1 Department of Dermatology and Center for Molecular Medicine, University of
Cologne (CMMC), Joseph-Stelzmann-Strasse 9, 50924 Cologne, Germany
2 Keratinocyte Laboratory, Cancer Research UK, 44 Lincoln's Inn Fields, London
WC2A 3PX, UK
* Author for correspondence (e-mail: Ingo.Haase{at}uni-koeln.de)
Accepted 10 April 2003
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Summary |
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Key words: IGF-1, EGF, Cell shape, Wound epithelialization, MAP kinase, PI-3 kinase
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Introduction |
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Epidermal growth factor (EGF) is a polypeptide involved in the maturation
of epithelia (Cohen and Elliott,
1963). It binds to the EGF receptor (EGFR) which is also a
receptor for EGF-related cytokines such as transforming growth factor
(TGF
) (Derynck, 1986
).
In normal adult epidermis, EGFR is predominantly expressed in basal
keratinocytes and signalling events elicited by it are known to affect their
proliferation, differentiation and migration
(Vassar and Fuchs, 1991
)
(reviewed in Nanney and King,
1996
). In healing skin wounds, EGFR expression is upregulated in
migrating and proliferating keratinocytes adjacent to the wound (reviewed in
Nanney and King, 1996
).
Clinical studies of the influence of treatment of skin wounds with soluble EGF
have revealed a stimulatory function of this growth factor in wound healing
(Brown et al., 1989
). An early
event after treatment of cultured epithelial cells with EGF is remodelling of
the actin cytoskeleton, which can lead to retraction of cells from the
substrate surface and rounding (Chinkers
et al., 1979
; Peppelenbosch et
al., 1993
). EGF also stimulates centrifugal outwards migration of
keratinocytes within colonies (Barrandon
and Green, 1987
).
Insulin-like growth factor 1 (IGF-1) is a peptide hormone with structural
homology to proinsulin. The major source of circulating IGF-1 in postnatal
life is the liver but IGF-1 can also be produced by many other tissues, where
it is thought to act in a paracrine fashion. In skin, IGF-1 produced by dermal
fibroblasts and macrophages (Han et al.,
1987; Rappolee et al.,
1988
) is thought to signal to basal epidermal keratinocytes that
express the IGF-1 receptor (Hodak et al.,
1996
). In keratinocytes, IGF-1 is thought to stimulate
proliferation (Tavakkol et al.,
1992
) and to contribute to hair follicle morphogenesis
(Liu et al., 1993
;
Philpott et al., 1994
;
Rudman et al., 1997
). Mice
with a targeted deletion of the IGF-1 receptor die shortly after birth from
respiratory failure and show an abnormally thin and translucent epidermis with
a decreased number of hair follicles (Liu
et al., 1993
). Until now, no function for IGF-1 in wound healing
has been clearly demonstrated; there is recent evidence, however, that levels
of IGF-1 are decreased in non-healing skin wounds of diabetic individuals
(Blakytny et al., 2000
).
Factors of the extracellular environment transduce information by specific
activation of intracellular signalling pathways. The classical
mitogen-activated protein kinase (MAPK or ERK) cascade is a module of three
protein kinases acting in a hierarchical order: the MAPKs (ERKs) are activated
by a MAPK kinase (MEK-1) through phosphorylation of conserved threonines and
tyrosines within a TXY motif (Canagarajah
et al., 1997), and the MAPKK kinase (Raf) regulate MEK-1 activity.
The MAPK cascade is an important regulator of cell proliferation and
differentiation (Bennett and Tonks,
1997
; Cowley et al.,
1994
; Schramek et al.,
1997
). We have previously shown that MEK-1 activity inhibits
terminal differentiation in primary human keratinocytes
(Haase et al., 2001
;
Zhu et al., 1999
). It also
exerts a crucial function in cell migration through phosphorylation of myosin
light chain kinase (MLCK) (Klemke et al.,
1997
; Nguyen et al.,
1999
). Signals stimulating the activity of the MAPK cascade are
mediated by growth factors (e.g. EGF)
(Gotoh et al., 1990
;
Takishima et al., 1991
),
cytokines (e.g. IL-1) (Saklatvala et al.,
1993
) and cell-matrix and cell-cell adhesion molecules (e.g.
integrins, ICAM-1) (Holland and Owens,
1997
; Miyamoto et al.,
1996
; Zhu et al.,
1999
). In several systems, including murine keratinocytes, IGF-1
is also able to stimulate ERK activation
(Vasioukhin et al., 2001
). The
phosphorylation of ERK by MEK-1 at threonine 183 and tyrosine 185
(Rossomando et al., 1992
) is
necessary and sufficient for its activation and can be detected using
phosphorylation specific antibodies. Exchange of serine residues 217 and 221
of MEK-1 against glutamic acid uncouples the enzyme from upstream regulating
events and leads to its constitutive activation
(Cowley et al., 1994
).
