MOLECULAR AND CELLULAR BIOLOGY, May 2005, p. 3648–3657
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 25, No. 9
Integrin-Linked Kinase Mediates Bone Morphogenetic Protein
7-Dependent Renal Epithelial Cell Morphogenesis
Chungyee Leung-Hagesteijn,1Ming Chang Hu,2Ahalya S. Mahendra,1,3,4
Sunny Hartwig,2,5Henry J. Klamut,6† Norman D. Rosenblum,2,3,5,7,8
and Gregory E. Hannigan1,3,4*
Cancer Research Program,1Program in Developmental Biology, Research Institute,2Division of Nephrology,5
and Department of Pediatric Laboratory Medicine,3Hospital for Sick Children, Ontario Cancer
Institute, University Health Network,6and Departments of Laboratory Medicine
and Pathobiology,4Physiology,7and Paediatrics,8University
of Toronto, Toronto, Ontario, Canada
Received 26 October 2004/Returned for modification 22 November 2004/Accepted 1 February 2005
Bone morphogenetic protein 7 (BMP7) stimulates renal branching morphogenesis via p38 mitogen-activated
protein kinase (p38MAPK) and activating transcription factor 2 (ATF-2) (M. C. Hu, D. Wasserman, S. Hartwig,
and N. D. Rosenblum, J. Biol. Chem. 279:12051–12059, 2004). Here, we demonstrate a novel role for integrin-
linked kinase (ILK) in mediating renal epithelial cell morphogenesis in embryonic kidney explants and identify
p38MAPKas a target of ILK signaling in a cell culture model of renal epithelial morphogenesis. The spatial and
temporal expression of ILK in embryonic mouse kidney cells suggested a role in branching morphogenesis.
Adenovirus-mediated expression of ILK stimulated and expression of a dominant negative ILK mutant
inhibited ureteric bud branching in embryonic mouse kidney explants. BMP7 increased ILK kinase activity in
inner medullary collecting duct 3 (IMCD-3) cells, and adenovirus-mediated expression of ILK increased
IMCD-3 cell morphogenesis in a three-dimensional culture model. In contrast, treatment with a small molecule
ILK inhibitor or expression of a dominant negative-acting ILK (ILKE359K) inhibited epithelial cell morpho-
genesis. Further, expression of ILKE359Kabrogated BMP7-dependent stimulation. To investigate the role of
ILK in BMP7 signaling, we showed that ILK overexpression increased basal and BMP7-induced levels of
phospho-p38MAPKand phospho-ATF-2. Consistent with its inhibitory effects on IMCD-3 cell morphogenesis,
expression of ILKE359Kblocked BMP7-dependent increases in phospho-p38MAPKand phospho-ATF-2. Inhi-
bition of p38MAPKactivity with the specific inhibitor, SB203580, failed to inhibit BMP7-dependent stimulation
of ILK activity, suggesting that ILK functions upstream of p38MAPKduring BMP7 signaling. We conclude that
ILK functions in a BMP7/p38MAPK/ATF-2 signaling pathway and stimulates epithelial cell morphogenesis.
Renal branching morphogenesis (RBM), is defined as
growth and branching of the ureteric bud (UB) and its daugh-
ter collecting ducts. RBM is dependent on inductive tissue
interactions with the surrounding metanephric mesenchyme,
or blastema, which secretes growth factors that induce ureteric
bud invasion and branching (38). Reciprocal signals are elab-
orated by the epithelial bud, thereby providing survival and
differentiation signals for cells of the nephrogenic mesen-
chyme. One such signal implicated in RBM is bone morpho-
genetic protein 7 (BMP7) (11, 31), a member of the transform-
ing growth factor ? superfamily. Expression of BMP7 in the
ureteric bud and metanephric blastema is consistent with a
physiological role during RBM (12, 39); moreover, BMP7 null
mice display profound defects of RBM (11, 30).
While the presence of reciprocal tissue interactions limits
the ability to interpret the primary effects of BMP7 in vivo, we
have identified BMP7 functions in the inner medullary collect-
ing duct 3 (IMCD-3) cell culture model of RBM (37, 39).
IMCD-3 cells form tubule progenitors (i.e., morphogenesis)
within 48 h of being cultured in collagen gels and respond to
serum or purified growth factors in a manner identical to
branching observed in embryonic kidney explants (3, 39).
BMP7 acts to control IMCD-3 cell morphogenesis in a com-
plex manner. High doses (?0.5 nM) inhibit morphogenesis in
an Smad1-dependent manner, whereas low doses (?0.5 nM)
stimulate progenitor formation in a Smad1-independent man-
ner via effects on cell proliferation (37, 39). We have also dem-
onstrated that stimulatory doses of BMP7 activate p38 mitogen-
activated protein kinase (p38MAPK) and its target, activating
transcription factor 2 (ATF-2), and that inhibiting p38MAPKac-
tivity blocks IMCD-3 morphogenesis (22). Our demonstration
that Smad1 activity and p38MAPK/ATF-2 activation are inversely
related suggests a cellular mechanism that integrates stimulatory
and inhibitory BMP7 signal transduction pathways.
