1 Program in Developmental Biology, Research Institute, The Hospital for Sick
Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada
2 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 13 March 2003
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SUMMARY |
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Key words: Renal dysplasia, ALK3, SMAD1, ß-catenin, Signaling
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INTRODUCTION |
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Normal kidney development is dependent on inductive interactions between
the metanephric blastema, a mesenchymal tissue and the ureteric bud, an
epithelial structure (reviewed by Saxen,
1987). Under the control of signals secreted by the metanephric
blastema, the ureteric bud grows as a lateral extension of the Wolffian Duct,
and invades the metanephric blastema at 5 weeks gestation in the human and
E10.5 in the mouse. Cells of the metanephric blastema adjacent to the tip of
the ureteric bud are induced by the ureteric bud to undergo a
mesenchymal-epithelial transformation. In reciprocal fashion, the metanephric
blastema signals the ureteric bud to grow and branch, a process termed
branching morphogenesis. Ongoing reciprocal inductive tissue interactions
result in the formation of proximal epithelial nephron segments (glomerulus,
proximal and distal tubules, loop of Henle) from blastemal progenitors, and
cortical and medullary collecting ducts, the terminal components of the
nephron, from the ureteric bud. Beginning at E15.5-E16.5, the murine kidney
becomes further patterned into an outer cortex (comprising glomeruli, proximal
and distal tubules, and cortical collecting ducts), and an inner medulla
composed of Henle's loop (a blastema-derived structure) and medullary
collecting ducts. During the final stages of development in utero, the first
five branches of the ureteric bud undergo transformation into the pelvis and
calyces.
Human renal malformations are typically classified as aplasia, hypoplasia
or dysplasia. Renal hypoplasia is defined as a reduction in the number of
normally formed nephrons. Since each ureteric bud branch induces a discrete
subset of blastemal cells to form a nephron, it is hypothesized that ureteric
bud branching is proportional to the final number of nephrons that are formed.
Consistent with this concept, diminution of ureteric bud branching is thought
to represent one pathogenic pathway during the genesis of renal hypoplasia. By
contrast, renal dysplasia is characterized by variable degrees of tissue
malformation in the renal cortex, medulla or both (reviewed by
Bernstein, 1992). In its most
extreme form, the dysplastic kidney either fails to form (renal aplasia) or
consists of undifferentiated blastema associated with primitive ureteric bud
branches with or without cystic transformation, the so-called multicystic
dysplastic kidney. Less severe forms of dysplasia are characterized by
discrete foci of malformed tissue elements, or dysplasia limited to the
peripheral cortex and medulla, or restricted to the medulla, alone. Each of
these forms has been associated with a variety of clinical entities, including
lower urinary tract obstruction and multi-organ malformation syndromes.
Mutations in genes encoding a variety of molecules, including transcription
factors, growth factors, growth factor receptors and heparan sulfate
proteoglycans, cause renal dysplasia (reviewed by
Piscione and Rosenblum, 1999).
Among these crucially important gene products are members of the bone
morphogenetic protein (BMP) family of secreted growth factors. Several members
of the BMP family are expressed in overlapping but distinct domains during
murine kidney development. The spatial and temporal expression of BMP2, and
its cell-surface receptor, activin-like kinase 3 (ALK3; BMPR1A Human
Gene Nomenclature Database), suggests a role for this secreted growth factor
and receptor during mesenchymal-epithelial interactions in the embryonic
kidney (Dewulf et al., 1995
;
Dudley and Robertson, 1997
;
Flanders et al., 2001
). The
early embryonic lethality observed in Bmp2-/- and
Alk3-/- mice (Mishina
et al., 1995
; Tremblay et al.,
2001
; Zhang and Bradley,
1996
) precluded analysis of their respective roles during renal
development. However, our studies in in vitro models of branching
morphogenesis have defined an inhibitory role for the BMP2/ALK3 signaling
through the cytoplasmic effector SMAD1 (MADH1 Human Gene Nomenclature
Database). BMP2 inhibits ureteric bud branching in embryonic kidney explants
and is bound to the surface of collecting duct cells by ALK3
(Piscione et al., 1997
).
