The Polycystic Kidney Disease 1 Gene Product Modulates Wnt
Signaling*
Emily
Kim
§,
Thierry
Arnould¶,
Lorenz K.
Sellin¶,
Thomas
Benzing¶,
Melinda J.
Fan
,
Wolfram
Grüning¶,
Sergei Y.
Sokol
,
Iain
Drummond**, and
Gerd
Walz¶
From the
Laboratory of Molecular and Developmental
Neuroscience, Massachusetts General Hospital, Harvard Medical School,
Boston, Massachusetts 02114, the ¶ Renal Division and the
Department of Molecular Medicine, Beth Israel Deaconess Medical
Center, Harvard Medical School, Boston, Massachusetts 02215, and the
** Renal Unit, Massachusetts General Hospital, Harvard Medical School,
Boston, Massachusetts 02114
 |
ABSTRACT |
Two distinct signaling pathways, involving Wnt
signaling and polycystin, have been found to be critical for normal
kidney development. Renal tubulogenesis requires the presence of
certain Wnt proteins, whereas mutations in polycystin impede the
terminal differentiation of renal tubular epithelial cells, causing the development of large cystic kidneys that characterize autosomal dominant polycystic kidney disease. Polycystin is an integral membrane
protein, consisting of several extracellular motifs indicative of
cell-cell and cell-matrix interactions, coupled through multiple transmembrane domains to a functionally active cytoplasmic domain. We
report here that expression of the C-terminal cytoplasmic domain of
polycystin stabilizes soluble endogenous
-catenin and stimulates TCF-dependent gene transcription in human embryonic kidney
cells. Microinjection of the polycystin C-terminal cytoplasmic domain induces dorsalization in zebrafish. Our findings suggest that polycystin has the capacity to modulate Wnt signaling during renal development.
 |
INTRODUCTION |
The kidney is widely used as a model system to study the
intricacies of tissue induction underlying vertebrate organogenesis. Kidney development begins with the condensation of mesenchyme around
the ureteric bud in response to a signal from the ureter. Through
reciprocal interactions between the ureteric bud and the mesenchyme,
the metanephric mesenchyme evolves into the tubular epithelium that,
together with a glomerulus, constitutes the mature nephron of the
mammalian kidney (reviewed in Ref. 1). Embryonic kidney induction is
thought to require the presence of Wnts, a highly conserved family of
developmentally important secreted signaling molecules involved in
embryonic induction, generation of cell polarity, and the specification
of cell fate (reviewed in Refs. 2 and 3). The proposed pathway for Wnt
signaling involves the inhibition of glycogen synthase kinase
(GSK)1-3
and the
consequent posttranslational stabilization of soluble
-catenin,
leading to its accumulation in the cytoplasm and nucleus (reviewed in
Ref. 3). In the nucleus,
-catenin interacts with members of the
TCF/LEF family of transcription factors to regulate gene expression (4,
5). Certain Wnt family members appear to mediate renal morphogenesis.
Wnt-1, a Wnt family member not expressed in the kidney, nevertheless
induces metanephric mesenchyme to differentiate into glomerular and
renal tubular epithelia (6), whereas contact with the metanephric
mesenchyme maintains Wnt-11 expression at the tip of the ureteric bud
(7). Mesenchymal expression of Wnt-4 is required for kidney
tubulogenesis; Wnt-4
/
mice fail to form pretubular cell
aggregates, a requisite stage of early tubule formation (8). Thus, in
the developing kidney, Wnts appear to be involved in the reciprocal
interactions between the ureteric bud and surrounding mesenchyme that
enable the latter to differentiate into epithelial tubule cells.
Polycystin, the gene product of PKD1, is an integral
membrane protein with 11 putative transmembrane helixes, an N-terminal extracellular region that contains motifs characteristic of cell-cell and cell-matrix interactions, and a C-terminal cytoplasmic domain of
226 amino acids (9, 10). Mutations of polycystin account for the
majority of patients with autosomal dominant polycystic kidney disease,
a common hereditary disease (1:1,000) of slowly progressive epithelial
cyst formation. Mice lacking polycystin die perinatally with massively
enlarged cystic kidneys. Analysis of PKD1
/
mice
indicates that polycystin is required during renal development for the
elongation and maturation of tubular structures (11). Further
functional characterization of polycystin and the genetic manipulation
of full-length polycystin has been hindered by its complexity. To gain
insight into the function of polycystin, we have begun to characterize
the signaling pathways that are activated by the C-terminal domain of
polycystin, and have recently demonstrated that the C-terminal 226 amino acids of polycystin trigger activation of the transcription
factor AP-1 (12). This study demonstrates that the C-terminal
cytoplasmic domain of polycystin activates Wnt signaling.
