RESEARCH ARTICLE 3233
Development 137, 3233-3243 (2010) doi:10.1242/dev.052845
© 2010. Published by The Company of Biologists Ltd
Integrin-linked kinase regulates p38 MAPK-dependent cell
cycle arrest in ureteric bud development
Joanna Smeeton5,*, Xi Zhang1,2,*, Nada Bulus1,*, Glenda Mernaugh1, Anika Lange6, Courtney M. Karner7,
Thomas J. Carroll7, Reinhard Fässler6, Ambra Pozzi1,2,4, Norman D. Rosenblum5 and Roy Zent1,2,3,4,†
SUMMARY
The integrin-linked kinase (ILK), pinch and parvin ternary complex connects the cytoplasmic tails of 1 integrins to the actin
cytoskeleton. We recently showed that constitutive expression of ILK and alpha parvin in both the ureteric bud and the
metanephric mesenchyme of the kidney is required for kidney development. In this study, we define the selective role of ILK in
the ureteric bud of the mouse kidney in renal development by deleting it in the ureteric cell lineage before the onset of
branching morphogenesis (E10.5). Although deleting ILK resulted in only a moderate decrease in branching, the mice died at 8
weeks of age from obstruction due to the unprecedented finding of intraluminal collecting duct cellular proliferation. ILK
deletion in the ureteric bud resulted in the inability of collecting duct cells to undergo contact inhibition and to activate p38
mitogen-activated protein kinase (MAPK) in vivo and in vitro. p38 MAPK activation was not dependent on the kinase activity of
ILK. Thus, we conclude that ILK plays a crucial role in activating p38 MAPK, which regulates cell cycle arrest of epithelial cells in
renal tubulogenesis.
INTRODUCTION
Murine kidney development begins at embryonic day (E) 10.5
when the ureteric bud (UB) grows into the metanephric
mesenchyme (MM) in response to inductive cues mediated by
growth factors (Reidy and Rosenblum, 2009). The UB undergoes
many iterations of branching morphogenesis to create the intrarenal
collecting system comprising collecting ducts (CD), calyces and
the pelvis, as well as forming the ureters and the bladder trigone.
The MM gives rise to the nephrons.
Integrin-linked kinase (ILK) is a cytoplasmic protein that is a
component of the ILK-pinch-parvin macromolecular protein
complex situated at focal adhesions. It comprises a pseudokinase
domain at the C-terminus, a pleckstrin homology-like domain and
ankyrin repeats at the N-terminus (Fukuda et al., 2009; Lange et al.,
2009). The C-terminus of ILK binds either directly or indirectly to
the cytoplasmic tail of 1- and 3-integrins. ILK plays a key role in
organizing the actin cytoskeleton by recruiting actin binding and
actin regulatory proteins, such as pinch, parvin, paxillin and kindlin
(Legate and Fassler, 2009; Legate et al., 2006; Legate et al., 2009),
and although it lacks intrinsic kinase activity (Fukuda et al., 2009;
Lange et al., 2009), it can regulate the activation of cell signaling
molecules such as protein kinase B (PKB/AKT) (Hannigan et al.,
2007; McDonald et al., 2008; Wang et al., 2008; White et al., 2006).
1
Division of Nephrology, Departments of Medicine, 2Cancer Biology, 3Cell and
Developmental Biology, Nashville, TN 37232, USA. 4Department of Medicine,
Veterans Affairs Hospital, Nashville, TN 37201, USA. 5Program in Developmental and
Stem Cell Biology, The Hospital for Sick Children, Department of Laboratory
Medicine and Pathobiology, University of Toronto, Toronto, ON M5G 1X8, Canada.
6
Max-Planck Institute of Biochemistry, Department of Molecular Medicine,
Martinsried, D-82152 Germany. 7Department of Internal Medicine, Division of
Nephrology, Department of Molecular Biology, University of Texas Southwestern
Medical Center, Dallas, Texas, TX 75390, USA.
*These authors contributed equally to this work
†
Author for correspondence ([email protected])
Accepted 20 July 2010
There is limited knowledge about the role of ILK in renal
development. Deletion of ILK in mice results in early embryonic
lethality due to defects in epiblast polarity and F-actin organization
(Sakai et al., 2003). However, it is clear that ILK is required for
renal development as we recently demonstrated that mice with
point mutations in the conserved lysine residue of the potential
ATP-binding site of the pseudokinase domain of ILK, which is
essential for -parvin binding, die perinatally owing to renal
agenesis (Lange et al., 2009). This in vivo phenotype and our in
vitro organ culture data demonstrating failure of the UB to respond
to GDNF, which is a crucial growth factor required for induction
of UB branching morphogenesis, suggested an intrinsic defect in
UB cells. The mechanisms by which ILK deficiency abrogates or
limits UB branching remain undefined.
The selective role of ILK in the developing collecting system of
the kidney has only been investigated in vitro. In these studies a
dominant-negative ILK (R211A) inhibited branching
morphogenesis in kidney organ cultures and overexpressing a
‘kinase-dead’ ILK mutant (E359K) and treatment with an ILK
inhibitor decreased the ability of inner medullary CD cells to
undergo BMP7-induced branching morphogenesis by inhibiting
phosphorylation of p38 MAPK and ATF2. These results suggested
that ILK functions upstream of p38 MAPK during BMP7 signaling
and that ILK plays a role in the BMP7-p38MAPK-ATF2 signaling
pathways, which regulates epithelial cell morphogenesis (LeungHagesteijn et al., 2005).
The only cell type in the kidney where ILK has been specifically
deleted is in podocytes of the glomerulus and these mice develop
massive proteinuria and end-stage renal failure resulting in death
at approximately six to eight weeks of age (Dai et al., 2006; ElAouni et al., 2006; Kanasaki et al., 2008). The phenotype is very
similar, although less severe, to when 1-integrin is deleted in
podocytes (Kanasaki et al., 2008; Pozzi et al., 2008), suggesting
that 1-integrin-dependent signaling is at least in part dependent on
the ILK.
