From the Renal Division and the
Cardiology Division, Department of
Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School,
Boston, Massachusetts 02215 and the ¶ Laboratory of Molecular and
Developmental Neuroscience, Massachusetts General Hospital, Harvard
Medical School, Boston, Massachusetts 02114
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ABSTRACT |
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Autosomal dominant polycystic kidney
disease (ADPKD) is a common hereditary disorder that accounts
for 8-10% of end stage renal disease. PKD1, one of two recently
isolated ADPKD gene products, has been implicated in cell-cell and
cell-matrix interactions. However, the signaling pathway of PKD1
remains undefined. We found that the C-terminal 226 amino acids of PKD1
transactivate an AP-1 promoter construct in human embryonic kidney
cells (293T). PKD1-induced transcription is specific for AP-1; promoter
constructs containing cAMP response element-binding protein, c-Fos,
c-Myc, or NFB-binding sites are unaffected by PKD1. In
vitro kinase assays revealed that PKD1 triggers the activation of
c-Jun N-terminal kinase (JNK), but not of mitogen-activated protein
kinases p38 or p44. Dominant-negative Rac-1 and Cdc42 mutations
abrogated PKD1-mediated JNK and AP-1 activation, suggesting a critical
role for small GTP-binding proteins in PKD1-mediated signaling.
Several protein kinase C (PKC) inhibitors decreased PKD1-mediated
AP-1 activation. Conversely, expression of the C-terminal domain of
PKD1 increased PKC activity in 293T cells. A dominant-negative PKC
,
but not a dominant-negative PKC
or
, abrogated PKD1-mediated
AP-1 activation. These findings indicate that small GTP-binding
proteins and PKC
mediate PKD1-induced JNK/AP-1
activation, together comprising a signaling cascade that may regulate
renal tubulogenesis.
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INTRODUCTION |
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Mutations within the PKD1 gene on human chromosome 16p13.3 account for 85% of all diagnosed cases of ADPKD.1 PKD1 was recently cloned and found to be broadly expressed (1-5). The predicted PKD1 protein is a large glycoprotein with a signal peptide and multiple transmembrane domains. Its large extracellular region of approximately 2557 amino acids contains multiple protein motifs that are associated with cell-cell and cell-matrix interactions, such as leucine-rich repeats, a C-type lectin domain, immunoglobulin-like repeats, and four type III fibronectin-related domains (reviewed in Ref. 6). The short C-terminal cytoplasmic tail consists of a novel domain of 226 amino acids.
In normal human adult kidneys, cellular proliferation is a rare and unusual event. The bulk of renal mitogenesis and morphogenesis occurs before birth, with the remainder completed within the first years of life. In early development, PKD1 appears to regulate the proliferation and maturation of tubular epithelial cells. PKD1 is highly expressed in fetal kidney in areas of rapid proliferation and developmental induction, such as the ureteric bud and the condensating mesenchyme (5, 7). In renal cysts of ADPKD patients, absence of PKD1 is associated with the increased expression of proto-oncogenes and growth factor receptors such as c-fos, c-myc, c-K-ras, and c-erb B2 (8-12). Similar abnormalities have been observed in several animal models of polycystic kidney disease (13). These studies have established a critical role for PKD1 in signaling pathways controlling proto-oncogene expression, cellular proliferation, and differentiation of tubular epithelial cells.
Insight into the function of PKD1 has been hampered by its complexity. Full-length PKD1 has not yet been expressed in vivo (14), curtailing genetic manipulation of the intact protein. Recently, we have shown heterodimeric interaction between PKD1 and PKD2 fusion proteins (15), using a heterologous integral membrane protein fused to the C-terminal cytoplasmic domain of PKD1 and PKD2. This approach has been used to delineate the effector mechanisms of various cytoplasmic domains (16-21). In the present study, we demonstrate that the C-terminal cytoplasmic domain of PKD1 triggers the activation of AP-1, a transcription factor that modulates a variety of cellular programs, including growth response and apoptosis (reviewed in Ref. 22). Furthermore, PKD1-induced AP-1 activity was mediated at least in part through activation of protein kinase C and c-Jun N-terminal kinase (JNK). Thus, we have shown that PKD1, a protein essential for normal tubulogenesis, activates a signaling cascade involved in cellular proliferation, differentiation, and apoptosis.