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EXPERIMENTAL PROCEDURES |
Reagents and Plasmids--
MG132 (ProScript), lithium acetate
(Sigma), cycloheximide (Sigma), and human epidermal growth factor (EGF)
(Clonetics) were used at concentrations as indicated. The C-terminal
domains of polycystin were expressed as membrane-bound fusion proteins
using CD16-CD7 domains or the leader sequence of CD5 fused to the CH2 and CH3 domain of human IgG1 followed by the transmembrane
region of CD7 as described previously (12, 16). The AP-1 and Siamois promoter constructs have been recently described (12, 13). The
His6-tagged c-Jun and hemagglutinin (HA)-tagged ubiquitin were kindly provided by D. Bohmann, the HA-tagged GSK-3
by J.R. Woodgett, and the EGF receptor HER453 by A. Ullrich. The cDNAs encoding the CD16.7-polycystin fusions were cut with XbaI
and inserted in frame into the Spe1 site of the pXT7 vector,
a derivative of pGEM4Z (Promega) and pSP64T. The GST-Axin 495-590
fusion protein contains amino acids 495 to 590 of mouse Axin fused to
glutathione S-transferase (GST) and was generated by
polymerase chain reaction, utilizing a myc-tagged full-length mouse
axin as a template (kindly provided by F. Costantini).
Western Blot Analysis--
HEK 293T cells seeded in 6-well
plates were transiently transfected by the calcium phosphate method.
After incubation for 24 h, cells were lysed in sample buffer,
fractionated on SDS-PAGE, and electroblotted to polyvinylidene
difluoride membrane (NEN Life Science Products). For the time course,
cells were incubated with cycloheximide for the indicated durations
after pretreatment with lithium acetate or MG132 for 3 h. Western
blot analysis was performed with M2 anti-Flag monoclonal antibody or
anti-
-catenin followed by incubation with horseradish
peroxidase-coupled sheep anti-mouse immunoglobulin (Dako). Immobilized
antibodies were detected by chemiluminescence (Pierce).
Subcellular Fractionation--
HEK 293T cells seeded in 10-cm
plates were transiently transfected with the indicated constructs.
After incubation for 24 h, cells were homogenized in 0.5 ml of 250 mM sucrose, 10 mM HEPES (pH 7.4), 2 mM MgCl2, 1 mM EGTA, 0.5 mM EDTA, 2 mM Na3VO4,
containing 44 µg/ml phenylmethylsulfonyl fluoride, and protease
inhibitor mixture (Boehringer Mannheim). After two successive
centrifugations at 1,000 × g for 10 min at 4 °C,
the anucleated supernatant was centrifuged at 100,000 × g at 4 °C for 1 h. The supernatant (S100 soluble
fraction) was removed and the pellet (P100 membrane fraction) was
resuspended in 0.1 ml of solubilization buffer (homogenization buffer
containing 1% Triton X-100 and 1% SDS). After determining protein
concentrations by the Bio-Rad DC protein assay, subcellular fractions
containing equal amounts of protein were concentrated by acetone
precipitation, eluted by heating in SDS-PAGE sample buffer, and
subjected to SDS-PAGE and immunoblot analysis using an anti-
-catenin
monoclonal antibody (Santa Cruz Biotechnology, Inc.).
Ubiquitination Assay--
HEK 293T cells seeded in 10-cm plates
were transiently transfected with the indicated constructs. After
incubation for 24 h, cells were incubated with MG132 or medium
alone for 3 h, lysed in 6 M guanidinium-HCl, 0.1 M
Na2HPO4/NaH2PO4 (pH
8.0), 5 mM imidazole, and sonicated for 1 min. After
centrifugation for 15 min at 4 °C, the His6-tagged c-Jun
conjugates were precipitated from the cleared lysate with
Ni2+-NTA-agarose (Qiagen) for 2 h at room temperature.
Complexes were washed with 8 M urea, 0.1 M
Na2HPO4/NaH2PO4 (pH
6.3), 0.01 M Tris, pH 8.0, and resuspended in sample
buffer. Proteins were fractionated on SDS-PAGE, electroblotted to
polyvinylidene difluoride membrane, and immunostained with rabbit
polyclonal anti-HA serum (Santa Cruz) or rabbit polyclonal
anti-Jun/AP-1 serum (Santa Cruz), followed by incubation with
horseradish peroxidase-coupled anti-rabbit immunoglobulin (Amersham
Pharmacia Biotech). Immobilized antibodies were detected by
chemiluminescence (Pierce).