Integrins mediate essential cell-extracellular matrix (ECM)
interactions during mammalian development but also trans-
duce signals regulating cell proliferation, differentiation, and
survival. In addition to growth factors, integrins contribute to
epithelial-mesenchymal interactions during kidney organogen-
esis. Genetic ablation of the ?8 integrin precludes formation of
functional ?8?1 integrin in the mesenteric mesenchyme, re-
sulting in severe defects of ureteric bud branching (34). The
* Corresponding author. Mailing address: Cancer Research Pro-
gram, Research Institute, Hospital for Sick Children, 555 University
Ave., Toronto, Ontario M5G 1X8, Canada. Phone: (416) 813-8149.
Fax: (416) 813-8883. E-mail: email@example.com.
† Present address: Musculoskeletal Disease Center, Jerry L. Pettis
Memorial VA Medical Center Department of Medicine, Loma Linda
University, Loma Linda, Calif.
integrin-linked kinase (ILK) is an intracellular protein serine/
threonine kinase that coordinates signaling by integrins and
growth factors (6–8, 19), including insulin-like growth factor 1
(IGF-1) (29, 32), nerve growth factor (33), platelet-derived
growth factor (2), and vascular endothelial growth factor (24,
44), in a variety of cell types. ILK binds directly to the cyto-
plasmic domains of ? integrin subunits (19). Additional pro-
tein interactions may function to physically link ILK with re-
ceptor tyrosine kinases (RTKs). ILK binds to PINCH, a LIM-
only adaptor protein (46, 49). PINCH binds to another adaptor
protein, Nck2, an SH2/SH3-containing protein that associates
with ligand-activated epidermal growth factor (EGF) and
platelet-derived growth factor receptors (47). In addition, li-
gand activation of these RTKs stimulates phosphoinositide
3?-OH kinase (PI 3-K) activity, and genetic or pharmacologic
inhibition of PI 3-K abolishes both RTK- and integrin-medi-
ated ILK activation (6, 7). The mechanism of ILK activation
involves the major lipid product of PI 3-K activity, phospho-
inositol-3,4,5-phosphate, which likely activates ILK by binding
to the pleckstrin homology-like domain (9). Thus, PI 3-K de-
pendent ILK activity exerts contextual effects governing epi-
thelial-mesenchymal transition (43), myoblast differentiation,
neurite outgrowth, and vascular morphogenesis.
BMP7 and ?8 integrin regulate RBM in vivo, suggesting
integrated growth factor and cell adhesion signaling during
collecting duct morphogenesis. Here, we report that ILK dis-
plays an expression pattern in the embryonic mouse kidney
that is consistent with a role in RBM. Infection of embryonic
mouse kidney explants with adenovirus (Ad) expressing a dom-
inant negative mutant of ILK markedly impaired formation of
collecting ducts, further implicating ILK signaling in RBM. We
therefore investigated the role of ILK in mediating BMP7-
dependent renal epithelial cell morphogenesis, using the
IMCD-3 model system. Treatment of IMCD-3 cells with stim-
ulatory concentrations of BMP7 rapidly (?15 min) induced
activation of ILK. Moreover, adenovirus-mediated expression
of ILK markedly stimulated the formation of tubule progeni-
tors and increased levels of phospho-p38MAPKand phospho-
ATF-2. Conversely, a small molecule inhibitor of ILK, KP-392,
abrogated IMCD-3 morphogenesis in three-dimensional cul-
tures. In addition, infection with a dominant negative ILK
mutant blocked BMP7-induced morphogenesis and inhibited
the phosphorylation of p38MAPKand ATF-2. Our results iden-
tify a novel BMP7/ILK/p38MAPK/ATF-2 signaling pathway
controlling epithelial cell morphogenesis.
MATERIALS AND METHODS
Immunohistochemistry. Embryonic day 13.5 (E13.5) mouse kidney sections
were processed for immunohistochemistry as described previously by us, with
slight modifications (21). Paraffin-embedded sections were deparaffinized and
heated in 10 mM citrate (pH 6.0) with a microwave oven for antigen retrieval.
After quenching endogenous peroxidase activity with H2O2for 10 min, sections
were incubated overnight at 4°C with ILK primary antibody (Upstate Biotech-
nology, Inc.; catalogue no. 06-592) at a 1:35 dilution.
Embryonic mouse kidney explant culture. Embryonic kidneys were surgically
dissected from E13.5 pregnant mice and cultured as previously described (22).
To evaluate the effect of ILK on RBM, adenoviruses expressing green fluores-
cent protein (GFP), ILK, or a dominant negative variant of ILK (28) were added
to culture medium at 106, 105, and 104PFU/ml, respectively, for 5 days. To verify
infection efficiencies, fluorescent photos were taken just before explants were
fixed for Dolichos biflorus agglutinin (DBA) staining. Selective staining of ure-
teric bud-derived structures in the whole-mount kidney specimens was achieved
with fluorescein isothiocyanate-conjugated DBA (20 ?g/ml) (Vector Laborato-
ries, Burlington, Ontario, Canada) as previously described (16).
