenous ALK7 in
vivo is limited at present, our observations do suggest that this
receptor may have effects on cell differentiation beyond the control of
proliferation, in particular, in the regulation of cytoskeletal
rearrangements and cell morphology.
Unexpectedly, despite the similarities in signaling between ALK7 and
ALK5, the latter was unable to induce the kind of morphological changes
promoted by ALK7 in PC12 cells. This difference could not have been due
to different levels of expression or signaling intensity between the
two systems, because all other effects of ALK7 and ALK5 activation were
fully comparable in magnitude and duration across several different
assays, including Smad and MAP kinase activation, induction of reporter
activity, and inhibition of DNA synthesis. On the other hand, the
evidence obtained suggests a qualitative difference in the signaling of
these two receptors in PC12 cells. Although the underlying mechanism is
unclear at the moment, these results suggest that different type I
receptors may have distinct biological effects, even if they activate
the same complement of Smad proteins.
In conclusion, the orphan receptor serine/threonine kinase ALK7 appears
to utilize many of the same signaling mechanisms activated by TGF- type I
receptor ALK5. Expression of activated ALK7 in PC12 cells resulted in
activation of Smad2 and Smad3, but not Smad1, as well as the
mitogen-activated protein kinases extracellular signal-regulated kinase
and c-Jun N-terminal kinase. Reporter assays demonstrated that ALK7
activation stimulates transcription from the Smad-binding element of
the Jun-B gene, the plasminogen activator inhibitor-1 gene, and AP-1
elements. In addition, ALK7 activation induced expression of endogenous gene products, including Smad7, c-fos mRNA, and
plasminogen activator inhibitor-1. Thymidine incorporation assays
revealed an anti-proliferative effect of ALK7 activation in PC12 cells,
which correlated with increased transcription from the promoters of
cycline-dependent kinase inhibitors p15INK4B
and p21. Unexpectedly, ALK7 signaling produced a remarkable change in
cell morphology characterized by cell flattening and elaboration of
blunt, short cell processes. Interestingly, no such changes were
observed upon induction of activated ALK5. The alterations in cell
morphology upon ALK7 activation were more pronounced in cultures grown
in full serum, were accompanied by rearrangements of actin filaments,
and were maintained for several days after withdrawal of treatment.
PC12 cultures that had been "primed" in this way showed an
accelerated and augmented differentiation response to nerve growth
factor. These results indicate that ALK7 may participate in the control
of proliferation of neuronal precursors and morphological
differentiation of postmitotic neurons.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
superfamily
constitutes the largest group of polypeptide growth factors known,
including the TGF-
s, activins, bone morphogenetic proteins (BMPs),
nodal, and growth and differentiation factors (GDFs). Members of the TGF-
superfamily exhibit an extensive array of biological
activities, regulating proliferation, lineage determination,
differentiation, migration, adhesion and apoptosis during development,
homeostasis, and repair in practically all tissues from flies to
humans. TGF-
ligands signal through a complex of two transmembrane
receptor serine/threonine kinases belonging to two distinct
subfamilies: the type I receptors of ~55 kDa and the type II
receptors of ~70 kDa. Both receptors cooperate with ligand binding,
type II receptors phosphorylate type I receptors, and type I receptors
activate members of the Smad family of signal transducers, which then
translocate to the nucleus where they take part in a number of DNA
binding complexes (1-3). Receptor-specific Smads transiently interact with and become phosphorylated by type I receptors. Smad2 and 3 participate in downstream signaling from TGF-
/activin receptors, and
Smad1, 5, and 8 participate in downstream signaling from BMP/GDF receptors. Smad4 form complexes with receptor-specific Smads and is a
common mediator of the nuclear responses of TGF-
/activin and
BMP/GDF. Smad 6 and 7, on the other hand, are inhibitory Smads that
compete with receptor-specific Smads for association with type I
receptors. Once in the nucleus, activated Smads may bind DNA on their
own or, most commonly, in association with other DNA-specific factors.
A number of general transcription factors, coactivators, and
corepressors have been found to associate with Smads during TGF-
signaling (1-3). TGF-
has also been shown to activate several other
cytoplasmic signaling pathways in addition to the Smads, including
several members of the MAP kinase family such as extracellular
signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) (4-8).
The signaling events leading from the activated receptors to these
signaling components remain for the most part undefined.
s
and activins, respectively. Despite this similarity, the extracellular
domain of ALK7 is very divergent from other type I receptors,
indicating that it binds a distinct member of the TGF-
superfamily.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]CTP-labeled cRNA probes using a
kit from Ambion according to the manufacturer's instructions.
Protected bands were visualized and quantified using a STORM840
PhosphorImager and ImageQuant software (Molecular Dynamics).