DEVELOPMENT
KEY WORDS: Kidney, Branching morphogenesis, Growth factor receptors, Mouse
3234 RESEARCH ARTICLE
Development 137 (19)
To define whether ILK regulates similar pathways to 1-integrin
in UB development in vivo, we selectively deleted it in the ureteric
bud cell lineage starting before the onset of UB branching
morphogenesis. Unlike the similar phenotypes observed when ILK
or 1-integrin was deleted in podocytes, there was a significant
difference between mice where these proteins were deleted in the
UB. In contrast to the severe branching morphogenesis abnormality
in mice lacking 1 expression in the UB, mice lacking ILK
expression have a moderate branching phenotype. Despite this,
these mice die at 8 weeks of age from severe obstruction caused by
cell accumulation within the tubule lumens. Although multiple
signaling pathways were dysregulated in the CDs of 1-null mice,
only p38MAPK signaling is disrupted in the collecting system of
ILK-null mice. In vitro studies show that ILK-null CD cells are
unable to undergo contact inhibition owing to their inability to
activate p38 MAPK, which occurs independent of the role for ILK
as a kinase. These data suggest that the obstruction in the CDs of
the ILK-null mice in vivo is due to the key role of ILK as a
regulator of epithelial cell contact inhibition, which is mediated by
activating p38 MAPK. Thus, deleting 1 and ILK in the UB results
in markedly different phenotypes because ILK only transduces a
distinct subset of signaling pathways activated by 1-integrin.
PCR reaction mix contained a cDNA sample and primers against ILK.
Real-time PCR amplification was performed using the Applied Biosystems
7900 HT Fast RT-PCR system. Relative levels of mRNA expression were
determined using the standard curve method. Individual expression values
were normalized by comparison to -2 microglobulin.
MATERIALS AND METHODS
Generation of ILK-null cell line
All experiments were approved by the Vanderbilt University Institutional
Animal Use and Care Committee. ILKflox mice (Terpstra et al., 2003) were
crossed with the HoxB7Cre (generous gift of Dr A. McMahon, Harvard
University) (Kobayashi et al., 2005) or Hoxb7creEGFP mice (generous gift
from Dr C. Bates, University of Pittsburgh) (Zhao et al., 2004). Mice were
a F4-F6 generation toward the C56/Black6 background. Aged-matched
littermates homozygous for the floxed ILK gene, but lacking Cre (ILKflox
mice), were used as controls.
Morphologic analysis
For morphological and immunohistochemical analysis, kidneys were
removed at different stages of development and (1) fixed in 4%
formaldehyde and embedded in paraffin; (2) embedded in OCT compound
without fixation and stored at –80°C until use; or (3) fixed in 2.5%
glutaraldehyde, post-fixed in OsO4, dehydrated in ethanol and embedded
in resin. Paraffin tissue sections were stained with either Hematoxylin and
Eosin or Periodic Acid Schiff’s (PAS) for morphological evaluation by
light microscopy. For electron microscopy, ultrastructural assessments of
thin kidney sections were performed using a Morgagni transmission
electron microscope (FEI). UB branching was imaged in whole kidneys by
fluorescence microscopy for the expression of GFP.
For immunofluoresence of ILK and 1-integrin expression, 8 m thick
cryosections of E11.5, E12.5 and E14.5 embryos were fixed for 1 hour in
3% PFA at 4°C and frozen in OCT (Thermo Shandon). Sections were
permeabilized with 0.1% Triton X-100 for 10 minutes, blocked with 3%
BSA for 1 hour and incubated overnight with the primary antibody,
followed by incubation with the secondary antibody. The fluorescent
images were collected by laser scanning confocal microscopy (DMIRE2;
Leica) using Leica Confocal software, version 2.5, Build 1227. The
primary antibodies were an rabbit anti-ILK antibody (Cell Signaling,
3862), rabbit anti-1 antibody (Millipore, AB1952) and a rat anti-nidogen
antibody (Millipore, MAB1883).
Immunohistochemistry for GSK3 was performed on 5 m kidney
sections of E12.5 embryos that were cut from paraffin blocks using a rabbit
monoclonal antibody (Cell Signaling, 27C10).
Ureteric bud isolation and real-time reverse-transcriptase PCR
Kidneys from E11.5 HoxB7Cre;ILKflox and ILKflox mice were dissected as
previously described (Cain et al., 2009). RNA was isolated using the
RNAqueous-Micro RNA Isolation Kit (Ambion) and cDNA was generated
using First Strand cDNA Synthesis (Invitrogen) from total RNA. Real-time
Embryonic kidney tissue was formalin-fixed and paraffin-embedded prior
to sectioning (4 m). Terminal deoxynucleotidyl transferase (TdT)mediated dUTP nick-end labeling (TUNEL) was performed as described
in the manufacturer’s instructions (Promega). Cell proliferation was
assayed by incorporation of 5-bromo-2-deoxyuridine (BrdU, Roche
Molecular Biochemicals), as previously described (Cano-Gauci et al.,
1999). Briefly, a single intraperitoneal injection of BrdU (100 mg/g body
weight) was given to pregnant females 2 hours prior to sacrifice. BrdUpositive cells were identified using anti-BrdU peroxidase-conjugated
antibody (Boehringer Mannheim). Immunoreactivity was visualized using
aminoethyl carbazole horseradish peroxidase chromogen and substrate
solution (Zymed Laboratories).
In situ mRNA hybridization
Whole embryos were fixed in 4% PFA in PBS for 16 hours at 4°C. In situ
hybridization was performed as described (Ding et al., 1998) on paraffinembedded sections (4 m) using DIG-labeled cDNA probes encoding
Wnt4 and Wnt9b.
CD cells were isolated from ILK1flox mice following the methodology
described by Husted et al. (Husted et al., 1988) and ILK was deleted by
infecting the cells with an adenocre virus in vitro. Deletion of ILK was
verified by immunoblotting. Generation of the CD cells expressing the ILK
mutants was described previously elsewhere (Lange et al., 2009).
Cell adhesion
Cell adhesion assays were performed in 96-well plates, as described (Chen
et al., 2004). Briefly plates were coated with different concentrations of
ECM components and blocked with BSA. 1⫻105 cells were placed in each
well in serum-free DMEM for 60 minutes; non-adherent cells were
removed and the remaining cells were fixed, stained with Crystal Violet,
solubilized and the optical density of the cell lysates was read at 540 nm.
Four independent experiments were performed in triplicate.
Cell migration
Cell migration was assayed as previously described (Chen et al., 2004).
Briefly, transwells with 8 m pores were coated with different ECM
components and 1⫻105 cells were added to the upper well in serum-free
medium. The cells that migrated through the filter after 4 hours were
counted. Three random fields were analyzed per each treatment. Four
independent experiments were performed in triplicate.
Cell proliferation
5⫻103cells were plated per well in 96-well plates on collagen I and
maintained in DMEM (10% FBS). After 12 hours, the cells were incubated
in DMEM (2% FBS) for 24 hours and then pulsed with 1 Ci/well [3H]
thymidine (PerkinElmer Life Sciences). Twenty-four hours later, the cells
were solubilized and radioactivity was measured using a scintillation
counter.