Interruption of ALK3 in an in vitro model of collecting tubule formation
abrogates the inhibitory functions of BMP2 and constitutive signaling of an
activated form of ALK3 is inhibitory
(Gupta et al., 2000
;
Piscione et al., 2001
).
Collectively, these observations suggest that the ALK3 can function downstream
of ligands such as BMP2 to inhibit branching morphogenesis in vivo.
To gain insight into the functions of the ALK3 pathway in vivo, we generated transgenic mice expressing a constitutive active form of ALK3 in the ureteric bud lineage and observed a novel medullary cystic dysplastic phenotype. We investigated the pathogenesis of this phenotype and demonstrated decreased branching morphogenesis during early embryonic renal development, which is consistent with our previous findings in vitro. However, in contrast to our prediction that decreased branching morphogenesis would lead to renal hypoplasia, we observed renal medullary cystic dysplasia with increased penetrance in homozygous transgenic mice. In experiments to identify mechanisms by which ALK3 signaling could generate this phenotype, we observed: increased ß-catenin expression in affected mice; increased expression of a TCF transcriptional reporter in compound transgenic progeny of Tcf-gal and TgALK3QD/QD mice; and molecular complexes consisting of ß-catenin and SMAD1, an ALK3 effector, in kidney tissue from transgenic mice. Our finding that SMAD1 and ß-catenin are overexpressed in dysplastic human kidney tissue suggests that dysregulation of BMP and WNT signaling effectors is pathogenic in human renal dysplasia.
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MATERIALS AND METHODS |
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Antibodies and tissue
For immunohistochemistry, the following antibodies were used: anti-HA (1:10
dilution; Roche Molecular Biochemicals-Boehringer Mannheim, Laval, P.Q.);
anti-ß-catenin (1:25 dilution; Upstate Biotech, Lake Placid, NY);
anti-E-cadherin (1:25 dilution; Santa Cruz Biotechnology, CA); and
anti-phospho-SMAD1 (1:10; Cell Signaling Tech, Beverly, MA). For western
analysis, these primary antibodies were diluted 1:500 as were antibodies for
MYC (Santa Cruz, CA), HA (Amersham Pharmacia Biotech, Piscataway, NJ) and
SMAD1 (Upstate Biotech, Lake Placid, NY). Chemiluminescence was performed
using commercially available reagents (ECL kit; Amersham Pharmacia Biotech,
Piscataway, NJ). Human kidney tissue was kindly provided by Dr G. Ryan with
the approval of the Research Ethics Boards at Mount Sinai Hospital, Toronto
and The Hospital for Sick Children, Toronto.
Immunohistochemistry
Paraffin wax-embedded sections (4 µm) of adult or embryonic kidney
tissue were pre-treated by microwave heating in 0.01 M citrate buffer (pH 6.0)
in a microwave pressure cooker for 14 minutes, including a boiling period of
1.5 minutes. After a 10 minute incubation in 1% H2O2 to
quench endogenous peroxidase activity, tissue sections were incubated with
primary antibody, followed by ABC complex, and then developed with peroxidase
substrate AEC (Zymed Laboratories, San Francisco, CA). Ureteric bud-derived
structures were identified with Dolichos Biflorus Agglutinin (DBA; Vector
Labs, Burlington, ON). Slides were imaged by brightfield and
immunofluorescence microscopy. Human tissues incubated with anti-phospho-SMAD1
antibody were treated with 0.01% pepsin in 10 mM HCl for 30-45 minutes at
37°C after treatment with H2O2 and prior to
incubation with antibodies.
