Glycogen synthase kinase-3 regulates cytoskeleton and translocation of Rac1 in long cellular extensions of human keratinocytes

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Experimental Cell Research 293 (2004) 68–80

Glycogen synthase kinase-3 regulates cytoskeleton and translocation of

Rac1 in long cellular extensions of human keratinocytes

Leeni Koivisto,a Lari Hakkinen,a Kazue Matsumoto,b Christopher A. McCulloch,c

Kenneth M. Yamada,b and Hannu Larjavaa,*

aDepartment of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z3bNational Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-4370, USA

cUniversity of Toronto, Toronto, ON, Canada M5S 3E2

Received 3 July 2003

Abstract

Wound keratinocytes form long cellular extensions that facilitate their migration from the wound edge into provisional matrix. We have

previously shown that similar extensions can be induced by a long-term exposure to EGF or rapidly by staurosporine in cultured cells.

This morphological change depends on the activity of glycogen synthase kinase-3 (GSK-3). Here, we have characterized the cytoskeletal

changes involved in formation of these extended lamellipodia (E-lam) in human HaCaT keratinocytes. E-lams contained actin filaments,

stable microtubules and keratin intermediate filaments. E-lam formation was prevented by cytochalasin D, colchicine and low

concentrations of taxol and nocodazole, suggesting that actin and microtubule organization and dynamics are essential for E-lam

formation. Staurosporine induced recruitment of filamentous actin (F-actin), cortactin, filamin, Arp2/3 complex, Rac1 GTPase and

phospholipase C-g1 (PLC-g1) to lamellipodia. Treatment of cells with the GSK-3 inhibitors SB-415286 and LiCl2 inhibited E-lam

formation and prevented the accumulation of Rac1 and Arp2/3 complex at lamellipodia. The formation of E-lams was dependent on

fibronectin-binding integrins and normally regulated Rac1, and expression of either dominant-negative or constitutively active forms of

Rac1 prevented E-lam formation. Overexpression of either RhoA or Cdc42 GTPases suppressed E-lam formation. We conclude that

extended lamellipodia formation in keratinocytes requires actin and tubulin assembly at the leading edge, and this process is regulated by

Rac1 downstream of GSK-3.

D 2003 Elsevier Inc. All rights reserved.

Keywords: Lamellipodia; Actin; Tubulin; Rho GTPases

Introduction In keratinocytes, microtubules are responsible for the

During re-epithelization of wounds, keratinocyte migra-

tion on the fibronectin-rich, provisional wound matrix starts

with the formation of long lamellipodia that extend into the

wound [1–4]. Coordinated changes in the organization of

the cytoskeleton are required for lamellipodia formation and

cell migration.

0014-4827/$ - see front matter D 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.yexcr.2003.09.026

Abbreviations: E-lam, extended lamellipodium; F-actin, filamentous

actin; GSK-3, glycogen synthase kinase-3; PLC-g1, phospholipase C-g1;

PP1, protein phosphatase 1; VSV, vesicular stomatitis virus.

* Corresponding author. Department of Oral Biological and Medical

Sciences, Faculty of Dentistry, University of British Columbia, 2199

Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3. Fax: +1-604-822-3562.

E-mail address: [email protected] (H. Larjava).

orientation and polarization of the cell [5]. Actin is involved

in regulating cell shape and motility, whereas keratin inter-

mediate filaments provide structural integrity and also

participate in organization of actin filaments and micro-

tubules [5,6]. Cell migration requires interplay between the

actin and microtubule cytoskeletal systems and modulation

of their organization [7,8]. The organization of filamentous

actin (F-actin) during cell motility can affect microtubule

assembly and conversely, microtubules may provide a

vector for directing sites of F-actin nucleation and assembly

[9].

The small GTPases of the Rho family, namely RhoA,

Rac1 and Cdc42, have been identified as key molecules

regulating morphological changes as they differentially reg-

ulate the formation of actin stress fibers, lamellipodia and

filopodia, respectively [10,11]. Only a limited amount of

L. Koivisto et al. / Experimental Cell Research 293 (2004) 68–80 69

information about functions of the small GTPases relates to

keratinocytes and other epithelial cells. As the function and

regulation of Rho-family GTPases are highly cell-type-

specific, many previous results may not be directly applica-

ble to keratinocytes. Rho-family GTPases function often as

cascades. In fibroblasts, activation of Cdc42 leads to sequen-

tial activation of Rac1 and RhoA. Others have reported that

Rac1 downregulates RhoA activity [12–14], and conversely,

RhoA can inhibit Rac1 and/or Cdc42 activation [15]. In

fibroblasts, both Cdc42 and Rac1 are required for cell

spreading on fibronectin [16]. Fibroblasts migrate as indi-

vidual cells, and microtubule growth occurs at the leading

edge and microtubule shortening at the rear of a migrating

cell, thereby contributing to the activation of small GTPases

Rac1 and RhoA, respectively [9,17]. Lamellar protrusion at

the leading edge is dependent on actin filament assembly,

myosin and the formation of adhesive contacts with the

extracellular matrix [18,19]. RhoA regulates cell contraction

and detachment at the rear of the cell that leads to translo-

cation of the cell body. However, epithelial cell migration as

a sheet appears to be only dependent on Rac1, whereas the

activity of RhoA is not a prerequisite [20].

