1 Program in Developmental Biology, Research Institute, The Hospital for Sick
Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada
2 Program in Developmental Biology, Research Institute, The Hospital for Sick
Children and Division of Nephrology, Department of Paediatrics, University of
Toronto, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada
* Author for correspondence (e-mail: norman.rosenblum{at}sickkids.ca)
Accepted 3 November 2004
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SUMMARY |
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Key words: Smad1, ß-catenin, Tcf4, renal dysplasia, cystogenesis, Myc
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Introduction |
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The stereotypic pattern of kidney development suggests that the
morphogenetic pathways that regulate renal formation are tightly regulated.
Indeed, investigation of the signaling pathways that control renal branching
morphogenesis has demonstrated the existence of pathways that stimulate or
inhibit epithelial tubule growth and branching in a coordinated manner
(Piscione and Rosenblum,
2002). Members of the bone morphogenetic protein (Bmp) family
inhibit ureteric bud growth and branching in in vitro models of branching
morphogenesis and in vivo (Hu et al.,
2003
; Miyazaki et al.,
2000
; Piscione et al.,
1997
). Recently, we have described a novel model of renal
medullary cystic dysplasia in TgAlk3QD mice, while
investigating the functions of the bone morphogenetic protein cell surface
receptor, activin-like kinase (Alk) 3, during renal embryogenesis
(Hu et al., 2003
).
Alk3 is expressed in both the metanephric mesenchyme and the ureteric
bud during the early stages of kidney development and at lower levels towards
the end of gestation (Dewulf et al.,
1995
). In TgAlk3QD mice, overexpression of a
constitutive active form of Alk3 (Alk3QD) in the ureteric bud
lineage inhibits branching morphogenesis during the early stages of renal
development and causes cystic malformation of medullary collecting ducts later
in utero during the stage of cortico-medullary patterning. The epithelial
cells that populate collecting duct cysts are remarkable for loss of
E-cadherin, a marker of epithelial cell differentiation, increased cell
proliferation and expression of the proto-oncogene Myc. As inhibition of
aberrant Myc expression in a model of polycystic kidney disease ameliorates
the cystic phenotype (Ricker et al.,
2002
), discovery of molecular mechanisms that control Myc
expression is likely to provide insights into epithelial cell differentiation
in normal and dysplastic tissues.
Our investigations of pathogenic mechanisms controlling the formation of a dysplastic kidney in TgAlk3QD mice led us to discover a marked increase in the expression of ß-catenin, an intracellular effector in the Wnt signaling pathway, in the medulla of dysplastic kidney tissue. Moreover, we identified molecular interactions between ß-catenin and Smad1, an intracellular effector of Bmp-Alk signaling in TgAlk3QD kidney tissue. The presence of consensus binding sequences for both Smads and Tcfs, transcription partners for ß-catenin, in the Myc promoter led us to hypothesize that Smad1 and ß-catenin, together with its Tcf partners, interact to control Myc and epithelial cell differentiation. Here, we investigated these interactions within a 1490 nucleotide segment of the Myc promoter that contains adjacent regions containing a cluster of Tcf and Smad consensus binding sequences. Our analysis of renal tissue derived from TgAlk3QD and wild-type mice demonstrate increased levels of Smad1/ß-catenin/Tcf4 molecular complexes and novel associations between these effectors and the Myc promoter in dysplastic tissues. Our investigation of the functional significance of these interactions reveals that each of Tcf4, ß-catenin and Smad1 are required for Bmp-dependent stimulation of Myc transcription. These results provide novel insights into the significance of crosstalk among signaling pathways during tissue development and, specifically, during the genesis of renal dysplasia.
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Materials and methods |
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Antibodies and Bmp2
Immunohistochemistry (IHC) was performed using paraffin wax-embedded tissue
sections (4 µm) generated from adult kidney tissue as described
(Hu et al., 2003) with the
following antibodies: rabbit anti-phospho-Smad1 (1:10; Cell Signaling,
Beverly, MA), mouse anti-Tcf4 (1:10 dilution; Upstate Biotech, Lake Placid,
NY), rabbit anti-ß-catenin (1:25 dilution; Upstate Biotech, Lake Placid,
NY) and mouse anti-ß-catenin (1:25 dilution; BD Transduction
Laboratories, Mississauga, ON, Canada). Biotinylated secondary antibodies were
used in a biotin-avidin complex assay (Vector Laboratories, Burlingame, CA).