Cell spreading
Cells were plated onto slides coated with collagen I (10 g/ml) for 1 hour,
after which they were fixed, permeabilized and exposed to Rhodamine
Phallodin (1:5000) and visualized under a microscope.
Cell polarity
Cells were grown on transwells comprising polyvinylpyrolidone-free
polycarbonate filters with 0.4 m pores. After reaching confluency, cells
were fixed in 4% formaldehyde and incubated with anti-ZO1 (1:200; BD
Transduction Laboratories) antibodies followed by the appropriate FITCconjugated secondary antibody. Chamber slides were mounted and viewed
using a confocal microscope.
DEVELOPMENT
Generation of HoxB7Cre;ILKflox mice
In situ TUNEL and BrdU incorporation assays
ILK and the ureteric bud
RESEARCH ARTICLE 3235
Tubulogenesis assays
CD cells were placed in 3D gels comprising rat tail collagen I and Matrigel
(Becton Dickinson) and Dulbecco’s Minimal Essential Media (DMEM)
containing 20 mM HEPES (pH 7.2) as previously described (Pozzi et al.,
2006). Briefly, 1.5⫻103 cells were suspended in 100 l of gel and plated
onto 96 wells. After 1 hour at 37°C, an equal volume of DMEM
supplemented with 10% FBS was added to the gels. The cells were allowed
to grow for 7 days, at which time the gels were stained with Rhodamine
Phalloidin and photographed on a confocal microscope.
Cell viability assays
One thousand CD cells were seeded in 96-well plates and grown in
medium with 15% FBS. At the timepoints indicated, 5 mg/ml MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] stock
solution was added to each well in an amount equal to 10% of the culture
volume in 8 replicates and incubated for 4 hours at 37°C. After the
incubation period, the resulting formazan crystals were dissolved in MTT
solvent (0.1 M HCl in anhydrous isopropanol) in an amount equal to the
original cell culture volume. Absorbance was measured at a wavelength of
570 nm and background absorbance at 690 nm was subtracted.
Immunoblotting
The replating assays were performed as previously described (Abair et al.,
2008). In brief, serum-starved CD cells were plated in serum-free medium
on collagen I (20 g/ml) for 0, 30 and 60 minutes after they were lysed and
subjected to immunoblotting.
The effects of growth factors on CD cells were examined as previously
described (Zhang et al., 2009). In brief, cells were trypsinized into serumfree DMEM and then plated on collagen I or vitronectin (10 ug/ml) for 45
minutes. Growth factors (FGF or GDNF, 10 ng/ml) were added to the
medium and the cells were lysed at different timepoints following growth
factor stimulation.
For analysis on kidney tissues, the medullas were removed and lysed
with RIPA buffer. Lysates were clarified by centrifugation and 30 g total
protein was electrophoresed onto a 10% SDS-PAGE and subsequently
transferred to nitrocellulose membranes. Membranes were blocked in 5%
milk with TBS Tween and then incubated with the different primary
antibodies followed by the appropriate HRP-conjugated secondary
antibodies.
Immunoreactive bands were identified using enhanced
chemiluminescence according to the manufacturer’s instructions. AKT
(1:1000; 9272), pAkt (1:1000; 9271S), ERK (1:1000; 9102), pERK
(1:1000; 9101S), p38 MAPK (1:1000; 9212), pp38 MAPK (1:1000;
9211S), cyclin D1 (1:1000; 2922), p27 KIP1 (1:1000; 2552) and phosphoCDK2 (1:1000; 2561S) were purchased from Cell Signaling. FAK (sc-558)
and pFAK (sc-16662-R) antibodies were purchased from Santa Cruz
Biotechnology.
Fig. 1. HoxB7Cre;ILKflox mice develop end-stage renal failure due
to obstruction in the CDs. (A)Deletion of ILK in the HoxB7Cre;ILKflox
mice was confirmed by immunoblotting the kidney medulla of
newborn mice with an anti-mouse ILK antibody (right panel).
(B,C)Gross appearance of kidneys of 6-week-old HoxB7Cre;ILKflox and
ILKflox mice. (D,E)Microscopy of Hematoxylin and Eosin (H&E) stained
kidney slides showing destruction of the medulla and corticomedullary
junction in HoxB7Cre;ILKflox kidneys but not in the ILKflox mice (40⫻).
The arrows denote the regions of the kidney that are shown at higher
power in panels F-I. (F-I)The collecting ducts (CDs) of the
HoxB7Cre;ILKflox mice are disorganized and hypercellular (G,I) when
compared with ILKflox controls (F,H). F,G, 100⫻; H,I, 400⫻.
The Student’s t-test was used for comparisons between two groups and
analysis of variance using Sigma Stat software was used for statistical
differences between multiple groups. P<0.05 was considered statistically
significant.
RESULTS
Deletion of ILK in the UB results in obstruction of
the collecting system
To define the role of ILK in the developing ureteric bud (UB),
we crossed HoxB7Cre mice (Zhang et al., 2009), which express
Cre in the wolffian duct and UB from E10.5, with ILKflox mice
(Terpstra et al., 2003). The mice were born in the predicted
Mendelian ratio; however, all the HoxB7Cre;ILKflox mice died by
eight weeks of age. ILK deletion was confirmed in mutant mice
by performing immunoblotting on isolated papillae of
HoxB7Cre;ILKflox and ILKflox mice with an antibody directed at
ILK (Fig. 1A). The predominant phenotype was that of
progressive obstruction within the papilla of the kidney resulting
in destruction of the cortex and medulla (Fig. 1B-E).
Approximately 73% (22/30) of the kidneys displayed evidence
of gross obstruction, whereas 27% (8/30) were severely
hypoplastic. In HoxB7Cre;ILKflox mice that were sacrificed prior
to developing end-stage renal failure, there was preservation of
the cortex with severe tubular abnormalities within the medulla
(Fig. 1E), whereas no abnormalities were seen in the ILKflox mice
(Fig. 1D). When compared with ILKflox mice (Fig. 1D), the
number of tubules found within the renal papilla of the
HoxB7Cre;ILKflox mice was consistent with a moderate
branching morphogenesis abnormality (Fig. 1E). However, when
the papillae were viewed at higher magnification (areas denoted
by the arrows), the architecture of the collecting ducts (CDs)
within the papilla of the HoxB7Cre;ILKflox mice was highly
atypical, with evidence of increased luminal cellularity and loss
of normal tubular architecture (Fig. 1G,I) compared with ILKflox
mice (Fig. 1F,H).