-glycerophosphate, 2 mM dithiothreitol) and then
incubated at 37 °C for 20 min in 30 µl of kinase buffer containing
2 µCi of [
-32P]ATP, 20 µM rATP, and 5 µM protein kinase A inhibitor. The reaction was stopped
by adding SDS/
-mercaptoethanol sample buffer, and the samples were
then boiled, fractionated by SDS-PAGE, and blotted onto polyvinylidene
difluoride membranes (Amersham Pharmacia Biotech). The membranes were
exposed to phosphorscreens and subsequently scanned in a STORM840
PhosphorImager (Molecular Dynamics). JNK activity was quantified using
ImageQuant software. After exposure, the membranes were probed with
anti-JNK antibodies.
-mercaptoethanol were added, and the remaining extracellular matrix
proteins on the plate were transferred to an Eppendorf tube for
subsequent SDS-PAGE. After fixing in 40% methanol, 7% acetic acid,
the gel was dried, and the 45-kDa PAI-1 band was visualized
using a STORM840 PhosphorImager.
th of its normal strength). Cultures were
examined and photographed under phase contrast illumination in a Zeiss
Axiovert microscope.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-specific reporter assays
(10).2 Signaling by TGF-
ligands is known to have profound effects on cell proliferation,
survival, and differentiation. To avoid potential problems caused by
constitutive signaling during clone expansion and to allow the study of
acute effects of ALK7 signaling, an inducible expression system based
on the tetracycline-responsive operon (14) was utilized to generate
stable cell lines. As a cellular host for inducible expression of
constitutively active ALK7, we chose the rat pheochromocytoma PC12.
This cell line has been extensively used as model system in studies of
signal transduction, primarily because of its ability to develop a
striking neuronal phenotype upon treatment with NGF (22). For
comparison with a better characterized signaling system, we also
generated PC12 cells with inducible expression of a constitutively
active form of the TGF-
type I receptor ALK5, generated, as
previously described, by replacement of Asp for Thr204
(T204D) (21). A C-terminal His6 tag was placed in ALK7, and a C-terminal HA tag was placed in ALK5. It should be noted that parental PC12 cells do not express ALK7 and express only moderate levels of endogenous ALK5 (data not shown).
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Fig. 1.
Inducible expression of constitutively active
ALK7 and ALK5 receptors in PC12 cells. A, dose-response
to doxycycline (Dox) of the induction of ALK7 mRNA
expression in ALK7-PC12 cells analyzed by RNase protection assay
48 h after doxycycline treatment (top panel). The
middle panel shows the signal obtained with a GAPDH
riboprobe and demonstrates comparable amounts of RNA in all the lanes.
The histogram (bottom panel) shows the quantification of the
ALK7 mRNA signals relative to GAPDH obtained by phosphorimaging.
B, time course of the induction of ALK7 mRNA expression
in ALK7-PC12 cells treated with 1 µg/ml doxycycline analyzed by RNase
protection assay (top panel). The middle panel
shows the signal obtained with a GAPDH riboprobe and demonstrates
comparable amounts of RNA in all the lanes. The histogram (bottom
panel) shows the quantification of the ALK7 mRNA signals
relative to GAPDH obtained by phosphorimaging. C, time
course of the induction of ALK7 (upper panels) and ALK5
(lower panels) expression in PC12 cells treated with 1 µg/ml doxycycline analyzed by Western blotting with
anti-His6 antibodies (ALK7) or anti-HA antibodies (ALK5).
The lower blots in each panel show reprobing of the same
filters with anti-CREB antibodies and demonstrate comparable amounts of
protein in all the lanes.
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Fig. 2.
Activation of Smad proteins and ERK and JNK
kinases in response to ALK7 and ALK5 signaling in PC12 cells.
A, nuclear extracts of KAL7- and ALK5-PC12 cells treated
with doxycycline (Dox) for the indicated periods of time
were analyzed by Western blotting with antibodies against
phospho-Smad1, phospho-Smad2, and Smad3 as indicated. Lysates were
normalized in protein content prior to the analysis. The
numbers below the lanes indicate the fold increase relative
to control. ALK7, similar to ALK5, causes accumulation of phospho-Smad2
and Smad3 in the nucleus but not phospho-Smad1. B, total
extracts of KAL7- and ALK5-PC12 cells treated with doxycycline for the
indicated periods of time were analyzed by Western blotting with
antibodies against P-ERK (left panels) or by a JNK in
vitro kinase assay (right panels). Lower
panels show reprobing of the corresponding blots with the
indicated antibodies and demonstrate comparable amounts of protein in
all the lanes. The numbers below the lanes indicate the fold
increase relative to control normalized to the total level of ERK or
JNK in each lanes. P-, phospho-.
and activin receptors ALK5 and
ALK4, signal transduction by the ALK7 receptor also utilizes the
pathway-specific Smads 2 and 3, as well as the MAP kinases ERK and
JNK.