Immunofluorescence (IF) was performed using anti-mouse or anti-rabbit
fluorescein- or rhodamine-conjugated secondary antibodies (Jackson
ImmunoResearch Laboratories, Wester Glove, PA). Immunoblotting was performed
using anti-Smad1 (1:250 dilution), anti-Tcf4 (1:250 dilution), anti-Smad4
(1:250 dilution), anti-ß-catenin (1:250 dilution) anti-ß-actin
(1:3000 dilution, Sigma, St Louis, MO) and anti-acetyl Histone 4 (1:250
dilution, Upstate) antibodies. Bmp2 was provided by Wyeth as per a material
transfer agreement.
Fractionation of cytosolic and nuclear proteins
Cytosolic and nuclear proteins were prepared from P30 kidneys using
published methods (Saifudeen et al.,
2002). Cytosolic or nuclear proteins (1 mg) were subjected to
immunoprecipitation. Proteins (40 µg) were directly subjected to SDS-PAGE
and transferred to PVDF membrane.
Identification of proteins associated with chromatin
Proteins associated with chromatin were identified by cisplatin
crosslinking (Chichiarelli et al.,
2002). Nuclear proteins prepared from a single P30 kidney were
added to 10 ml of 1 mM cisdiammineplatinum (II) dichloride (cisplatin) (Sigma,
Saint Louis, MO) and then incubated at 37°C for 2 hours while rotating.
The protein pellet was washed with 5 mM thiourea for 5 minutes at room
temperature to inactivate free cisplatin. Pellets were resuspended in 5 ml of
nuclear lysis buffer at 4°C for 30 minutes. The crosslinked sample (4 mg)
was mixed with 1 g of pre-equilibrated hydroxyapatite resin (HTP) (DNA grade
bio-gel HTP, BioRad, CA) at 4°C for 1 hour. After centrifugation, the
precipitated mixture was washed twice with lysis buffer to remove free
protein. To release the protein from chromatin, 5 ml of reverse buffer was
incubated with the HTP resin pellet overnight at 4°C. After
centrifugation, the supernatant was dialyzed against distilled water for 24
hours at 4°C (Spectra/Por #2, MW cutoff 12,000
14,000, Spectrum
Medical Industries, Houston, TX). The dialyzed solution was concentrated using
a Centricon YM-10 filter (Millipore Corporation, Bedford, MA). The retained
solution was stored at -70°C.
Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) was performed using commercially
available reagents (Upstate Biotech) and published modifications
(Weinmann and Farnham, 2002).
Briefly, nuclear extracts were treated with formaldehyde (1% final
concentration) to crosslink proteins to DNA. The reaction was stopped by the
addition of 125 mM glycine. Soluble chromatin with an average size of
200-500 bp was prepared by sonication
(Weinmann et al., 2001
). After
immunoprecipitation, molecular complexes consisting of chromatin, protein and
antibody were precipitated with a pre-prepared mixture of Salmon sperm DNA and
protein A agarose (Upstate), and then lysed with 1% SDS in 0.1 M
NaHCO3. Proteinase K was added to digest protein in complexes.
Supernatants were used for PCR. The 185 bp TBE-A region (-1322 to -1160)
(Fig. 3) was amplified using
primers: sense, 5'-CAAGCTTTAATTAGCTTAACACA-3'; anti-sense,
5'-GGAGCCTGCAGAGACCCTA-3' at an annealing temperature of 52°C
for 30 seconds. The 229 bp SBE-A region (-1056 to -946) was amplified using
primers: sense, 5'-CGCGTCTAGCCTTGATTTTC-3'; anti-sense,
5'-GGCTCTTTCCCCCTGTAGGTC-3' at an annealing temperature of
52°C for 30 seconds. The 579 bp SBE-B region (-572 to -13) was amplified
using primers: sense, 5'-GCAATTTTAATAAAATTCCAGACA-3'; anti-sense,
5'-AGACCCCCGGAATATAAAGG-3' at an annealing temperature of 54°C
for 30 seconds. The 104 bp RNA polymerase II core promoter region (-40 to -24)
was amplified using primers: sense, 5'-AAACGAGGAGGAAGGAAA-3';
anti-sense, 5'-GCAGACCCCCCGGAATATAA-3' at an annealing temperature
of 53°C for 40 seconds.