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EXPERIMENTAL PROCEDURES |
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Reagents and Plasmids--
Genistein (Calbiochem), staurosporine
(Calbiochem), calphostin C (Calbiochem), BAPTA-acetoxymethyl ester
(Molecular Probes), wortmannin (Calbiochem), and human epidermal growth
factor (Clonetics) were used at concentrations as indicated. The
C-terminal domain of PKD1 was expressed as a CD16-CD7 fusion protein
(CD16.7.PKD1) (15). The control vector (CD16.7) encodes the
extracellular domain of CD16 followed by the CD7 transmembrane domain
and a short cytoplasmic tail. The AP-1 luciferase reporter construct of
four AP-1-binding sites (kindly provided by D. Moore), the collagenase
promoter reporter construct containing a single AP-1-binding site at
73/63 (kindly provided by N. H. Colburn), the Jun2TRE construct
of three 12-O-tetradecanoylphorbol-13-acetate-response elements (TRE) from the c-Jun promoter second TRE (kindly provided by
S. Lewis and S. Hyman), the NF
B construct of eight NF
B-binding sites (kindly provided by B. Seed), and the c-myc construct
of four c-myc-binding sites (derived from a construct kindly
provided by R. N. Eisenman) all contain DNA-binding sites followed
by either a minimal thymidine kinase or Rous sarcoma virus promoter
directing the expression of luciferase. The c-Fos promoter construct
(kindly provided by Bender GmBH) and the cAMP-response element promoter construct (kindly provided by K. Mayo) contain promoter regions of
c-Fos and
-inhibin, respectively. The hemagglutinin (HA)-tagged p38
and p44 were kindly provided by J. Pouysségur, HA-JNK1 was kindly
provided by M. Karin, the dominant-negative mutants of Rac-1
(Rac-1(N17)) and Cdc42 (Cdc42(N17)) were kindly provided by J. S. Gutkind, a dominant-negative form of PKC
was kindly provided by D. Rosson, and a dominant-negative form of PKC
II m217 was kindly
provided by C. E. Chalfant. The dominant-negative form of PKC
was generated through site-directed mutagenesis replacing the lysine at
377 with arginine (23).
Luciferase Assay--
293T cells seeded in 6-well plates were
transiently transfected with a luciferase reporter construct, a
-galactosidase expression vector (kindly provided by C. Cepko), and
a vector directing the expression of CD16.7.PKD1. Total DNA amount was
1-2.5 µg/well. Cells were serum starved for 24 h, harvested in
cold phosphate-buffered saline, and lysed in 200 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.
Pharmacological inhibitors were added for 8 h before the
assay.
In Vitro Kinase Assays--
The immune complex kinase assays
were carried out after co-transfecting 293T cells with HA-tagged MAP
kinases and the CD16.7.PKD1 construct at a 1:1 ratio. 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, the HA-tagged kinases were
immunoprecipitated from the cleared lysate with 5 µg of monoclonal
anti-HA antibody (Boehringer Mannheim) for 2 h at 4 °C. Immune
complexes were immobilized by adding 30 µl of Gamma-Bind Sepharose
(Pharmacia Biotech Inc.). The immune complexes were washed three times
in lysis buffer and twice in kinase reaction buffer (25 mM
Hepes, 20 mM MgCl2, 2 mM
dithiotreitol, 0.1 mM Na3VO4, pH
7.6). The immunoprecipitates were resuspended in 30 µl of kinase reaction buffer containing 3 µg of substrates phosphorylated heat- and acid-stable protein-1 (Stratagene) for HA-p38 and HA-p44 or GST-c-Jun (1-79) (Stratagene) for HA-JNK1. The assay was carried out
in the presence of 20 µM unlabeled ATP and 10 µCi of
[-32P]ATP for 30 min at 30 °C, stopped by the
addition of 30 µl of SDS sample buffer, and boiled for 5 min. The
reaction mixture was fractionated on a 12% SDS-PAGE. Phosphorylated
substrates were visualized by autoradiography and quantitated by
scanning with a Molecular Dynamics densitometer. JNK immunoprecipitates were analyzed by SDS-PAGE and Western blot, using the anti-HA monoclonal antibody in combination with a goat anti-mouse horseradish peroxidase antibody (Dako) and enhanced chemiluminescence (Pierce).