Migration of keratinocytes on the wound matrix is

mediated by integrins [21–23]. During wound healing and

in culture, keratinocytes are induced to express at least three

different fibronectin-binding integrins, a5h1, avh1 and

avh6 [23–26]. The roles of the cytoskeleton, small

GTPases of the Rho family and fibronectin-binding integ-

rins in the formation of extended lamellipodia in keratino-

cytes are poorly described. We have recently shown that the

protein-serine/threonine kinase inhibitor staurosporine pro-

motes a unique keratinocyte phenotype with long extended

lamellipodia (E-lam) similar to those induced by epidermal

growth factor and resembling the cellular projections

expressed in wound keratinocytes [4]. The morphological

change from stationary to migratory phenotype was depen-

dent on glycogen synthase kinase-3 (GSK-3) [4]. Since

GSK-3 did not seem to regulate the formation of conven-

tional lamellipodia that are associated with keratinocyte

spreading on fibronectin, we hypothesized that E-lams are

structurally distinct from classical lamellipodia. In the

present report, we demonstrate that E-lam formation is

dependent on organization of both actin and tubulin as well

as on normally regulated Rac1 GTPase. Staurosporine

induced strong accumulation of F-actin and Rac1 to lamel-

lipodial ruffles together with cortactin, filamin, Arp2/3

complex and phospholipase C-g1 (PLC-g1). These mole-

cules did not accumulate in lamellipodia in untreated

keratinocytes that were spreading on fibronectin, suggesting

that staurosporine-induced E-lams are indeed distinct from

typical lamellipodia in keratinocytes. Inhibition of GSK-3

activity prevented the accumulation of Rac1 in staurospor-

ine-induced lamellipodia. Since Rac1 regulates actin assem-

bly, our results suggest a novel function for GSK-3 as an

upstream modulator of Rac1 in the formation of extended

lamellipodia in keratinocytes.

Materials and methods

Reagents

Staurosporine, cytochalasin D, taxol and nocodazole

were obtained from Sigma (St. Louis, MO, USA). Colchi-

cine was obtained from Biomol (Plymouth Meeting, PA,

USA). Y-27632 and SB-415286 were obtained from Tocris

Cookson Inc. (Ellisville, MO, USA).

Cell culture

The immortalized epidermal keratinocyte cell line

HaCaT (a generous gift from Dr. Norbert Fusenig, German

Cancer Center, Heidelberg, Germany) was maintained in

Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL

Life Technologies, Rockville, MD, USA) supplemented

with 23 mM sodium bicarbonate, 20 mM HEPES, anti-

biotics (50 Ag/ml streptomycin sulfate, 100 U/ml penicillin)

and 10% heat-inactivated fetal calf serum (FCS; Gibco

BRL). The HaCaT cell line mimics many of the properties

of normal epidermal keratinocytes, is not invasive and can

differentiate under appropriate experimental conditions [27].

Cells were always subcultured 2 days before experiments to

maintain consistency throughout the study.

Cell spreading assays

Cell spreading experiments were performed essentially

as described previously [26]. Cell culture plates were

coated with fibronectin (2 Ag/cm2; from bovine plasma;

Chemicon, Temencula, CA, USA). Unoccupied sites were

blocked with 1% heat-denatured bovine serum albumin.

HaCaT cells (30,000/cm2) were seeded on plates in serum-

free DMEM containing 50 AM cycloheximide to prevent

de novo protein synthesis. The cells were allowed to attach

and spread for 120 min before adding the modulators of

the cytoskeleton or protein kinase inhibitors to the cells. To

quantify the effects of the agents on cell spreading and E-

lam formation, the cells were fixed by carefully adding 2�formaldehyde fixative [8% formaldehyde and 10% sucrose

in phosphate-buffered saline (PBS) containing calcium and

magnesium]. After fixation, the wells were filled with

distilled water to the top, covered with a glass plate for

observation by phase-contrast microscopy, and the percen-

tages of spread cells and cells with E-lams were counted.

To study the role of integrins in E-lam formation,

inhibitory antibodies against various integrin subunits,

namely, a5 (BIIG2; hybridoma supernatant diluted 1:5)

[28], h1 (mAb13; 20 Ag/ml) [29], av (L230; 20 Ag/ml)

[30] and h6 (MAB2077Z; 20 Ag/ml; Chemicon) were

used alone or in various combinations together with

staurosporine. Because these antibodies can differentially

affect HaCaT cell spreading on fibronectin [26], they were

added to cells that had already attached and spread.

Antibody L230 was purified from cell culture superna-

L. Koivisto et al. / Experimental Cell Research 293 (2004) 68–8070

tants of hybridoma cells grown in our laboratory (ATCC

HB8448) and antibody BIIG2, developed by Dr. Caroline

Damsky, was obtained from the Developmental Studies

Hybridoma Bank maintained by The University of Iowa,

Department of Biological Sciences, Iowa City, IA, USA.

The cells were treated with the antibodies for 60 min,

fixed and analyzed for cell spreading and E-lam formation

as above.