Another important kinase involved in growth factor mediated signal
transduction is phosphatidylinositol-3-kinase (PI-3K). Being a lipid kinase,
this enzyme phosphorylates the inositol ring of phosphatidylinositols in their
3' position, creating docking sites at the plasma membrane for
pleckstrin homology (PH) domain containing proteins. This is thought to
organize the interactions of signalling molecules spatially, thereby
facilitating the formation of signalling complexes (reviewed in
Czech, 2000). The PI-3K
holoenzyme consists of two subunits, p85 and p110. The p85 regulatory subunit
contains Src-homology 2 and 3 domains, and can bind to tyrosine kinase growth
factor receptors, thus targeting the p110 catalytic subunit to the plasma
membrane, where its substrates reside. Membrane targeting of p110 is
sufficient for its activation because a chimeric protein consisting of a
transmembrane receptor (CD2) fused to p110 exhibits constitutively activated
PI-3K activity (Reif et al.,
1996
).
Among the factors that signal through PI-3K are platelet-derived growth
factor (PDGF), insulin and IGF-1 (Kotani
et al., 1994; Way and Mooney,
1993
). PI-3K has been implicated in the regulation of cell
proliferation, transformation, protein trafficking, actin cytoskeletal
organization and apoptosis by activation of different effectors (reviewed in
Toker and Cantley, 1997
). One
effector is the product of the akt oncogene, protein kinase B, which
becomes phosphorylated and activated upon stimulation of PI-3K
(Burgering and Coffer, 1995
;
Franke et al., 1995
).
Phosphorylation of protein kinase B/Akt at serine 473 can be detected using
phosphorylation specific antibodies (Toker
and Newton, 2000
).
PI-3K has been shown to regulate actin cytoskeletal reorganization. This
function is thought to be mediated by the small GTPase Rac, which induces
protrusion of the plasma membrane and formation of lamellipodia by so far
unidentified mechanisms (Nobes and Hall,
1995; Nobes and Hall,
1999
; Reif et al.,
1996
).
Although the effects of individual growth factors on keratinocyte
proliferation, differentiation and migration have been studied in culture,
neither the signalling mechanisms by which these factors exert their functions
nor the interactions between the respective signalling pathways are well
understood. There is increasing evidence for a role of the actin cytoskeleton
in the transduction of growth-factor and matrix-derived signals that regulate
basic cellular functions, and it is thought that such functions could be
crucial to wound epithelialization, morphogenesis, tissue modelling and
regulation of proliferation (Martin,
1997). We therefore carried out experiments in which we analysed
the potential of two soluble epidermotropic factors to induce actin-dependent
changes of keratinocyte shape and motility, and the mechanisms involved.
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Materials and Methods |
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Keratinocyte culture and retroviral infection
Primary human keratinocytes were isolated from foreskins and cultured on a
3T3 fibroblast feeder layer in FAD medium as described previously
(Watt, 1998). 3T3 fibroblasts,
strain J2, were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10%
foetal calf serum (FCS). FAD medium and DMEM with low calcium concentration
(FAD low Ca2+ and DMEM low Ca2+) had essentially the
same composition as FAD medium and DMEM, respectively, but contained only 50
µM Ca2+. From FCS and Ham's F12 medium, Ca2+ was
removed by incubating a volume of 50 ml with 2 g Chelex resin (Biorad, Munich,
Germany) overnight on a rotating shaker followed by sterile filtration.
The constitutively active mutant of MEK1 (MAPKK1) with glutamic acid
substitutions at serines 217 and 221
(Cowley et al., 1994) and
cloned into pBabe puro was a generous gift of C. Marshall (Institute of Cancer
Research, London, UK). The plasmid vector pEF-Bos containing the cDNA of a
fusion protein between the extracellular domain of the rat CD2 receptor and
the catalytic p110 subunit of PI-3K, rCD2p110
(Reif et al., 1996
) was a kind
gift of D. Cantrell (London). The rCD2p110 construct was subcloned into the
XhoI site of the retroviral vector pLXSN (Clontech, Heidelberg,
Germany). Correct insertion was confirmed by direct DNA sequencing. Ecotropic
producer lines were generated by transfecting GP+E packaging cells
(Markowitz et al., 1988b
)
using FUGENE (Roche, Mannheim, Germany) reagent and subsequent selection in 1
mg ml-1 G418 (Life Technologies, Karlsruhe, Germany) for 14 days.
Supernatants were used to infect AM-12 amphotropic packaging cells
(Markowitz et al., 1988a
).
Clones of amphotropic packaging cells producing each retroviral vector were
generated and isolated as described previously
(Zhu and Watt, 1996
). The
viral titres were as follows: 1.9x106 pfu ml-1
(MAPKK1); 4x105 pfu ml-1 (rCD2p110). Keratinocytes
were infected with retrovirus by using producer cells as feeders for the first
3 days (Zhu and Watt,
1996
).