IMCD-3 cell culture, growth factor treatment, and morphogenesis assays. The
IMCD-3 cell line is derived from the terminal inner medullary collecting duct of
the simian virus 40 transgenic mouse. The IMCD-3 cell line retains several
differentiated characteristics of the nephron, as previously described (40), and
has been used as an in vitro model of collecting duct morphogenesis. IMCD-3
cells were grown in monolayers and maintained in Dulbecco’s modified Eagle’s
medium–Ham’s F12 medium (DMEM-F12) supplemented with 5% fetal bovine
serum (Sigma), penicillin (100 U/ml), and streptomycin (100 U/ml) in 5% CO2
To assay IMCD-3 cell morphogenesis, cells were suspended in type I collagen
gels in 96-well tissue culture plates as previously described (39). Collagen gels
were prepared on ice by mixing 4 ?l of 1 M N-2-hydroxyethylpiperazine-N-2-
ethanesulfonic acid (Sigma), 8 ?l of 1 M NaHCO3, 40 ?l of DMEM-F12, 200 ?l
of 3.5-mg/ml rat type I collagen (Collaborative Biomedical Products), and 50,000
IMCD-3 cells. Aliquots (each, 50 ?l/well) were seeded in 96-well culture plates.
Gels were solidified at 37°C, and then 100 ?l of medium containing 5% fetal
bovine serum was added to each well. Cultures were maintained at 37°C in 5%
CO2. BMP7 and epidermal growth factor stock solutions were prepared in
DMEM-F12 and added at the indicated concentrations to the 5% fetal bovine
serum-containing culture medium of newly established cultures of IMCD-3 cells.
Cells were cultured in monolayers for biochemical studies or embedded in
collagen gels for morphogenetic studies. After 48 h, gels were fixed in 4%
formaldehyde in phosphate-buffered saline for 10 min at room temperature.
Fixed gels were then washed four times in phosphate-buffered saline and imaged
by differential interference contrast microscopy. Eight microscopic fields of
equivalent dimensions were randomly selected and photographed at 100? mag-
nification. Morphogenesis was quantified by counting the number of elongated,
linear structures in these fields, presented as the number of independent linear
structures per area of standard dimension. Assays were standardized for the
number of cells seeded into the each collagen gel, as we have previously pub-
lished (37, 39). The statistical significances between treatment differences were
determined by a two-tailed, nonpaired Student’s t test, using Prism software,
version 3.0 (GraphPad Software, Inc.).
ILK immune complex kinase assay. ILK immune complex kinase assays were
carried out as described previously (19, 29). Protein concentrations were deter-
mined by Bradford assays (Bio-Rad, Richmond, Calif.). Cell lysates (0.25 to 1.0
mg of protein) were immunoprecipitated with 1 ?g of affinity purified rabbit
anti-ILK (Upstate Biotechnology, Inc.; catalogue no. 06-592) overnight at 4°C
with rotation. Protein A-Sepharose (Sigma), preswollen in NP-40 lysis buffer
(150 mM NaCl, 1% [vol/vol] NP-40, 0.5% [wt/vol] sodium deoxycholate, 50 mM
HEPES [pH 7.4], 1 ?g of leupeptin/ml, 1 ?g of aprotinin/ml, 3 mM phenylmeth-
ylsulfonyl fluoride) was added for 2 h at 4°C to capture the antibodies. After two
washes with NP-40 lysis buffer and two washes with kinase wash buffer (10 mM
MgCl2, 10 mM MnCl2, 50 mM HEPES [pH 7.5], 0.1 mM sodium orthovanadate,
1 mM dithiothreitol), assays were performed directly on the protein A beads in
a 25-?l reaction volume containing 10 mM MgCl2, 10 mM MnCl2, 50 mM
HEPES (pH 7.5), 0.1 mM sodium orthovanadate, 2 mM sodium fluoride, 5 ?Ci
of ?-32P (Amersham, Piscataway, N.J.) and 2.5 ?g of myelin basic protein (MBP)
as substrate (Upstate Biotechnology, Inc.). Incubation was for 30 min at 30°C.
The reaction was stopped with 10 ?l of sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) nonreducing stop buffer and heated for 5 min
at 95°C. Phosphorylated MBP bands were visualized by autoradiography of dried
SDS–10% PAGE gels, followed by quantitation in a PhosphorImager (Molecular
Adenovirus expression constructs. Adenoviruses expressing either wild-type
or dominant negative ILK were constructed for expression in kidney explants and
in IMCD-3 cells. Briefly, the full-length ILK coding sequence or the dominant
negative point mutant, E359K, including a C-terminal myc-His epitope tag, was
amplified from a pcDNA3.1 construct. EcoRV was used to digest the ILK–myc-
His expression fragments for subcloning into pAd Trac (pAd Easy kit; Clontech).
Bicistronic GFP and ILK expression allowed infection efficiencies to be con-
firmed visually by fluorescent microscopy. Viruses were amplified and CsCl
gradient purified and titers were determined on HEK293 cells as described
Antibodies and Western blotting. For analysis of protein levels by Western
blotting, SDS-PAGE gels were transferred to polyvinyldifluoride membranes
(Immobilon-P; Millipore, Bedford, Mass.) in transfer buffer (25 mM Tris, 192
mM glycine, 20% methanol). Membranes were blocked in 5% milk in TBST (20
mM Tris [pH 7.5], 500 mM NaCl, 0.1% Tween 20). Affinity-purified primary
polyclonal or monoclonal antibodies were used at a concentration of 1 ?g/ml in
Tris-buffered saline with 1% (wt/vol) bovine serum albumin (Fraction V; Sigma).
VOL. 25, 2005ILK MEDIATES EPITHELIAL CELL MORPHOGENESIS3649
Secondary antibodies were goat anti-mouse or anti-rabbit coupled to peroxidase,
used at a dilution of 1/20,000 in TBST. Protein bands were visualized by chemi-
luminescence (ECL; Amersham) and exposure to Kodak X-Omat film, and
signals were quantified with Kodak ID digital imaging software, version 2.0.2.