Immunocytochemistry

Cells were allowed to spread on fibronectin-coated

SonicSealk slides (Nunc, Rochester, NY, USA) or on glass

coverslips and then treated with staurosporine as described

above. The cells were formaldehyde-fixed and permeabi-

lized with PBS containing 0.5% Triton X-100. Immunoloc-

alization was performed as described previously [31] with

antibodies recognizing h-tubulin (MAB3408; Chemicon),

acetylated a-tubulin (6-11B-1; Sigma), cytokeratin 14

(LL002; Serotec LTD, Oxford, UK), cortactin (H-191; Santa

Cruz Biotechnology, Santa Cruz, CA, USA), filamin

(MAB1680; Chemicon), Arp3 (H-110; Santa Cruz Biotech-

nology), Rac1 (C-14; Santa Cruz Biotechnology) or phos-

pho-phospholipase-g1 (pY783; P6111; Sigma). Anti-mouse

or anti-rabbit VectastainR ABC and VectorR VIP peroxi-

dase substrate kits (Vector laboratories Inc., Burlingame,

CA, USA) were used for immunodetection. For actin

filaments, the cells were stained with biotin-XX-phalloidin

(Molecular Probes, Eugene, OR, USA) followed by detec-

tion with VectastainR ABC and VectorR VIP peroxidase

substrate kits.

Plasmid constructs and transfections

Plasmids containing wild-type or mutated cDNAs encod-

ing the small GTPases Rac1, Cdc42 and RhoA were kindly

provided by J. Silvio Gutkind (National Institute of Dental

and Craniofacial Research, Bethesda, MD, USA) [32]. The

cDNA inserts from these original pCEF-AU5 vectors were

subcloned as HindIII–EcoRI, BamHI–EcoRI or BamHI–

XbaI fragments into a pRK5-based plasmid expression

system containing a cytomegalovirus promoter, Kozak con-

sensus sequence, and two adjacent vesicular stomatitis virus

(VSV) epitope sequences [33]. Mutagenesis was performed

using Stratagene (La Jolla, CA, USA) QuikChange muta-

genesis kits to generate a complete set of wild-type, consti-

tutively activated and dominant-negative expression

plasmids corresponding to each of the three Rho-family

GTPases, for which integrity of each cDNA insert was

confirmed by complete DNA sequencing.

To analyze the involvement of Rac1, RhoA and Cdc42

in E-lam formation, HaCaT cells were transiently trans-

fected with plasmids expressing dominant-negative

[pRKVSVRacT17N (RacN17) , pRKVSVRhoT19N

(RhoN19), pRKVSVCdc42T17N (Cdc42N17)], wild type

[pRKVSVRacWT (RacWT), pRKVSVRhoWT (RhoWT),

pRKVSVCdc42WT (Cdc42WT)] or constitutively active

[pRKVSVRacQ61L (RacQL), pRKVSVRhoQ63L (RhoQL),

pRKVSVCdc42Q61L (Cdc42QL)] forms of these GTPases.

HaCaT cells were transfected using LipofectAMINE PLUS

Reagent (Gibco BRL). The transfected cells were allowed to

spread on fibronectin-coated glass coverslips with or without

staurosporine as above. The cells were then immunostained

with Cy3-conjugated anti-VSV glycoprotein antibody (Sig-

ma). The percentage of VSV-positive cells that were spread

and had formed E-lams was compared to the percentage of

negative cells on the same coverslip. Only cells with clearly

distinguishable Cy3 staining were considered as positive.

The transfection efficiency in the experiments was approx-

imately 12%.

Statistical analysis

All experiments were repeated at least three times. The

spreading assays each used two to four replicate wells, and

four fields of cells for each well were counted (n = 8–16).

Statistical analysis was performed using one-way ANOVA,

and statistical significance was set at P < 0.05. The signifi-

cance of difference between control and treated samples was

analyzed using Dunnett’s posttest.

Results

Staurosporine induces E-lam formation on fibronectin

We have described elsewhere that protein-serine/threo-

nine kinase inhibitors including staurosporine exert a unique

effect on keratinocyte cell morphology on fibronectin as the

cells form thin, long, straight lamellipodia that mimic

wound-edge keratinocyte lamellipodia [4]. Staurosporine

was able to induce E-lams to the same extent in spreading

cells and in cells that had already spread [4]. This charac-

teristic allowed us to investigate signaling mechanisms of E-

lam formation that were independent of cell spreading.

Typical staurosporine-induced keratinocyte morphology is

shown in Fig. 1B.

Both actin and tubulin organization are critical for E-lam

formation

As changes in cell shape require rearrangement of the

cytoskeleton, we examined the distribution of cytoskeletal

elements in E-lams. Microtubules, F-actin and keratin inter-

mediate filaments were immunolocalized in cells spread on

fibronectin. Microtubules were detected by immunostaining

with anti-h-tubulin antibody. In untreated control keratino-

cytes, an extensive microtubule network was found through-

out the cell including the lamellipodia (Fig. 2A). In cells

treated with 50 nM staurosporine, long microtubule bundles

extended through the stem of the E-lam and separated in the

lamellar tip to cover the entire lamellipodium (Fig. 2B).

