From the Departments of Physiology and Pharmacology
and of
Paediatrics, Child Health Research
Institute and Lawson Health Research Institute, University of Western
Ontario, London, Ontario N6A 5C1, Canada and the ¶ Department of
Cell and Developmental Biology, State University of New York Upstate
Medical University, Syracuse, New York 13210
Received for publication, August 14, 2002, and in revised form, January 23, 2003
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ABSTRACT |
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Integrin complexes are necessary for proper
proliferation and differentiation of epidermal keratinocytes.
Differentiation of these cells is accompanied by down-regulation of
integrins and focal adhesions as well as formation of intercellular
adherens junctions through E-cadherin homodimerization. A central
component of integrin adhesion complexes is integrin-linked kinase
(ILK), which can induce loss of E-cadherin expression and
epithelial-mesenchymal transformation when ectopically expressed in
intestinal and mammary epithelia. In cultured primary mouse
keratinocytes, we find that ILK protein levels are independent of
integrin expression and signaling, since they remain constant during
Ca2+-induced differentiation. In contrast,
keratinocyte differentiation is accompanied by marked reduction in
kinase activity in ILK immunoprecipitates and altered ILK subcellular
distribution. Specifically, ILK distributes in close apposition to
actin fibers along intercellular junctions in differentiated but not in
undifferentiated keratinocytes. ILK localization to cell-cell borders
occurs independently of integrin signaling and requires
Ca2+ as well as an intact actin cytoskeleton. Further, and
in contrast to what is observed in other epithelial cells, ILK
overexpression in differentiated keratinocytes does not promote
E-cadherin down-regulation and epithelial-mesenchymal transition. Thus,
novel tissue-specific mechanisms control the formation of ILK complexes
associated with cell-cell junctions in differentiating murine epidermal keratinocytes.
The epidermis is formed by keratinocytes at different stages of
differentiation (1, 2). Undifferentiated keratinocytes reside in the
basal cell layer and are attached to a basement membrane that separates
them from the dermis (3-5). Proliferation, adhesion, and motility of
undifferentiated keratinocytes are regulated by integrins and
associated proteins, which form focal adhesions (6). Upon receiving
appropriate signals, basal keratinocytes initiate terminal
differentiation, characterized by loss of proliferative capacity,
decrease in integrin expression, detachment from the basement membrane,
and migration upward to form postmitotic suprabasal keratinocyte
layers. In culture, primary keratinocytes can be induced to
differentiate by elevating the extracellular Ca2+
concentration. Induction of differentiation by Ca2+ is
accompanied by loss of integrin expression and formation of epithelial
sheets. Intercellular adhesion in differentiated keratinocytes occurs
through tight junctions, cadherin-mediated formation of adherens
junctions, and desmosomes (6-9). Epidermal integrity and barrier
function depend on these cell-cell junctions.
Originally identified as a The regulation and function of ILK have yet to be explored in
keratinocytes. Given that keratinocyte differentiation involves marked
changes in both integrin signaling and cytoskeletal organization, we
postulate that ILK must be involved in these processes. We now show
that ILK kinase activity, but not abundance, is substantially reduced
during Ca2+-induced keratinocyte differentiation. Further,
we report a novel link between ILK and the actin cytoskeleton in areas
of intercellular contact, which involves distribution of ILK to areas
adjacent to cell borders specifically in differentiated keratinocytes. Finally, and in contrast to its effects on other epithelial cell types,
exogenous ILK expression in differentiated keratinocytes does not
induce epithelial-mesenchymal transition through loss of E-cadherin
expression and cell-cell adhesion.
Cell Culture and Infections--
Primary keratinocytes were
isolated from 1-2-day-old CD-1 mice and cultured in medium containing
0.05 mM Ca2+ as described (13). To induce
differentiation, keratinocytes were cultured in medium with 1.0 mM Ca2+ for 24 h. Viral infections were
conducted by incubating keratinocytes with appropriate adenovirus at a
multiplicity of infection of 10 for 5 h in serum-free medium,
followed by 24 h in the presence or absence of 1.0 mM
Ca2+ prior to cell processing. For Ca2+
withdrawal experiments, cells were incubated for 24 h in medium containing 1.0 mM Ca2+. The cells were washed
in Ca2+-free PBS three times and cultured for an additional
12 h in medium containing 0.05 mM Ca2+. In
these experiments, control cultures were incubated in 0.05 or 1.0 mM Ca2+ for a total of 24 h. In
experiments in which the cytoskeleton was disrupted, cells were
incubated with cytochalasin D (8 µM final concentration)
15 min prior to Ca2+ addition and a further 24 h prior
to processing for immunofluorescence microscopy. Human HEK293 cells
were purchased from the American Type Culture Collection and cultured
with Dulbecco's modified minimal essential medium containing 8% fetal
bovine serum.
Recombinant Adenoviruses and Plasmids--
cDNAs encoding
V5-tagged wild-type ILK and the kinase mutant ILK E359K (14) were
isolated to generate the corresponding recombinant adenoviruses using
the pAdEasy system (15). Viral stocks were amplified and titered by
dilution assay in HEK293 cells.
Anti-ILK Antibodies--
The mouse monoclonal anti-ILK antibody
(611803) from Transduction Laboratories (hereafter termed ILK(TL)) was
obtained using as immunogen the C terminus of ILK (12). The polyclonal
goat anti-ILK antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) (sc7516, denoted as ILK(SC)) was raised against a peptide sequence
in the C terminus of ILK. The two rabbit polyclonal anti-ILK antibodies
(06-550 and 06-552) from Upstate Biotechnology, Inc. (Lake Placid, NY)
(ILK(USB)) were obtained using a purified, bacterially produced GST
fusion containing the entire ILK protein as immunogen (16).