Coating of cellware and spreading assay
Permanox chamber slides (Invitrogen, Karlsruhe, Germany) with two chambers
(for spreading assays) or bacteriological dishes (for contraction assays) were
coated with a solution of 3-5 µg ml-1 bovine collagen I and 20
µg ml-1 poly-L-lysine (PLL) or a solution of 20 µg
ml-1 collagen I, respectively, in PBS overnight at 4°C followed
by 2 hours at 37°C. Prior to plating of cells chamber slides and dishes
were washed with PBS.
Feeders were removed from preconfluent cultures of infected or uninfected
keratinocytes by incubation in 0.02% EDTA in PBS for 5-10 minutes.
Keratinocytes were trypsinized, resuspended in 500 µl complete FAD medium
and stirred into suspension medium (complete FAD medium containing 1.75%
methylcellulose). Preparation of the suspension medium was as described by
Adams and Watt (Adams and Watt,
1989). Cells were kept in suspension culture in a cell culture
incubator for 4 hours and then harvested by diluting the suspension medium
1:10 with PBS and centrifuging. The cell pellet was washed twice in DMEM
without supplements and then adjusted to a density of 105 cells
ml-1. Cells were plated onto precoated chamber slides at a density
of 2x104 cells per chamber in the absence or presence of
growth factors and inhibitors and incubated at 37°C for 40 minutes. To
inhibit Rho GTPase activity, toxin B was added to the culture medium 16 hours
prior to the experiment as well as to the suspension medium at a concentration
of 20 ng ml-1. Cells were fixed with 4% formaldehyde in PBS and
permeabilized with 0.4% Triton X-100 in PBS for 5 minutes at room temperature.
Slides were blocked with 50% FCS for 30 minutes at 37°C. The actin
cytoskeleton was then stained with TRITC-labelled phalloidin and digital
images were acquired using a Nikon Eclipse E800 microscope attached to a
Basler A 113 monochrome video camera at a power of 200. For determination of
cell size, a binary image was created and the area covered by each single cell
was calculated using the computer program LUCIA. 200 cells per sample were
analysed and the results plotted as histograms using Excel.
Determination of cell motility
Keratinocytes were harvested and plated onto bacteriological dishes coated
with 10 µg ml-1 fibronectin (Becton Dickinson, Heidelberg,
Germany) or 20 µg ml-1 collagen I (Sigma) at a density that
allowed tracking of individual cells. Frames were taken every 2 minutes using
Olympus IMT1 or IMT2 inverted microscopes driven by Broadcast Animation
Controllers (BAC 900) and fitted with monochrome CCD cameras and video
recorders (Sony M370 CE and PVW-2800P, respectively). Recordings were
digitized and the sequence of all frames was run on a PC. 20-23 individual
cells per sample were tracked manually and speed was calculated using
Mathematica.
Wound healing assays
Keratinocytes were cultured on 60 mm cell culture dishes in the presence of
3T3 feeder cells or on collagen-I-coated chamber slides without feeders. When
the cultures were confluent, remaining feeder cells were removed with PBS/EDTA
and keratinocytes were kept in complete FAD medium containing 50 µM calcium
ions (FAD low Ca2+) for 24 hours. Cultures were treated with 4
µg ml-1 mitomycin C in DMEM containing 50 µg ml-1
calcium ions (DMEM low Ca2+) without FCS for 2 hours, then washed
in PBS and the monolayer wounded with a tip of a glass pipette. Cells were
incubated in DMEM low Ca2+ in the absence or presence of growth
factors and inhibitors for 4 hours. Cultures were fixed, permeabilized and
stained with TRITC-labelled phalloidin. Digital images were acquired as
described.
Western blot analysis of ERK and Akt phosphorylation
To assess the effect of growth factor treatment on activation of ERK and
PI-3K, dishes of preconfluent keratinocytes were starved overnight in
serum-free medium after removing their feeder layers. Cells were then
stimulated with DMEM supplemented with 10 ng ml-1 EGF or 100 ng
ml-1 IGF-1, or fresh medium alone for 15 minutes before lysis.
Keratinocytes were lysed in situ in modified RIPA buffer containing 5 mM EDTA, 1% Triton X-100, 20 µM leupeptin, 1 mM PMSF, 0.5 mg ml-1 soybean trypsin inhibitor, 0.5 mM NaVO3 and 10 mg ml-1 p-nitrophenylphosphate, scraped from the dishes and sonicated for 30 seconds at full power. Lysates were centrifuged at 14,000 g for 10 minutes and the supernatant was used for western blot analysis. Equal amounts of protein were separated by SDS-PAGE and blotted onto Hybond-P PVDF membranes (Amersham, Freiburg, Germany). ERK phosphorylation was detected with antibodies specific for phosphorylated ERK1/2 (Santa Cruz Biotechnology, Heidelberg, Germany). As readout for PI-3K activity, phosphorylation of the protein kinase Akt was determined using an antibody that specifically detects phosphorylated Ser 473 of Akt (New England Biolabs, Beverly, USA). Blots were reprobed with antibodies to ERK2 (Santa Cruz Biotechnology) to check for equal loading of the lanes. Protein bands were visualized with horseradish peroxidase (HRP) coupled secondary antibodies on Hyperfilm using enhanced chemiluminescence (ECL, Amersham).