Fig. 1. Staurosporine-induced E-lam formation in HaCaT keratinocytes. HaCaT cells were allowed to spread on fibronectin for 2 h and were then treated with

50 nM staurosporine for 1 h (B) or left untreated (A). The cells were fixed, stained with crystal violet and photographed. Stem regions of extended lamellipodia

are marked with arrows.


Acetylation of a-tubulin is linked to microtubule stabiliza-

tion [34]. To investigate whether staurosporine could induce

alterations in microtubule stability, HaCaT cells were immu-

nostained with an antibody recognizing acetylated a-tubu-

lin. This antibody strongly recognized the microtubules that

were present in the extended part of the E-lam while those at

the lamellar tips were less pronounced (Fig. 2D). This

staining pattern suggests that the subset of microtubules

that is present in the stem of an E-lam may be more stable

than those that are present in lamella.

Actin filaments were not well organized and were prac-

tically undetectable in untreated control cells, as keratino-

cytes do not typically form prominent actin stress fibers (Fig.

2E) [5]. Treatment of keratinocytes with staurosporine in-

duced accumulation of F-actin in lamellipodia, and actin

became clearly visible in the stems of E-lams (Fig. 2F).

Cytokeratin 14 is a major component of keratin intermediate

filaments in basal epithelial keratinocytes and is also

expressed by keratinocytes in culture [35,36]. Extensive

networks of keratin filaments containing cytokeratin 14 were

present in both untreated and staurosporine-treated cells

except for the outer lamellipodia (Figs. 2G and H). Keratin

filaments were, however, concentrated in the stem of the E-

lam (Fig. 2H). Thus E-lam extension seemed to involve the

assembly of all major cytoskeletal components—actin fila-

ments, stable microtubules and intermediate filaments.

To test the importance of actin filaments and micro-

tubules for keratinocyte morphology and E-lam formation,

we treated the cells with a microtubule-destabilizing agent,

colchicine, and an F-actin-disrupting agent, cytochalasin D,

on fibronectin in the presence and absence of staurosporine.

Untreated and staurosporine-treated cells were differentially

sensitive to these agents. Control cells rounded up after

treatment with colchicine, whereas they were relatively

resistant to disruption of actin filaments and remained

spread (Fig. 3A). In contrast, cells treated with 50 nM

staurosporine were sensitive to cytochalasin D treatment

but resistant to the treatment with colchicine (Fig. 3A). The

results suggest that the spread phenotype of untreated

keratinocytes on fibronectin is dependent mainly on their

microtubule network, while staurosporine-treated were more

dependent on actin filaments. Both colchicine and cytocha-

lasin D totally prevented E-lam formation (Fig. 3B), indi-

cating that both actin and microtubule organization were

needed for E-lam formation.

Since E-lam formation depends on the activity of GSK-3

[4], we tested whether the inhibition of GSK-3 with a

specific inhibitor, SB-415286 [37], affects the staurospor-

ine-induced change in the cytoskeleton. SB-415286 alone

did not affect cell spreading on fibronectin, but strongly

inhibited colchicine-induced cell contraction to a rounded

morphology in control keratinocytes (Fig. 3C). SB-415286

also blocked staurosporine-induced sensitization of kerati-

nocytes to cytochalasin D (Fig. 3C). These observations

suggest that GSK-3 participates in the regulation of cyto-

skeleton in keratinocytes.

Colchicine (1 AM) totally breaks down the assembled

microtubule networks in cells impeding the interpretation of

the importance of microtubule assembly for E-lam forma-

tion [8]. Therefore, we studied the effects of low concen-

trations of taxol and nocodazole, which dampen microtubule

dynamics without disrupting the existing microtubule net-

works on E-lam formation [8]. In agreement with not

breaking down the existing microtubules, neither agent

affected the spread morphology of untreated control cells

(Figs. 4A and B). Both agents, however, caused a dose-

dependent, statistically significant inhibition in staurospor-

ine-induced E-lam formation (Figs. 4A and B), supporting

the importance of dynamic microtubule assembly for E-lam

formation.

E-lam formation requires normally regulated Rac1, whereas

the activities of RhoA and Cdc42 are suppressive

The Rho-family small GTPases RhoA, Rac1 and Cdc42

are involved in processes regulating cell shape such as actin

stress fiber formation, formation of lamellipodia and filo-

podia, respectively [10]. To explore the involvement of the

small GTPases Rac1, RhoA and Cdc42 in E-lam formation

in keratinocytes, HaCaT cells were transiently transfected

with plasmids expressing dominant-negative (RacN17,

RhoN19, Cdc42N17), wild-type (RacWT, RhoWT, Cdc42WT)


Fig. 4. The effect of nocodazole and taxol in E-lam formation. HaCaT cells

were allowed to spread on fibronectin for 120 min and were then treated

with nocodazole (A; 0–200 nM) or with taxol (B; 0–100 nM) in the

presence or absence of 50 nM staurosporine for 60 min. The cells were

fixed, and four representative fields in triplicate wells were examined by

phase-contrast microscopy using a 20� objective (n = 12). The percentage

of cells that were spread or forming E-lams within each field was calculated

(mean F SD). (*P < 0.05; **P < 0.01).