PKC Activity-- PKC activities were determined using a colorimetric PKC assay with neurogranin as a dye-labeled synthetic peptide substrate (Pierce). Cells were harvested and lysed on ice for 10 min in 45 µl of cold hypotonic buffer (1 mM Hepes, 5 mM MgCl2, 25 µg/ml leupeptin, 25 µg/ml pepstatin). Isotonicity was reestablished by adding 5 µl of Hepes (200 mM, pH 7.4) and 25 µl of an equilibrium buffer (20 mM Hepes, 5 mM MgCl2, 1 mM NaF, 0.1 mM Na3VO4). The PKC reaction was performed at 30 °C for 30 min using 10 µl of cleared lysates following the instructions of the manufacturer. The absorbance of the phosphorylated substrates was spectrophotometrically determined at 570 nm, and a microprotein assay (Bio-Rad) was used to normalize the PKC activities for the protein content.
Western Blot Analysis-- 293T cells were transiently transfected by the calcium phosphate method. After incubation for 24 h, cells were lysed in sample buffer, fractionated on SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (NEN Life Science Products). Western blot analysis was performed with an anti-CD16 monoclonal antibody followed by incubation with horseradish peroxidase-coupled goat anti-mouse immunoglobulin (Dako). Immobilized antibodies were detected by chemiluminescence (Pierce).
Statistical Analysis-- Results were expressed as means ± S.D. Analysis of variance with a subsequent Scheffe's test was used to determine significant difference in multiple comparisons. Values of p < 0.05 were considered to be significant.
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RESULTS AND DISCUSSION |
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Previous studies indicate that ADPKD epithelial cells arrest in a less than terminally differentiated state, leading to cystogenesis. To identify potential signaling pathways of PKD1, we coexpressed the cytoplasmic tail of PKD1 with luciferase reporter constructs transactivated by defined DNA-binding sites. The C-terminal 226 amino acids of PKD1 were fused to the extracellular domain of CD16 and the transmembrane domain of CD7. The combination of CD16 together with a CD7 transmembrane domain has previously been shown to target cytoplasmic domains to the plasma membrane without altering effector function (18-21) or protein-protein interaction, such as between PKD1 and PKD2 (15). The expression of CD16.7.PKD1, monitored by Western blot analysis, consistently triggered a 5-10-fold activation of the AP-1 reporter construct containing four AP-1-binding sites (Figs. 1, 3, and 5), and a more than 5-fold activation of a collagenase promoter construct containing a single AP-1 site (24) (Fig. 1). PKD1-mediated AP-1 activation was highly specific, involving only AP-1 and the Jun2TRE (Fig. 1). Cytoplasmically expressed FLAG-tagged PKD1, lacking both leader sequence and transmembrane domain, completely lost the ability to activate AP-1 (data not shown), suggesting that membrane localization of PKD1 is requisite for AP-1 activation. Likewise, a control protein of the extracellular domain of CD16 fused to the transmembrane domain of CD7 had no effect (Fig. 1). AP-1 activation induced by PKD1 exceeded activation by both epidermal growth factor and serum by 2-fold (data not shown). AP-1 is a transcriptional activator composed of Jun, Fos, or activating transcription factor 2 homodimers and heterodimers that bind to a common DNA sequence, the TRE. AP-1 is activated by a large variety of extracellular stimuli, including growth hormones and cytokines; its activity is controlled both at the transcriptional level and through post-translational modifications of c-Fos and c-Jun (reviewed in Ref. 22). Several kinase cascades have been demonstrated to regulate AP-1 activity (25), including the Hog1p homolog p38, the mitogen-activated kinases p42 and p44, and members of the JNK family (reviewed in Ref. 26).