Antibodies specific to phospho-glycogen synthase kinase 3? (phospho-GSK3?)
(pSer9), protein kinase B (PKB) (Ser473), p38MAPK, ATF-2, phospho-p38MAPK
(Thr180/182), and phospho-ATF-2 (Thr69/71) were purchased from Cell Signal-
ing Technology, and antibodies recognizing total GSK3?/? and PKB were ob-
tained from Transduction Labs. A monoclonal antibody to the c-myc epitope was
purchased from Santa Cruz Biotechnology.
Immunohistochemical analysis of embryonic mouse kidney
revealed strong ILK immunoreactivity in the uretric bud and
metanephric mesenchyme of E13.5 kidneys (Fig. 1). By E17.5,
ureteric bud cells have differentiated into collecting duct cells
in the cortex and medulla. At this stage as well, ILK is strongly
expressed throughout the collecting duct system. Thus, the
temporal and spatial pattern of ILK in embryonic kidney is
consistent with a functional role for ILK during RBM. To test
whether ILK activity plays a role in renal morphogenesis, we
infected embryonic mouse kidney explants with adenoviruses
carrying wild-type ILK (Ad-ILKWT), or a dominant negative
ILK mutant (Ad-ILKE359K). We confirmed that control, Ad-
ILKWT, and Ad-ILKE359Kviruses infected the kidney explants
efficiently, judging by the expression of virally encoded GFP in
whole-mount explants (Fig. 2A). Interestingly, we noted that
Ad-ILKWT-infected kidneys appeared slightly larger and those
infected with Ad-ILKE359Kappeared smaller than control virus
(AdCONTROL)-infected explants. This is consistent with a role
for ILK in regulating cell proliferation in the intact kidney.
After 5 days in culture, formation of collecting ducts was
readily apparent in the control virus-infected kidneys. Infec-
tion with the Ad-ILKWTvirus stimulated an increase in the
number of UB branches formed, and UB formation was mark-
edly inhibited in kidneys that had been infected with Ad-
ILKE359K(Fig. 2B). These expression and functional data sug-
gest an important role for ILK signaling in RBM.
The cellular complexity and dynamic epithelial-mesenchy-
mal interactions in the developing kidney make it difficult to
discriminate direct and indirect effects of growth factors. To
directly examine a role of ILK in renal cell morphogenesis, we
first tested whether BMP7 induced ILK activity in IMCD-3 cell
cultures. We used EGF as a positive control for ILK stimula-
tion and induction of tubule progenitors, since it potently in-
duces ILK activity in other epithelial cells (unpublished data).
Growth factor-dependent morphogenesis was quantified by
culturing the IMCD-3 cells in three-dimensional collagen gels
containing 5% serum, 0.25 nM BMP7, or 20 ng of EGF/ml. In
this morphogenetic assay system, IMCD-3 cells forms struc-
tures at 48 h comprising two or more cells, with cellular pro-
FIG. 1. ILK is expressed in the UB and metanephric mesenchyme of E13.5 kidneys and in the collecting duct system at E17.5. E13.5 and E17.5
mouse kidneys were sectioned and stained with nonimmune immunoglobulin G (control) or ILK antibody (anti-ILK). Sections demonstrate
ureteric bud (UB), glomerular progenitors (GP), cortex (Ctx), medulla (Med), collecting duct (CD), and glomeruli (G). E13.5 fields shown are
magnifications of ?400, and E17.5 fields are magnifications of ?100.
3650 LEUNG-HAGESTEIJN ET AL.MOL. CELL. BIOL.
cesses that form the basis for multicellular branches, which are
observed after 5 to 7 days in culture. Again, both these growth
factors enhanced IMCD-3 morphogenesis significantly over
serum-induced (control) levels. BMP7 induced a 1.4-fold in-
crease, and EGF induced a 2-fold increase, over serum-in-
duced levels (Fig. 3A). We then assayed for growth factor
activation of ILK, using immune complex kinase assays. BMP7
(0.25 nM) and EGF (20 ng/ml) induced ILK activity to from
five- to sixfold over unstimulated levels (Fig. 3B). Thus, ILK
activation is an early event during BMP7-dependent IMCD-3
cell morphogenesis, and these results therefore raised the
question of whether ILK acts to stimulate morphogenesis. We
directly tested whether ILK promotes morphogenesis by in-
fecting IMCD-3 cells with Ad-ILKWT. Infection with Ad-
ILKWTstimulated tubule progenitor formation 2.8-fold rela-
tive to serum-induced levels (Fig. 4), indicating that ILK is a
positive mediator of IMCD-3 morphogenesis. We also noted
an increase in cell numbers in Ad-ILKWT-infected cultures;
thus, like BMP7, ILK controls both proliferation and morpho-
genesis of renal epithelial cells. To test whether morphogenesis
requires ILK activity, we inhibited ILK by infecting IMCD-3
cells with adenovirus expressing a dominant negative ILK mu-
tant, ILKE359K. We (28, 32) and others (33, 45) have shown
that the ILKE359Kmutant exerts dominant inhibition of growth
FIG. 2. ILK controls ureteric bud morphogenesis in embryonic kidney explant cultures. (A) Embryonic (E13.5) mouse kidneys were cultured
as explants as described in Materials and Methods. Adenoviruses biscistronically expressing GFP and ILK, dominant negative ILK (GFP-dnILK),
or control GFP virus were added to explant cultures at a concentration of 105PFU/ml. Fluorescent microscopic analysis of GFP expression in
whole-mount preparations at 5 days postinfection indicated efficient infection by each virus. (B) Embryonic explants were infected at increasing
doses with ILK adenoviruses, as in panel A. At 5 days postinfection, explants were fixed and stained with DBA for specific visualization of ureteric
bud branches. Images in both panels are representative of 20 explants examined for each viral infection group.