Analysis of ureteric bud branching in embryonic kidneys
Kidneys were surgically dissected from embryonic (E) day 13.5 pregnant mice
and cultured as described previously
(Piscione et al., 1997).
Ureteric bud-derived structures were identified in whole-mount kidney
specimens with fluorescein isothiocyanate (FITC)-conjugated DBA (20 µg/ml;
Vector Labs, Burlington, ON) as described. Ureteric bud branch points were
defined as the intersection between two connected branches
(Grisaru et al., 2001
).
In situ cell proliferation assay
Ureteric bud cell proliferation in embryonic kidney was assayed with
5-bromo-2'-deoxyuridine, as described previously
(Cano-Gauci et al., 1999).
Ureteric bud cell proliferation was calculated as the number of BrdU-labeled
cells within the population of ureteric bud/collecting duct cells identified
by DBA. Cell proliferation in postnatal mouse kidney was assayed by
immunohistochemistry using anti-Ki-67 antibody (1:10 dilution; Roche Molecular
Biochemicals-Boehringer Mannheim, Laval, P.Q.). The number of positive cells
and the number of tubule cross-sections were counted in ten randomly selected
areas imaged at 400x magnification.
TCF-reporter activity in kidney tissue
TCF-reporter activity was assayed in Tcf-gal mice, kindly provided
by Dr B. Alman (Cheon et al.,
2002). Tcf-gal mice express the lacZ gene
downstream of a FOS minimal promoter and three consensus TCF-binding motifs.
ß-Galactosidase activity was assayed using published methods
(Godin et al., 1998
).
Co-immunoprecipitation assay
Cell or tissue lysates were subjected to immunoprecipitation with
anti-SMAD1 or anti-ß-catenin antibody, followed by adsorption to protein
G plus-agarose (IP04; Oncogene, Boston, MA). Immunoprecipitated proteins were
washed, separated by SDS-polyacrylamide gel electrophoresis and transferred to
PVDF membrane.
RT-PCR assay
First-strand cDNA was synthesized using total RNA as a template. PCR was
then performed using the following primers encoding highly conserved regions
within WNT11, WNT4, WNT2B, WNT5B, WNT7B and WNT6: sense,
5'-GAGTGCAAGTGTCACGGGGT-3'; and antisense,
5'-CAGCACCAGTGGAACTTGCA-3'. Thirty cycles of PCR amplification
were peformed using the following protocol: 30 cycles of 92°C for 1
minute, 59°C for 1 minute and 72°C for 1 minute.
Data analysis
The pixel density of protein bands identified by immunoblotting was
adjusted for the density of a control protein (i.e. actin, SMAD1 or
ß-catenin) in that sample. The adjusted values of protein were analyzed
using the Stat-View statistical analysis program (version 4.01; Abacus
Concepts, Berkeley, CA). Mean differences were examined by Student's
t-test (two-tailed) or by ANOVA. Significance was taken at a value of
P<0.05.
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RESULTS |
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Constitutive expression of activated ALK3 causes postnatal mortality
associated with renal aplasia/dysplasia and renal medullary cystic
dysplasia
To analyze the viability and renal developmental status of
ALK3QD Tg mice, we examined litter size and viability, and
performed a gross anatomic analysis of the kidney
(Table 1). Seven out of 161
(4.3%) mice in Tg Line 1 died within several days of birth. Postmortem
examination revealed bilateral renal aplasia in one mouse, and bilateral renal
malformations consisting of a combination of aplasia and severe dysgenesis in
six other mice. Southern analysis of genomic DNA revealed that these seven
affected mice were homozygous Tg. All mice in Line 2 survived beyond the
neonatal period. Thirty-four mice (31 in Line 1 and three in Line 2)
demonstrated severe growth retardation and died between one and two months of
age. All affected mice were homozygous Tg. Three of these mice were observed
to have bilateral aplasia/dysgenesis. Histological examination in the
remaining 31 showed medullary cystic dysplasia of variable severity (see
below). Mice that survived beyond two months of age (n=144)
demonstrated no obvious abnormalities up to at least 7 months of age, by which
time all were sacrificed. However, histological analysis revealed medullary
cystic dysplasia in 89 of these mice. Thus, we observed renal
aplasia/dysgenesis or medullary cystic dysplasia in 5% (10/185) and 65%
(120/185) of Tg mice, respectively.