|
Electrophoretic mobility gel shift assay
Electrophoretic mobility gel shift assays (EMSA) were performed as
described (Saifudeen et al.,
2002) with modifications. The following oligonucleotides encoding
regions of the mouse Myc promoter were used: a 36 bp oligonucleotide (-1294 to
-1258, 5'-GGCAAAAATGTAACGTTACTTTGATCTGATCAGGGC-3') containing a
Tcf consensus sequence (TTTGATCT), a mutant 36 bp oligonucleotide containing
mutated Tcf consensus sequence (TTTGGCCT), a 46 bp oligonucleotide (-939 to
-893, 5'-GTGGAGGTGTATGGGGTGTAGACCGGCAGAGACTCCTCCCGGAGGAG-3')
containing two Smad-binding sequences (AGAC), and a mutant 46 bp
oligonucleotide containing mutant Smad-binding sequences (GAGT). Protein (10
µg) derived from nuclear extract was incubated with 32P-labeled
oligonucleotide probes and/or unlabeled probes and in some experiments with 1
µg of anti-Smad1, anti-Tcf4 or anti-ß-catenin antibody. The migratory
characteristics of the radiolabeled probes were analyzed in 5% non-denaturing
polyacrylamide/bisacrylamide gels.
RNA interference
RNAi interference (RNAi) was performed using the pSUPER RNAi system
(OligoEngine, Seattle, WA) (Brummelkamp et
al., 2002) and synthetic 64 bp double-stranded oligonucleotides
designed to encode 19 nucleotide target sequences and their reverse complement
separated by a spacer region. Oligonucleotide exact-match sequences (19 mer)
corresponding to sequences in ß-catenin, Smad1 and Tcf4 were selected
based on their respective mRNA sequences (GenBank Accession Numbers: mouse
ß-catenin, NM007614; mouse Smad1, AH010073; mouse Tcf4, AJ22230770).
Based on their efficiency in decreasing endogenous mRNA and protein levels,
the following sequences were used: ß-catenin,
5'-163GAAGATGTTGAACACCTCCC-3'; Smad1,
5'-243GGGACTACCTCATGTCATT-3'; and Tcf4,
5'-1149CGACAGCTTCACAATGCAGC-3'. Oligonucleotides (64
mer) were annealed and cloned into pSuper at the BglII and
HindIII sites. Positive clones were identified by DNA sequencing.
Luciferase assay
The mouse Myc promoter (-1490 to -1), amplified by PCR, was
inserted into a luciferase expression vector upstream of a minimal
Fos promoter (kindly provided by Dr B. Alman, The Hospital for Sick
Children, Toronto, Canada). Transient transfection was performed using Fugene
6 (Roche Diagnostics Corporation, Indianapolis, IN) and lacZ as a
transfection control. Cell lysates were prepared for a luciferase activity
assay using commercial reagents (Promega, Madison, WI). Luminescence was
recorded using a Monolight 2010 Luminometer.
Data analysis
Mean differences between groups were examined by Student's t-test
(two-tailed) or by ANOVA using commercially available software (Statview,
version 4.01; Abacus Concepts, Berkeley, CA). Statistical significance was
taken at a value of P<0.05.
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Results |
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Smad1, ß-catenin and Tcf4 interact with Smad and Tcf binding elements in the Myc promoter
Smad and ß-catenin/Tcf consensus binding sites have been identified
previously in the murine Myc promoter (He
et al., 1998; Yagi et al.,
2002
). Inspection of the 1500 nucleotide sequence upstream of the
transcription start site (GenBank M12345) reveals that Tcf and Smad consensus
binding elements are organized in groups in three discrete regions
(Fig. 3A). We designated these
regions TBE (Tcf-binding region)-A, SBE (Smad-binding region)-A and SBE-B. The
geographic organization of these elements, specifically TBE-A and SBEA, in
close proximity to each other, provided a basis for examining
Smad1/ß-catenin/Tcf4 interactions at this promoter. First, we determined
the association of these proteins with their respective binding regions using
ChIP and specific antibodies (Fig.