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To further delineate the signaling pathway through which PKD1 may
generate AP-1, we examined the activity of several kinase cascades in
293T cells expressing the C terminus of PKD1. HA-tagged p38, p44, and
JNK1 were co-expressed with the C-terminal domain of PKD1 fused to
CD16.7 (CD16.7.PKD1) or the control vector (CD16.7). After serum
starvation for 16 h, HA-tagged kinases were immunoprecipitated, and the activity of the different kinases was determined using an
in vitro kinase assay. Expression of the cytoplasmic domain of PKD1 increased JNK activity, but not that of p38 or p44 (Fig. 2). Activation of JNK was specific for
PKD1 and was not detectable in 293T cells expressing full-length PKD2
(data not shown). These results together with our findings that PKD1
triggers the Jun2TRE, but not the c-Fos promoter, suggests that
PKD1-induced AP-1 activation may be mediated through the formation of
c-Jun and activating transcription factor 2 dimers. JNKs are typically
activated by growth factors, cytokines, osmotic stress, or UV light,
triggering c-Jun dimerization through phosphorylation at residues
Ser-63 and Ser-73 (27, 28). Many of these conditions result in the concomitant activation of p38 and JNK, although selective activation of
JNK can occur. MEK kinases, specific mixed lineage kinases, transforming growth factor- activated kinase, tumor progression locus 2 (Tpl-2), and p21-activated kinases have all been demonstrated to activate the JNK pathway (reviewed in Ref. 29), whereas selective activation of JNK has been reported through the specific activation of
MKK4 by germinal center kinase (30). It appears that protein-activated kinases, MEK kinases, mixed lineage kinase 3, and Raf are regulated upstream by low molecular weight GTP-binding proteins (25, 31-35). Recently, two members of this family, Rac-1 and Cdc42, have been demonstrated to activate JNK (33).
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To determine the role of small GTP-binding proteins Rac-1 and Cdc42 in the PKD1-mediated activation of JNK, we co-expressed the C-terminal domain of PKD1 with the dominant-negative mutants Rac-1(N17) and Cdc42(N17) (33). Although wild-type Rac-1 and Cdc42 had no affect on the PKD1-mediated JNK activation (data not shown), both molecules nearly abrogated the PKD1-mediated JNK activation (Fig. 3A). A comparable inhibition of PKD1-mediated AP-1 activation by Rac-1(N17) and Cdc42(N17) was observed (Fig. 3B). Thus, Rho family members Rac-1 and Cdc42 appear to play a central role in the signaling pathway triggered by the C terminus of PKD1.
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To define additional downstream components of PKD1-mediated signaling,
we examined the ability of various pharmacological agents to disrupt
PKD1-mediated AP-1 activation. Staurosporine, BAPTA, and calphostin C
inhibited PKD1-induced AP-1 activation in a dose-dependent
fashion (Fig. 4), whereas genistein and
wortmannin had no effect (data not shown). Consistent with the profound
effect of staurosporine and calphostin C, PKD1 caused an elevation of total protein kinase C activity; a comparable increase in PKC activity
was obtained after stimulation of 293T cells with phorbol 12-myristate
13-acetate (1 µM) for 60 min (Fig.
5A). Because PKD1-mediated PKC
activation was inhibited by both staurosporine (200 nM) and BAPTA (10 µM), we speculated that PKD1-induced AP-1
activation is mediated by a calcium-dependent PKC
isoenzyme. PKC is a family of proteins with at least 13 different
members (36), but only the conventional PKCs, ,
I,
II, and
, are regulated by calcium. A dominant-negative form of the
calcium-dependent PKC
(37) suppressed PKD1-mediated
AP-1 activation, whereas equally well expressed dominant-negative forms
of PKC
II and PKC
had no effect (Fig. 5B). These
findings suggest that PKC
may mediate PKD1 signaling. PKC
has
been reported to activate MAP kinase and TRE/AP-1 in other signaling
pathways (38-40), perhaps by dephosphorylating c-Jun at one or more of
three critical serine/threonine residues that negatively regulate its
DNA binding activity (41).