To identify the dysplastic renal phenotype in embryos and postnatal mice, we performed a histopathological analysis (Fig. 2). By E18.5, the crucial events during epithelial morphogenesis and patterning have occurred in the kidney. Homozygote Tg mice demonstrated marked abnormalities compared with control mice. The diameter of the outer cortex was decreased (compare boxed area in Fig. 2D with Fig. 2A, and Fig. 2E with Fig. 2B). The outer cortex is the site at which glomerular progenitors, termed comma and S-shaped bodies, form. Although these progenitors appeared normal qualitatively, the decreased number of elements suggested a primary or secondary defect in the number of inductive events. The medulla in TgALK3QD mice was remarkable because of a marked reduction in the number of medullary tubules (compare Fig. 2D,F with Fig. 2A,C), and an increase in interstitial matrix (Fig. 2D,F). These changes were accompanied by the formation of epithelial cysts in the inner and outer regions of the medulla and in the cortex (Fig. 2D,E). These histological abnormalities were also observed in heterozygous Tg mice, although the phenotype was observed in a minority of mice and was less severe (data not shown). At P10, the dysplastic phenotype could be readily identified in hemizygous Tg mice (Fig. 2H), and was more severe in homozygous Tg mice (Fig. 2I,J). In contrast to control mice (Fig. 2G), 50% of hemizygous Tg mouse kidneys were characterized by a decreased number of tubules in the medulla, increased intervening extracellular matrix, underdevelopment of the papilla, and the formation of cortical and medullary cysts (Fig. 2H). Abnormalities of a greater severity were observed in 74% of homozygous Tg mice (Fig. 2I,J). In these mice, the medulla was occupied by large cysts and extracellular matrix, and the cortex was reduced to a thin layer, perhaps as a result of compression caused by cysts. Analysis of cyst epithelium with DBA revealed positive staining, which suggests that ALK3-HA expressing cysts are derived from the ureteric bud lineage (Fig. 2K,L). Taken together, these findings suggest a pathological diagnosis of medullary cystic dysplasia with cortical cyst formation.
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Association of Phospho-SMAD1 and ß-catenin in molecular
complexes is increased in dysplastic TgALK3QD mouse
kidney
Molecular interactions between the ß-catenin binding partner,
LEF1/TCF, and the TGF-ß SMAD signaling effectors, SMAD2, SMAD3 and SMAD4
have been demonstrated in Xenopus
(Nishita et al., 2000) and
cultured mammalian cells (Fischer et al.,
2002a
; Labbe et al.,
2000
). By contrast, no interactions between SMAD1 and
ß-catenin or TCF have yet been demonstrated. Our finding of increased
transcriptional activity in QD/QD; Tcf-gal mice suggested
possible molecular interactions between ALK3 signaling effectors and
ß-catenin. We investigated interactions between SMAD1, SMAD4 and
ß-catenin in renal tissue lysates isolated from P10 mice by
co-immunoprecipitation and immunoblotting
(Fig. 7). Whereas low levels of
ß-catenin were detected in association with SMAD1 in the renal tissue of
controls, ß-catenin levels associated with SMAD1 were increased ninefold
in kidney lysates from homozygous Tg mice
(Fig. 7A). Similarly, a
ninefold elevation of phospho-SMAD1 in association with ß-catenin was
also detected in homozygous Tg mouse kidney compared with that of controls
(Fig. 7B). Formation of
molecular complexes between phospho-SMAD1 and the common SMAD, SMAD4, before
translocation to the nucleus, suggested that elevated levels of phospho-SMAD1
associated with ß-catenin would be accompanied by increased amounts of
SMAD4. Our results indicate a twofold elevation in levels of SMAD4 associated
with ß-catenin (Fig. 7C).