Fig. 3. The role of microtubules and actin in E-lam formation. HaCaT cells

were allowed to spread on fibronectin for 120 min and were then treated

with 1 AM cytochalasin D (CC-D) or with 1 AM colchicine in the presence

or absence of staurosporine (0, 10 or 50 nM) for 60 min. The cells were

fixed, and four representative fields in triplicate wells were examined by

phase-contrast microscopy using a 20� objective (n = 12). The percentage

of cells that were spread (A) or forming E-lams (B) within each field was

calculated (meanF SD). Control cells were treated with the vehicle for CC-

D or colchicine (0.05% ethanol). (C) The experiment was performed and

quantitated as in A, except that the cells were treated with 1 AMcytochalasin D or with 1 AM colchicine in the presence or absence of

staurosporine (STP; 50 nM), SB-414286 (SB; 30 AM) or a combination of

staurosporine and SB-415286 (STP + SB).


and constitutively active (RacQL, RhoQL, Cdc42QL) forms of

these GTPases, and allowed to spread in the presence or

absence of staurosporine on fibronectin. The morphology of

successfully transfected cells was compared to that of

negative, non-transfected cells within the same sample.

None of the transfected or control cells formed E-lams

without treatment with staurosporine (data not shown).

Fig. 2. Immunolocalization of cytoskeleton in E-lam formation. Cells were allo

staurosporine or left untreated for 60 min. To immunolocalize the major cytoskeleta

(A, C, E, G) were immunostained for h-tubulin (A, B), acetylated a-tubulin (C

lamellipodia and the presence of cytoskeletal components in the stem of the E-lam

cell shape, partial outlines of cells are marked with dashed lines. Enlarged details

The cells transfected with RacWT, RhoN19 and Cdc42N17

and treated with 50 nM staurosporine behaved similarly to

the non-transfected cells and showed equivalent cell spread-

ing and E-lam formation (Figs. 5A–C). Cells transfected

with any of the other six constructs and treated with

staurosporine failed to form E-lams (Figs. 5B and C).

Staurosporine-treated cells expressing RacQL spread equally

well as the non-transfected cells, whereas the spreading of

cells that were left untreated was reduced by about 75%

(Fig. 5A). Transfection with RacN17 reduced the spreading

of both staurosporine-treated and untreated cells by 70–

80% (Fig. 5A). The cells expressing either RacQL or RacN17

and treated with staurosporine were nonpolar and lacked E-

lams; RacQL-expressing cells were surrounded by lamellar

cytoplasm, whereas RacN17-expressing cells showed long,

hair-like cellular extensions consistent with Cdc42-induced

filopodia (Fig. 5C, panels c, g and h). These results suggest

that the activities of these two GTPases might be mutually

exclusive in keratinocytes. The expression level of Rac1 did

wed to spread on fibronectin for 120 min and then treated with 50 nM

l components, the cells with (B, D, F, H) or without staurosporine treatment

, D), actin (E, F) and cytokeratin 14 (G, H). The accumulated F-actin in

are marked with arrowheads and arrows, respectively. To help visualize the

of lamellar edges are shown in the insets. Scale bar = 10 Am.

Fig. 5. The role of GTPases in E-lam formation. Cells were transiently transfected with plasmid constructs expressing wild type (WT), constitutively active

(QL) and dominant-negative (N) forms of Rac1, RhoA and Cdc42 GTPases. The cells were allowed to spread on fibronectin in the presence or absence of

staurosporine, immunostained with Cy3-conjugated anti-VSV antibody to identify the transfected cells, and analyzed for cell spreading (A), E-lam formation

(B) and cellular morphology (C). The percentage of VSV-positive cells that were spread and had formed E-lams was determined and compared to the

percentage for the negative cells on the same coverslip. Mean F SD of the ratios of these percentages is presented (n = 16). No difference between VSV-

positive and -negative cells would be calculated as 1. (C) The typical morphology of staurosporine-treated transfected cells expressing wild type (a, d, g),

constitutively active (b, e, h) and dominant-negative (c, f, i) forms of Rac1 (a–c), RhoA (d– f) and Cdc42 (g– i) GTPases on fibronectin.


not seem to be critical for the process of cell spreading and

E-lam formation. Overexpression of RacWT, however, in-

duced branched E-lams (Fig. 5C, panel a). The expression

levels of RhoA and Cdc42 appeared to be more critical,

since overexpression of these two exogenous wild-type

GTPases was detrimental to cell spreading and E-lam

formation. We conclude that Rac1 activity that is under

normal cellular control is needed for keratinocyte cell

spreading and E-lam formation, whereas activities of RhoA

and Cdc42 are suppressive.

Staurosporine can also inhibit Rho-associated kinase

(Rho-kinase), a downstream effector of RhoA [38,39].

When the Rho-kinase inhibitor Y-27632 was added to the

cells together with staurosporine, the numbers of E-lams

were strongly enhanced (data not shown). These results

support the notion that RhoA activity is suppressive for E-

lam formation.