Other Antibodies and
Chemicals--
Anti- Cell Lysates, Immunoblotting, and Kinase Assays--
Total
keratinocyte extracts were obtained by harvesting keratinocytes at
indicated times after the addition of Ca2+, followed by
suspension in lysis buffer T (20 mM Hepes, 450 mM NaCl, 4 M EDTA, 25% glycerol, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml each leupeptin and aprotinin, 0.4 mM
each NaF and Na3VO4), followed by three cycles
of freeze-thawing. The lysates were cleared by centrifugation
(12,000 × g, 15 min) and used immediately or stored at
In vitro kinase activity in ILK immunoprecipitates was
determined using in each sample 100 µg of protein from keratinocyte lysates as described (16), with the following modifications. Cell
lysates were precleared by incubation with a mixture of protein A- and
protein G-Sepharose beads for 2 h, prior to ILK
immunoprecipitation using the mouse monoclonal anti-ILK antibody from
Transduction Laboratories. Kinase assays were conducted for 30 min at
30 °C in the presence of 5 µg of MBP and 10 µCi of
[ Immunoprecipitation--
Keratinocytes were cultured, infected
with ILK-encoding adenovirus, and induced to differentiate with 1.0 mM Ca2+ for 24 h. The monolayers were
rinsed with ice-cold PBS, harvested, and lysed in immunoprecipitation
buffer (10 mM Tris-HCl, pH 7.6, 50 mM NaCl, 1%
Nonidet P-40, 10% glycerol, 2 µg/ml aprotinin, 2 µg/ml leupeptin,
1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, 1 mM Na3VO4). Samples containing
500 µg of protein were precleared with protein A/G-Sepharose for
2 h at 4 °C. Following centrifugation, lysates were incubated
overnight at 4 °C with anti-V5 antibodies (1 µg/sample). Immune
complexes were isolated by the addition of protein A/G-Sepharose (2 h,
4 °C) and washed five times with immunoprecipitation buffer.
Proteins bound to the Sepharose beads were released by boiling in
SDS-PAGE sample buffer for 5 min, resolved by SDS-PAGE, transferred to
polyvinylidene difluoride membranes, and probed with antibodies
indicated in individual experiments.
Indirect Immunofluorescence and Confocal
Microscopy--
Keratinocytes were rinsed once in PBS prior to
fixation. For the experiments shown in Figs. 5, 7, and 8, cells were
permeabilized (0.2% Triton X-100 in PBS, 4 °C, 15 min) and fixed
with 2% PFA (4 °C, 40 min), followed by quenching with glycine (100 mM in PBS). In indicated experiments, cells were fixed with
cold methanol (
Confocal microscopy was conducted with a Zeiss LSM metalaser-scanning
microscope. For the analysis of undifferentiated keratinocytes shown in
Fig. 6, cells were fixed with 4% PFA, 0.1% Triton X-100 at 22 °C
for 15 min. Blocking and incubation with antibodies was conducted as
described above, and confocal images were obtained with a Zeiss 63 × 1.25 Plan-Neofluor oil immersion lens. The Ca2+-treated
keratinocytes shown in Fig. 6 were first permeabilized (0.2% Triton
X-100 in PBS, 4 °C, 15 min) and then fixed with 2% PFA (4 °C, 40 min) prior to blocking and incubation with antibodies. Differentiated
keratinocyte confocal images were obtained with a Zeiss 100 × 1.3 Plan-Neofluor oil immersion lens.
Identification of ILK in Keratinocyte Lysates--
ILK has been
reported as a protein with a molecular mass of 59 kDa in a variety of
cell types when detected on immunoblots using rabbit polyclonal
antibodies raised against the entire protein (16). However, ILK has
also been detected as a protein with molecular mass of 51 kDa in
lysates from multiple rat tissues, mouse fibroblasts, and epithelial
HeLa and COS-7 cells, using monoclonal antibodies raised against the C
terminus of ILK (12). Thus, we first sought to determine the apparent
molecular mass of ILK in mouse keratinocyte extracts analyzed with the
available anti-ILK antibodies. Using either the mouse monoclonal
antibody (ILK(TL)) raised against the C terminus of ILK or a goat
polyclonal antibody raised against a synthetic peptide in the C
terminus of ILK(ILK(SC)), we observed a single species of about 51 kDa on immunoblots, corresponding to endogenous ILK (Fig.
1). We also included in this analysis
cell lysates from keratinocytes infected with recombinant adenoviruses
encoding wild-type or E359K mutant V5-tagged ILK. We observed that the
exogenous ILK exhibited a slightly lower mobility than the endogenous
protein and an apparent mass of ~56 kDa. The mobility of the
exogenously expressed ILK was confirmed using antibodies directed
against the V5 epitope tag (Fig. 1). In all of the blots probed with
the mouse and goat antibodies, the 56-kDa protein from infected cells
overlapped with that of the exogenously expressed ILK forms, identified
with the anti-V5 antibody (Fig. 1, V5 panel),
indicating that the presence of the V5 tag slightly reduces the
mobility of ILK on SDS-PAGE. Detection of both endogenous and exogenous
ILK proteins was inhibited when the blots were probed with the goat
polyclonal ILK(SC) antibody in the presence of the corresponding
blocking peptide (Fig. 1), thus confirming the specificity of this
antibody. These observations are consistent with the notion that, in
mouse keratinocytes, endogenous ILK is expressed as a polypeptide of
apparent molecular mass of 51 kDa. In contrast, when we probed those
immunoblots with the rabbit polyclonal antibody ILK(UB)), raised
against a purified, bacterially produced glutathione
S-transferase fusion protein containing the entire ILK
sequence, we were barely able to detect the V5-tagged exogenous ILK at
56 kDa and could not visualize the endogenous ILK protein at 51 kDa
(Fig. 1). Rather, the ILK(UB) antibody detected a doublet at about 60 kDa, which does not correspond to any of the ILK species detected by
the other antibodies and appears to represent an as yet unidentified
protein (Fig. 1, complex A). As a result of these
experiments, subsequent studies were conducted with either the mouse
monoclonal or the goat polyclonal antibody, which specifically
recognize ILK in primary mouse keratinocytes.