In Vitro Binding
Assay--
[35S]Methionine-labeled GSK-3
was
generated in a 40-µl reaction mix, using the Promega TNT system
following the instructions of the manufacturer. 10-µl of the reaction
mix was then incubated with 2 µg of GST-PKD 115-226 or GST-Axin
495-590, and immobilized on glutathione-Sepharose in the presence of
450 µl reaction buffer (50 mM potassium phosphate, pH
7.5, 150 mM KCl, 1 mM MgCl2, 10% (v/v) glycerol, 1% Triton X-100, and protease inhibitors). The reaction mix was incubated for 2 h, washed three times in reaction buffer, and separated on a 10% SDS acrylamide gel. Radiolabeled GSK-3
was detected by autoradiography. The amount of bound GSK-3
was compared with 5% of labeled input protein equivalent to 0.5 µl
of the labeling mix.
In Vivo Coimmunoprecipitation--
HEK 293T cells were
transiently transfected with 5 µg of plasmid encoding HA-tagged
GSK-3
and 5 µg of plasmids encoding sIg.7, sIg.7-PKD 115-226
(16), or myc-Axin (kindly provided by F. Costantini) by the calcium
phosphate method. After incubation for 24 h, cells were washed
twice with phosphate-buffered saline and then lysed in 1 ml of 1%
Triton X-100, 0.5% Nonidet P-40 buffer containing 150 mM
KCl, 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, and
protease inhibitors. Cell lysates containing equal amounts of total
protein were incubated for 4 h at 4 °C with 30 µl of protein
G-Sepharose beads (Amersham Pharmacia Biotech); the myc-Axin containing
lysate was additionally incubated with 5 µg of anti-myc mouse
monoclonal antibody (Calbiochem). The beads were washed extensively
with lysis buffer, and bound proteins were fractionated by 10%
SDS-PAGE. Western blot analysis was performed with anti-HA rabbit
polyclonal antibody (Santa Cruz) followed by incubation with
horseradish peroxidase-coupled donkey anti-rabbit immunoglobulin
(Amersham Pharmacia Biotech). Immobilized antibodies were detected by
chemiluminescence (Pierce).
GSK-3 Kinase Assay--
HEK 293T cells were transfected with
plasmids encoding CD16.7-PKD 115-226 or control CD16.7 as indicated.
Total DNA amount was titrated to 10 µg with control vector. Cells
from one 10-cm dish were lysed 24 h after transfection in 1 ml of
cold lysis buffer containing 1% Triton X-100, 150 mM NaCl,
10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4,
and a protease inhibitor mixture (Boehringer Mannheim). After
centrifugation for 15 min at 4 °C, GSK-3
was immunoprecipitated
from the cleared lysate with 5 µg of monoclonal anti-GSK-3
antibody (Transduction Laboratories) for 2 h at 4 °C. Immune
complexes were immobilized by adding 40 µl of Gamma-Bind-Sepharose
(Amersham Pharmacia Biotech) and washed twice with 800 µl of lysis
buffer. During the second wash, 200 µl of resuspended beads were
removed for Western blot analysis. Immunoprecipitates were washed with
500 µl of kinase reaction buffer (20 mM Tris-HCl (pH
7.5), 10 mM MgCl2, 5 mM
dithiothreitol) and resuspended in 50 µl of kinase reaction buffer
containing 50 µM of cAMP-responsive element binding
phosphopeptide (NEB). The assay was carried out in the presence of 20 µM unlabeled ATP and 10 µCi [
-32P]ATP
for 30 min at 30 °C. A 25-µl aliquot was applied to a
phosphocellulose membrane spin column (Pierce), washed with 500 µl of
75 µM phosphoric acid, and assayed by scintillation
spectrophotometry. GSK-3
immunoprecipitates, removed before the
kinase assay, were analyzed by SDS-PAGE and Western blot, using the
anti-GSK-3
monoclonal antibody in combination with a goat anti-mouse
horseradish peroxidase antibody (Dako) and enhanced chemiluminescence (Pierce).
Luciferase Assay--
HEK 293T cells seeded in 12-well plates
were transiently transfected with a luciferase reporter construct, a
-galactosidase expression vector (kindly provided by C. Cepko), and
vectors directing the expression of CD16.7 fusion proteins, Wnt
signaling components, and the EGF receptor HER453 as indicated. Total
DNA amount was 3 µg/well. Cells were serum starved for 24 h,
harvested in cold phosphate-buffered saline, and lysed in 100 µl of
reporter lysis buffer (Promega) for 15 min at room temperature. Lysates
were centrifuged at 14,000 rpm for 3 min to remove insoluble material. Luciferase activity was determined using a commercial assay system (Promega) following the manufacturer's instructions, and normalized for
-galactosidase activity to correct for the transfection
efficiency. EGF at 20 ng/ml was added for 8 h before the assay.