VOL. 25, 2005 ILK MEDIATES EPITHELIAL CELL MORPHOGENESIS3651
factor- or ECM-induced ILK activity in a number of cell lines.
Infection of IMCD-3 cells with the Ad-ILKE359Kvirus inhib-
ited tubule progenitor formation by approximately twofold
over serum control levels (Fig. 4). Thus, ILK is sufficient to
induce IMCD-3 morphogenesis, and inhibition of ILK blocks
We next tested whether ILK mediates BMP7-dependent
morphogenesis by quantifying BMP7-induced formation of tu-
bule progenitors in Ad-ILKE359K-infected cultures. BMP7-in-
duced morphogenesis in Ad-ILKE359K-infected cells was de-
creased by approximatelysixfold,
AdCONTROLvirus-infected cells. Similarly, EGF-stimulated
morphogenesis was decreased about threefold by Ad-
ILKE359K, relative to that of control virus-infected cultures
(Fig. 5A). These results demonstrate that ILK activity medi-
ates morphogenetic signaling by BMP7.
As a second, independent method of inhibiting ILK we used
KP-392, a small molecule that selectively inhibits ILK activity
(18). IMCD-3 cells were cultured in collagen gels supple-
mented with 5% serum or 20 ng of EGF/ml, with or without 5
?M KP-392 (50). After 48 h, morphogenesis was visually quan-
tified (Fig. 5B). Serum stimulated morphogenesis was inhibited
by about 2 fold, and EGF stimulation was inhibited by 4.5 fold
in KP-392-pretreated cells. We could not determine an effect
of KP-392 on BMP7-dependent morphogenesis, since treat-
ment with a dimethyl sulfoxide vehicle at the appropriate con-
trol concentration abrogated BMP7 activity. These results,
coupled with those showing stimulation of tubule progenitors
by Ad-ILK, indicate that ILK activity plays a role in IMCD-3
ILK activation, by growth factors or integrin-mediated cell
adhesion, is dependent on the activity of PI 3-K. Thus, genetic
or pharmacologic inhibition of PI 3-K signaling blocks activa-
tion of ILK by a variety of stimuli in epithelial cells, platelets,
and myoblasts (9, 13, 32, 35, 36). To test for the involvement of
PI 3-K activity in the BMP7 stimulation of ILK, we used the
selective PI 3-K inhibitor LY294002. We pretreated IMCD-3,
cultured in collagen gels, with LY294002 and then quantified
induction of tubule progenitors by BMP7 and EGF. LY294002
pretreatment inhibited BMP7 induction of IMCD-3 tubule
progenitors by 2.3 fold and inhibited EGF induction by 3.3 fold
(Fig. 6A), suggesting that PI 3-K activity is required in BMP7-
and ILK-dependent morphogenesis. We therefore tested
whether LY294002 inhibited BMP7-dependent ILK activation
by two independent assays of ILK activity. LY294003 effec-
tively suppressed BMP7-activated GSK3? Ser9 phosphoryla-
tion, a known cellular target of ILK (Fig. 6B). Similarly, pre-
treatment of IMCD-3 cells with LY294002 inhibited ILK
activity as measured by ILK immune complex kinase assays
(Fig. 6B). Our results indicate that BMP7 stimulation of both
morphogenesis and ILK activity requires PI 3-K activity.
We recently reported that stimulatory doses of BMP7 acti-
vate p38MAPKin IMCD-3 cells and that the selective p38MAPK
inhibitor, SB203580, blocks BMP7-dependent morphogenesis
(22). In the light of our present results showing ILK-dependent
IMCD-3 morphogenesis, we examined whether ILK stimulates
p38MAPKactivity and whether blocking ILK signaling with a
dominant negative ILK mutant blocks stimulation of p38MAPK
by BMP7. Ligand-induced phosphorylation on Thr180 and
Tyr182 activates p38MAPK. To test for ILK-dependent stimu-
lation of p38MAPK/ATF-2, we assayed levels of phospho-
p38MAPKin IMCD-3 cultures infected with AdCONTROL, Ad-
ILKWT, or the dominant negative Ad-ILKE359Kmutant virus
(Fig. 7). Forty-eight hours after infections, cells were harvested
and analyzed by Western blotting with phosphospecific anti-
bodies to p38MAPK(Thr180/Tyr182). Cells were treated for 1 h
with BMP7 to induce p38MAPKphosphorylation (22). As a
positive control, we assessed ILK-dependent GSK3? Ser9
relative to thatof
FIG. 3. BMP7 and EGF induce morphogenesis and ILK activ-
ity.(A) IMCD-3 cells were cultured in three-dimensional collagen gels
containing 5% fetal calf serum, 0.25 nM BMP7, or 20 ng of EGF/ml.
After 48 h, tubule progenitors were quantitated as described in Mate-
rials and Methods. The mean numbers of progenitors (? standard
deviation) per microscope field are as follows: serum stimulated, 55 ?