3B). The association of Tcf4 with TBE-A was increased 4.7-fold in
TgAlk3QD kidney tissue. Similarly, the association of
Smad1 with each of SBE-A and SBE-B was increased 2.6-fold. Having demonstrated
associations between Tcf4 and Smad1, we used ChIP to determine whether Tcf4
interacts with the SBE (SBE-A) adjacent to TBE-A. Indeed, after
immunoprecipitation of proteins crosslinked to DNA using anti-Tcf4 antibody,
we were able to amplify SBE-A. Moreover, amplification of SBE-A was detected
only in TgAlk3QD tissue, consistent with our detection in
TgAlk3QD tissue, but not in wild-type tissue, of
Tcf4/Smad1 protein associations (Fig.
2C). The specificity of the interaction of Tcf4 with SBE-A was
demonstrated by a failure to amplify the more distant SBE-B region in
experiments conducted in parallel.
Our results using ChIP, together with those using immunoprecipitation and immunoblotting of nuclear extracts, suggested that Smad1 and ß-catenin/Tcf4 associate in a molecular complex at the Myc promoter region encoding Tcf-A and SBE-A. We tested this possibility using EMSA and oligonucleotide duplexes encoding the Tcf consensus binding sequences within TBE-A and the Smad consensus binding sequences within SBE-A (Fig. 4). The migration of a radiolabeled oligo-duplex encoding a 36 nucleotide sequence within TBE-A was retarded by nuclear extract prepared from either wild-type or TgAlk3QD tissue. However, the intensity of the retarded band was greater with TgAlk3QD nuclear extract consistent with increased binding of the oligo-duplex by components of the extract. Addition of anti-Tcf4 antibody to nuclear extract generated from TgAlk3QD or wild-type tissue generated a marked supershift of the oligo-duplex, indicating binding of the oligo-duplex by Tcf4 resident in the nuclei of TgAlk3QD and wild-type tissues. However, the greater intensity of the supershifted band indicated higher levels of Tcf4 in TgAlk3QD extracts. Moreover, the mobility of the supershifted band was slower in TgAlk3QD samples, suggesting that the molecular composition of the Tcf4-containing molecular complexes differs between TgAlk3QD and wild-type tissues. Addition of anti-ß-catenin antibody to nuclear extract also caused a supershift with greater intensity of the supershifted band in TgAlk3QD extracts, consistent with known interactions between ß-catenin and Tcfs. The mobility of the supershifted bands in TgAlk3QD and wild-type tissues differed as observed in samples treated with anti-Tcf4 antibody. Addition of anti-Smad1 antibody also generated a supershift. However, this effect was observed only in dysplastic kidney tissue (Fig. 4A, left panel). The mobility of the supershifted band was identical to that observed in TgAlk3QD nuclear lysates treated with anti-Tcf4 antibody or anti-ß-catenin antibody. These results suggest that the molecular complex that associates with TBE in TgAlk3QD nuclei consists of Smad1, ß-catenin and Tcf4, while that in wild-type nuclei does not contain Smad1. The requirement for Tcf consensus sequences in these interactions was shown by lack of binding by nuclear extracts in experiments in which a mutant TBE oligo-duplex was substituted for the corresponding wild-type oligo-duplex (Fig. 4A, right panel). The specificity of the interaction between oligo-duplex and nuclear lysates was demonstrated by loss of the shifted band after addition of unlabeled probe (Fig. 4B). The specificity of protein-specific antisera in generating supershifted bands was shown by the absence of these bands when non-immune serum was substituted (Fig. 4C). Taken together, these results provide further evidence favoring the existence of a Smad1/ß-catenin/Tcf4 molecular complex associated with TBE-A in dysplastic renal tissue.
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Bmp2 induces formation of ß-catenin/P-Smad1 molecular complexes and Myc expression in collecting duct cells in vitro
We developed an in vitro model aimed at investigating the functional
consequences of Alk3-dependent Smad1/ß-catenin/Tcf4 interactions with the
Myc promoter. Previously, we have demonstrated that treatment of inner
medullary collecting duct (mIMCD-3) cells with Bmp2 activates Alk3 and Smad1
(Gupta et al., 1999). Further
investigation of Bmp2-mediated effects demonstrated simultaneous increases in
the cellular levels of ß-catenin and P-Smad1/ß-catenin molecular
complexes 1.9-fold and 2.3-fold, respectively
(Fig. 5A,B). These increases
were associated with a 2.4-fold increase in Myc
(Fig. 5C). Moreover, Bmp2
treatment of mIMCD-3 cells transfected with a plasmid encoding luciferase
under the control of the 1490-nucleotide segment of the Myc promoter increased
luciferase activity by as much as 26-fold after addition of Bmp2
(Fig. 5D). Together, these
observations provided a basis for testing the functional consequences of
Smad/ß-catenin interactions on Myc promoter function.