Embryo Microinjection--
Zebrafish embryos were injected at
the 1-2 cell stage with 10-20 pg of either DNA expression vectors or
capped mRNA. For DNA expression vector studies, embryos were
injected with CD16.7-PKD 115-226 or the control vector CD16.7 in 200 mM KCl and 0.1% phenol red as a tracer. Capped mRNA
for injection was synthesized using a MessageMachine kit (Ambion) and
T7 polymerase. RNA was injected in a solution of 0.1% phenol red.
Embryos were examined during epiboly, 5-10 h post-fertilization (hpf),
and at 24 h.
 |
RESULTS |
Polycystin Stabilizes Soluble
-Catenin--
Recent studies
indicate that Wnt signal transduction involves the inhibition of
GSK-3
activity, leading to a posttranslational stabilization of
soluble
-catenin that is commonly used as an indicator for Wnt
signaling. We found that the C-terminal cytoplasmic domain of
polycystin significantly elevated total
-catenin steady-state protein levels (Fig. 1a) in
HEK 293T cells that were transiently cotransfected with a Flag-tagged
-catenin plasmid together with cDNA encoding the C-terminal
cytoplasmic tail of polycystin, CD16.7-PKD 1-226. The C-terminal
domain of polycystin was fused to the extracellular domain of CD16 and
the transmembrane domain of CD7, forming a heterologous integral
membrane protein. This approach has been used to delineate effector
functions and protein-protein interactions of various receptor
cytoplasmic domains by targeting them to the plasma membrane (14-16).
The accumulation of
-catenin was further localized to the C-terminal
113 amino acids of polycystin, whereas two controls, CD16.7, a
construct with a stop codon shortly after the transmembrane domain and
CD16.7-PKD 1-92, a construct containing the N-terminal 92 amino acids
of the C-terminal cytoplasmic domain of polycystin, had no detectable
effect on
-catenin levels. As a positive control, exposure to
lithium, a known inhibitor of GSK-3
activity (17, 18), stabilized
-catenin in CD16.7-transfected HEK 293T cells. Cells were
cotransfected with a constant amount of plasmid DNA encoding
Flag-tagged green fluorescent protein (F.gfp) to control for
loading and transfection efficiency.

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Fig. 1.
The C-terminal domain of polycystin
increases -catenin stability and inhibits
c-Jun ubiquitination. a, the C-terminal domain of
polycystin increases -catenin levels. Cell lysates of HEK 293T cells
transiently cotransfected with Flag-tagged -catenin, F.gfp, and
CD16.7 or CD16.7-PKD 1-92 (controls), CD16.7-PKD 1-226, or CD16.7-PKD
115-226 were immunoblotted with an anti-Flag antibody. b,
-catenin is stabilized by MG132, lithium, and polycystin. HEK 293T
cells were transiently transfected with control plasmids CD16.7 or
CD16.7-PKD 115-226 and incubated with 40 µg/ml cycloheximide for 0, 1, 2, 4, 6, and 8 h after pretreatment with 40 µM
MG132, 20 mM lithium acetate, or medium alone for 3 h
as indicated. Cell lysates were immunoblotted with an anti- -catenin
antibody. In all experiments, coexpression of F.gfp was utilized to
monitor transfection efficiency and equal loading. F.gfp was stable
over the course of 8 h, and not affected by lithium, MG132, or
polycystin. A representative Western blot analysis of F.gfp expression
is shown at the bottom of the panel. c, the
C-terminal domain of polycystin increases soluble but not membrane
-catenin levels. HEK 293T cells transiently transfected with CD16.7,
CD16.7-PKD 1-226, or CD16.7-PKD 115-226 were lysed, anucleated, and
fractionated at 100,000 × g. Lysates of subcellular
fractions were immunoblotted with an anti- -catenin antibody
(top). Amounts of cytoplasmic -catenin (black
bars) were quantified by densitometric tracing after normalizing
for membraneous fractions (white bars) (bottom).
d, c-Jun ubiquitination is reduced in the presence of
C-terminal polycystin. Cell extracts of HEK 293T cells transiently
cotransfected with CD16.7 control, CD16.7-PKD 115-226,
His6-tagged c-Jun, HA-tagged ubiquitin constructs as
indicated and incubated with 40 µM MG132 or medium alone
for 3 h. His6-tagged c-Jun conjugates were purified by
nickel-chelate affinity chromatography and immunoblotted with an
anti-HA antibody to detect c-Jun-ubiquitin conjugates. The lower
panel shows the same blot developed with a polyclonal antibody
against c-Jun.