DEVELOPMENT
Statistics
3236 RESEARCH ARTICLE
Development 137 (19)
Deletion of ILK in the UB results in a moderate
branching morphogenesis defect and impaired
metanephric mesenchyme induction
We previously showed that the HoxB7Cre;1flox mice have a severe
branching morphogenesis phenotype that is present as early as
E11.5 (Zhang et al., 2009). We therefore determined the expression
pattern of ILK and 1-integrin during early kidney development by
performing immunostaining on E11.5 kidneys. Both ILK and 1integrin are expressed in the developing UB and the metanephric
mesechyme (MM) at E11.5 (Fig. 2A,B) and this expression is
increased at day E12.5 and E14.5, respectively (Fig. 2C,D). Thus,
ILK is expressed in the UB at E11.5 and its expression levels
increase with time.
To define the temporal relationship of branching abnormalities
in the HoxB7Cre;ILKflox mice, we studied embryonic kidneys
starting from E11.5. In contrast to the HoxB7Cre;1flox mice, at this
developmental stage, both HoxB7Cre;ILKflox and ILKflox mice had
developed T-shaped UBs (Fig. 2E,F). It was only at E12.5 that a
significant branching phenotype was observed in HoxB7Cre;ILKflox
mice, with an average of 6 branch points compared with the 13
branch points present in the ILKflox mice (Fig. 2G-I). A similar
branching defect was seen when HoxB7Cre;ILKflox and ILKflox
E12.5 kidneys were cultured in vitro (data not shown). In addition
to the branching phenotype, at E13.5, there was less comma- and
S-shaped body formation in the HoxB7Cre;ILKflox kidneys (Fig.
2J,K) and these defects were even more conspicuous at E14.5 (Fig.
2L,M).
To further define the UB branching defect as well as the
mechanism for defective induction of the MM in the
HoxB7Cre;ILKflox kidneys, we performed proliferation and
apoptosis assays on the embryonic kidneys of HoxB7Cre;ILKflox
and ILKflox mice. There was no difference in proliferation or
apoptosis in E12.5 kidneys in the different genotypes (data not
shown); however, there was a significant decrease in
proliferation in E13.5 HoxB7Cre;ILKflox embryonic kidneys,
which was predominantly found in the MM (Fig. 2N). There was
also a significant increase in apoptosis in the E14.5
HoxB7Cre;ILKflox kidneys, which was also mainly found in the
MM (Fig. 2O). Thus, deleting ILK early in UB development
results in a moderate branching morphogenesis phenotype
DEVELOPMENT
Fig. 2. HoxB7Cre;ILKflox mice have a moderate
branching morphogenesis defect and impaired
metanephric mesenchyme induction. (A-D)ILK
expression in wild-type embryonic kidneys at days
E11.5 to E14.5 (A,C,D) and additionally 1-integrin
expression at E11.5 (B; 200⫻). Arrows indicate the
ureteric bud (UB). (E-I)Kidneys were isolated from
embryos of HoxB7Cre;ILKflox and mice at E11.5 and
E12.5 and the GFP expressed in the UB was
visualized under a fluorescent microscope (E-H). The
number of branches from 6 kidneys in each
genotype was quantified. Differences between
HoxB7Cre;ILKflox and ILKflox mice (**) were
significant (P<0.01) when quantified (I). (J-M) H&E
staining revealed a moderate UB branching
phenotype with impaired metanephric mesenchyme
induction in HoxB7Cre;ILKflox mice when compared
with ILKflox mice at E13.5 (J,K 100⫻), which was
even more severe at E14.5 (L,M; 100⫻). (N)BrdU
staining was performed on E13.5 kidneys from
HoxB7Cre;ILKflox and ILKflox mice and quantified.
Differences between HoxB7Cre;ILKflox and ILKflox
mice (*) were significant (P<0.05). (O)TUNEL
staining was performed on E14.5 kidneys from
HoxB7Cre;ILKflox and ILKflox mice and quantified.
Differences between HoxB7Cre;ILKflox and ILKflox
mice (**) were significant (P<0.01).
characterized by both decreased proliferation and increased
apoptosis mainly in the MM, which commence at days E13.5
and 1E4.5, respectively.
Owing to the unexpected finding that the abnormalities in kidney
development only commenced at E12.5, we examined whether ILK
was being efficiently deleted in the UB at the initiation of UB
development. To do this, E11.5 UBs were dissected out of
HoxB7Cre;ILKflox kidneys and quantitative real-time PCR was
performed. Almost no ILK mRNA transcripts were present in the
HoxB7Cre;ILKflox UBs (Fig. 3A), suggesting that the Cre was
effectively deleting the ILK gene at this timepoint. Owing to the
low turnover rate of ILK (Lorenz et al., 2007), we performed
immunofluoresence to define the expression levels of ILK in E12.5
kidneys from HoxB7Cre;ILKflox mice. In contrast to the almost total
absence of ILK mRNA in the UB at E11.5, there was still
expression of ILK in the UB in the HoxB7Cre;ILKflox mice,
although it was decreased compared with ILKflox mice (Fig. 3B,C).
Thus, the moderate branching phenotype of the UB at E12.5 in the
HoxB7Cre;ILKflox mice correlates with the moderately decreased
expression of ILK in the UB at this timepoint.
ILK regulates multiple signal pathways including the WNT
signaling pathways (Oloumi et al., 2010; Oloumi et al., 2006),
which are required for renal development (Pulkkinen et al., 2008).
As both WNT4 (Chi et al., 2004; Itaranta et al., 2006; Naillat et al.,
2010; Shan et al., 2009) and WNT9b (Carroll et al., 2005; Karner
et al., 2009) play vital roles in renal development, we assessed
whether deleting ILK in the UB affects their expression by
performing in situ hybridization on E12.5 kidneys from
HoxB7Cre;ILKflox and ILKflox mice. No differences in WNT4 or
WNT9b expression were seen in the HoxB7Cre;ILKflox (Fig. 3D,F)
and ILKflox (Fig. 3E,G) mice. A key component in mediating WNTdependent signaling is GSK3 (Pulkkinen et al., 2008), which has
been shown to be regulated by ILK (Hannigan et al., 2007). We
therefore determined whether there were any alterations in GSK3
expression in the E12.5 kidneys of HoxB7Cre;ILKflox mice. When
immunohistochemistry using antibodies directed against GSK3
was performed, equal expression of this protein was observed in
E12.5 kidneys of HoxB7Cre;ILKflox (Fig. 3H) and ILKflox (Fig. 3I)
mice. Thus, the defects in kidney development caused by deleting
ILK in the UB at the initiation of its development are not due to
alterations in WNT4, WNT9b or GSK3 expression in the kidney.