Regulation of ILK Protein Expression during Keratinocyte
Differentiation--
Primary keratinocytes are a useful model of
epidermal development. When cultured in medium containing low
Ca2+ concentration (0.05-0.07 mM), these cells
remain undifferentiated and retain proliferative capacity. Increases in
extracellular Ca2+ (
Undifferentiated keratinocytes express various integrins, including
Culturing keratinocytes as a single-cell suspension in the presence of
low extracellular Ca2+ has a dual effect. It initially
prevents integrin ligation and subsequently results in differentiation
and reduction in integrin abundance (22, 23). To determine whether ILK
protein levels are regulated by integrin ligation, we also cultured
keratinocytes in suspension for 24 h in medium containing 0.05 mM Ca2+. Similar to the Ca2+
treatments, culture in suspension for 24 h did not appreciably alter cellular ILK levels (Fig. 2C). Thus, ILK protein
expression in cultured epidermal keratinocytes is not associated with
differentiation status and is independent of integrin Regulation of ILK-associated Kinase Activity in Differentiated
Keratinocytes--
ILK exhibits weak kinase activity and has been
reported to phosphorylate substrates such as protein kinase B/Akt and
myosin light chain in some cell types (24, 25). ILK kinase activity increases upon integrin stimulation by fibronectin (16) and upon
phosphoinositide 3-kinase stimulation (11). Whereas
Ca2+-induced keratinocyte differentiation triggers integrin
down-regulation, it also induces activation of phosphoinositide
3-kinase (17). Therefore, we examined whether kinase activity
associated with ILK is altered in Ca2+-treated cells. We
immunoprecipitated ILK from undifferentiated or differentiated
keratinocyte lysates and observed that ILK immunoprecipitates from
undifferentiated keratinocyte lysates efficiently phosphorylated MBP
in vitro (Fig. 3A).
In contrast, the kinase activity in ILK immunoprecipitates from
differentiated cells was substantially reduced, although the total ILK
protein levels were comparable in all lysates (Fig. 3, B and
C). Thus, differentiation in keratinocytes triggers a
reduction in kinase activity associated with ILK immunoprecipitates, suggesting kinase-independent roles for ILK in this cell type.
Distribution of ILK in Fractionated Keratinocyte Lysates--
In
addition to its function as a kinase, ILK localizes to focal and
fibrillar adhesions, interacting with several proteins and serving as a
cytoskeletal adapter between integrins and the actin cytoskeleton (10,
11). To address the possibility of a scaffolding role for ILK in
keratinocytes, we first examined its subcellular localization using
fractionated cell extracts. We found that ILK is abundant in
cytoplasmic fractions, irrespective of the differentiation status of
the cells (Fig. 4A). ILK was also present in the Triton X-100-soluble fraction obtained after extraction of the cytoplasmic component as well as in the Triton X-100-insoluble fraction (Fig. 4A). To verify the efficiency
of our fractionation, we sequentially probed our blots with marker proteins for each component (Fig. 4A). We observed that
Integrins are essential for keratinocyte migration, adhesion, and
proliferation (20, 29, 30). In undifferentiated keratinocytes, integrins concentrate in focal adhesions, connecting the actin cytoskeleton with the extracellular matrix. Focal adhesions also associate with the scaffolding protein paxillin, which interacts with
ILK and various other proteins in nonepithelial cell types (12). The
presence of ILK in Triton X-100-insoluble fractions, together with the
known interactions between ILK and paxillin at focal adhesions,
prompted us to investigate whether ILK and paxillin associate in
undifferentiated keratinocytes. Thus, we isolated ILK
immunoprecipitates from undifferentiated keratinocytes and probed them
for the presence of paxillin. We observed that paxillin is a component
of ILK immunoprecipitates from undifferentiated keratinocytes.
Conversely, we were also able to demonstrate the presence of ILK in
paxillin immunoprecipitates (Fig. 4, B and C),
demonstrating the existence in undifferentiated keratinocytes of
complexes containing both ILK and paxillin.
As mentioned above, differentiation induces marked decreases in
integrin levels and focal adhesions in keratinocytes. Consequently, we
investigated whether integrin down-regulation is associated with loss
of ILK-paxillin interactions in differentiating keratinocytes. Using
similar co-immunoprecipitation assays as those described for
undifferentiated cells, we found that the interaction between ILK and
paxillin is maintained in differentiated keratinocytes (Fig. 4,
B and C), indicating its independence from
integrin levels and differentiation status in these cells.
ILK Localizes to Cell-Cell Borders in Differentiated
Keratinocytes--
To further investigate the regulation of ILK during
keratinocyte maturation, we focused on the subcellular distribution of ILK, inducing expression of ILK by adenovirus-mediated gene transfer in
undifferentiated and differentiated cells. We then visualized the
exogenous protein by immunofluorescence microscopy. Undifferentiated keratinocytes cultured in 0.05 mM Ca2+ exhibit
abundant focal adhesions but do not form adherens junctions (31). We
observed that ILK immunofluorescence was detectable throughout the cell
(Fig. 5) in samples fixed with cold
methanol. Methanol fixation interferes with proper detection of several structures associated with the cytoskeleton, including focal adhesions. When we fixed the cells with PFA in the presence of Triton X-100 to
better preserve cytoskeletal components, we observed that wild-type ILK
exhibited a punctate pattern throughout the cell in undifferentiated keratinocytes (Fig. 6).