-responsive Promoter Elements Following ALK7
Signaling--
Downstream transcriptional responses to ALK7 signaling
were investigated using the luciferase reporter gene coupled to
regulatory elements from different genes known to be responsive to
TGF-
superfamily proteins, including the genes of
cycline-dependent kinase (cdk) inhibitors
p15INK4B (23) and p21 (24), the PAI-1 gene (i.e.
p3TP-Lux, which also includes three TPA-responsive sequences) (25),
four tandem copies of the Smad-binding element from the Jun-B gene
((SBE)4) (26), AP-1 elements, and nine tandem copies of the
Smad-binding element from the PAI-1 promoter (CAGA)9 (27).
(SBE)4 is activated by both TGF-
/activin and BMP
ligands, whereas (CAGA)9 is only activated by
Smad3-mediated signaling (27). ALK7-PC12, ALK5-PC12, and parental PC12
cells were transiently transfected with reporter plasmids, and reporter
gene activity was assayed in cell lysates 48 h following
doxycycline treatment. ALK7 signaling stimulated transcription from all
the reporters (Fig. 3A).
Similar results were obtained in an independently isolated ALK7-PC12
cell clone (data not shown). With the exception of the p21 cyclin
inhibitor reporter, which appeared down-regulated after doxycycline
treatment, similar results were also observed in ALK5-expressing cells
(Fig. 3B). No changes were detected in parental PC12 cells
after doxycycline stimulation (data not shown). Thus, with the possible
exception of p21, ALK7 signaling resembles that of the type I TGF-
receptor ALK5 in PC12 cells, including the robust activation of the
Smad3-specific reporter (CAGA)9.
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Fig. 3.
Activation of
TGF- -responsive promoter elements by ALK7
signaling. Luciferase reporter constructs as indicated were
transiently transfected into ALK7-PC12 cells (A) or
ALK5-PC12 cells (B). Samples from control (open
bars) or doxycycline-treated cells (solid bars) were
assayed 48 later. The results indicate corrected luciferase values
expressed relative to control (averages ± S.D.) and were
reproduced in several other independent experiments. Dox,
doxycycline.
and Activin signaling pathways. Expression of the inhibitory Smad7 is induced by TGF-
via
direct interaction of Smad3 and 4 with the smad7 gene
promoter (17). In PC12 cells, ALK7 activation induced expression of
Smad7 with a peak of protein expression between 12 and 24 h
following Dox treatment (Fig.
4A), indicating that, similar
to other TGF-
family members, ALK7 signaling is also regulated
through a feedback loop of inhibitory Smad proteins. Expression of
immediate early genes is also regulated by TGF-
signaling, and the
fact that activation of ALK7 induced phosphorylation of Erk proteins
prompted us to examine the levels of c-fos mRNA in these
cells. Doxycycline stimulation stimulated c-fos mRNA
levels in ALK7-PC12 with a peak at 24 h of treatment, declining
back to basal levels by 72 h (Fig. 4B). Finally, we
examined the expression of PAI-1, an extracellular matrix component
which is up-regulated by TGF-
and activin in several cell types
(19). We found that in ALK7-PC12 cells treated with doxycycline, the
levels of PAI-1 were elevated compared with untreated cells (Fig.
4C), indicating that ALK7 activation regulates target genes
responsible for some of the phenotypic changes induced by TGF-
proteins.
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Fig. 4.
Induction of endogenous genes by ALK7
signaling in PC12 cells. A, induction of Smad7
expression was assessed by Western blotting of total extracts of cells
stimulated with doxycycline for the indicated periods of time.
B, induction of c-fos mRNA expression was
determined by RNase protection assay. A GAPDH probe was included in the
same reaction as loading control. C, induction of PAI-1
expression was assessed by direct SDS-PAGE and autoradiography of cell
matrix extracts prepared from metabolically labeled cells. The two most
prominent bands (arrows) correspond to the PAI-1 protein.
Dox, doxycycline.
receptor ALK5, has an anti-proliferative effect on PC12 cells.
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Fig. 5.
Anti-proliferative effects of ALK7 activation
in PC12 cells. A, assay of cell viability. The
indicated cells were pulsed with [3H]thymidine and then
cultured for 48 h with the indicated concentrations of
doxycycline. The results are expressed relative to control as
averages ± S.D. B, assay of DNA synthesis. The
indicated cells were cultured for 48 h with the indicated
concentrations of doxycycline and then pulsed with
[3H]thymidine. The results are expressed relative to
control as averages ± S.D.
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Fig. 6.
Effects of ALK7 signaling on PC12 cell
morphology, actin filaments, and neuronal differentiation.