HoxB7Cre;ILKflox mice develop obstruction of the
CDs due to intraluminal cell growth
To further define the obstruction phenotype in the mice, we
sacrificed mice every day during embryonic development and at
birth (Fig. 4A-I). We found a variable phenotype in newborn
HoxB7Cre;ILKflox mice from a moderate branching phenotype in
approximately 48% of mice (10/21; Fig. 4E) to evidence of severe
obstruction with a destroyed medulla and markedly dilated CDs in
the medullary rays in 52% (11/21; Fig. 4D,F). Evidence of excessive
cellularity and morphological abnormalities of the CDs was evident
even when the renal phenotypes were relatively mild (Fig. 4G,H).
This phenotype became evident at approximately E17 (Fig. 4I).
To determine whether the morphological abnormalities were due
to alterations in epithelial cell polarity, we performed staining for
epithelial cell markers on newborn mice. No differences in ZO1,
-catenin (Fig. 4J,K), Par3 or E-cadherin (Fig. 4L,M) localization
was present in the HoxB7Cre;ILKflox and ILKflox mice. There were
also no differences in membrane localization of E-cadherin
between the two genotypes, as determined by immunoblotting of
membrane and nuclear fractions of isolated medullas from the two
RESEARCH ARTICLE 3237
Fig. 3. HoxB7Cre;ILKflox mice have decreased ILK expression in
the UB by E12.5 but normal expression of WNT4, WNT9B and
GSK3. (A)Quantitative RT-PCR of UBs isolated out of HoxB7Cre;ILKflox
demonstrated significantly decreased mRNA for ILK when compared
with ILKflox mice. Differences between HoxB7Cre;ILKflox and ILKflox mice
(**) were significant (P<0.01). (B,C)Less ILK protein was expressed in
E12.5 HoxB7Cre;ILKflox (B) when compared with ILKflox (C) mice;
however, there was still expression of the protein within the UBs.
Arrows indicate the ureteric bud (UB). (D-I)In situ hybridization (D-G) or
immunostaining (H,I) revealed no differences in Wnt4 (D,E), Wnt9b
(F,G) or GSK3 (H,I) expression in E12.5 kidneys isolated from
HoxB7Cre;ILKflox (D,F,H) and ILKflox (E,G,I) mice.
genotypes (data not shown), further suggesting no abnormalities in
cellular polarity in the HoxB7Cre;ILKflox mice. To obtain highdefinition visualization of the CDs, we performed electron
microscopy on the CDs. As suggested by the polarity marker
staining, there were no obvious morphological differences in the
cells in the HoxB7Cre;ILKflox and ILKflox mice; however, it was
clear that the tubules were dilated and that the epithelial cells grew
on top of each other within the lumen of the CDs (Fig. 4N,O).
When cell proliferation in the medulla was determined in newborn
mice by Ki67 staining, there was more than double the number of
proliferating cells in the HoxB7Cre;ILKflox mice (Fig. 4P,R). By
contrast, only a few apoptoic cells were seen in the lumens of the
CDs of HoxB7Cre;ILKflox mice, whereas none were present in the
ILKflox mice (data not shown). These results suggest that the renal
tubular cells lost their ability to stop proliferating after the tubules
were fully formed, resulting in luminal obstruction.
ILK-deficient renal CD cells have adhesion,
migration and proliferation defects and are
unable to undergo tubulogenesis in vitro
We undertook two approaches to define the role of ILK in renal
tubule cell tubulogenesis. ILK was depleted in a UB cell line
utilizing siRNA, and CD cells were isolated from 6-week-old mice
DEVELOPMENT
ILK and the ureteric bud
3238 RESEARCH ARTICLE
Development 137 (19)
Fig. 4. HoxB7Cre;ILKflox mice develop intraluminal
obstruction of the CDs owing to epithelial cell
hyperproliferation. (A-I)Wholemount dissections of
the urinary system of newborn HoxB7Cre;ILKflox and
ILKflox mice, demonstrating obstructed kidneys in the
HoxB7Cre;ILKflox mice with no obstruction in the ureters
or the bladder (A,D). Kidneys dissected from mice were
stained with H&E. Some kidneys from newborn
HoxB7Cre;ILKflox mice (E) are hypoplastic and dysplastic,
whereas others are more obstructed (F), when
compared with ILKflox kidneys (B; 25⫻). There is
evidence of interaluminal hypercellularity and
obstruction of the CDs (arrows) in newborn (G,H;
100⫻) and E17.5 HoxB7Cre;ILKflox (I; 200⫻) mice when
compared with ILKflox mice (C; 100⫻). (J-M)There were
no differences in polarity between HoxB7Cre;ILKflox and
ILKflox when assessed with polarity markers Zo1 and catenin (-cat; J,K) or Par3 and E-cadherin (E-cad; L,M).
Developing renal ducts were stained with the lectin
dlichus biflorus (DBA). (N,O)Electron microscopy of CDs
demonstrates dilatation of the tubules in P1
HoxB7Cre;ILKflox mice with cells within the tubular
lumen. (P-R)Proliferation of CD cells in the
HoxB7Cre;ILKflox mice was present as shown by Ki67
staining. Differences between HoxB7Cre;ILKflox and
ILK1flox mice (**) were significant (P<0.01) when
quantified.
from CD cells results in a decrease in cell adhesion, spreading,
migration and proliferation, and inhibits in vitro tubulogenesis
without altering the ability of the cells to polarize.
ILK is required for specific integrin- and growthfactor-dependent signaling required for UB
branching morphogenesis
ILK plays a central role in regulating cell signal transduction
induced by integrin ligation and growth factors (Legate and Fassler,
2009; Legate et al., 2006; Legate et al., 2009). Owing to the
moderate adhesion, migration and spreading defects observed in
the ILK–/– CD cells, we investigated which signaling pathways
were altered when these cells were plated onto collagen I (Fig. 6A)
or laminin 111 (data not shown). Consistent with the decreased
ILK–/– CD cell adhesion, there was a decrease in FAK
phosphorylation 20 and 40 minutes after cell plating (Fig. 6A);
however, there was no alteration in AKT or ERK phosphorylation.
Surprisingly, the most dramatic change in signaling was the
inability of ILK–/– CD cells to phosphorylate p38 MAPK when
plated on collagen I.
We next defined whether this signaling abnormality was specific
to integrin ligation or whether it was also present when cells were
activated by growth factors known to be important for UB
DEVELOPMENT
and infected with adeno Cre recombinase to delete ILK in vitro
(Fig. 5A). As the results of siRNA-depleted UB and ILK–/– CD
cells are virtually identical, we only show findings obtained for the
ILK–/– CD cells.