Fluorescence-activated cell sorting analysis
Keratinocytes were trypsinized, kept in suspension medium for 4 hours and
then harvested. The pellet was resuspended in DMEM containing 10% FCS. Primary
antibodies against EGFR (Santa Cruz Biotechnology) and IGF-1 receptor
(Oncogene Science, Cambridge, USA) were diluted 1:100 and incubated on ice for
30 minutes with occasional agitation. Cells were washed twice in PBS and
resuspended in DMEM containing 10% FCS and a goat polyclonal antibody against
mouse IgG coupled to the dye Alexa 488 at a dilution of 1:500 (Molecular
Probes, Eugene, Oregon, USA). Cells were incubated for 30 minutes, washed
three times in PBS and analysed using a FACS Calibur (Becton Dickinson).
Measurement of intracellular calcium
Keratinocytes were trypsinized and kept in suspension for 4 hours, washed
out and loaded with 10 µM FURA 2-AM in Ca2+-free Hanks buffered
salt solution (HBSS) supplemented with 15 mM HEPES for 1 hour at room
temperature. All subsequent measurements were carried out in
Ca2+-free HBSS. Cells were washed three times and the cell density
was adjusted to 106 cells ml-1. A quartz cuvette with
stirring bar was mounted in the stirred cuvette holder of a Perkin Elmer LS50B
fluorescence photometer and filled with 2 ml cell suspension. The excitation
wavelength was set to 380 nm, fluorescence was recorded at 509 nm.
Keratinocytes were pre-incubated with inhibitors for 20 minutes. Bradykinin
(10 µM) or IGF-1 (100 ng ml-1) were added directly to the
stirred cell suspension in the cuvette.
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Results |
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We conclude that IGF-1, but not EGF, induces actin polymerization-dependent spreading of single primary human keratinocytes plated onto collagen-coated surfaces.
Treatment with EGF has been described previously to lead to cell size
reduction in A431 cells (Peppelenbosch et
al., 1993). We adjusted the conditions of our experiment to assess
a possible influence of growth factors on cell size reduction. Keratinocytes
were allowed to spread for 1 hour in DMEM without supplements. Cells were then
treated with fresh DMEM without supplements or DMEM containing 10 ng
ml-1 EGF or 100 ng ml-1 IGF-1. Cells were fixed after 30
minutes and cell size was determined as described. We found that cell size was
reduced in a high proportion of keratinocytes treated with EGF compared with
the control (Fig. 1C, top).
Treatment with IGF-1 did not have this effect
(Fig. 1C, bottom).
IGF-1 and EGF use distinct signal transduction pathways to regulate
keratinocyte shape
EGF is known to stimulate signalling via the classical MAPK cascade (e.g.
Zhu et al., 1999). When we
treated preconfluent cultures of primary human keratinocytes with 10 ng
ml-1 EGF a transient increase of MAPK phosphorylation (and hence
activation) was detectable in cells analyzed after 15 minutes and 30 minutes.
This stimulation was suppressed when cells were preincubated with 10 µM of
the specific MEK-1 inhibitor PD98059 (Fig.
2A). EGF has also been reported to stimulate activity of PI-3K in
various cell types (Carter and Downes,
1992
; Hill et al.,
2000
). We used western blot analysis with an antibody specific to
phosphorylated Akt as a readout for PI-3K activity
(Datta et al., 1996
). Under
our experimental conditions EGF did not stimulate activity of PI-3K in
keratinocytes (Fig. 2B). Notice
that, in these cultures, basal activity of MAPK is increased compared with the
cells analysed in Fig. 2A. This
is probably due to increased levels of paracrine growth factors in
preconfluent cultures.
|
IGF-1 can activate PI-3K-mediated signalling in other cell types
(Way and Mooney, 1993).
Stimulation of preconfluent human keratinocytes with 100 ng ml-1
IGF-1 resulted in pronounced phosphorylation of Akt but not of MAPK. This
phosphorylation was suppressed when keratinocytes were pretreated with 25
µM of a specific inhibitor of PI-3K, LY294002
(Fig. 2B).
We then tested whether the effects of EGF and IGF-1 on cell rounding and
spreading that we had observed were mediated by MAPK and PI-3K, respectively.
We carried out experiments in which we used either specific inhibitors or
constitutively active mutants of PI-3K (rCD2p110) or MEK-1 (MAPKK1).
Constitutive activation of PI-3K signalling by a fusion protein consisting of
the extracellular and transmembrane domains of the rat CD2 receptor and the
catalytic subunit of PI-3K (rCD2p110) has been described previously
(Reif et al., 1996).
Stimulation of MAPK signalling by an activating mutant of MEK-1 (MAPKK1) has
been shown by Cowley et al. (Cowley et
al., 1994
). Both mutant kinases were expressed using retroviral
gene transfer and subsequent selection with puromycin and G418, respectively.