Increases in extracellular Ca2+ to 1-2 mM in
keratinocyte cultures trigger the formation of intercellular junctions,
which involves homotypic interactions of E-cadherin molecules localized
on adjacent cells (18). In this system, there is an initial stage of
adherens junction formation within 1 h of Ca2+
addition, followed by maturation and stabilization of the cell contacts
within 20 h. Cultured keratinocytes infected with the ILK-encoding
adenovirus in 1 mM Ca2+ for 24 h exhibited
striking changes in ILK distribution to regions juxtaposed to points of
cell-cell contact, which in some cells comprised the entire cell
diameter (Fig. 5). Localization to cell-cell borders in
Ca2+-treated keratinocytes was observed with exogenously
expressed ILK (e.g. Figs. 5
and 6), as well as with the endogenous protein (e.g. Figs. 7
and 8). Importantly, ILK localization to
cell-cell borders was not apparent during the first 12 h after
Ca2+ addition but was readily detected in the mature
junctions present after 24 h (Fig. 5). These results indicate that
ILK movement to areas associated with intercellular junctions is not an
immediate response to increased extracellular Ca2+ or
initial formation of adherens junctions. Rather, ILK becomes associated
with cell borders with kinetics comparable to those of junction
maturation.
ILK localization to focal adhesions and interaction with paxillin
requires a region in ILK that encompasses its putative kinase domain
(32). Indeed, an E359K mutant of ILK that behaves as a dominant
negative form in some cells but still exhibits kinase activity in
vitro neither associates with paxillin nor localizes to focal
adhesions (16, 32-34). To begin to explore the molecular determinants
for ILK localization in keratinocytes, we investigated whether an
intact kinase domain is required for normal distribution, analyzing the
subcellular localization of the E359K ILK mutant. In
undifferentiated keratinocytes, ILK E359K exhibited a diffuse distribution throughout the cell, distinct from the punctate pattern shown by the wild-type protein, indicating a requirement for the kinase
domain in the normal distribution of this protein (Fig. 5). In
contrast, this mutant was able to localize to cell-cell junctions in
keratinocytes induced to differentiate with Ca2+ as
efficiently as wild-type ILK (Fig. 5). This indicates that the
mechanisms responsible for ILK association with paxillin and characteristic subcellular distribution in undifferentiated
keratinocytes are dissociable from those that induce ILK
localization to intercellular borders in differentiated cells. ILK
localization to cell borders in Ca2+-treated cells does not
appear to depend on the presence of a fully functional kinase domain or
on direct association with paxillin.
ILK Subcellular Distribution and Actin Filament
Organization--
Undifferentiated primary keratinocytes form actin
networks of stress fibers that appear as thin cobweb-like structures
(Fig. 6). F-actin stress fibers in undifferentiated keratinocytes can end at focal adhesion sites or participate in the formation of filopodia or leading edges in migrating cells (18, 35). In these cells,
we observed areas of endogenous ILK localization in close proximity to
the edges of actin bundles in addition to the previously described
punctate distribution throughout the cell (Fig. 6). The formation of
intercellular junctions in keratinocytes triggered by increases in
extracellular Ca2+ also requires actin reorganization and
polymerization to seal cell-cell borders (18). This results in the
formation of cortical actin fibers that run parallel to the plasma
membrane (Fig. 6). When we examined the relationship between these
cortical actin filaments and ILK located along intercellular junctions,
we observed that ribbons of ILK immunoreactivity occur along the cell
membrane, running parallel and in close apposition to the actin fibers
(Fig. 6). The localization patterns of ILK and actin fibers prompted us
to examine whether ILK would still segregate to cell borders in
cultures in which the cytoskeleton was disrupted by treatment with
cytochalasin D. We found that disassembly of F-actin filaments in cells
cultured in the presence of cytochalasin D and 1 mM
Ca2+ was accompanied by loss of ILK from the cell periphery
(Fig. 7), indicating that actin polymerization is an essential
requirement for ILK redistribution in differentiated keratinocytes.
Ca2+ Dependence of ILK Localization to Intercellular
Junctions--
The development of adherens junctions in keratinocytes
cultured in 1-2 mM Ca2+ also requires the
accumulation at cell-cell borders of E-cadherin complexes associated
with the actin cytoskeleton and that contain Modulation of ILK Abundance and Kinase Activity during Keratinocyte
Differentiation--
The epidermis is a complex epithelial tissue,
which consists of undifferentiated, proliferative cells and their
terminally differentiated progeny. A notable characteristic of primary
murine epidermal keratinocytes is their ability to undergo terminal
differentiation in response to increases in extracellular
Ca2+ concentrations. Differentiation in primary cultured
keratinocytes is accompanied by loss of integrin expression and focal
adhesions. Because ILK is an important component of integrin signaling
and focal adhesion complexes, we investigated if ILK is also regulated during Ca2+-induced keratinocyte differentiation. We found
that, whereas ILK protein levels do not appreciably change in
differentiated keratinocytes, kinase activity in ILK immunoprecipitates
is substantially decreased. The ILK immune complexes we isolated from
keratinocytes also contained other cellular proteins and, consequently,
it remains to be determined whether the kinase activity we measured in
the keratinocyte extracts arises from ILK itself or from another
protein present in ILK immunoprecipitates. Regardless, the decreased
ILK-associated kinase activity triggered by Ca2+ treatment
in keratinocytes is significant, since it demonstrates the existence of
tight regulatory mechanisms for this pathway in response to
differentiation signals in epidermal tissue.