We initially determined whether there were differences in the
ability of the ILKflox/flox and ILK–/– CD to adhere, migrate or
proliferate on 1-integrin-dependent matrices. Deleting ILK
resulted in a decrease in all three of these cell functions on
collagen I (Fig. 5B-D). Similar results were observed for laminin
111 (data not shown). The cells also demonstrated a marked
spreading defect on collagen I (Fig. 5E,F) and laminin 111 (data
not shown). No differences in integrin expression were detected
(data not shown). To determine the role for ILK in the regulation
of CD cell polarity, cells were grown on transwells until
confluent and stained with ZO1 (Fig. 5G,H) and E-cadherin
(data not shown) antibodies. Confocal microscopy revealed no
difference between the two cell lines in ZO1 or E-cadherin
localization on Z-sectioning. We also determined the effects of
deleting ILK on in vitro CD cell tubulogenesis in either 3D
collagen I (data not shown) or collagen and Matrigel gels (Fig.
5I,J). In contrast to the ILKflox CD cells, which formed welldefined branching tubules with lumens, the ILK–/– cells were
only able to form small cyst-like structures. Thus, deleting ILK
ILK and the ureteric bud
RESEARCH ARTICLE 3239
Fig. 5. Deleting ILK from CD cells results in
abnormal adhesion, migration and proliferation
on 1-integrin-dependent substrates.
(A)Immunoblotting was performed on ILKflox/flox and
ILK1–/– CD cells to verify ILK deletion. (B)CD cell
populations were allowed to adhere to different
concentrations of collagen I and cell adhesion was
evaluated 1 hour after plating. Values are the mean
± s.d. of three experiments performed in triplicates.
Asterisk denotes statistically significant difference
(P<0.01) between ILKflox/flox and ILK–/– CD cells.
(C)CD cells were plated on transwells coated with
collagen I (10g/ml) and migration was evaluated
after 4 hours. Values are the mean ± s.d. of three
experiments performed in triplicates. Asterisk
denotes statistically significant difference (P<0.01)
between ILKflox/flox and ILK–/– CD cells. (D)The CD cell
populations were plated on collagen I. After 24
hours, cells were treated with 3H-Thymidine and
incubated for a further 24 hours. 3H-Thymidine
incorporation was then determined as described in
the Materials and methods. Values are the mean ±
s.d. of three experiments performed in triplicates.
Asterisk indicates statistically significant difference
(P<0.01) between ILKflox/flox and ILK–/– CD cells.
(E,F)CD cell populations were plated on collagen I
and allowed to spread for 1 hour, after which they
were stained with Rhodamine Phalloidin. (G,H)CD
cell populations were grown on transwells and
stained with antibodies directed against ZO1.
(I,J)ILKflox/flox and ILK–/– CD cells were placed into 3D
collagen and Matrigel gels and allowed to undergo
tubulogenesis over 7 days in the presence of 5%
FBS. They were stained with Rhodamine Phalloidin
and visualized by confocal microscopy.
system in vivo. When isolated collecting systems from newborn
HoxB7Cre;ILKflox and ILKflox mice were immunoblotted for FAK,
ERK, p38 MAPK and AKT, only p38 MAPK phosphorylation was
markedly decreased in the HoxB7Cre;ILKflox mice (Fig. 6C).
Taken together, these results suggest that deleting ILK in cells
derived from the collecting system of the kidney either in vivo or
in vitro has profound effects on p38 MAPK activation.
Inability of ILK–/– CD cells to activate p38 MAPK
results in the loss of CD cell contact inhibition
The intraluminal cellular filling of the CDs and the inability to
activate p38 MAPK were the most striking phenotypes observed
in the HoxB7Cre;ILKflox mice. Because ILK–/– cells proliferate
slower than ILKflox CD cells in vitro and there is a marked
proliferative defect in the HoxB7Cre;ILKflox kidneys during the
rapid phase of UB development, we hypothesized that ILK
regulation of p38 MAPK was required to induce cell cycle arrest
in the developing UB. A study demonstrating that p38 MAPK
activation is required for cells to undergo contact inhibition in
vitro (Faust et al., 2005) further supported this hypothesis. When
non confluent, ILK–/– CD cells were placed in 10% FBS and they
grew slower than ILKflox/flox cells (data not shown); however,
they did not stop proliferating at confluence and grew on top of
each other (Fig. 7A), although they were not transformed as they
failed to grow in soft agar (data not shown). To formally test the
ability of ILK–/– CD cells to exit the cell cycle, we performed
MTT assays on ILKflox/flox and ILK–/– CD cells grown in 10%
DEVELOPMENT
branching morphogenesis. GDNF is crucial for the initial branching
phase of the UB, whereas the FGFs are required for later phases of
branching. The requirement of ILK for GDNF and FGF10
signaling was assessed on the 1-integrin-dependent matrix
collagen I, as well as plastic and the 1-integrin-independent
matrix, vitronectin. ILKflox/flox and ILK–/– CD cells were placed on
these matrices for 2 hours, after which they were stimulated with
GDNF, FGF10 or FGF2. No differences in signaling were observed
between ILKflox/flox and ILK–/– CD cells when they were plated on
collagen I (Fig. 6B), vitronectin (data not shown) or plastic (data
not shown) and exposed to GDNF. When placed on either
vitronectin (Fig. 6B) or plastic (data not shown) and exposed to
FGF10, both ILKflox/flox and ILK–/– CD cells were able to
phosphorylate FAK, AKT and ERK equally. However, the ILK–/–
CD cells did not phosphorylate p38 MAPK, whereas ILKflox/flox
cells showed robust p38 MAPK phosporylation. Similar results
were observed with FGF2 (data not shown). When placed on
collagen I, the ILK–/– CD cells could not phosphorylate p38 MAPK
and there was also decreased phosphorylation of AKT and ERK
when compared with ILKflox/flox CD cells. These results suggest that
ILK is not required for normal GDNF-dependent signaling of CD
cells; however, it differentially regulates certain FGF-dependent
signaling pathways on different matrices, with the biggest effect
seen on the p38 MAPK pathway.