We have shown previously that expression of MAPKK1 results in constitutive
activation of MAPK signalling in primary human keratinocytes
(Haase et al., 2001
;
Zhu et al., 1999
). Infection
of primary keratinocytes with retroviruses encoding rCD2p110 led to expression
of the fusion protein as shown by detection of a band at approximately 120 kDa
in western blots with a monoclonal antibody against the extracellular domain
of rat CD2 (Ox-34) (see also Reif et al.,
1996
). The band indicated by an arrowhead in
Fig. 3A represents the rCD2p110
fusion protein; it was not present in keratinocytes infected with the empty
vector neo only (Fig. 3A). We
also detected expression of rCD2p110 on the surface of keratinocytes
transduced with retroviruses encoding for the construct but not in
keratinocytes transduced with empty vector neo. Immunofluorescent staining
using the antibody Ox-34 revealed that rCD2p110 specifically localized to
protruding areas of the cell membrane (Fig.
3B,C), which were characterized by a fine mesh of thin actin
fibres, indicating that these protrusions were relatively young.
|
Treatment of keratinocytes with 25 µM or 50 µM LY294002 prior to stimulation with IGF-1 led to a reduction of cell spreading at 40 minutes or 3 hours after plating compared with control cells pretreated with DMSO only (Fig. 4A and data not shown). Conversely, activation of PI-3K signalling by expression of rCD2p110 resulted in enhanced spreading of keratinocytes compared with the neo control (Fig. 4B).
|
We next tested whether the effect of EGF on keratinocyte size reduction was mediated by MAPK. When spreading keratinocytes were treated with 10 µM PD98059 prior to EGF stimulation, the reduction in cell size was less pronounced than in keratinocytes pretreated with DMSO only (Fig. 4C). Conversely, keratinocytes expressing MAPKK1 were smaller than controls (Fig. 4D). From these experiments, we conclude: (1) that activation of PI-3K is sufficient to induce spreading of human keratinocytes and partly mediates IGF-1-stimulated spreading; and (2) that activation of MAPK is sufficient to induce rounding of human keratinocytes and the reduction of keratinocyte size induced by EGF is mediated by MAPK.
Crucial downstream effectors of IGF-1-stimulated, PI-3K-dependent Schwann
cell motility are the small GTP-binding proteins Cdc42 and Rac
(Cheng et al., 2000). We
therefore tested the involvement of Rho GTPases in IGF-1-stimulated spreading
by inhibiting their function with the exotoxin B of the bacterium
Clostridium difficile (toxin B)
(Just et al., 1995
).
Preincubation of primary human keratinocytes with toxin B inhibited
IGF-1-induced spreading (Fig.
5G). We conclude that Rho GTPase function is essential for
spreading of keratinocytes.
|
Another signalling pathway known to regulate cell movements involves
phospholipase C (PLC
) (Chen
et al., 1994
). Activity of PLC results in the enzymatic hydrolysis
of phosphatidylinositol-4,5 bisphosphate and the generation of inositol-1,4,5
trisphosphate, which releases Ca2+ from intracellular stores
(Berridge and Irvine, 1984
).
Intracellular Ca2+ concentration after agonist stimulation can
therefore be used as a measure of PLC activity. We used a fluorimetric method
with the Ca2+-binding fluorescent dye FURA-2 to test inhibition of
PLC activity by the inhibitor U73122. U73343, an isomer of U73122 that is less
efficient at inhibiting PLC, was used as a control. Keratinocytes were loaded
with FURA-2 and intracellular fluorescence of suspended keratinocytes in
Ca2+-free Hanks buffered salt solution was measured at 509 nm.
Under these conditions, stimulation with 100 ng ml-1 IGF-1 did not
result in a detectable increase of the intracellular calcium concentration
(Fig. 5B). Stimulation with 10
µM bradykinin resulted in a rapid increase in fluorescence
(Fig. 5A). Cells were then
pre-incubated with different concentrations of U73122 and U73343, and
stimulated with 10 µM bradykinin. Although incubation with 1 µM or 5
µM of both U73122 and U73343 reduced the amplitude of fluorescence after
bradykinin stimulation, this suppression was more pronounced in cells
pre-incubated with U73122 (Fig.
5C-F).
Using inhibitor concentrations of 1-10 µM U73122 and U73343, we tested
whether PLC contributes to IGF-1-stimulated spreading in human keratinocytes.
In four out of five experiments, there was consistent inhibition of cell
spreading at concentrations from 1 µM to 10 µM U73122
(Fig. 5H). At concentrations of
1-4 µM, U73343 had no detectable influence on the degree of IGF-1-induced
cell spreading (Fig. 5I). At 10
µM, both substances inhibited spreading to a similar degree, suggesting
that reduced spreading at this inhibitor dose was not due to specific
inhibition of PLC (data not shown). Inhibitors were not toxic as determined by
analysis of lactate dehydrogenase (LDH) release (data not shown). We conclude,
based on experiments relying on pharmacologic inhibition, that PLC
activity is required for IGF-1-stimulated spreading of human
keratinocytes.