ILK as a Cytoskeletal Adapter in Undifferentiated
Keratinocytes--
ILK appears to have dual functions as a kinase and
as a scaffold in multiprotein complexes. Specifically, ILK localizes to focal and fibrillar adhesions, serving as a cytoskeletal adapter between integrins and the actin cytoskeleton, which is essential for
muscle development in C. elegans and D. melanogaster (10, 11). Central for the adapter functions of ILK
are its interactions with factors such as PINCH, paxillin, and
actopaxin (12, 32, 37). In undifferentiated keratinocytes, ILK can be
found in Triton X-100-insoluble fractions and in complexes that contain paxillin. In these cells, ILK is distributed with a punctate pattern. It remains to be determined whether ILK is directly associated with
focal adhesions in undifferentiated keratinocytes. The different patterns of ILK cellular distribution in undifferentiated
versus differentiated keratinocytes suggest that ILK may
play multiple roles in adhesion, migration, and/or proliferation in
these cells through a combination of signaling and scaffold functions.
ILK and Formation of Cell-Cell Junctions--
Primary
keratinocytes cultured in 1-2 mM Ca2+ form
stratified layers with extensive cell-cell contacts (38). Under these
conditions, integrins do not play a major role in intercellular
adhesion (8, 21). The formation of adherens junctions in keratinocytes
cultured in 1-2 mM Ca2+ is accompanied by
accumulation of E-cadherin complexes containing
We have determined that, in differentiated keratinocytes, ILK is
present in cytosolic, Triton X-100-soluble and -insoluble fractions.
This contrasts with its primarily cytoskeletal distribution in other
cell types (24, 34). Notably, induction of differentiation by
Ca2+ in keratinocytes is accompanied by marked changes in
subcellular location, characterized by ILK concentration to cell-cell
borders. The keratinocyte response to Ca2+ may be
tissue-specific, since ILK appears to be excluded from intercellular
adhesion sites in other cell types (37). Ca2+ also triggers
accumulation of E-cadherin and
It has been demonstrated that Ca2+ plays a dual role in
keratinocyte adhesion; it stimulates homotypic cadherin engagement and activates a pathway that leads to actin polymerization at contact sites (18). Our experiments suggest that both events are necessary for
ILK localization to cell junctions. This is evidenced by the loss of
ILK from intercellular adhesion sites following either disruption of
E-cadherin ligation by blocking antibodies2 or disruption
of actin polymerization by cytochalasin D treatment. In keratinocytes
induced to differentiate by Ca2+, groups of cortical actin
bundles are formed in a Ca2+-dependent manner
parallel to the cell borders (Fig. 6) in a process that coincides with
the appearance of E-cadherin localization at intercellular junctions
(41). We have identified ILK as an additional component in those
multiprotein complexes found adjacent to the sealed cell borders in keratinocytes.
A tight relationship between ILK and the actin cytoskeleton is also
indicated by the close localization of ILK to cortical actin fibers at
cell-cell borders in differentiated keratinocytes (e.g. Fig.
6). Taken together with the known down-regulation of integrins that
occurs upon keratinocyte differentiation (42), our data suggest that
ILK may uniquely participate in the establishment and/or maintenance of
cell-cell contacts in differentiated epidermal keratinocytes that
is unrelated to integrin function.
In addition to cell-cell borders, we also observed ILK
immunofluorescence in inner cell regions in differentiated
keratinocytes. Our analysis of ILK immunoprecipitates from
differentiated keratinocytes has shown the presence of paxillin and
actopaxin in these complexes (Fig. 4B).2
However, the capacity of the mutant E359K to segregate to cell-cell borders despite its inability to interact with paxillin (12) suggests
that the ILK-paxillin complexes detected in differentiated keratinocytes may serve a separate function. Suprabasal epidermal layers containing differentiated keratinocytes do not exhibit focal
adhesions, although they form extensive cell-cell contacts. In these
regions, paxillin is not found in foci corresponding to areas of
intercellular contact (21).
ILK Does Not Induce Epithelial-Mesenchymal Transition in
Differentiated Keratinocytes--
We observed E-cadherin formation of
adherens junctions and ILK localization to cell-cell borders in
Ca2+-treated keratinocytes, irrespective of whether the
cells exogenously expressed ILK. The fact that in epidermal
keratinocytes ILK overexpression does not induce epithelial-mesenchymal
transitions through E-cadherin degradation and loss of intercellular
adhesion contrasts sharply with the reported responses to ectopic ILK
expression in mammary and in intestinal epithelial cell lines (40, 43).
Indeed, our results are consistent with the proposal that ILK may serve a unique role in intercellular adhesion, cytoskeletal organization, and
maintenance of barrier function in differentiated keratinocytes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 integrin-binding protein,
ILK1 also interacts with
paxillin, actopaxin, and affixin in focal adhesions. Accumulating
evidence from a variety of cell types indicates that ILK can function
as a kinase and as a scaffold protein that mediates interactions
between integrins and the actin cytoskeleton through interaction with
multiple proteins (10-12). Such interactions are essential for the
formation of proper attachment sites of muscle fibers in
Caenorhabditis elegans and Drosophila melanogaster.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1-integrin (610468) and
anti-E-cadherin (610405) antibodies were purchased from Transduction
Laboratories. Anti-V5 (R96025) and anti-
-catenin (C2206) antibodies
were purchased, respectively, from Invitrogen and Sigma. The monoclonal
E7 anti-
-tubulin antibody was from the Iowa State University
Hybridoma Bank. Rabbit anti-GAPDH (4699-9555) was purchased from
Biogenesis. The anti-caveolin 1 (610057) and anti-paxillin (610051)
antibodies were from Transduction Laboratories. Secondary antibodies
conjugated to fluorescein isothiocyanate, Cy3, or Cy5 were purchased
from Jackson Immunochemicals. All other chemicals were purchased from Sigma.