Compared with SMAD1, the lower amount of SMAD4 associated with ß-catenin
suggests that the ratio of SMAD4:SMAD1 may not be 1:1 in molecular complexes
containing of ß-catenin, and that SMAD1/ß-cateinin complexes may
function in a manner distinct from those containing SMAD4. Taken together,
these results are the first demonstration of molecular associations among
SMAD1, SMAD4 and ß-catenin during the genesis of dysplasia during
mammalian organogenesis.
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DISCUSSION |
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A subset of human renal dysplasias is characterized by less severe abnormalities that are either more focal in spatial distribution, or are restricted to one or more tissue elements, such as medullary collecting ducts. Because the tissue damage in these phenotypes is less pervasive and severe, they are associated with some degree of renal survival during fetal and postnatal life. With a view towards human disease, it is crucial to understand the pathogenesis of these forms of dysplasia because the knowledge gained may lead to strategies aimed at modifying aberrant developmental pathways and ameliorating disease. In this paper, we report a mouse model of medullary cystic dysplasia in which nephrogenesis is qualitatively intact but quantitatively deficient, and in which decreased branching morphogenesis is associated with a decreased mass of medullary collecting ducts and with cystic degeneration of these tubules.
The renal phenotype of TgALK3QD mice is distinct from
that of other mouse models characterized by misdevelopment of the renal
medulla. These include mice deficient for angiotensinogen (Ang),
angiotensin receptor-1 (Agtr1) and -2 (Agtr2), glypican 3
(Gpc3), p57KIP2 and BMP4. Ang-/- and
Agtr1-/- mice demonstrate progressive dilatation of the
calyx, and atrophy of the papilla and underlying medulla after birth. These
defects appear to be caused by decreased proliferation of the smooth muscle
layer lining the pelvis, which results in decreased thickness of this layer in
the proximal ureter (Miyazaki et al.,
1998; Niimura et al.,
1995
). Agtr2 inactivation results in a spectrum of renal
dysplasia and lower urinary tract abnormalities termed CAKUT. These
malformations appear to be caused by misdevelopment of the proximal ureter and
by secondary effects on the development of the renal parenchyma
(Nishimura et al., 1999
). GPC3
deficiency causes medullary cystic dysplasia owing to massive apoptosis of
medullary collecting ducts. In contrast to TgALK3QD mice,
in Gpc3-mutant mice, medullary dysplasia is preceded by an increase
in branching morphogenesis and ureteric bud cell proliferation
(Cano-Gauci et al., 1999
;
Grisaru et al., 2001
). The
renal phenotype of p57KIP2-null mice is similar to that
observed in Gpc3-null mice. Although the pathogenic mechanisms have
not been elucidated, the function of p57KIP2 as an inhibitor of
cyclin-dependent kinases suggests that it may act in a pathway shared by
Gpc3. Although Bmp4, a Bmp2 homolog, can signal
through ALK3, Bmp4+/- mice exhibit a phenotype distinct
from that observed in TgALK3QD mice. Renal dysplasia in
Bmp4+/- mice appears to be caused by abnormal budding of
the ureteric bud at its origin at the Wolffian Duct, and, consequently,
abnormal interactions between one or more ureteric buds and the metanephric
blastema (Miyazaki et al.,
2000
). This, in turn, results in focal dysplasia consisting of a
major interruption of nephrogenesis with cystic dysplasia and hydronephrosis.
Thus, the unique renal phenotype observed in TgALK3QD mice
suggests the existence of novel pathogenic mechanisms not previously
discovered in other models of renal dysplasia. The similarity of this
phenotype to that observed in some fetuses with associated lower urinary tract
dilatation suggests that knowledge of these mechanisms will provide further
insight into the pathogenesis of human dysplasia
(Al Saadi et al., 1984
;
Daïkha-Dahmane et al.,
1997
).