Conceivable mechanisms for MAPK-mediated keratinocyte contraction include
stimulation of myosine light chain kinase (MLCK)
(Klemke et al., 1997) or the
activation of calpain, a protease implicated in the degradation of focal
adhesion components (Glading et al.,
2000
). When we stained keratinocytes with antibodies against
ß1 integrin and vinculin in order to visualize focal adhesions, we
noticed that MAPKK1 expressing cells seemed to have fewer focal adhesions than
control cells (data not shown). We used the MLCK inhibitor ML-7 and the
calpain inhibitor MDL28170 at concentrations of 1-10 µM in order to test
the involvement of these enzymes. Under these conditions, the inhibitors
showed no influence on EGF-stimulated keratinocyte contraction (data not
shown).
MAPK mediates EGF-stimulated keratinocyte motility
EGF and insulin are among the growth factors that stimulate keratinocyte
migration (Barrandon and Green,
1987; Benoliel et al.,
1997
). MAPK has been shown to regulate the motility of several
cell types by direct phosphorylation of MLCK
(Klemke et al., 1997
;
Nguyen et al., 1999
). We
therefore analysed the influence of MEK-1 mutants and the specific MEK-1
inhibitor PD98059 on keratinocyte motility. To exclude toxic effects of the
chemical inhibitor PD98059 on keratinocytes during the prolonged observation
period for cell migration (24 hours), we also used a dominant negative mutant
of MEK-1, MANA (Cowley et al.,
1994
), to inhibit MAPK activity. The effect of this construct on
suppression of MAPK activity has been shown previously
(Cowley et al., 1994
;
Haase et al., 2001
;
Zhu et al., 1999
).
Keratinocyte motility was monitored by time lapse video microscopy on fibronectin and collagen coated dishes. Similar results were obtained for both substrata. Data shown in Fig. 6A,B were obtained with fibronectin coated dishes. Data sets shown in Fig. 6C,D were obtained with collagen coated dishes (two experiments) and fibronectin coated dishes. Constitutive activation of MAPK by expression of MAPKK1 led to an increase in the total distance moved by single cells (Fig. 6A,B) and a twofold increase in average cell speed (Fig. 6C). Fig. 6C shows the results of four separate experiments in which the speeds of cells expressing the empty vector, puro, or MAPKK1 were compared. The number of cells moving at the same speed within each experiment is represented by the width of the bars. Analysis of variance revealed that the difference between puro expressing (16.9x1.8 µm h-1) and MAPKK1 expressing (32.8x1.2 µm h-1) keratinocytes was statistically significant (P<0.01).
|
Consistent with previous findings
(Barrandon and Green, 1987),
incubation of keratinocytes with 10 ng ml-1 EGF led to a threefold
increase in cell speed (P<0.001; compare puro expressing cells in
Fig. 6C,D). In the presence of
10 ng ml-1 EGF, inhibition of MAPK activity with 25 µM PD98059
or by introduction of MANA reduced keratinocyte motility. The
histogram in Fig. 6D shows the
speed of all cells analysed in five independent experiments in which MEK-1
activity was inhibited. Analysis of variance revealed a significant difference
in speed between the puro/DMSO and the MANA/PD98059 groups
(P<0.001). The direction of all cell movement observed in these
experiments was always completely random
(Fig. 6A,B) and neither
activation nor inhibition of MAPK signalling had any influence on the
directionality of cell movement.
IGF-1 enhances keratinocyte random migration independently of PI-3K
activity
We also investigated the influence of IGF-1 on keratinocyte motility. In
three independent experiments, treatment of primary human keratinocytes with
100 ng ml-1 IGF-1 stimulated random cell migration by a factor of
1.5 to 2 (Fig. 6E). Two
experiments were carried out with fibronectin coated dishes and one experiment
with collagen coated dishes, yielding similar results. We further tested the
requirement of PI-3K activity for spontaneous and IGF-1 induced migration.
Treatment with 25 µM LY294002 slowed down spontaneous keratinocyte
movements in the absence of IGF-1 but did not reduce IGF-1 stimulated
migration even when added at a concentration of 50 µM
(Fig. 6E,F and data not shown).
During the 24 hour period of observation, no signs of cytotoxicity were
detected in the keratinocytes. We conclude that PI-3K activity can regulate
keratinocyte motility but it is not required for IGF-1 stimulated keratinocyte
migration in vitro.