5; BMP7, 75 ? 7; and EGF, 115 ? 9. The statistical significance of
BMP7 and EGF stimulations is indicated, as determined by a two-
tailed Student’s t test. B. IMCD-3 cells were grown in standard two-
dimensional cultures and serum starved for 18 h prior to stimulation
for 15 min with 0.25 nM BMP7 or 20 ng of EGF/ml. ILK activity was
assayed by ILK immune complex kinase assays with exogenous MBP
substrate. Relative quantitation was by densitometry of bands. Results
are from three independent determinations, and error bars indicate
standard errors of the mean. Inset is a representative blot of
incorporation into MBP in the kinase assays.
3652LEUNG-HAGESTEIJN ET AL.MOL. CELL. BIOL.
phosphorylation in the same lysates. Phospho-p38MAPKlevels
were increased in Ad-ILKWT- but not Ad-ILKE359K- or
AdCONTROL-infected cultures. Infection with Ad-ILK also
stimulated phosphorylation of GSK3? on Ser9, whereas cells
infected with AdCONTROLor Ad-ILKE359Kshowed no increase
in pSer9 levels (not shown). BMP7 also stimulated p38MAPK
phosphorylation in control, but not in Ad-ILKE359Kinfected
cells. As a positive control, phosphorylation of the known ILK
target, PKB Ser473, was stimulated by Ad-ILKWTand blocked
by Ad-ILKE359k(Fig. 7A). These data indicate that ILK can
activate p38MAPKindependently of ligand and that induction
of p38MAPKby BMP7 is ILK dependent.
FIG. 4. ILK activity induces IMCD-3 tubule progenitors. IMCD-3 cells were infected with Ad-ILKWT, dominant negative Ad-ILKE359K, or
“empty” AdCONTROLviruses (multiplicity of infection [MOI] ? 5). At 24 h postinfection, cells were cultured in collagen gels containing 5% serum,
and formation of tubule progenitors was quantified 48 h after seeding. Bars represent data from five independent determinations. White arrows
indicate linear structures that were enumerated as tubule progenitors. The inset shows a Western blot with expression of myc-tagged ILK constructs
at different MOI values. Significant differences between Ad-ILKWTcultures and control or E359K-infected cultures were determined by a
two-tailed Student’s t test.
VOL. 25, 2005 ILK MEDIATES EPITHELIAL CELL MORPHOGENESIS3653
Activating phosphorylation of the transcription factor,
ATF-2, on Thr71 is mediated by p38MAPK. Our previous work
showed that BMP7 stimulates ATF-2 phosphorylation in
IMCD-3 cells and that pretreatment of cells with the p38MAPK
inhibitor SB203580 blocks ATF-2 phosphorylation (22). We
treated IMCD-3 cells with BMP7 for 15 and 60 min and found
that although ATF-2 phosphorylation was evident at 60 min, it
was not appreciably induced at 15 min (data not shown). We
reasoned that BMP7 signaling at this relatively early time point
would be more robust in ILK-expressing cells and therefore
tested whether BMP7-dependent ATF-2 phosphorylation was
affected by increased ILK expression. IMCD-3 cells infected
with either Ad-ILKWTor Ad-ILKE359Kviruses were treated
for 15 min with BMP7 and assayed for ATF-2 phosphorylation.
Ad-ILKWTbut not Ad-ILKE359Kstimulated ATF-2 Thr71
phosphorylation (Fig. 7B), similar to p38MAPK(Fig. 7A).
BMP7 treatment for 15 min potentiated ATF-2 phosphoryla-
tion in Ad-ILKWT- but not in Ad-ILKE359K-infected cells (Fig.
7B). Thus, increased ILK expression accelerated BMP7-in-
duced ATF-2 phosphorylation, further indicating that ILK acts
in the BMP7/p38/ATF-2 signaling axis.
Our data suggested that ILK lies upstream of p38MAPKin
FIG. 5. Dominant negative ILK mutant blocks BMP7-induced
IMCD-3 morphogenesis. (A) Cells were cultured in collagen gels con-
taining 5% serum, 0.25 nM BMP7, or 20 ng of EGF/ml, and induction
of morphogenesis was quantitated after 48 h. BMP7-induced morpho-
genesis in AdCONTROL-infected cells was 121 ? 6 per microscope field,
decreasing to 21 ? 2 tubule progenitors per field in Ad-ILKE359K-
infected cells. EGF-stimulated morphogenesis was similarly decreased,
from 143 ? 5 to 52 ? 8 per field, by Ad-ILKE359Kinfection. The
statistical significance (n ? 6; P ? 0.05) between AdCONTROLand
Ad-ILKE359Kin all treatment cases was determined by a two-tailed
Student’s t test. (B) Cells were seeded into collagen gels in the pres-
ence of 5% serum, with or without 5 ?M KP-392 ILK inhibitor. After
48 h, morphogenesis was visually quantified (Fig. 4B). Serum-stimu-
lated morphogenesis was inhibited from 28 ? 2 to 12 ? 4 progenitors/
field, and EGF stimulation was inhibited from 45 ? 6 to 10 ? 1
progenitors/field by the KP-392 ILK inhibitor. The dimethyl sulfoxide
vehicle controls were at the same dilution as in the inhibitor-treated
gels. The significant difference in EGF stimulation between vehicle
and KP-392 treated cultures is indicated, calculated by a Student’s t
test as described in Materials and Methods.