80 °C. To obtain fractionated extracts, keratinocytes were
harvested and suspended in ice-cold lysis buffer A (20 mM Tris, pH 7.5, 2 mM EDTA, 2 mM EGTA, 0.1%
-mercaptoethanol, 0.4 mM NaF and sodium orthovanadate,
0.5 mM phenylmethylsulfonyl fluoride, 1 mM
dithiothreitol, 2 µg/ml each leupeptin, pepstatin, and aprotinin) and
sonicated for 10 s on ice. After centrifugation (100,000 × g, 1 h), the supernatant (cytosolic fraction) was
removed. The pellet was suspended in buffer A containing 1.2% Triton
X-100, followed by centrifugation (12,000 × g, 15 min). After removal of the Triton X-soluble supernatant, the pellet was
suspended in SDS buffer (50 mM Tris, pH 6.8, 1% SDS, 10%
glycerol). Gel electrophoresis and immunoblotting were conducted with
18-50 µg of protein/sample, as described (13). Immunoblots were also probed for
-tubulin or GAPDH to normalize for protein loading. No
substantial differences in loading were noticed between samples in a
given immunoblot using this method. The results shown in the
immunoblots are representative of experiments conducted at least three times.
-32P]ATP. Kinase assay complexes were resolved by
SDS-PAGE, and phosphorylated products were visualized by autoradiography.
20 °C, 15 min) in place of Triton X-100
permeabilization and PFA fixation. After two PBS washes, cells were
blocked in PBS containing 5% nonfat dry milk and 5% goat serum (16 h,
4 °C), followed by incubation with primary antibody (1 h, 22 °C).
After three 20-min PBS washes, the cells were probed with the
appropriate Cy3- or Cy5-labeled secondary antibody, at 22 °C for
1 h. Following removal of the secondary antibody, the cells were
rinsed twice, incubated with Hoescht 33258 (10 µg/ml) in PBS for 5 min at 22 °C, rinsed five times, and mounted in anti-fade medium
(Dako). Photomicrographs were obtained with a Leica DMIRBE microscope equipped with an Orca II digital camera (Hamamatsu), using Openlab 3.0 software (Improvision). The results shown are representative of
experiments conducted on duplicate samples at least three times.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Identification of ILK as a 51-kDa protein in
epidermal keratinocytes. Lysates were prepared from uninfected
undifferentiated primary mouse keratinocytes (U) or from
keratinocytes infected 24 h prior to harvesting with a recombinant
adenovirus encoding V5-tagged wild-type (Wt) or E359K mutant
(Mt) ILK. Whole-cell lysates (50 µg/lane) were resolved by
SDS-PAGE and blotted onto polyvinylidene difluoride membranes. The
blots were probed with the following antibodies: ILK(TL), Transduction
Laboratories mouse monoclonal anti-ILK (611803); ILK(SC), Santa Cruz
Biotechnology goat polyclonal anti-ILK (sc7516), in the absence or
presence of blocking peptide, as indicated; V5, Invitrogen anti-V5
(R96025); and ILK(UB), Upstate Biotechnology rabbit polyclonal anti-ILK
(06-550). The positions of exogenous V5-tagged ILK and endogenous ILK
are indicated. Complex A denotes the ~59-kDa
species exclusively detected by the ILK(UB) antibody.
0.1 mM) elicit terminal
differentiation within 24 h, characterized by changes in gene
expression and cytoskeletal organization, as well as formation of
E-cadherin-containing adherens junctions involved in intercellular
contact (17, 18).
6
4 and
5
1,
and signaling through these integrins is necessary for normal
keratinocyte proliferation (19, 20). Induction of differentiation in
keratinocytes in vivo or in culture is accompanied by
integrin down-regulation and exit from the cell cycle (21). Given the
association between ILK and
1 integrin function, we
first investigated whether ILK expression was regulated during
Ca2+-induced keratinocyte maturation. We found that
cellular ILK protein levels were
unaltered throughout the time course of keratinocyte differentiation
(Fig. 2A), although
1 integrin
expression was substantially reduced by 24 h following the
addition of Ca2+ (Fig. 2B).
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Fig. 2.
ILK protein expression during keratinocyte
differentiation. A, primary keratinocytes cultured in
0.05 mM Ca2+ were induced to differentiate by
increasing extracellular Ca2+ levels to 1.0 mM.
Cell lysates (50 µg/lane) were prepared at the indicated times after
Ca2+ addition, resolved by SDS-PAGE, and probed with
ILK(TL) or -tubulin antibodies. B, lysates from
undifferentiated keratinocytes (lane 1) or from
cells differentiated by culture in 1.0 mM Ca2+
for 24 h (lane 2) were resolved by SDS-PAGE,
and immunoblots were probed with the indicated antibodies.
C, adherent keratinocytes were maintained as
undifferentiated cells (lane 1), were induced to
differentiate with 1.0 mM Ca2+ (lane
2), or were trypsinized and cultured in suspension at low
cell density in normal growth medium (0.05 mM
Ca2+) containing 1.45% methyl-cellulose for 24 h
(lane 3). Cell lysates were prepared, resolved by
SDS-PAGE, and transferred to membranes, which were probed with the
indicated antibodies.
-Tubulin and GAPDH signals were used to
normalize for differences in loading.
1
expression or ligation.
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Fig. 3.
