Hyperosmotic stress induces phosphorylation of cytosolic

phospholipase A2 in HaCaT cells by an epidermal growth factor

receptor-mediated process

Isabel Rodrıguez a, Marietta Kaszkin b, Andreas Holloschi c, Kirsten Kabsch a,Margarita M. Marques a, Xiaohong Mao a, Angel Alonso a,*

aDeutsches Krebsforschungszentrum, Im Neuenheimer Feld-242, Heidelberg 69120, GermanybPharmazentrum Frankfurt, University Hospital, Frankfurt/Main, Germany

cMannheim University of Applied Sciences, Mannheim, Germany

Received 22 December 2001; accepted 20 February 2002

Abstract

Cytosolic phospholipase A2 (cPLA2) is an enzyme involved in the formation of proinflammatory mediators by catalyzing the release of

arachidonic acid, thereby mediating eicosanoid biosynthesis. Using HaCaT keratinocytes as a model system, we present experimental

evidence that in these cells, cPLA2 is constitutively phosphorylated and that the degree of phosphorylation dramatically increases in cells

under hyperosmotic stress induced by sorbitol. In parallel, a rapid release of arachidonic acid followed by prostaglandin E2 formation was

detected. Elucidating the mechanism of cPLA2 upregulation, we observed that it is mediated via epidermal growth factor receptor (EGFR)

activation, since tyrphostin AG1478, a selective inhibitor of EGFR tyrosine kinase, completely inhibited cPLA2 phosphorylation.

Furthermore, addition of PD98059, which is an inhibitor of MEK1 activation, but not of SB203580, which is an inhibitor of p38 stress

kinase, inhibited cPLA2 phosphorylation, indicating that the ras–raf–MEK cascade is the major signalling pathway involved in cPLA2

phosphorylation. In addition, depletion of the cells from intracellular calcium does not prevent sorbitol-elicited cPLA2 phosphorylation,

suggesting that this process is independent of the presence of calcium. Together, our results demonstrate that hyperosmotic stress

phosphorylates cPLA2 in human keratinocytes by an EGFR-mediated process. D 2002 Elsevier Science Inc. All rights reserved.

Keywords: Phospholipids; Signal transduction; EGF; Growth factor receptors

1. Introduction

Human skin keratinocytes are cells that are confronted

with multiple external stress conditions. UV light, heat,

mechanical effects or osmotic pressure due to application

of drugs are some of these stress conditions to which the

human skin may be exposed. In particular, osmotically

active nonionic substances such as sorbitol or mannitol are

used as stabilizers or preservatives with antibacterial proper-

ties for drugs, which are applied topically to the skin as gels

containing antibiotics or glucocorticoids or as transdermal

therapeutic systems. In this respect, sorbitol is thought to be

metabolically inactive and thus to be an inert compound.

However, sorbitol by itself may exert hyperosmotic stress

effects in the skin, which might lead to inflammation-like

reactions and changes in the epidermal homeostasis. Little

information is available about the mechanisms by which

epidermal keratinocytes respond to such stress conditions.

So far, it is known that sorbitol elevates intracellular calcium

concentrations in HaCaT keratinocytes, which may induce

considerable changes in signal transduction processes and

proliferation or differentiation [1].

One important parameter of proinflammatory processes

in the skin is the activation of cytosolic phospholipase A2

(cPLA2 or Group IV PLA2) [2,3]. cPLA2 has been shown to

hydrolyze preferentially those phospholipids containing

arachidonic acid at the sn-2 position, thereby releasing

arachidonic acid into the extracellular milieu. The functions

of the cPLA2 rely mainly in the production of arachidonic

0898-6568/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved.

PII: S0898 -6568 (02 )00031 -1

Abbreviations: cPLA2, cytosolic phospholipase A2; EGF, epidermal

growth factor; EGFR, epidermal growth factor receptor; GST–ATF2,

fusion proteins GST– transcription factor ATF2; MBP, myelin basic protein.* Corresponding author. Tel.: +49-6221-423-215; fax: +49-6221-424-

932.

E-mail address: A.Alonso@dkfz.de (A. Alonso).

www.elsevier.com/locate/cellsig

Cellular Signalling 14 (2002) 839–848

acid, a precursor of proinflammatory mediators like prosta-

glandins and leukotrienes. In addition, after acute keratino-

cyte damage, the 2-lysophospholipids produced during

phospholipid hydrolysis may be utilized to form platelet-

activating factor as another important inflammatory media-

tor [4]. Moreover, it seems now established that arachidonic

acid by itself is a signalling molecule [5–7].

Three different isoforms of cPLA2 have already been

described [8–10]. They possess different activation proper-

ties in terms of calcium requirement, with the 85-kDa form

(cPLA2-a) being the most analysed so far. Activation of

cPLA2 has been shown to be dependent on the cellular

location and phosphorylation of the protein at serine-505

[3,11,12]. After phosphorylation, cPLA2 translocates mainly

to perinuclear membranes and this translocation has been

shown in CHO cells to be dependent on calcium ions [13].