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Our results indicate that Smad1 is a positive regulator of Myc
transcription in the context of Tcf4 and ß-catenin. As Smads have been
shown previously to inhibit Myc transcription
(Yagi et al., 2002), we
investigated the function of SBE-A to determine whether it is a positive or
negative regulator of Myc. Using site-directed mutagenesis, we introduced
nucleotide substitutions into the Tcf-A or SBE-A regions of the Myc promoter
and then determined the function of the 1490 nucleotide promoter segment in
mIMCD-3 cells in the presence or absence of Bmp2. Mutagenesis of a single Tcf
consensus binding site (AT to GC) within Tcf-A decreased the basal activity of
the promoter and largely inhibited Bmp2-dependent promoter activity,
consistent with the functions for Tcf and ß-catenin elucidated using
RNAi. Mutagenesis of SBE-A via nucleotide substitutions in two distinct
consensus sequences significantly increased basal Myc promoter activity,
suggesting that these sequences act to inhibit Myc during a state in which
Tcf4/ß-catenin/Smad1 complex formation is not stimulated. By contrast, in
a condition in which this molecular complex is induced (Bmp2 treatment), an
intact SBE-A was required for a full stimulatory response
(Fig. 6D). These results
suggest that Smad1-SBE-A function is converted from an inhibitory to a
stimulatory function in a state during which interactions between Smad1 and
ß-catenin/Tcf4 occur.
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Discussion |
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The data described in this paper provide further insight into how
Smad1/ß-catenin interactions may control epithelial dedifferentiation and
cyst formation focusing on Myc, a paradigm for genes that control these
pathological processes. Myc is involved in a wide range of cellular processes,
including proliferation, differentiation and tumorigenesis
(Grandori et al., 2000). Of
relevance to the TgAlk3QD model, the general observation
that Myc expression strongly correlates with growth and proliferation is
consistent with our finding of Myc expression in epithelia marked by an
increased rate of proliferation (Hu et
al., 2003
). Our finding that Myc expression is associated with
loss of epithelial differentiation markers is in agreement with previous
reports showing that Myc overexpression inhibits terminal differentiation in a
wide variety of cell types. The relevance of our observations regarding Myc is
further supported by the recognition that Myc is misexpressed in collecting
duct cells in murine recessive polycystic kidney disease (PKD)
(Cowley et al., 1987
), that
overexpression of Myc induces PKD in mice
(Trudel et al., 1998
) and that
antisense-mediated downregulation of Myc ameliorates PKD in mice
(Ricker et al., 2002
). Thus,
investigation of mechanisms that control Myc gene regulation is fundamental to
understanding tissue maldevelopment and epithelial cell differentiation.
Control of Myc transcription by Smad1, ß-catenin and Tcf4
The recognition that the Myc promoter contains Smad-binding elements as
well as Tcf finding elements (Yagi et al.,
2002) led us to investigate the nature of Smad/ß-catenin
interactions at the level of gene transcription. Several of our observations
suggested that these interactions are, at the very least enhanced, if not
induced, in dysplastic TgAlk3QD compared with wild-type
tissue. First, molecular complexes consisting of Smad1, ß-catenin and
Tcf4 are detectable in significantly higher amounts in
TgAlk3QD kidney tissue compared with wild-type tissue.
Second, interactions between Tcf4 and a Smad-binding element contiguous with a
Tcf-binding element in the Myc promoter were observed only in dysplastic
tissue. Third, interaction between Smad1, ß-catenin and Tcf4 with Smad-
or Tcf-binding sequences were observed at high levels in nuclear lysates
generated from dysplastic renal tissue but were not detectable in wild-type
renal tissues. Our studies in a collecting duct cell model of Bmp-dependent
Smad1/ß-catenin interactions extend these observations at a functional
level. Using RNAi and site-directed mutagenesis, we demonstrate that not only
Tcf4 and ß-catenin but also Smad1 are required for Bmp-dependent
stimulation of Myc. Consistent with these results, examination of the function
of the Smad-binding region adjacent to the Tcf-binding region revealed that
these sequences subserve a positive role in the context of Bmp-stimulated Myc
expression but an inhibitory function in the absence of these conditions.