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To demonstrate that the accumulation of
-catenin was mediated
through the stabilization of
-catenin, we examined the effect of
polycystin on the half-life of endogenous
-catenin while inhibiting protein synthesis. HEK 293T cells were transfected with a control plasmid CD16.7 or the construct CD16.7-PKD 115-226 and treated with
the protein synthesis inhibitor cycloheximide (Fig. 1b). HEK
293T cells transfected with CD16.7 showed a gradual decline of
-catenin protein levels over the course of 8 h.
-catenin is
subject to ubiquitin-dependent proteasome degradation, a
process downstream of GSK-3
that can be retarded by inhibiting
ubiquitination or the proteasome (19). Preincubation of
CD16.7-transfected cells with lithium, an inhibitor of GSK-3
and
ubiquitination, or MG132, a proteasome inhibitor, prolonged the
half-life of
-catenin. A similar prolongation of
-catenin
half-life was seen in HEK 293T cells transfected with CD16.7-PKD
115-226. To determine whether polycystin increased the cytoplasmic
fraction of
-catenin, transfected HEK 293T cells were fractionated
and endogenous
-catenin was visualized. An increase in cytoplasmic
-catenin levels was observed in HEK 293T cells transfected with
CD16.7-PKD 1-226 and 115-226, but not control transfected cells (Fig.
1c).
Polycystin Inhibits Ubiquitination of c-Jun--
The observed
stabilization of
-catenin by polycystin prompted us to examine
whether polycystin inhibits the ubiquitination of other proteins
regulated by GSK-3
. The transcription factor c-Jun is regulated at
the posttranslational level by ubiquitination and subsequent
proteolysis (20). Both the stability and activity of c-Jun are
regulated by mitogen-activated protein kinases and GSK-3
(18, 21,
22). To investigate whether polycystin affects the ubiquitination of
c-Jun in vivo, we coexpressed the C-terminal 112 amino acids
of polycystin with histidine-tagged c-Jun and HA-tagged ubiquitin in
HEK 293T cells. After purification of c-Jun under denaturing
conditions, multi-ubiquitinated forms of c-Jun were detected by
immunoblotting. We found that polycystin reduced the
multi-ubiquitination of c-Jun, even in the presence of the proteasome
inhibitor MG132 (Fig. 1d). This is consistent with a
mechanism where polycystin suppresses the ubiquitination and subsequent
degradation of c-Jun.
Polycystin Inhibits GSK-3
Activity--
To further discriminate
the mechanism through which the C terminus of polycystin stabilized
-catenin, we examined the relationship of polycystin and GSK-3
.
Polycystin does not directly interact with GSK-3
in vitro
or in vivo. Bacterially expressed recombinant PKD 115-226
fused with GST did not bind GSK-3
in vitro, although recombinant GST-Axin, a known binding partner, clearly interacted with
GSK-3
(Fig. 2a). Similarly,
in vivo, a HA-tagged GSK-3
coimmunoprecipitated with
Axin, but not with the C terminus of polycystin in transfected HEK 293T
cells (Fig. 2b). However, polycystin inhibited endogenous
GSK-3
in a cellular system, suggesting that polycystin requires an
intermediary molecule to modulate GSK-3
activity. In HEK 293T cells
transfected with CD16.7-PKD 115-226, GSK-3
kinase activity was
decreased by 33-45% compared with cells transfected with the controls
CD16.7 or CD16.7-PKD 1-92 (Fig. 2c). This inhibition of GSK
activity is comparable with that reported for Wg in mouse fibroblasts
which is sufficient to promote Wnt signaling (23), and to the
inhibitory effect of insulin-like growth factor-1 or insulin in muscle
cells (24).

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Fig. 2.