As p38 MAPK activation was markedly impaired in CD cells
following integrin ligation and exposure to FGFs, we determined
whether this pathway was altered in the developing collecting
Fig. 6. ILK–/– CD cells and HoxB7Cre;ILKflox mouse renal medulla is
unable to activate P38 MAPK. (A)ILKflox/flox and ILK–/– CD cells were
either left in suspension or plated onto collagen I, after which they
were lysed at 20 and 40 minutes, respectively, and immunoblotted for
pFAK, pAKT, perk and pP38 MAPK. An immunoblot of total FAK is
shown to verify equal protein loading. (B)ILKflox/flox and ILK–/– CD cells
were allowed to adhere to collagen I or vitronectin for 1 hour, after
which they were treated with the growth factor designated for various
times. The cells were then lysed and 20g of total cell lysates were
analyzed by western blot for levels of pFAK, pAKT, pERK and pP38
MAPK. An immunoblot of total FAK is shown to verify equal protein
loading. A representative experiment is shown. (C)Medullas of
newborn ILKflox/flox and HoxB7Cre;ILKflox/flox kidneys were lysed and
20g of total protein was analyzed by western blot for levels of
phosphorylated and total FAK, ERK, p38 MAPK and AKT.
FBS. As shown in Fig. 7B, 6 days after reaching confluence,
almost a twofold-higher density saturation was achieved by the
ILK–/– compared with the ILKflox/flox CD cells. As expected, the
ILK–/– CD cells were unable to activate p38 MAPK when they
reached confluence (Fig. 7C). Consistent with the inability to
undergo contact inhibition, the ILK–/– CD cells had increased
expression of cyclin D1 and CDCK2 phosphorylation, as well as
decreased p27Kip1 accumulation (Fig. 7C). To confirm that the
decreased contact inhibition was due to decreased p38 MAPK
activation, ILKflox/flox cells were grown to confluence in the
presence and absence of the p38 MAPK inhibitor SB203580
(Fig. 7D). The inhibitor abrogated contact inhibition of these
cells when they reached confluence at day 6. These results
suggest that ILK–/– CD cells are unable to undergo contact
inhibition by exiting the cell cycle owing to their inability to
activate p38 MAPK.
We next defined whether the putative ILK kinase activity is
required for ILK to activate p38 MAPK by transfecting the ILK–/–
CD cells with wild-type ILK or point mutants including a serine
(S343A) kinase-dead mutant of the potential autophosphorylation
site (Persad et al., 2001), as well as a lysine 220 mutant to alanine
Development 137 (19)
Fig. 7. Confluent ILK–/– CD cells are unable to undergo contact
inhibition or activate P38 MAPK. (A)ILK–/– CD cells grow on top of
each other under normal culture conditions in 10% FBS. (B)ILK–/– CD
cells proliferate when cultured under confluent conditions as assessed
by the MTT assay. Values are the mean ± s.d. of three experiments
performed in triplicates. (C)Under confluent conditions, ILK–/– CD cells
are unable to activate P38 MAPK. They also have increased expression
of cyclin D1 and decreased expression of p27, as well as increased
pCDK2. An immunoblot of total p38 MAPK is shown to verify equal
protein loading. (D)ILKflox/flox CD cells lose the ability to undergo
contact inhibition, as assessed by the MTT assay when treated with the
p38 MAPK inhibitor SB203580. (E)ILK–/– CD cells stably expressing
S343A, K220A and K220M mutants proliferate like ILKflox/flox CD cells
when cultured under confluent conditions, as assessed by the MTT
assay. (F)S343A-, K220A- and K220M-expressing ILK–/– CD cells
activate p38 MAPK to similar levels as ILKflox/flox CD cells.
(K220A) or methionine (K220M) in the ATP-binding site of ILK
reported to lead to a kinase-dead ILK associated with diminished
binding to -parvin. We recently demonstrated that none of these
mutants altered the kinase activity of ILK with respect to
phosphorylating MBP (Lange et al., 2009). When MTT assays
were performed on the mutant cells at 6 days of confluency, they
all underwent contact inhibition (Fig. 7E) and phosphorylated p38
MAPK-like ILK–/– CD cells constituted with full-length ILK (Fig.
7F). We also found that ILK does not coimmunoprecipitate with
p38 MAPK (data not shown). Together, these results suggest that
ILK and p38 MAPK do not form a complex and, in line with
previous findings, we show that ILK-dependent p38 MAPK
activation is not due to ILK kinase activity.
DEVELOPMENT
3240 RESEARCH ARTICLE
DISCUSSION
We recently demonstrated that ILK is required for kidney
development. In these studies, mice expressing constitutive K220A
or K220M point-mutations in the -parvin binding site of ILK
developed severe renal agensis and/or dysgenesis due to intrinsic
abnormalities of the UB and MM, as well as defective inductive
tissue interactions between them (Lange et al., 2009). In the current
study, we explored the specific role of ILK expression in the UB
in renal collecting system formation. Unlike studies where ILK and
1-integrins were deleted in podocytes, the HoxB7Cre;ILKflox mice
did not phenocopy the HoxB7Cre;1flox mice (Dai et al., 2006; ElAouni et al., 2006; Kanasaki et al., 2008; Zhang et al., 2009).
HoxB7Cre;1flox kidneys demonstrate a lethal branching phenotype
starting at UB induction (Zhang et al., 2009); however, the
branching defect in the HoxB7Cre;ILKflox mice is only moderate
and starts at E12.5. Both genotypes die at a similar age but the
predominant abnormality in the HoxB7Cre;ILKflox mice is
obstruction due to cells that have grown into the tubular lumen of
the CDs. We further show that, in contrast to 1–/– CD cells, which
are unable to activate any signals induced by HGF, FGF and
GDNF (Zhang et al., 2009), ILK–/– CD cells transduce signals from
select growth factors and the signaling defects are primarily
restricted to the P38 MAPK pathway. Thus, ILK appears to
mediate the transduction of a limited number of signals activated
by 1-integrin that underly the major phenotpyical differences in
UB development.
The moderate branching defect that was delayed until E12.5 in
the HoxB7Cre;ILKflox mice was unexpected. In light of our recent
data that renal agenesis occurs in the ILK (K220A) and (K220M)
mice (Lange et al., 2009), it is highly unlikely that ILK expression
in the UB is not required for the initial induction and branching of
the UB (Costantini, 2006; Costantini and Shakya, 2006). The
explanation for the moderate early defects is most likely the slow
turnover of ILK following ILK gene deletion, which was also
observed in other organs such as skin (Lorenz et al., 2007). We
could clearly rule out an inefficient deletion by the HoxB7Cre
mouse because there was almost no ILK message in UBs isolated
from E11.5 kidneys.