Staurosporine-induced membrane ruffling and the

accumulation of Rac1 to the cell periphery can be

prevented by inhibition of GSK-3

Next, we examined the localization of several molecules

that are involved in or regulate F-actin assembly, namely,

cortactin, filamin, Arp2/3 complex and Rac1 GTPase [40–

42], in E-lams, and how the inhibition of GSK-3 affects the

cellular localization of these molecules. Additionally, we

analyzed whether the localization of tyrosine-phosphorylat-

ed and presumably activated phospholipase C-g1 (p-PLC-

g1), which we have previously shown to colocalize with the


sites of F-actin in E-lams [4], is affected by the inhibition of

GSK-3. F-actin, cortactin, Arp2/3 complex, Rac1 and p-

PLC-g1 did not localize to the cell periphery in control cells

(Figs. 6A, 6E, 6M and 7A, 7E), but some positive staining

for filamin was detected in lamellipodia of control cells (Fig.

6I). Staurosporine induced the accumulation of F-actin,

cortactin, filamin, Rac1 and p-PLC-g1 to the lamellipodial

ruffles (Figs. 6B, 6F, 6J and 7B, 7F). Arp3 staining was

punctate, but it was also enriched in peripheral regions of E-

lams (Fig. 6N). Treatment of HaCaT cells with the GSK-3

Fig. 6. The effect of GSK-3 inhibition on actin assembly. To determine the effect

cells were allowed to spread on fibronectin for 120 min and then treated with

combination of both agents (D, H, I, P), or left untreated (A, E, I, M) for 60 min. Th

G, H), filamin (I, J, K, L) or Arp3 (M, N, O, P). Enlarged details of lamellar edg

inhibitor SB-415286 did not alter the localization of any of

these molecules compared to control cells (Figs. 6C, 6G,

6K, 6O and 7C, 7G). In the presence of SB-415286, E-lam

formation by staurosporine was blocked. Interestingly, how-

ever, the cells expressed hair-like filopodia that contained

actin, cortactin, filamin and p-PLC-g1 (Figs. 6D, 6H, 6L

and 7H). Significantly and in agreement with the importance

of Rac1 in E-lam formation, SB-415286 prevented staur-

osporine-induced accumulation of Rac1 and Arp2/3 com-

plex into lamellipodia (Figs. 7D and 6P, respectively). Thus,

of GSK-3 inhibition on staurosporine-induced actin filament assembly, the

50 nM staurosporine (B, F, J, N), 30 AM SB-425286 (C, G, K, O) or a

e cells were fixed and immunostained for actin (A, B, C, D), cortactin (E, F,

es are shown in the insets. Scale bar = 10 Am.

Fig. 7. The effect of GSK-3 inhibition on localization of Rac1 and p-PLC-g1. HaCaT cells were allowed to spread on fibronectin for 120 min and then treated

with 50 nM staurosporine (B, F), 30 AM SB-425286 (C, G) or a combination of both agents (D, H), or left untreated (A, E) for 60 min. The cells were fixed and

immunostained for Rac1 (A, B, C, D) or tyrosine-phosphorylated PLC-g1 (E, F, G, H). Enlarged details of lamellar edges are shown in the insets. Scale bar =

10 Am.

Fig. 8. The effect of inhibitory anti-integrin antibodies on E-lam formation.

HaCaT cells were allowed to spread on fibronectin and then treated with

inhibitory antibodies against a5, h1, av and h6 integrins in the presence of

50 nM staurosporine. The percentage of spread cells (A) or cells forming E-

lams (B) of total cell number (mean F SD, n = 8) was calculated. The

statistical significance of the inhibition in cell spreading and E-lam

formation by anti-integrin antibodies compared to cells treated with

staurosporine only was also calculated (*P < 0.05; **P < 0.01).


inhibition of GSK-3 specifically prevented Rac1 localiza-

tion to lamellipodia and its action in F-actin assembly that is

required for E-lam formation, yet still allowed the formation

of filopodia, which are presumably Cdc42-mediated. These

data collectively suggest that GSK-3 could function up-

stream of Rac1 in E-lam formation. To confirm this novel

finding, Rac1 was also prevented from localizing to lamel-

lipodia by LiCl2 (data not shown), another GSK-3 inhibitor

that blocks E-lam formation [4,43].