Regulation of ILK-associated kinase activity
during keratinocyte differentiation. A, phosphorylation
of MBP by ILK immunoprecipitates from keratinocytes. ILK was
immunoprecipitated from lysates (100 µg of protein/sample) obtained
from keratinocytes cultured in 1.0 mM Ca2+ for
the indicated times. Immune complexes were assayed for kinase activity
using MBP as substrate and [ -32P]ATP. Phosphorylated
MBP was resolved by SDS-PAGE and visualized by autoradiography.
B, to verify the levels of ILK present in the
immunoprecipitates used for kinase assays, replicate samples were
prepared, resolved by SDS-PAGE, and immunoblotted with ILK antibodies.
ILK migrates just under the IgG heavy chain, as indicated on the blot.
C, a darker exposure of the ILK signal shown in
B.
95% of the cytosolic marker 14-3-3
was present in the cytoplasmic
fractions, in agreement with its reported distribution in a variety of
cell types (26). Similarly, we used Na+/K+
ATPase as a marker for Triton X-100-soluble proteins (26) and found
that it was exclusively present in this fraction. We also probed the
membranes with
-actinin and caveolin 1, which are two proteins that
generally segregate to Triton X-100-insoluble fractions (27, 28). We
found that caveolin 1 was present exclusively in the Triton
X-100-insoluble portion of extracts from both undifferentiated and
differentiated keratinocytes. We also observed that, whereas
-actinin was exclusively present in Triton-insoluble fractions in
undifferentiated keratinocyte lysates, a small proportion of
-actinin was present in cytosolic and Triton X-100-soluble fractions from differentiated cell extracts (Fig. 4A). We conclude
that different pools of ILK are present in primary murine
keratinocytes, which suggests that this protein probably fulfills
multiple cellular functions in these cells.
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Fig. 4.
ILK subcellular fractionation and association
with paxillin. A, primary mouse keratinocytes
were cultured for 24 h in medium containing 0.05 mM
Ca2+ (Low) or 1.0 mM
Ca2+ (High). Cell lysates containing cytoplasmic
(C), Triton X-100-soluble (S), or Triton
X-100-insoluble (I) fractions were prepared, resolved by
SDS-PAGE (50 µg of protein/lane), and transferred to a polyvinylidene
difluoride membrane. The membrane was sequentially probed with the
indicated antibodies. The positions of molecular mass markers are
indicated on the left. The membrane was probed for
-actinin just prior to probing for Na+/K+
ATPase. The signal corresponding to
-actinin was still visible when
the membrane was probed with the Na+/K+ ATPase
antibody, as indicated. B, keratinocytes infected with an
ILK-encoding adenovirus were cultured in the presence of 0.05 mM Ca2+ (lane 1) or 1.0 mM Ca2+ (lane 2) for
24 h. Cells were harvested and lysed, and paxillin was
immunoprecipitated (IP) with a specific antibody. Immune
complexes were resolved by SDS-PAGE, blotted, and analyzed for the
presence of ILK. Control samples included cell lysates incubated with
Sepharose beads in the absence of anti-paxillin antibody
(lane 3) or 50 µg of whole cell lysate
from undifferentiated cells to confirm the presence of ILK in the
extracts (lane 4). C, keratinocytes
expressing exogenous V5-tagged ILK were cultured in the presence of
0.05 mM Ca2+ (lane 1) or
1.0 mM Ca2+ (lane 2) for
24 h. Cells were harvested and lysed, and ILK was
immunoprecipitated using an anti-V5 antibody. Immune complexes were
resolved by SDS-PAGE, blotted, and analyzed for the presence of
paxillin. Control samples included cell lysates incubated with protein
A- and protein G-Sepharose in the absence of anti-V5 antibody
(lane 3) and 50 µg of whole cell lysate from
undifferentiated cells as positive control for paxillin
(lane 4).
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Fig. 5.
Localization of ILK to cell-cell borders in
differentiated keratinocytes. A, keratinocytes were
infected with adenovirus encoding wild-type ILK (WT) 24 h prior to induction of differentiation by culture in medium containing
1.0 mM Ca2+. Cells were fixed with cold
methanol and processed for immunofluorescence using an anti-ILK
antibody at measured intervals following the addition of
Ca2+, as indicated in individual micrographs.
B, keratinocytes infected with a recombinant adenovirus
encoding a V5-tagged E359K ILK mutant were induced to differentiate by
culture in medium containing 1.0 mM Ca2+. The
cells were processed to visualize ILK immunofluorescence using Triton
X-100 permeabilization, followed by fixation in 2% PFA, as described
under "Materials and Methods." The numbers in
individual micrograph panels indicate
the time of fixation after the addition of Ca2+. The
No Ab panels represent negative
controls containing infected cells cultured in medium with 1.0 mM Ca2+ for 0 or 24 h, as indicated, and
processed for immunofluorescence microscopy excluding incubation with
anti-V5 antibodies.
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Fig. 6.
Ca2+-induced changes in
distribution of ILK and actin filaments. Primary mouse
keratinocytes were infected with V5-tagged ILK-encoding adenovirus, and
cultured in growth medium containing 0.05 µM
(Low Ca2+) or 1.0 mM
Ca2+ (High Ca2+) for 24 h prior to processing for confocal laser-scanning microscopy.
Actin and exogenous ILK were visualized, respectively, using Cy3- or
fluorescein isothiocyanate-labeled phalloidin and an anti-V5 antibody.
Insets represent higher magnification images of the areas
outlined in the High Ca2+
panel. Scale bars, 8 µm.
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Fig. 7.