C-terminal polycystin inhibits GSK-3 activity
in vivo, but does not associate with
GSK-3 in vitro or in
vivo. a, Axin but not C-terminal polycystin binds
GSK-3 in vitro. The amount of
[35S]methionine labeled GSK-3 that bound to
glutathione-Sepharose (GS), to the last 112 amino acids of
polycystin fused to glutathione S-transferase (GST-PKD
115-226), or to GST-Axin was compared with 5% of labeled input
protein. The GST fusion proteins were immobilized on
glutathione-Sepharose and incubated with a mixture of equal amounts of
35S-GSK-3 . b, Axin but not C-terminal
polycystin binds GSK-3 in vivo. The C terminus of
polycystin was fused to sIg.7, a construct containing the leader
sequence of human CD5 followed by the CH2 and
CH3 domain of human IgG1, and the transmembrane
region of human CD7. HEK 293T cells were transiently transfected with
HA-tagged GSK-3 and sIg.7 (control), sIg.7-PKD 115-226,
or myc-Axin. Lysates were immunoprecipitated with protein G and
blotted with anti-HA polyclonal antibody. The myc-Axin-containing
lysate was additionally incubated with 5 µg of anti-myc mouse
monoclonal antibody before immunoprecipitation with protein G. C, C-terminal polycystin inhibits GSK-3 activity in
vivo. HEK 293T cells, transfected with control CD16.7, CD16.7-PKD
1-92, or CD16.7-PKD 115-226 (left panel), were lysed and
immunoprecipitated with an anti-GSK-3 antibody, and assayed for
kinase activity in two independent transfections. The amounts of
precipitated endogenous GSK-3 were demonstrated by Western blot
analysis. HEK 293T cells, transfected with control CD16.7 and
increasing amounts of CD16.7-PKD 115-226 (right panel),
were lysed and immunoprecipitated with an anti-GSK-3
antibody and assayed for kinase activity. Equal
amounts of precipitated endogenous GSK-3 were demonstrated by
Western blot analysis.
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Polycystin Induces TCF-dependent Gene
Transcription--
To establish that the inhibition of GSK-3
activity by polycystin functionally couples to downstream events of Wnt
signaling, the formation of transcriptional active
-catenin·TCF
complexes was assayed by contransfecting a Xenopus Siamois
luciferase reporter construct (
833pSiaLuc) into HEK 293T cells. The
promoter of the dorsalizing homeobox gene Siamois is a
direct target for the
-catenin·TCF complex (25). Both CD16.7-PKD
1-226 and CD16.7-PKD 115-226, but not CD16.7-PKD 1-92, activated the
Siamois promoter 10-12-fold (Fig.
3a). In contrast, CD16.7-PKD
1-226 and CD16.7-PKD 1-92, but not CD16.7-PKD 115-226 induced
AP-1-dependent transcription (Fig. 3b).
Wnt-dependent activation of the Siamois promoter
requires the most proximal TCF-binding site (13). A truncated
Siamois promoter construct (
245pSiaLuc) with a mutation of
the proximal activating TCF-binding site showed significantly decreased
responsiveness to polycystin (Fig.
4b) compared with the
unmutated, truncated Siamois promoter (Fig. 4a),
indicating that polycystin-mediated activation of the
Siamois promoter depends upon the binding of
-catenin·TCF complexes. The differences in signaling mediated between polycystin and EGF are demonstrated in Fig. 4c. EGF
is a GSK-3 inhibitor that does not activate Wnt signaling. EGF
stimulation of HEK 293T cells expressing EGF receptor did not activate
the Siamois, but activated the AP-1 promoter 3-4-fold. In
contrast, polycystin mediated a 10-12-fold induction of both AP-1 and
Siamois promoter constructs. Thus, unlike growth factors
such as EGF, which inhibit GSK-3
without activating the Wnt
signaling pathway, polycystin both inhibits GSK-3
and activates Wnt
signaling, a signaling pattern resembling that of canonical Wnt
signaling molecules. Furthermore, polycystin augmented the activity of
four Wnt signaling components, XWnt8, XDsh, rFz2, and
-catenin (Fig.
5).

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Fig. 3.
Siamois and AP-1 are differentially activated
by the C-terminal domain of polycystin. HEK 293T cells were
cotransfected with the indicated constructs, a Siamois or
AP-1 luciferase reporter construct, and a -galactosidase expression
vector. Transactivation was determined after 24 h of incubation
and expressed as relative light units (RLU) after
normalization for -galactosidase activity. a, the
Siamois promoter was activated by cotransfection with
CD16.7-PKD 1-226 or CD16.7-PKD 115-226, but not CD16.7-PKD 1-92;
b, in contrast, the AP-1 promoter was activated by
CD16.7-PKD 1-226 or CD16.7-PKD 1-92 but not by CD16.7-PKD
115-226.
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Fig. 4.