E-lam formation is integrin-mediated

Cell–matrix interactions were found to contribute to the

staurosporine-induced morphology, since staurosporine-in-

duced cellular extensions on collagen types I and IV were

shorter, branched and surrounded the whole cell perimeter

instead of being long, straight and directional on fibronectin

(data not shown). We have previously shown that human

keratinocytes express a5h1, avh6 and avh1 fibronectin

receptor integrins in culture and that function-blocking

antibodies against these integrins differentially inhibit cell

spreading on fibronectin [26]. To investigate whether staur-

osporine-stimulated and GSK-3-mediated E-lam formation

on fibronectin was mediated by a specific fibronectin-bind-

ing integrin, spreading assays were conducted in presence

of blocking antibodies against these integrins. To differen-

tiate the inhibitory effect of these antibodies on E-lam

formation from their effect on cell spreading, the antibodies

were added together with staurosporine to cells that had


already spread. In this experimental model, staurosporine

caused a slight but statistically significant increase in the

proportion of spread cells compared to untreated control cells

(Fig. 8A). Anti-a5 integrin antibody did not affect cell

spreading, whereas the addition of antibodies against av

and h6 integrins restrained cell spreading to control levels

(Fig. 8A). The antibody against h1 integrin subunit reduced

cell spreading by 35% (n = 8, P < 0.01) compared to cells

treated with staurosporine only (Fig. 8A). The combination

of anti-av and h1 integrin antibodies that is capable of

blocking all fibronectin-binding integrins in keratinocytes

caused a loss of cell spreading and rounding up of the cells

(Fig. 8A). The combination of anti-a5 and h6 integrin

antibodies aimed at revealing the remaining adhesive func-

tion due to the avh1 integrin [26] produced an effect similar

to anti-h1 integrin antibody (Fig. 8A). None of the inhibitoryantibodies alone was able to inhibit extended lamellipodia

formation in a statistically significant manner, whereas the

combination of anti-av and h1 integrin antibodies complete-

ly blocked E-lam formation (Fig. 8B). These findings

suggest that while E-lam formation obviously is integrin-

mediated, it is not mediated by any single specific receptor,

but that all of the fibronectin-binding integrins can inter-

changeably contribute to this phenomenon.

Discussion

We have shown previously that the activity of GSK-3 is

required for the formation of long lamellipodia and for cell

migration in keratinocytes [4]. In the present study, we

demonstrated a novel action for GSK-3 in the regulation

of the cytoskeleton and Rac1 translocation during E-lam

formation in cultured human keratinocytes.

HaCaT cells immunostained with anti-h-tubulin anti-

body showed long microtubule bundles extending through

the E-lam. The elongated part of the E-lam also consisted

of actin and keratin intermediate filaments. The presence

of keratin in the stem of the E-lam together with acety-

lated a-tubulin suggests that this extension may be a

stabilized structure. Staurosporine also induced accumula-

tion of prominent actin filaments to the tips of protruding

lamella. Indeed, E-lam formation required both microtu-

bule and F-actin assembly. E-lam formation was complete-

ly blocked by either colchicine or cytochalasin D, agents

that disrupt microtubules and actin, respectively. In addi-

tion, E-lam formation was inhibited by taxol and nocoda-

zole that prevent dynamic microtubule assembly without

breaking down existing microtubules [8]. Previous studies

have suggested that a factor, possibly Rac1, associated

with microtubules can be released and affects the assem-

bly of F-actin in lamellipodia formation [8]. Rac1 induces

cortical actin polymerization that results in membrane

ruffling and lamellipodia formation [44]. Notably, Rac1

showed a similar distribution as F-actin in staurosporine-

induced E-lams.

The formation of E-lams required Rac1 that was under

normal cellular regulation, that is, Rac1 apparently needs to

be activated and deactivated in a spatiotemporally controlled

fashion to allow the cell to acquire a polarized morphology.

Expression of either the dominant-negative or constitutively

active form of Rac1 caused loss of E-lams. In contrast,

overexpression of either Cdc42 or RhoA prevented E-lam

formation, but cells formed E-lams when they expressed

dominant-negative forms of these GTPases.

In its activated form, Rac1 binds tubulin directly in vitro

and colocalizes with microtubules in vivo, whereas tubulin

does not bind Cdc42 or RhoA [45]. In fibroblasts, Rac1

signaling depends on microtubule assembly, and polymer-

ization of microtubules is required for Rac1 activation [9].

On the other hand, Rac1 and/or Cdc42 may be able to

stabilize microtubules locally [45,46]. Activated Rac1 may

then be localized to the cell membrane where its activity

leads to de novo assembly of actin filaments and also

promotes microtubule growth into leading-edge protrusions

[9,47]. The cellular translocation of Rac1 from cytosol to

cytoskeleton and to cell membrane is likely a key event in

Rac1 function, and tubulin binding may permit translocation

along the microtubule network. Disruption of microtubules

by colchicine prevents Rac1-mediated membrane ruffling

[45]. In the present study, we showed that inhibition of

GSK-3, which specifically mediates E-lam formation in

keratinocytes [4], prevented Rac1 translocation to the cell

membrane, accumulation of Arp2/3 complex and F-actin

assembly. GSK-3 inhibition did not affect the staurosporine-

induced accumulation of various other molecules present in

E-lams, namely, cortactin, filamin and tyrosine-phosphory-

lated PLC-g1. GSK-3 regulates kinesin-based motility and

membrane-bound organelle release along stable microtu-

bules [48–50], providing one potential pathway by which

GSK-3 could regulate Rac1 translocation in keratinocytes.

GSK-3 binds to microtubules in vitro and in vivo and it

aligns with microtubules both in neuronal cells and in

fibroblasts [50,51]. We have previously shown that in

staurosporine-induced E-lams, GSK-3 is localized to the

stem of the E-lam [4]. As shown in the present report, the

stem of the E-lam is an area of extensive microtubule

elongation, bundling and acetylation indicating increased

microtubule stabilization. Stable microtubules may be im-

portant mediators of differentiative events since they are

typically found aligned with developing asymmetries in

cells undergoing morphogenesis [52–54]. Increased stabili-

zation of microtubules by staurosporine has been previously

reported in neuronal cells, but tubulin was excluded as a

direct cellular target for staurosporine action [55,56].