FIG. 6. BMP7 induction of morphogenesis and ILK activation are
PI 3-K dependent. (A) IMCD-3 cells were seeded in collagen gels
treated with BMP7 and EGF as shown in Fig. 4, with and without 10
?M LY294002. Tubule progenitors were quantified after 48 h. The
statistical significance level between vehicle and LY294002-treated
cultures is indicated. (B) After serum starvation for 18 to 20 h,
IMCD-3 cells were pretreated for 1 h with LY294002, as in panel A.
Cells were then treated for 15 min with BMP7, and cytoplasmic lysates
were analyzed for induced levels of GSK3? Ser9 phosphorylation.
Numbers below the panel indicate ratios of phospho-GSK3? to total
GSK3? by densitimetry. (C) IMCD-3 cells were treated as in the
experiment shown in panel B, and cytoplasmic lysates were then sub-
jected to ILK immune complex kinase assays, using MBP as exogenous
substrate. All results are representative of three independent experi-
3654 LEUNG-HAGESTEIJN ET AL.MOL. CELL. BIOL.
the BMP7 morphogenetic pathway; therefore, we tested the
effects of SB203580 p38MAPKinhibitor on BMP7-induced ILK
activity. Cells were pretreated for 60 min with 0 or 10 ?M
SB203580 and subsequently treated for 15 min with 0.25 nM
BMP7. As a measure of ILK activation, we assayed phospho-
GSK3? (pSer9) levels. SB203580 pretreatment did not inhibit
BMP7-induced Ser9 phosphorylation (Fig. 7C). These lysates
were also subjected to ILK immune complex kinase assays (28,
29), which showed lack of inhibition of BMP7-induced ILK
activity by SB203580, whereas LY294002 inhibited ILK activity
(Fig. 7D). These results place p38MAPKdownstream of ILK
activation by BMP7. Together with the results showing inhibi-
tion of BMP7-induced p38MAPKphosphorylation and morpho-
genesis by ILKE359K, these data identify a novel BMP7/ILK/
The results presented here implicate ILK as an effector of
BMP7-dependent epithelial cell morphogenesis. We find that
ILK is abundantly expressed in medullary collecting duct epi-
thelium of E13.5 and E17.5 mouse kidneys, indicating it could
mediate RBM. Such a role for ILK is further supported by our
observation that expressing dominant negative ILK inhibits
formation of ureteric bud branches in embryonic kidney ex-
plants. We have shown a direct effect of ILK in mediating renal
epithelial cell morphogenesis. BMP7 induces ILK activity dur-
ing in vitro morphogenesis of IMCD-3 collecting duct cells.
Inhibition of ILK by either a small molecule or a dominant
negative ILK mutant blocks IMCD-3 tubule progenitor forma-
tion. Consistent with this, adenovirus-mediated expression of
ILK promotes IMCD-3 morphogenesis, suggesting that ILK is
sufficient for induction of epithelial cell morphogenesis. Fur-
thermore, our data showed that ILK activity stimulates phos-
phorylation of p38MAPKand ATF-2 during BMP7-dependent
morphogenesis and that a dominant negative ILK mutant ef-
ficiently blocks p38MAPK/ATF-2 activation. Inhibition of
p38MAPKactivity does not affect BMP7-dependent activation
of ILK, consistent with ILK acting upstream of p38MAPKin this
Using both embryonic kidney explants and the IMCD-3 cell
culture model, we have previously shown opposite, dose-de-
pendent effects of BMP7 in the regulation of epithelial cell
morphogenesis (39). Low doses (?0.5 nM) stimulate and high
doses (?0.5 nM) inhibit formation of IMCD-3 tubule progen-
itors. Interestingly, induction of ILK activity in our hands is
FIG. 7. ILK mediates BMP7-dependent activation of p38MAPKand
ATF-2. (A) IMCD-3 cells were infected with the indicated adenovi-
ruses (MOI ? 5). At 24 h postinfection, cells were treated for 60 min
with 0.25 nM BMP7. Cells were harvested and cytoplasmic lysates were
analyzed in Western blots for phosphorylated and total protein levels
of p38MAPK(Thr180/Tyr182) and GSK3? (Ser 9). The panel below
indicates lack of p38MAPKphosphorylation by infection with empty
adenovirus and with untreated and Ad-ILK virus infection controls.
(B) IMCD-3 cells were infected as indicated in the legend to panel A.
Following 15 min of treatment with 0.25 nM BMP7, cells were har-
vested and cytoplasmic lysates were analyzed for levels of phospho-
ATF-2 (Thr71) and total ATF-2. (C) Cells were pretreated for 1 h with
or without 10 ?M SB203580 p38MAPKinhibitor and then treated with
or without 0.25 nM BMP7 for 15 min. Cytoplasmic lysates were ana-
lyzed by Western blotting for levels of total and phospho-GSK3?
(pSer9), as a measure of ILK activity. (D) IMCD-3 cells were pre-
treated for one hour with SB203580 or LY294002 as described above
and then treated with BMP7 for 15 min. ILK immune complex kinase
assays were performed on cytoplasmic lysates, as above. Numbers
below panels B and C are ratios of phospho/total protein, normalized
to the strongest signal. Numbers below panel D are relative densito-
metric units. All results shown in each panel (A to D) are represen-
tative of three to five independent experiments.