Polycystin-mediated Siamois activation
depends on the presence of a functional TCF binding site.
a, the truncated Siamois promoter construct
( 245pSiaLuc) containing only one activating TCF site was activated by
CD16.7-PKD 1-226 or CD16.7-PKD 115-226 in HEK 293T cells.
b, the polycystin-mediated responses were drastically
reduced in the 245pSiaLuc construct containing a point mutation in
the activating TCF site, demonstrating that transactivation of the
Siamois promoter by polycystin depends critically on functionally
active TCF binding sites. c, EGF stimulates the AP-1
promoter but fails to trigger the Siamois promoter. In this experiment,
HEK 293T cells were cotransfected with the human EGF receptor and
either the AP-1 or the Siamois promoter construct, followed by
stimulation with EGF (20 ng/ml for 8 h). In contrast, polycystin
(CD16.7-PKD 1-226) induces a greater than 10-fold activation of both
promoter constructs. The depicted results are the composite of several
experiments performed in triplicate, and represent mean ± S.D.
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Fig. 5.
Polycystin augments Wnt signaling.
a, cotransfection with CD16.7-PKD 115-226 augmented the
activation of the Siamois promoter by XWnt8, XDsh, rFz2, and
-catenin in HEK 293T cells; b, the augmentation of Wnt
signaling by polycystin was most pronounced for rFz2, resulting in a
greater than 3.5-fold increase of promoter activity. All experiments
were performed in triplicate; results represent mean ± S.D.
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Polycystin Dorsalizes Zebrafish Embryos--
Previous work has
shown that Wnt signaling is involved in specifying dorsal anterior
fates and antagonizing signals specifying ventral posterior cell fates
in the early embryonic axis (26-28). In zebrafish,
-catenin induces
an ectopic embryonic axis, whereas lithium produces hyperdorsalization
(29, 30). To examine whether polycystin has a similar activity during
early zebrafish development, embryos were injected at the 1-2 cell
stage with either the control DNA expression vector CD16.7 or
CD16.7-PKD 115-226. Control injected embryos showed no morphological
abnormalities at any stage of development indicating that expression of
the CD16.7 fusion protein does not disrupt normal development (Fig.
6a). In contrast, a marked
dorsalizing effect of CD16.7-PKD 115-226 DNA was observed in injected
embryos examined at 24 hpf (Fig. 6b). Embryos formed relatively normal head and brain tissue but invariably showed defects
in posterior trunk and tail development. Injections of RNA encoding the
same portion of the polycystin C terminus resulted in similarly
dorsalized embryos (Fig. 6c). In the most extreme cases the
embryo failed to complete epiboly resulting in a constriction around
the yolk cell and an exposed yolk cell mass at 24 hpf (Fig. 6c). Trunk muscle and pronephric duct development was
detectable although the form and organization of these tissues was
disrupted (data not shown). In the least severely affected embryos, the tail formed, but defects in the development of ventral tissue just
posterior to the yolk extension were always observed (Fig. 6d). The results indicate that when ectopically expressed in
the context of early zebrafish development, the polycystin C terminus can have hyperdorsalizing effects, similar to that seen with GSK-3
inhibition by lithium.

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Fig. 6.
Expression of C-terminal polycystin
dorsalizes zebrafish embryos. Embryos were injected at the 1-2
cell stage. a, control vector CD16.7 injection has no effect
on zebrafish development at 24 hpf; b, injection of
CD16.7-PKD 115-226 severely disrupts trunk and tail development,
whereas dorsal anterior structures are less affected. In two separate
experiments, injection of CD16.7-PKD 115-226 RNA also resulted
in a failure in posterior development. c and d,
in a representative experiment, five of eight embryos did not develop
posterior tissue (c) and two failed to complete epiboly
resulting in embryos with exposed yolk at 24 hpf
(arrows, c). The remaining three embryos
showed similar but less severe defects in the development of ventral
posterior tissue (d).
|
|
 |
DISCUSSION |
Autosomal dominant polycystic kidney disease, the leading genetic
cause of renal failure, is primarily caused by mutations in polycystin.
Polycystin is required for normal tubulogenesis during renal
development, but its precise physiologic function is not yet defined.