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This study demonstrates that the cytoplasmic domain of PKD1 activates
AP-1 and JNK and that PKC , Cdc42, and Rac-1 are critical components
of these signaling events. The mediators immediately downstream of PKD1
that engage these proteins are yet to be delineated. Recent reports
indicate that the coiled-coil structure in the C-terminal domain of
PKD1 serves as a docking site for several proteins including PKD2 (15,
42-44). In addition to several potential PKC
/PKA
and tyrosine
phosphorylation sites, a putative 14-3-3-binding domain and a putative
SH3-binding domain in the C terminus of PKD1 may serve as docking sites
for cytoplasmic signaling proteins. Binding of 14-3-3 to the C terminus
of PKD1 has recently been demonstrated in the yeast two-hybrid system
(45); interestingly, 14-3-3 adapter proteins have been shown to
activate certain PKC isoenzymes (46, 47). Further mapping of the C
terminus of PKD1, such as point mutations that destroy the 14-3-3 and
SH3-binding domain, could distinguish discrete sites involved in
PKD1-mediated AP-1 activation and facilitate the identification of
pertinent docking proteins that modulate PKC, GTP-binding proteins, and other effectors. A role of Rho family GTP-binding proteins is to
regulate the organization of the actin cytoskeleton and influence cell
motility, shape, and adhesion (reviewed in Refs. 48 and 49). We
postulate that aberrant activation of several small GTP-binding
proteins could underlie some of the anomalies evident in ADPKD cysts,
such as abnormalities of cell polarity and protein sorting (50-55). In
this model of ADPKD, disregulation of PKD1 function would
pathologically affect the activation or recruitment of these small
GTP-binding molecules. The role of AP-1 during renal development
remains to be elucidated. We hypothesize that a burst of
transcriptional activity, mediated in part by PKD1, triggers cellular
proliferation and differentiation, followed by a decline in
proto-oncogene expression. In cells lacking PKD1, proto-oncogene levels
remain pathologically elevated. Indeed, certain genes implicated in
cystogenesis in ADPKD contain AP-1-binding sites. These include
1
(I) collagen and several matrix metalloproteinases, genes normally
involved in the synthesis and remodeling of the extracellular matrix
during normal tubulogenesis (56-60). Aberrant expression of these gene
products may underlie the accumulation of an abnormal extracellular
matrix in ADPKD (reviewed in Ref. 61). Another potential candidate of
AP-1 regulation is hepatic nuclear factor (HNF). HNF-1 and HNF-4 are
reduced in both cpk mouse and cy rat models of
cystic disease (reviewed in Ref. 62), indicating a certain minimal
requirement for these AP-1-dependent transcription factors
during renal development. Lastly, AP-1 has been implicated in the
induction of differentiation (63, 64) as well as apoptosis (reviewed in
Ref. 22); the latter appears to be tightly controlled by Bcl-2 (65).
Surprisingly, mice lacking Bcl-2 develop cystic disease despite massive
apoptosis, perhaps as the result of an inbalance between Bcl-2 and PKD1
activity. Our results suggest that the C-terminal domain of PKD1
triggers cellular functions important for normal renal development.
Future experiments will be necessary to analyze the PKD1 motifs and the nature of PKD1-binding adapter proteins involved in this
activation.
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FOOTNOTES |
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* 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 a Research Fellowship of the Belgian American Educational Foundation, Belgium.
Supported by Public Health Service Grant MH-01147.
** Supported by the Deutsche Forschungsgemeinschaft, Germany.
§§ To whom correspondence should be addressed: Renal Div., Dept. of Medicine, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215. Fax: 617-667-1610; E-mail: gwalz{at}bidmc.harvard.edu.
1
The abbreviations used are: ADPKD, autosomal
dominant polycystic kidney disease; JNK, c-Jun N-terminal
kinase; HA, hemagglutinin; MAP, mitogen-activated protein; PKC, protein
kinase C; TRE,
12-O-tetradecanoylphorbol-13-acetate-responsive element;
AP-1, activation protein 1; NFB, nuclear factor
B; PAGE,
polyacrylamide gel electrophoresis; BAPTA,
1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid; HNF, hepatic nuclear factor.
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REFERENCES |
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