ALK3 inhibits renal branching morphogenesis
The invariant structural organization of the mature kidney and the
relatively constant number of collecting ducts formed suggests that branching
morphogenesis is a tightly regulated process. We have hypothesized that
regulation of renal branching morphogenesis occurs through the integrated
actions of stimulatory signaling pathways, including GDNF/RET, and inhibitory
pathways downstream of BMP2 and its receptor ALK3
(Piscione et al., 1997).
Bmp2 is expressed in the metanephric mesenchyme adjacent to the tips
of ureteric bud branches (Dudley and
Robertson, 1997
). Alk3 is expressed in both ureteric bud
and blastemal cells during early stages of renal development, and at lower
levels later during gestation. Identification of the roles played by BMP2 and
ALK3 has been limited by the early embryonic lethality induced by mutational
inactivation of either gene. Although deletion of one allele of the BMP2
homolog BMP4 gives rise to a dysplastic renal phenotype, the distinct pattern
of BMP4 expression in the mesenchyme surrounding the Wolffian Duct and the
ureteric bud (Miyazaki et al.,
2000
) suggests a function different from that of BMP2, and limits
speculation regarding the function of ALK3 in regions adjacent to
Bmp2 expression. Previously, we demonstrated that BMP2 and ALK3
inhibit renal branching morphogenesis in embryonic kidney explants and in a
3-dimensional culture model of tubule formation. Our results in the
TgALK3QD mouse are consistent with these results and
provide the first demonstration in vivo that activation of ALK3 in the
ureteric bud inhibits branching morphogenesis. This effect probably underlies
the pathogenesis of renal aplasia in a small group of
TgALK3QD mice. The relatively small number of mice with
this malformation may be because of modifying factors, including the level of
expression of the transgene and the genetic background of the mice. Indeed,
our results demonstrated considerable variation in the effect on branch point
number within progeny derived from the same founder. In the majority of
TgALK3QD mice, the inhibitory effect of ALK3QD
is insufficient to abrogate ureteric bud growth. Rather, it inhibits
branching, thereby inducing hypoplasia of the collecting system and decreasing
the number of nephrons induced. Recognition of this effect suggests that ALK3
activity must be tightly regulated. The basis for this regulation remains
unknown. Because the ALK3QD transgene was mainly expressed
in the ureteric bud lineage, we are unable to interpret the role of ALK3 in
the metanephric mesenchyme. The development of mice expressing Cre
recombinase in specific kidney cell lineages under temporal control will
provide a means to study the actions of ALK3 in the ureteric bud and
metanephric blastema both separately and together.
Interactions between the BMP and WNT pathways during
organogenesis
Our analysis of TgALK3QD mice revealed genetic and
molecular interactions between effectors in the BMP and WNT pathways. Our data
expand the breadth of interactions previously observed during embryogenesis in
flies and frogs, and during tumor formation in mice. In Drosophila,
dpp, a BMP2/4 homolog, and wg, a WNT homolog, act
synergistically to regulate the expression of ultrabithorax (Ubx) in
the endoderm (Riese et al.,
1997). In Xenopus, SMAD4 and LEF1 cooperate to control
the expression of the homeobox gene Xtwn8 in Spemann's organizer
(Nishita et al., 2000
). In
mice, deficiency of one Smad4 allele synergizes with deficiency of
APC, a regulator of ß-catenin degradation, to increase the number and
invasiveness of intestinal tumors (Takaku
et al., 1998
). These findings have been extended to the level of
molecular interactions in Xenopus by the demonstration of
SMAD4/LEF1(TCF) and SMAD4/ß-catenin molecular complexes, and the
cooperative interaction of SMAD4 and LEF1/TCF at the XTWN promoter
(Nishita et al., 2000
). These
nature of these interactions has been further defined by the demonstration
that LEF1/TCF binds via its C-terminal domain to the TGF-ß effectors
SMAD2, SMAD3 and SMAD4 (Labbe et al.,
2000
). Our results reveal interactions between ß-catenin and
SMAD1, implicating the BMP pathway in interactions with this WNT effector.