VOL. 25, 2005 ILK MEDIATES EPITHELIAL CELL MORPHOGENESIS3655
maximal at the stimulatory dose (0.25 nM) of BMP7 (data not
shown). We previously reported that inhibitory doses of BMP7
induce phosphorylation of Smad1 and formation of Smad1/
Smad4 protein complexes and that a Smad1 dominant negative
mutant selectively blocks inhibitory signaling (21, 37). Inter-
estingly, the dominant negative Smad1 mutant potentiates
BMP7 activation of p38MAPK/ATF-2 (22), suggesting that
Smad1 acts to restrict ILK-p38MAPKsignaling. We have pre-
viously shown that stimulatory and inhibitory pathways func-
tion in parallel in IMCD-3 cells (17); thus, it is likely that
dose-dependent BMP7 signals are integrated at a point down-
stream of ILK-p38MAPK. However, a number of morphoge-
netic growth factors work through ILK in IMCD-3 cells, in-
cluding hepatocyte growth factor (HGF) (our unpublished
data), indicating that ILK is a point of stimulatory signal con-
vergence. We do not know if ILK regulates RBM in vivo, as
ILK null embryos die at E4.5, well before the onset of kidney
development (42). Based on the results presented here, we
speculate that developmentally regulated expression of mor-
phogens, such as BMP7 and/or HGF (5), determines ILK-
dependent ureteric bud branching during kidney organogene-
Our data place ILK upstream of p38MAPKin the BMP7
stimulatory pathway and suggest that BMP7 activates ILK in a
PI 3-K-dependent manner. As discussed above, PI 3-K activity
links a diverse complement of cell surface growth factor recep-
tors and integrins to ILK signal transduction (6, 7). Both in
vitro and in vivo studies indicate that activin-like ALK2 and
ALK3 receptors mediate inhibitory BMP signaling in kidney
epithelia (17, 21, 38); however, we do not know if stimulatory
signaling is downstream of these ALKs or is mediated by a
distinct receptor. Critical questions, thus, relate to the identify
of the BMP7 stimulatory receptor and of the molecules that
link it to PI 3-K activation. In this context, it will be important
to determine whether the signaling adaptor, Nck2, plays a role
in BMP7-ILK signaling.
Although ILK signaling has largely been studied in terms of
ILK-PKB or ILK-GSK3? interactions, studies have high-
lighted the importance of the ILK/p38MAPKsignaling axis in
regulating cell behavior. Indeed, the ILK/p38MAPKpathway is
implicated in different morphogenetic events, since (as with
IMCD-3 morphogenesis) inhibition of either kinase activity
blocks induction of neurite outgrowth in mouse neuroblastoma
cells (23). D’Amico et al. also showed that point mutation of an
ATF-2 binding site or expression of a dominant negative
ATF-2 mutant abolishes ILK-dependent transcriptional acti-
vation of the cyclin D1 gene in mammary epithelial cells (4).
We note that BMP7 induction of p38MAPKand ATF-2 follows
delayed kinetics relative to activation of ILK, suggesting indi-
rect stimulation of p38MAPKby ILK. Thus, ILK regulation of
p38MAPK/ATF-2 mediates diverse, context-dependent devel-
The overlapping expression patterns of BMP7 (12, 14, 15,
31), ?1 integrin (1, 27), p38MAPK(20, 22), and ILK (Fig. 1) in
embryonic kidneys suggest these molecules interact to regulate
epithelial-mesenchymal interactions. Integrin-mediated UB
cell-ECM interactions regulate branching in vitro and in vivo
(10, 25, 51), and the requirement for a three-dimensional col-
lagen matrix for IMCD-3 cell morphogenesis also implicates
?1 integrin signaling in this response. Functional integrity of
?1 integrins is required for proper development of the ureteric
bud and elaboration of the collecting duct system. Genetic
ablation of the ?8 subunit in mice abolishes expression of ?8?1
integrin, leading to profound defects of RBM (34). Loss of
?2?1 integrin expression inhibits both collagen interaction and
HGF-stimulated branching morphogenesis of MDCK kidney
epithelial cells (41). Accordingly, blocking ?1 integrin function
markedly inhibits branching of primary UB cells in three-di-
mensional collagen cultures and UB development in embry-
onic kidney explants (51). These results indicate the impor-
tance of ?1 integrin signaling in UB development, and our
results reveal a key function of ILK in coordinating integrin
and growth factor signaling during mammalian nephrogenesis.
Interestingly, blocking ?1 integrin function suppresses ductal
branching in the mammary epithelium of mice and inhibits
HGF-induced branching of mammary epithelial cells in vitro
(26). Moreover, expression of ILK in the mammary epithelium
of transgenic mice induces formation of ductal branching
structures (48), indicating that ILK signaling is likely to be of
broad relevance in mediating epithelial branching during
We acknowledge the expert technical assistance of Yunkai Yu, Perry
Mongroo for quantifying protein expression data, and S. Dedhar (Uni-
versity of British Columbia) for the gift of KP-392.
S.H. is the recipient of a studentship from the Research Training
Committee of the Hospital for Sick Children. This work was supported
by grants from the Canadian Institutes of Health Research (CIHR) (to
N.D.R. and G.E.H.), and the National Cancer Institute of Canada (to
G.E.H., with funds from the Terry Fox Run). G.E.H. was a Scholar of
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