Co-operative regulation of wound epithelialization by IGF-1 and EGF
dependent pathways
Epidermal wound healing is a typical situation during which keratinocyte
shape and migration are regulated and both EGF and IGF-1 levels are known to
be increased in skin wounds (Singer and
Clark, 1999). Because cell shape and random cell migration
represent only single functions of keratinocytes within the complex process of
wound epithelialization, we compared the effects of EGF and IGF-1 in a wound
epithelialization model of a keratinocyte monolayer in vitro. Keratinocytes
were grown on cell culture dishes in the presence of feeders (three
experiments) or on collagen coated dishes in the absence of feeders (two
experiments). Similar results were obtained under both conditions. In order to
exclude effects of either growth factor on cell-cell adhesion and on cell
proliferation, confluent sheets of keratinocytes in FAD low Ca2+
were treated with 4 µg ml-1 mitomycin C for 2 hours. This
treatment prevented the formation of Ca2+-dependent cell-cell
contacts and abrogated proliferation of keratinocytes (data not shown). We
then wounded the keratinocyte monolayer with a glass pipette tip, creating
wounds of constant size, and monitored wound closure over 4-6 hours. As
expected, treatment with 100 ng ml-1 or 10 ng ml-1 EGF
accelerated wound closure compared with the untreated control
(Fig. 7A,E and data not shown).
This was almost completely inhibited by treatment with 10 µM of PD98059
(Fig. 7E,F). Addition of IGF-1
at 100 ng ml-1 also accelerated wound closure
(Fig. 7A,C) and this effect
could be abrogated by inhibition of PI-3K activity using 25 µg
ml-1 LY294002 (Fig.
7C,D). When EGF and IGF-1 were added simultaneously to the wounded
cultures, re-epithelialization of the denuded area was faster than with either
growth factor alone (Fig.
7B).
|
We also investigated whether activation of MAPK or PI-3K were sufficient to stimulate wound epithelialization using confluent monolayers of keratinocytes infected with MAPKK1, rCD2p110 or empty vector controls on collagen coated dishes. Constitutive activation of MAPK signalling by expression of MAPKK1 accelerated wound closure dramatically (Fig. 7G,H). Activation of PI-3K by expression of rCD2p110 also stimulated re-epithelialization, but this effect was less pronounced than the effect of MAPK activation (Fig. 7I,J).
Using pharmacological inhibitors, we further analysed the contribution of different signalling molecules to IGF-1 and EGF stimulated wound epithelialization. Consistent with the results obtained in the spreading assay, preincubation with toxin B (an inhibitor of Rho activity) strongly inhibited IGF-1 induced wound closure (Fig. 8A,B). Whereas addition of MDL28170 and U73122 had no inhibitory effect, ML-7 delayed EGF stimulated wound closure at concentrations of 5 µM and 10 µM (Fig. 8C-E).
|
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Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In our study we have identified a new function of IGF-1 in epidermal
keratinocytes: IGF-1 regulates keratinocyte shape by stimulating plasma
membrane protrusion and spreading. This action of IGF-1 results in the
acceleration of wound epithelialization in vitro and it requires activity of
PI-3 kinase and of Rho GTPases (Ridley and
Hall, 1992; Ridley et al.,
1992
). In view of the importance of regulated shape changes of
epidermal cells during Drosophila development
(Harden et al., 1995
) this
mechanism could also serve as a potential explanation for the disturbance of
hair follicle formation in the skin of IGF-1-receptor deficient mice
(Liu et al., 1993
).
EGF, in contrast to IGF-1, induces rounding of the cell body, leading to a
decrease of the cell-substratum contact area. Similarly, in A431 cells EGF has
been shown to induce cytoskeletal rearrangements that lead to cell contraction
and rounding (Peppelenbosch et al.,
1993). Considering these results, it might seem contradictory that
treatment of keratinocyte colonies with EGF should lead to enlargement of
these colonies within minutes (Barrandon
and Green, 1987
). This enlargement, however, does not directly
reflect cytoskeletal changes induced by EGF but is caused by translocation of
keratinocytes from the colony centre to its periphery because of increased
migration. The most likely interpretation, combining both observations, is
that cell body contraction is part of the EGF stimulated migration
process.
The observed differences in the regulation of keratinocyte shape led us to
investigate intracellular pathways that mediate EGF and IGF-1 functions in
more detail. As has been described previously in keratinocytes and in other
cell types, EGF treatment resulted in activation of the classical MAPK pathway
(Gotoh et al., 1990;
Zhu et al., 1999
). Treatment
of cultured primary human keratinocytes with IGF-1-stimulated phosphorylation
of the protein kinase Akt, indicating activation of PI-3 kinase, but not the
classical MAPK pathway. This result is different from data obtained in murine
keratinocytes, in which IGF-1 treatment led to an increase in MAPK activity
(Vasioukhin et al., 2001
).
This difference might reflect species-specific responses to IGF-1 or
variations in culture conditions. Although treatment with IGF-1 did not
stimulate MAPK activity above background levels, pretreatment with LY294002, a
specific inhibitor of PI-3K, completely abrogated MAPK activity
(Fig. 2B). This might indicate
that activity of PI-3K could be required by keratinocytes in order to enable
MAPK activation by autocrine signalling. Inhibition of basal MAPK activity by
LY294002 could also account for the reduction of spontaneous, IGF-1
independent migration observed in keratinocytes treated with this inhibitor
(Fig. 6E).