There are big differences in the renal phenotypes between the
ILK (K220A) and (K220M) mice and the HoxB7Cre;ILKflox
mice. Renal agenesis is the principal defect in the K220A or M
mutant ILK mice, which is due to intrinsic abnormalities of both
the UB and MM and defective inductive tissue interactions
between them (Lange et al., 2009). By contrast, renal agenesis
did not occur in the HoxB7Cre;ILKflox mice because ILK is
present in the UB at the initiation of UB development. The
principal findings of excessive intraluminal cell growth in the
collecting ducts in the HoxB7Cre;ILKflox mice is not seen in the
ILK (K220A) and (K220M) mice (Lange et al., 2009). This
suggests that this phenotype is not due to the interaction of ILK
with -parvin, as this is disrupted by the K220A and K220M
mutations (Lange et al., 2009). We do not currently have a
clear molecular explanation for the phenotype of the
HoxB7Cre;ILKflox mice; however, ILK binds more than ten
proteins (McDonald et al., 2008) and many of these interactions
have physiological significance (Grashoff et al., 2004; Legate et
al., 2006; Wickstrom et al., 2010). We are in the process of
defining the molecular mechanism for this phenotype by making
specific point ILK mutations expressed exclusively in the UB in
vivo as well as by generating mice where ILK-binding partners
are deleted in the UB and comparing their phenotypes with the
HoxB7Cre;ILKflox mice.
RESEARCH ARTICLE 3241
The branching and growth abnormalities observed in the kidneys
after E12.5 is consistent with the defects in proliferation, adhesion
and migration, which are crucial processes required for UB
branching morphogenesis and tubule formation (Chen et al., 2004).
The defects in the HoxB7Cre;ILKflox mice and ILK–/– CD cells are
less marked than those found in the HoxB7Cre;1flox mice and
1–/– CD cells respectively, which is likely to be explained by our
observations that ILK only regulates restricted signaling pathways,
whereas all growth factor signaling was abrogated in 1–/– CD cells
and HoxB7Cre;flox/flox mice (Zhang et al., 2009). p38 MAPK
pathway is the major signaling pathway regulated by ILK in UB
development and it only has a moderate effect on regulating UB
branching in in vitro organ cultures (Pozzi et al., 2006).
Another interesting observation in the HoxB7Cre;ILKflox kidneys
was the relatively severe defect in metanephric mesenchyme
induction compared with the UB branching abnormality. The
reasons for this are unclear; however, we demonstrated that it is not
due to alterations in WNT4 or WNT9b expression in the
embryonic kidney. These vital paracrine pathways required for
renal development might have been ILK-dependent as ILK can
regulate WNT signaling (Oloumi et al., 2010; Oloumi et al., 2006).
It is probable that ILK affects expression of any one of the many
crucial UB-derived growth factors that induce the MM and we are
in the process of defining them; however, these studies are beyond
the scope of this manuscript.
The continued intraluminal cellular proliferation in the CDs,
which leads to intrarenal obstruction, is unprecedented. Its
correlation with decreased p38 MAPK activation in vivo suggests
that a defect in this signaling pathway is at least in part the
mechanism for the observed phenotype. Supporting this was our in
vitro evidence that integrin ligation, together with FGF treatment,
was unable to activate p38 MAPK in ILK–/– CD cells and that
ILK–/– CD cells were unable to undergo contact inhibition or
activate p38 MAPK. Furthermore, inhibiting p38 MAPK decreased
the ability of ILKflox CD cells to undergo contact inhibition. p38
MAPK activity is increased in confluent human fibroblast cultures
compared with proliferating cultures and p38 MAPK–/– fibroblasts
show a high saturation density, which is reversed by reconstituted
expression of p38 MAPK (Faust et al., 2005). p38 MAPK also
regulates both the G2/M as well as G1/S cell cycle checkpoints
(Lavoie et al., 1996; Thornton and Rincon, 2009). Consistent with
our ILK–/– CD cells, which have increased cyclin D1 expression,
p38 MAPK regulates G1/S cell cycle checkpoints by reducing the
levels of cyclin D1 at the level of transcription and by increasing
cyclin D1 ubiquitination and proteosomal degradation. p38 MAPK
phosphorylates and activates p53, which leads to the inactivation
of CDK2 and induction of a p53-dependent G2/M checkpoint
(Huang et al., 1999; Thornton and Rincon, 2009). Together, these
data strongly argue that although ILK–/– CD cells proliferate slower
than ILKflox/flox CD cells during early UB development, they fail to
undergo cell cycle arrest when tubulogenesis is complete because
they cannot activate p38 MAPK.
Almost nothing is known about the relationship between ILK
and p38 MAPK. In N1E-115 cells, p38 MAPK activation was
reported to be involved in ILK-mediated signal transduction
leading to integrin-dependent neurite outgrowth (Ishii et al., 2001).
In other studies utilizing wild-type embryonic kidney organ
cultures and IMCD cells transfected with mutants that were
proposed to alter the kinase activity of ILK, ILK was shown to be
required for BMP7-induced branching morphogenesis and a
dominant-negative
ILK
decreased
BMP7-dependent
phosphorylation of p38 MAPK and phospho-ATF2. Our in vitro
DEVELOPMENT
ILK and the ureteric bud
studies show that the kinase activity of ILK is not required for p38
MAPK activation. In addition, activation of p38 MAPK does not
appear to require ILK--parvin interactions as do the K220A and
K220M ILK mutants, which cannot interact with -parvin but are
able to activate p38 MAPK like wild-type ILK. There is also no
physical interaction between p38 MAPK and ILK as they could not
be coimmunoprecipitated. Together, these data suggest that either
ILK or one of its binding proteins might be required to target p38
MAPK to focal adhesions, where it is activated by one of the many
kinases present in this signaling hub.
In conclusion, we demonstrated that ILK and 1-integrins have
distinct roles in mediating UB development. Furthermore, deleting
ILK in renal tubular epithelial cells results in their inability to
activate p38 MAPK and exit the cell cycle, giving rise to a unique
obstructive renal phenotype. Thus, we have demonstrated a
functionally significant phenotype associated with a novel role for
ILK as a cell cycle regulator.
Acknowledgements
We would like to thank Shoukat Dedhar for the ILKflox mice, as well as Andy
McMahon and Carlton Bates for giving us the HoxB7Cre mouse. We thank
Sean Schaffer for technical help with confocal microscopy. This work was
supported by NIH grants DK065123 (A.P. and R.Z.); DK075594 (R.Z.);
DK65123 (R.Z.); DK083187 (R.Z.); AHA established investigator award (R.Z.); a
Merit award from the Department of Veterans Affairs (A.P. and R.Z.) and the
George O’Brien Center Grant (A.P. and R.Z.) and grants from the Canadian
Institutes of Health Research and Canada Research Chairs Program (to N.D.R.).
Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
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