Nevertheless, cPLA2 activation is most probably a rather

complex phenomenon, since it has been reported that in

several cellular systems, an increase in calcium concentra-

tion is not necessary for translocation and that phosphor-

ylation is necessary but not sufficient for phospholipid

hydrolysis [14–16]. As kinases involved in cPLA2 phos-

phorylation at serine-505, the mitogen-activated protein

(MAP) kinases erk1/2 as well as p38 stress kinase have

been identified [11]. The kinase responsible for activation of

cPLA2 under defined conditions seems to be cell type- and

stimulus-dependent [17–20].

To study signal transduction mechanisms involved in

hyperosmotic stress reactions, we used HaCaT cells, an

immortalized, nonmalignized human skin keratinocyte cell

line that still retains the possibility to proliferate and to

differentiate [21]. We report experimental results showing

that hyperosmotic stress on HaCaT cells with nonionic

solutes such as sorbitol results in an enhanced phosphor-

ylation of the cPLA2 as well as arachidonic acid release and

PGE2 formation. cPLA2 phosphorylation is mediated by the

epidermal growth factor receptor (EGFR), which is acti-

vated after sorbitol treatment and which switches on the

ras–raf–MAP kinase cascade. This activation parallels

translocation of cPLA2 to perinuclear membranes, as

observed by immunofluorescence. Finally, we show that

sorbitol-mediated phosphorylation was not abolished when

the cells were depleted from intra- and extracellular calcium.

2. Materials and methods

2.1. Cell culture

HaCaT cells [21] were cultured routinely in Dulbecco’s

modified Eagle’s medium containing 10% FCS and pen-

icillin/streptomycin. For experiments, cells were transferred

to plastic dishes (3.5 cm in diameter) and grown until 80%

confluency. Then cells were serum-starved for 24–48 h

before any treatment. Hyperosmotic stress was produced by

adding sorbitol to 600 mM concentration in the culture

medium. Inhibitors used were PD98059 (10 AM), SB-

203580 (20 AM) and tyrphostin AG-1478 (1 AM; all from

Calbiochem, Schwalbach, Germany).

2.2. Immunoprecipitations

For immunoprecipitations, cells were grown as described

before, and treated with the corresponding inhibitors and

sorbitol for the time points indicated. After washing twice

with cold PBS, cells were extracted with RIPA buffer

containing protease and phosphatase inhibitors (Sigma,

Munchen, Germany) for 15 min on ice, scrapped into an

Eppendorf tube and passed through a Shredder column to

shear DNA (Qiagen, Hilden, Germany). RIPA buffer con-

tains 10 mM sodium phosphate buffer, pH 7.2, 2 mM

EDTA, 1% Triton X-100, 0.5% sodium deoxycholate and

0.1% SDS. An amount of 500 Ag of proteins was precipi-

tated with 4 Ag of cPLA2 antibody (sc-545; Santa Cruz,

Heidelberg, Germany) at 4 jC overnight. Immunoprecipi-

tation of EGFRs was performed using 200 Ag of protein

extracts in RIPA buffer, as described above, and 4 Ag of

antibody sc-03 (Santa Cruz). Protein Sepharose A or G was

added (50 Al) and incubated for further 120 min. After

centrifugation and washing, immunoprecipitates were sepa-

rated on 10% polyacrylamide gels. Proteins were blotted

onto PVDF membranes and immunoblotted with the same

antibody. Reactions were detected with the ECL system

(Amersham-Pharmacia, Freiburg, Germany).

Western blot analyses were also performed with anti-

bodies detecting the EGFR (sc-03; Santa Cruz), phospho-

tyrosine (4G10; Upstate Biotechnology, Biozol, Munich,

Germany) and with specific antibodies to the unphosphory-

lated or the phosphorylated forms of p42/44 and p38 MAP

kinases (New England Biolaboratories, Bad-Schwalbach,

Germany). All blots were controlled for equal loading.

2.3. Arachidonic acid release

HaCaT cells were cultured in plastic Petri dishes (3.5 cm

in diameter) as described above and were serum-starved for

24 h in DMEM containing 0.1% fatty acid-free bovine

serum albumin (BSA). Then cells were prelabelled for an

additional 24 h with 1-[14C]arachidonic acid (0.4 ACi/ml).

During this period, 95% of the labelled fatty acid was

incorporated into the cells. After washing the cells twice

with DMEM plus 0.1% BSA, cells were then incubated for

the indicated time points with sorbitol. Supernatants were

collected and, from 500-Al aliquots, free fatty acids were

extracted with 2 ml of ethyl acetate and 50 Al of 1 N HCl.

After extraction of the lipids, the organic phase was dried in

a vacuum concentrator. Then the lipids were dissolved in 50

Al of ethylacetate and separated by thin layer chromatog-

raphy on silica gel 60 plates (Merck, Darmstadt, Germany)

using the organic phase of the ethylacetate/isooctane/acetic

acid/water mixture (110/50/20/100 by volume) as a solvent

system. The detection and quantification of the separated 1-

I. Rodrıguez et al. / Cellular Signalling 14 (2002) 839–848840

[14C]arachidonic acid were performed with a phosphorim-

ager BAS 1500 from Fuji (Raytest, Straubenhardt).