ILK localization to cell-cell borders
requires an intact actin cytoskeleton. A, primary
keratinocytes were cultured for the indicated times in medium
containing 1.0 mM Ca2+. To disrupt the actin
cytoskeleton, some cultures were incubated in the presence of
cytochalasin D (+cyto; 8 µM final
concentration) for 15 min prior to the addition of Ca2+
(1.0 mM final concentration) and cultured for 24 h.
Endogenous ILK was visualized using a goat anti-ILK antibody, and actin
was visualized using fluorescein isothiocyanate-labeled phalloidin.
B, controls containing cells infected with wild-type
ILK-encoding adenovirus and cultured in medium with 1.0 mM
Ca2+ for 0 or 24 h, as indicated, and processed for
immunofluorescence microscopy, but in the presence of IgG in place of
primary anti-ILK antibody.
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Fig. 8.
ILK localization and permanence adjacent to
cell-cell borders requires high extracellular Ca2+
levels. Primary keratinocytes were cultured in medium containing
0.05 mM Ca2+ (A) or 1.0 mM Ca2+ (B) for 24 h prior to
processing for fluorescence microscopy. Alternatively, the cells were
cultured for 24 h in medium containing 1.0 mM
Ca2+, washed thoroughly, and cultured for an additional
12 h in medium containing 0.05 mM Ca2+
(C). Endogenous ILK and E-cadherin were detected with the
corresponding antibodies, whereas actin was visualized using
fluorescein isothiocyanate-labeled phalloidin. ILK and actin were
detected by double labeling of a single set of cells, whereas
E-cadherin was visualized using replicate cell samples.
- and
-catenin (21,
36). Because homotypic E-cadherin interactions are
Ca2+-dependent, maintenance of adherens
junctions in differentiated keratinocytes requires the
continuous presence of high extracellular Ca2+ levels. To
further investigate the mechanisms directing ILK to intercellular
junctions in differentiated keratinocytes, we determined the calcium
requirements of this process. Thus, we first cultured cells for 24 h in 1 mM extracellular Ca2+ to induce ILK
migration to areas of cell-cell contact (Fig. 8, A and
B). Subsequently, we switched the culture medium from 1.0 to
0.05 mM Ca2+ and cultured the cells for an
additional 12-h period. As shown in Fig. 8C,
Ca2+ withdrawal in differentiated keratinocytes induced the
loss of E-cadherin localization to cell-cell borders, disrupting the
previously formed adherens junctions. Decreases in extracellular
Ca2+ levels also altered the cortical actin fibers
assembled parallel to the membrane, although other cytoskeletal actin
fibers throughout the cell remained assembled (Fig. 8C).
Ca2+ withdrawal also resulted in the disappearance or
substantial reduction of ILK immunoreactivity from cell borders,
similar to that observed for the cortical actin filaments. These
results demonstrate that the patterns of localization of F-actin, ILK, and E-cadherin along intercellular borders require the continuous presence of high extracellular Ca2+ concentrations. When we
conducted these experiments in cells exogenously expressing ILK by
adenovirus-mediated gene transfer, we found that the increased ILK
levels in differentiated keratinocytes did not interfere with formation
of cell junctions or with expression and localization of E-cadherin and
-catenin to cell borders in response to elevated extracellular
Ca2+ (e.g. Fig. 6 and data not shown),
indicating that elevated ILK does not induce mesenchymal transformation
in differentiated epidermal keratinocytes. Together, our results
demonstrate that ILK is tightly regulated at multiple
post-translational levels during keratinocyte differentiation and
suggest a potential novel role for this protein in the genesis of
cell-cell contacts in differentiated keratinocytes and in epidermal
barrier formation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
- and
-catenin.
Such complexes are associated with the actin cytoskeleton and exhibit
decreased solubility in Triton X-100 (21, 36). The development of
adherens junctions requires reciprocal interactions between E-cadherin
and the actin cytoskeleton. Specifically, initial intercellular
ligation of E-cadherin dimers can directly induce actin assembly at the
cell surface (39), but continuous actin reorganization and
polymerization is required to maintain sealed cell borders, as
evidenced by the disruption of cadherin-mediated cell-cell junctions in
cytochalasin D-treated cells (18).
-catenin at intercellular junctions
(18). We observed that
-catenin shows only partial co-localization
with ILK by immunofluorescence microscopy, but we have not detected
-catenin in ILK
immunoprecipitates,2
suggesting that ILK and
-catenin may participate in the formation of
distinct complexes associated with cell borders and/or the cytoskeleton.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. S. Dedhar for ILK and ILK E359K
cDNA constructs, R. Kothary for the anti--tubulin antibody, S. Ferguson for helpful comments on the manuscript, and L. Dale for help
with confocal microscopy.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Canadian Institutes of Health Research (to L. D.) and from the Kidney Foundation of Canada (to S. J. A. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of an Ontario Graduate Scholarship in Science and Technology.
Supported by National Institutes of Health Grant HL70244.
** To whom correspondence may be addressed: Dept. of Physiology and Pharmacology, Medical Sciences Bldg., University of Western Ontario, London, Ontario N6A 5C1, Canada. Fax: 519-661-3827; E-mail: sjdsouza@uwo.ca.
§§ A Cancer Research Society/CIHR Scholar. To whom correspondence may be addressed: Dept. of Physiology and Pharmacology, Medical Sciences Bldg., University of Western Ontario, London, Ontario N6A 5C1, Canada. Fax: 519-661-3827; E-mail: ldagnino@uwo.ca.
Published, JBC Papers in Press, January 23, 2003, DOI 10.1074/jbc.M208337200
2 A. Vespa, S. D'Souza, and L. Dagnino, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: ILK, integrin-linked kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MBP, myelin basic protein; PBS, phosphate-buffered saline; PFA, paraformaldehyde.
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