Using pharmacological inhibitors and mutants of MEK-1, we found that
activity of the MAPK cascade is both required and sufficient to induce
keratinocyte rounding. In a cell migration model proposed recently
(Cheresh et al., 1999), MAPK
acts as an opponent of p130CAS, cCrkII and the small GTPase Rac, which are
known to stimulate ruffling, by regulating the actin-myosin motor that
generates forces for cell contraction. Proposed mechanisms for the stimulatory
effect of MAPK on cell motility include phosphorylation and activation of MLCK
(Klemke et al., 1997
), and
activation of calpain, a protease implicated in the degradation of focal
adhesion components (Glading et al.,
2000
). We did not find evidence for the involvement of these
enzymes; the mechanism of MAPK mediated keratinocyte cell rounding therefore
remains unidentified here. One possibility that we have not addressed in our
study is the stimulation of matrix metalloproteinases through sustained
activation of MAPK (McCawley et al.,
1999
), which could both result in degradation of matrix molecules
and enhance the release of autocrine ligands for the EGFR from the
keratinocyte surface (Fan and Derynck,
1999
). Although MLCK activity was not required for EGF induced
cell rounding, the MLCK inhibitor ML-7 prevented wound epithelialization
stimulated by EGF. This indicates that other MLCK dependent cellular functions
are required for the stimulation of wound closure by EGF.
In contrast to MAPK, activation of PI-3K stimulated membrane protrusion,
whereas its inhibition delayed keratinocyte spreading. PI-3K has been
described to induce cytoskeletal rearrangements by stimulation of Rac activity
(Nobes et al., 1995) and we
show here the involvement of small GTPases of the Rho family in
IGF-1-stimulated keratinocyte spreading. Consistent with a putative role of
Rac, rCD2p110 localized to protruding areas of the cell membrane, where Rac is
normally active (Del Pozo et al.,
2002
; Nobes and Hall,
1995
).
In addition, we found a requirement for PLC activity for keratinocyte
spreading. Although IGF-1 did not stimulate PLC activity in suspended
keratinocytes, U73122 inhibited keratinocyte spreading when added at
concentrations that specifically suppress PLC activity. Efficient activation
of PLC by growth factors has been reported to depend on cell adhesion
(McNamee et al., 1993) and it
is therefore possible that PLC
is activated by IGF-1 in adherent, but
not in suspended, keratinocytes.
In adult skin, keratinocytes migrate by crawling
(Fenteany et al., 2000)
(reviewed in Jacinto et al.,
2001
). This form of movement requires extension of the plasma
membrane on one hand and contraction of the cell body on the other. The
relationship between cell shape changes and migration speed is so far unclear.
It has been suggested for different cell types, however, that activation of
PI-3K and Rac, which promote cell spreading, is sufficient to stimulate
motility (Keely et al., 1997
).
The fact that inhibition of PI-3K can delay IGF-1-induced keratinocyte
spreading but not inhibit IGF-1-stimulated motility suggests, however, that
IGF-1 uses multiple intracellular pathways to exert its effects on
keratinocyte shape and motility. This is further supported by the observed
inhibition of cell spreading with the PLC inhibitor U73122 and by results of
others showing the involvement of NF-
B-dependent signalling in the
regulation of insulin-stimulated motility of keratinocytes
(Benoliel et al., 1997
). The
finding that inhibition of PI-3K activity can reduce keratinocyte motility in
the absence of exogenous growth factors
(Fig. 6E) could reflect the
requirement for PI-3K activity to maintain a basal level of MAPK signalling
(Fig. 2B).
Although the PI-3K inhibitor LY294002 failed to affect IGF-1-stimulated
random motility of keratinocytes, it was able to inhibit spreading of
keratinocytes on collagen I and to delay wound closure in vitro. These
findings raise the possibility that the speed of random keratinocyte migration
is not the only determinant of wound epithelialization speed and suggest that
changes in the keratinocyte actin cytoskeleton are crucial for the stimulation
of wound epithelialization by IGF-1. Because wound epithelialization in vivo
starts from the wound edge, it is conceivable that disturbances of cell
spreading could delay or prevent the start of epithelial sheet locomotion over
the wound. This might occur in chronic wounds of diabetic individuals, in
which IGF-1 amounts are reduced (Blakytny
et al., 2000) and/or decreased levels of insulin hamper optimum
keratinocyte migration (Benoliel et al.,
1997
). The biological significance of cytokine-induced
cytoskeletal rearrangements has been illustrated in
ß5-integrin-transfected CS-1 melanoma cells in which IGF-1 leads to the
co-localization of
vß5 integrin with
-actinin and results
in enhanced tumour cell metastasis in vivo
(Brooks et al., 1997
).
The speed of epidermal wound healing is apparently regulated by an interplay of mechanisms involving different signalling molecules. Our data provide evidence for the existence of at least two relevant signalling pathways that are able to stimulate wound epithelialization in an additive manner: an EGF-stimulated, MAPK-dependent pathway and an IGF-1-stimulated, PI-3K/Rac-dependent pathway. Further dissection of these pathways will provide deeper insight into the mechanisms that regulate skin wound epithelialization.
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Acknowledgments |
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