Protein phosphatase 1 (PP1) binds to microtubules and is

involved in the maintenance of stable microtubules both in

fibroblasts and in kidney epithelial cells [56,57]. Based on

our unpublished results, PP1 is also involved in E-lam

formation in HaCaT cells. E-lam formation was totally

blocked by 2 nM calyculin A or by 500 nM okadaic acid.

This is in agreement with published IC50 values of calyculin


A (2 nM) and okadaic acid (60–500 nM) for PP1 [58].

Interestingly, GSK-3 is an activator of PP1, and conversely,

PP1 may contribute to GSK-3 activation [59–61].

Cdc42 has been shown to induce filopodia that require

actin polymerization [12]. Our results suggest that in kera-

tinocytes, Rac1 and Cdc42 compete with each other. The

expression of dominant-negative RacN17, activated Cdc42QL

and to lesser extent Cdc42WT by transfection resulted in

Cdc42-mediated filopodia formation and abolishment of

lamellipodia, whereas expression of RacWT, activated RacQL

and dominant-negative Cdc42N17 led to Rac1-mediated

lamellipodia formation. The overexpression of either Rac1

or Cdc42 may disturb the internal balance between these

GTPases leading to either lamellipodia or filopodia forma-

tion, respectively. Rho-family GTPases mediate the forma-

tion of dynamic actin structures through different WASP

(Wiscott-Aldrich syndrome protein) family proteins, and

these pathways intersect at the Arp2/3 complex [62,63].

The Arp2/3 complex nucleates actin filaments at cellular

margins. Proteins of the WASP-family activate Arp2/3 and

provide a potential link between Cdc42 (through N-WASP)

and Rac1 (through Scar/WAVE) and the actin cytoskeleton.

Thus, one potential converging point where Rac1 and

Cdc42 can functionally compete with each other is at the

actin branching point where Arp2/3 nucleates filament

growth. Interestingly, the inhibition of GSK-3 appeared to

shift the balance towards Cdc42-mediated filopodia forma-

tion, presumably by preventing the function of Rac1.

Recently, Cdc42-mediated signaling was shown to lead to

inhibition of GSK-3 activity [64], providing an alternative

mechanism for Cdc42-mediated inhibition of Rac1 function.

RhoA can inhibit Rac1 activation via the function of

Rho-kinase, and the inhibition of Rho-kinase leads to the

activation of Rac1 and/or Cdc42 [15,65]. In agreement with

these previous findings, our results suggest that RhoA

activity inhibits cell spreading, and that E-lam formation

is favored by suppression of RhoA and Rho-kinase. While

E-lam formation was enhanced by the inhibition of Rho-

kinase, it was not, however, caused simply by eliminating

RhoA function, because cells transfected with the dominant-

negative form of RhoA or treated with Rho-kinase inhibitor

did not form E-lams in the absence of staurosporine.

During lamella formation, the protruding lamellipodium

that is pushed out by the polymerizing actin becomes

anchored to extracellular matrix by integrins. Integrin clus-

tering seems to be required for spatial regulation of lamelli-

podium extension and stability [66]. Integrin clustering to

focal complexes is controlled by Rac1 and/or Cdc42

GTPases, and conversely, integrin-mediated adhesion regu-

lates the activities of Rho-family GTPases [16,67–69].

Integrin engagement with the matrix leads to reorganization

of the cytoskeleton, and it is required for changes in cell

morphology. Staurosporine-induced E-lam formation was

matrix- and integrin-dependent, but was not restricted to one

specific integrin, as none of the inhibitory anti-integrin

antibodies alone could prevent E-lam formation. E-lam

formation was, however, prevented by a combination of

antibodies that blocked all of the fibronectin-binding integ-

rins in HaCaT cells. Thus, in E-lam formation, HaCaT cells

could adaptively switch from one receptor to another, as we

have previously shown for cell spreading and migration on

fibronectin [26]. While the avh6 integrin is the dominant

fibronectin receptor mediating cell spreading in HaCaT cells

[26], blocking the function of this integrin showed the

weakest inhibition of E-lam formation. Interestingly, the

avh1 integrin was strongly effective in mediating E-lam

formation on fibronectin. We have previously shown that

avh1 integrin does not mediate HaCaT cell spreading on

fibronectin, but is involved in HaCaT cell migration on this

matrix [26]. This fact supports further the notion that

keratinocytes can utilize different fibronectin receptors for

different functions, and that the formation of E-lams may be

associated with cell migration [4,26].

In summary, E-lams are structurally distinct from classi-

cal lamellipodia that are associated with cell spreading on

fibronectin. The formation of extended cellular projections

in keratinocytes requires both actin and microtubule orga-

nization as well as engagement of fibronectin-binding

integrins with the matrix. Normally regulated Rac1 GTPase

and absence of RhoA and Cdc42 GTPase activation are

necessary for the organization of the cytoskeleton. We

propose that E-lam formation is controlled by Rac1 down-

stream of GSK-3.

Acknowledgments

This study was supported by grants from The Canadian

Institutes of Health Research and The Finnish Cultural

Foundation.

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