Although data reveal that these pathways interact cooperatively at the level
of a Tcf reporter in kidney tissue, further experiments will be
required to identify the functional consequences of SMAD1/ß-catenin
interactions. The recognition that the MYC promoter contains SMAD-binding
elements as well as TCF-binding elements
(Yagi et al., 2002
) provides a
basis for studying these interactions in the context of a gene that is
relevant to the pathogenesis of polycystic kidney disease
(Trudel et al., 1998
) and
cystic dysplasia (our data).
Interactions between the BMP and WNT pathways during the genesis of
renal disease
Our data from TgALK3QD mice indicate that ALK3
upregulates ß-catenin expression. Previous observations demonstrating
that ß-catenin is upregulated by polycystin 1, the gene product mutated
in type I autosomal dominant polycystic kidney disease
(Kim et al., 1999), and that
ß-catenin induces polycystic kidney disease when overexpressed in
transgenic mice (Saadi-Kheddouci et al.,
2001
) suggest that dysregulation of BMP signaling in the embryonic
kidney triggers a cystogenic pathway. Similarly, these findings, together with
those presented here, raise the possibility of dysregulation of the BMP
pathway in polycystic kidney disease. The mechanisms mediating interactions
between ALK3 and ß-catenin remain to be determined. One possibility is
that BMP signaling upregulates WNT expression, a positive effector of
ß-catenin expression. Although we were not able to associate the
expression of a subset of WNT proteins with ALK3 signaling in vivo, one or
more of the larger number of WNTs may be involved in this process.
Alternatively, ALK3 may control ß-catenin signaling in a manner that is
WNT-independent. The BMP family member TGF-ß controls cytoplasmic levels
of ß-catenin in cultured cells by stimulating redistribution of
ß-catenin from adherens junctions to the cytoplasm
(Tian and Phillips, 2002
).
BMP-dependent activation of MAP kinases, such as TAK1 (MAP3K7 Human
Gene Nomenclature Database), control ß-catenin signaling at the level of
transcription. TAK1 is an intracellular target of TGF-ß and BMPs
(Yamaguchi et al., 1995
), and
is required for BMP-dependent control of cardiomyocyte differentiation
(Monzen et al., 2001
). Acting
through its downstream targets, TAK1 modulates the transcriptional activity of
ß-catenin/TCF molecular complexes
(Ishitani et al., 1999
). Taken
together with our observations, these observations provide a basis for
identifying BMP-dependent molecules that modulate the intracellular levels of
ß-catenin and/or its transcriptional activity in conjunction with TCF
family members.
Our findings provide a basis for generating a two-phase model of medullary renal dysplasia caused by expression of activated ALK3 in the ureteric bud lineage. In the first phase, inhibition of branching morphogenesis inhibits nephron formation, leading to the formation of a thin outer cortex. During the second phase, ALK3 upregulates ß-catenin, alters the expression of genes crucial for epithelial cell differentiation and induces cystogenesis. Further experiments will be required to determine the specific elements of this model. These will include strategies to test the effect of SMAD1/ß-catenin complexes on the expression of genes such as E-cadherin and MYC, and the development of new mouse models in which the expression of ß-catenin is temporally and spatially controlled in the kidney. These approaches will need to be complemented by the analysis of a broad range of phenotypes in tissue samples taken from individuals with renal dysplasia to gain further insight into the general significance of our finding that phospho-SMAD1 and ß-catenin are markedly upregulated in dysplastic renal tissues.
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ACKNOWLEDGMENTS |
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