Our studies expand the breadth of signaling interactions between the Smad
and ß-catenin pathways previously described. Molecular interactions
between Bmp and Tcf signaling effectors have been described during the control
of Msx2 promoter activity in mouse embryonic stem cells
(Hussein et al., 2003). The
Msx2 promoter contains two SBEs and two TBEs upstream of the SBEs. As
expected, Bmp2 stimulation of Msx2 is dependent on the SBEs. However,
rather surprisingly, Bmp2 stimulatory activity is also dependent on the TBEs.
Consistent with this finding is the recognition that Bmp2 induces recruitment
of LEF1/Tcf to the promoter, association of Lef1/Tcf with the SBE and
association of Smad1 with the TBE. Molecular interactions between Smad and
ß-catenin effectors during normal embryogenesis have been observed
between Smad4 and Lef1/Tcf and between Smad4 and ß-catenin in
Xenopus, specifically involving cooperative interactions between
Smad4 and Lef1/Tcf at the Xtwn promoter
(Nishita et al., 2000
). The
nature of these interactions has been further elucidated in genetic cell
models. The Xtwn promoter contains a SBE and TBE within a 322 nucleotide
region. Lef1/Tcf stimulates transcription; TGFß, alone, does not
stimulate. However, TGFß stimulates in the presence of Lef1/Tcf and
induces binding of Smad3/Lef1 molecular complexes to the Xtwn promoter.
Indeed, the effect of Lef1/Tcf is enhanced in the presence of Smad3 and
mutagenesis of the SBE partially abrogates Smad-dependent enhancement of
Lef1/Tcf stimulation (Labbe et al.,
2000
). Our results in the TgAlk3QD model are
consistent with these reports, demonstrating the conversion of a Bmp-dependent
inhibitory function to a stimulatory effect that enhances Tcf signaling in a
manner that is associated with Smad/Tcf complexes and Tcf/SBE interaction and
is dependent on the SBE. Thus, our work demonstrates the relevance of Smad/Tcf
interactions to mammalian tissue morphogenesis in health and disease.
Our results in the TgAlk3QD model reveal a functional
role for ß-catenin in Smad/Tcf interactions, a finding that is different
than observations made during the analysis of Xtwn and Msx2.
Constitutive action of Alk3 increases ß-catenin levels and associations
between ß-catenin, Tcf and P-Smad1 in the nucleus. ß-catenin is
detected together with Smad1 and Tcf4 in nuclear extracts that bind to
sequences within SBE-A and TBE-A, suggesting a role for ß-catenin in
transcription. Such a function for ß-catenin is further suggested by our
finding that Bmp-dependent induction of Myc is abrogated when intracellular
levels of ß-catenin are lowered. Thus, in contrast to embryonic stem
cells (Hussein et al., 2003),
in dysplastic kidney tissue ß-catenin may serve to mediate Smad/Tcf
protein interactions via its property of binding to both Smads and Tcfs
simultaneously via separate domains (Labbe
et al., 2000
).
Model of Smad and ß-catenin/Tcf signaling in normal and dysplastic kidney tissue
Our analysis of Smad1, ß-catenin and Tcf4 signaling in the
TgAlk3QD model of renal dysplasia suggests a model of
Smad/ß-catenin signaling in normal and dysplastic renal tissues
(Fig. 7). This model suggests
that in the absence of Bmp-stimulated Smad1/ß-catenin interactions, Myc
expression is inhibited by Smad1 bound to Smad-binding regions. We have
identified two regions containing a minimum of seven Smad-binding elements
within the first 1400 nucleotides upstream of the Myc transcription start
site. It is possible that other Smad-binding elements exist within other
regulatory regions. In a manner elucidated for TGFß-dependent repression
of Myc, it is likely that Smad1 acts in concert with other transcription
factors to inhibit Myc (Chen et al.,
2002; Frederick et al.,
2004
). Our model further suggests that in pathological states
associated with recruitment of Smad1 to molecular complexes consisting of
ß-catenin and Tcf4, the Smad1-containing complex acts to stimulate. The
mechanisms that control this switch in Smad activity remain to be discovered
but probably involve recruitment of other transcription factors that bind to
Smad proteins and control transcription via direct or indirect mechanisms.
Further elucidation of these mechanisms will provide a basis for inhibiting
pathogenic signaling interactions in dysplastic tissue.
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ACKNOWLEDGMENTS |
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