Studies of PKD1
/
mutant mice indicate that early stages
of tubule morphogenesis, such as nephrogenic condensation and
epithelialization, remain intact (11). Expression of polycystin can be
detected at E14 in the ureteric bud, and subsequently in the S- and
Comma-shapes of the condensing mesenchyme (31-37). Cyst formation
begins at embryonic day 15.5 (E15.5) in proximal tubules and progresses
rapidly to replace the entire renal parenchyma. Wnt-4 is also required
for renal tubulogenesis during this time; at E15.5 the mesenchyme of
Wnt-4
/
mice fails to aggregate and differentiate,
lacking pretubular aggregates and more developed tubules (8). Kidneys
of Wnt-4
/
mice are growth retarded at E15.5 and agenic
at E18.5, consisting of undifferentiated mesenchyme interspersed with
branches of collecting duct epithelium. In the developing kidney, the
expression pattern of polycystin appears to overlap with at least two
known Wnt family members, Wnt-11, expressed in the ureteric bud (38),
and Wnt-4, expressed in mesenchymal aggregates undergoing epithelial
transition (8). Both spatially and temporally, Wnt and polycystin
localization and signaling events coincide, supporting the hypothesis
that polycystin may modulate Wnt signaling. Polycystin augmented the transcriptional activity of XWnt8, XDsh, rFz2, and
-catenin, indicating that polycystin may serve to reinforce or maintain Wnt
signaling during critical stages of renal tubulogenesis. The combinatorial interaction of Wnt signaling with other signaling pathways promotes functions that are different from the function of Wnt
molecules alone (reviewed in Ref. 2). Although Wnt family members
induce mesenchymal-epithelial conversion, cross-talk with polycystin
may be required at a later stage to maintain tubular structure,
polarity, and integrity. It is particularly interesting that
-catenin, a transcriptional mediator of Wnt signaling, recently has
been reported to regulate the reorganization of renal epithelial cell aggregates into long tubules (39).
Ectopic expression of many Wnt pathway components upstream of
-catenin can be mimicked by the overexpression of
-catenin in a
variety of systems, suggesting that the role of certain Wnt family
members is to increase levels of soluble
-catenin (40). Genetic and
biochemical studies indicate that secreted Wnts, through the family of
frizzled receptors, activate disheveled and inhibit glycogen synthase
kinase, to stabilize
-catenin and facilitate transcription of
certain target genes (reviewed in Refs. 2 and 3). In the absence of Wnt
signaling, cytoplasmic
-catenin is rapidly degraded in mammalian
cells by a process involving the adenomatous polyposis coli tumor
suppressor protein, ubiquitination, and proteasome (19, 41). Inhibition
of the ubiquitination-proteasome pathway or overexpression of
-catenin results in cytoplasmic accumulation of
-catenin and its
translocation into the nucleus (42). A modest inhibition of GSK-3
activity appears sufficient to transduce Wnt signaling, raising the
possibility that only a Wnt-sensitive subcellular GSK-3
pool is able
to regulate the stability of
-catenin (reviewed in Ref. 3). Our data
suggests that expression of the C-terminal domain of polycystin
stabilizes soluble
-catenin and increases the amount of
transcriptionally active
-catenin. Although GSK-3
activity was
reduced in the presence of polycystin, we were unable to detect a
direct interaction of the C terminus of polycystin with GSK-3
in vitro or in vivo, indicating that polycystin
does not directly inhibit GSK-3
kinase activity through a physical interaction.
A critical role for GSK-3
in early vertebrate pattern formation has
been clearly established in dorsoventral axis formation (43-45).
Lithium, an inhibitor of GSK-3
, dorsalizes zebrafish and
Xenopus (30, 46),
-catenin induces induction of a
complete secondary body axis in both species (29, 40), and some
upstream components of the Wnt-signaling pathway induce an ectopic
embryonic axis in Xenopus (reviewed in Ref. 3). A marked
dorsalizing effect of the terminal 112 amino acids of polycystin was
observed in zebrafish embryos. The polycystin phenotype strikingly
resembles the exaggerated dorsoanterior structures resulting from
lithium treatment, indicating that polycystin may similarly
inhibit GSK-3
during zebrafish development (30).
The ligand of polycystin remains unidentified; hence, it is still
unknown how extracellular events may influence the capacity of
polycystin to modulate Wnt signaling. We speculate that ligand-receptor interactions governing the activity of the C-terminal portion of
polycystin will be found to influence cytoplasmic-signaling cascades
during critical stages of renal tubular development.
 |
FOOTNOTES |
*
This work was supported by a grant from the Polycystic
Kidney Research Foundation (to G.W.).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 Public Health Service Grant MH-01147.

To whom correspondence should be addressed. Fax: 617-667-1610;
E-mail: gwalz{at}bidmc.harvard.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
GSK, glycogen synthase kinase;
PKD, polycystic kidney disease;
HEK, human
embryonic kidney cells;
PAGE, polyacrylamide gel electrophoresis;
AP-1, activation protein-1;
GST, glutathione S-transferase;
F.gfp, Flag-tagged green fluorescent protein;
EGF, epidermal growth factor;
HA, hemagglutinin;
hpf, hours post-fertilization.
 |
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