A, ALK7-PC12 cells were cultured for 4 days in the absence
(control) or presence (+Dox) of doxycycline and
then visualized by phase contrast illumination (left panels)
or stained with Alexa488-conjugated phalloidin (right
panels). Note the marked effects on cell morphology produced by
ALK7 activation (black arrows, left), paralleled
by a decrease in actin filament staining in the same cells (white
arrows, right). B, ALK5-PC12 cells,
in contrast, do not show alterations in cell morphology or actin
filament staining 4 days after doxycycline treatment.
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Fig. 7.
Accelerated neuronal differentiation in
response to NGF after activation of ALK7 signaling. ALK7-PC12
cells were cultured for 4 days in the absence (control) or
presence (Dox) of 1 µg/ml doxycycline prior to stimulation
with 50 ng/ml NGF for 12 h (left panels) or 16 days
(right panels). Note that many more cells (50%) had already
initiated neuronal differentiation in doxycycline-treated cultures
12 h after NGF addition compared with controls (10%). The extent
of differentiation 16 days after NGF treatment was also more pronounced
in cells exposed to ALK7 activity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, i.e. ALK4 and ALK5, anticipated further similarities in the signaling mechanisms of these receptors. In agreement with this, we saw very
comparable signaling profiles following ALK7 and ALK5 activation in
PC12 cells, both in the type of Smad proteins and MAP kinases activated
as well as in the transcriptional responses elicited on a variety of
TGF-
responsive reporters. A recent study failed to detect
activation of Smad2 by a constitutively active ALK7 receptor (28),
which is surprising given the universal role played by this Smad in
downstream signaling by receptors of the TGF-
and activin subfamily.
In addition to the cell type used, a major difference between the
present and previous studies that utilized constitutively active forms
of type I receptors lies in our ability to control the onset of
receptor activity by stimulation with doxycycline. This has allowed us
to study the acute effects of ALK7 activation, something that has not
been possible in previous experiments that relied on heterogeneous
populations of transiently transfected cells.
superfamily the ability to control the cell cycle and inhibit
cell division. Arrest of proliferation is in many neuronal systems a
prerequisite for cell differentiation. The effects seen here on PC12
cells suggest that ALK7 signaling may play a similar role in
vivo, for example, facilitating the differentiation of mitotically
active neuronal precursors. Intriguingly, a site of abundant expression
of ALK7 is the developing postnatal cerebellum (9), where the onset of
ALK7 expression coincides with cessation of mitosis and neuronal
differentiation of granule and Purkinje cells. Together, these
observations suggest the possibility that one or more cell types in the
developing cerebellum may be a source of ALK7 ligand that, upon
activation of the ALK7 receptor complex on precursor cells, induces or
facilitates cell cycle exit and neuronal differentiation.
has recently been found to regulate the
levels of Rho GTPases, and inhibition of Rho function was shown to
block the ability of TGF-
1 to induce cytoskeletal reorganization in
transformed fibroblasts (29). Although the precise mechanisms by which
TGF-
proteins affect the cytoskeleton remain unknown, these and
other studies offer possible biochemical links between type I receptor
signaling and actin reorganization.
and activin receptors. The biological effects of ALK7 signaling in PC12
cells suggest, however, that some of the signals transmitted by this
receptor are distinct, as illustrated by its unique ability to induce
morphological and cytoskeletal changes in these cells. Together with
its predominantly neuronal pattern of expression, these findings
indicate that ALK7 may participate in the control of proliferation of
neuronal precursors and morphological differentiation of postmitotic
neurons in several regions of the central nervous system.
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ACKNOWLEDGEMENTS |
---|
We are grateful to L. Attisano and J. L. Wrana for the T204D-ALK5 mutant, J. Massagué for the p3TP-Lux plasmid, and H. Bujard and M. Gossen for the tetracycline system vectors. We also thank Ann-Sofie Nilson and Anita Morén for help with protein purification, DNA sequencing, and generation of the Smad antibodies, Annika Ahlsén for additional technical assistance, and Xiaoli Li-Ellström for secretarial help.
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FOOTNOTES |
---|
* This work was supported by Swedish Cancer Society Grant 3474-B97-05XBC, by funds from Petrus & Agneta Hedlunds Stiftelsen, Göran Gustafssons Stiftelsen, Swedish National Board for Laboratory Animals, and the Karolinska Institute (to C. F. I.), and by European Union TMR Grant ERBFMRXCT980216 (to P. t. D.).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.
§ Supported by the Swedish National Network in Neuroscience.
To whom correspondence should be addressed. E-mail:
carlos@cajal.mbb.ki.se.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M005200200
2 H. Jörnvall, A. Blokzijl, P. ten Dijke, and C. F. Ibáñez, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: TGF, transforming growth factor; BMP, bone morphogenetic protein; GDF, growth and differentiation factor; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; ALK7, activin receptor-like kinase 7; NGF, nerve growth factor; HA, hemagglutinin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; MAP, mitogen-activated protein.
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