2.4. Determination of prostaglandin E2 (PGE2)

PGE2 was measured in aliquots of the cell culture super-

natants by enzyme immunoabsorbance assays (EIA) accord-

ing to the manufacturer’s instructions (Biotrend, Koln,

Germany).

2.5. Cell fractionation

For cell fractionation, cells were washed twice with cold

PBS and then scrapped with a rubber policeman in hypo-

tonic buffer (50 mM Tris–HCl, pH 7.6, 5 mM MgCl2, 0.5

mM EGTA, protease and phosphatase inhibitors). Cells

were broken using a Dounce homogenizer and centrifuged

for 15 min at 2000� g. The sediment, comprising the nuclei

nucleus-associated membranes, was taken into RIPA buffer.

The supernatant was further centrifuged at 100,000� g for

120 min to separate the membrane fraction (sediment) from

the cytosol fraction (supernatant) in a TL-100 ultracentri-

fuge (Beckman, Munich, Germany).

2.6. Intracellular calcium depletion and determinations

To measure intracellular calcium concentrations, HaCaT

cells were incubated in HBSS medium with or without

calcium in the presence or absence of 0.5 mM EGTA and, in

parallel, treated for 15 min with 600 mM sorbitol. HBSS

contains 137 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 20

mM Hepes and 0.1% glucose, pH 7.4, and was supple-

mented or not with 1.8 mM CaCl2. As a positive control for

the detection of intracellular calcium ions, cells were incu-

bated with 300 nM ionomycin, which is known to release

calcium from intracellular stores. In another set of experi-

ments, cells were preincubated for 30 min with ionomycin

and EGTA in calcium-free HBSS in order to completely

deplete intracellular calcium stores and to chelate released

extracellular calcium. After this incubation period, cells

were treated without medium change with sorbitol or

ionomycin. Calcium concentrations were measured by load-

ing cells with 5 AM fura-2-AM for 30 min as described [22].

Fluorescence (510 nm emission) at alternating excitation

wavelengths (340 and 380 nm) was recorded and analysed

with Ion Vision Software (Improvision; Ion Vision, Heidel-

berg, Germany). For calibration, the method of Grynkiewicz

et al. [23] was used.

2.7. Immunofluorescence

To demonstrate cPLA2 by immunofluorescence, cells

were grown on coverslips until 80% confluency and then

starved as indicated before. Sorbitol was added to 600 mM

and, after 30 min of incubation, cells were fixed in fresh 4%

paraformaldehyde and treated with 0.1% Triton X-100 in

PBS for 10 min. The cPLA2 antibody was added at a

dilution of 1:250 and incubated for 60 min. After washing,

an Alexa-labelled secondary antibody was added and further

incubated for 1 h. After washing, cells were mounted in

50% glycerol. Pictures were taken with a Leica TCS

confocal microscope.

2.8. Statistical analysis

Data represent the means of four independent experi-

ments F S.E.M. (n = 6). Statistical analysis was performed

by Student’s t test to determine significant differences

among two groups. A probability < .05 was considered as

significant.

3. Results

3.1. Effect of sorbitol on cPLA2 phosphorylation

To analyse the effect of osmotic stress on activation of

the cPLA2 in human keratinocytes, HaCaT cells were

treated with 600 mM sorbitol, protein extracts were immu-

noprecipitated with an antibody to cPLA2 and immunoblot-

ted with the same antibody. Activation of cPLA2 takes place

mainly by serine phosphorylation of residue serine-505 and

subsequent translocation to the cellular membranes [12].

This phosphorylation results in a migration shift of the

nonphosphorylated to the phosphorylated forms of the

enzyme during electrophoresis [11]. Western blot analysis

shows that in untreated cells, two bands representing the

unphosphorylated and the phosphorylated forms can already

be observed (Fig. 1A). The intensity of both bands is

similar, with slight variations between different experiments

in a total of eight experiments, indicating that about half of

the enzyme is already in the phosphorylated form in

unstimulated cells. Treatment with sorbitol for 10 min

markedly increased the amount of the upper, slower-migrat-

ing band, indicating that sorbitol was able strongly to

increase phosphorylation of the enzyme early after addition.

Similar results were also obtained with mannitol as another

osmotically active substance (data not shown).

As a positive control, cells were treated for 10 min with

EGF, which is known to activate cPLA2 in several cell types

[24]. As shown in Fig. 1A, EGF treatment results in a

complete shift from the lower, nonphosphorylated band to

the upper, phosphorylated band. From these experiments,

we concluded that hyperosmotic stress induced by sorbitol

produces a strong increase in the phosphorylation of cPLA2

in human keratinocytes.

3.2. Effect of sorbitol on EGFR tyrosine phosphorylation

To investigate the possible signalling mechanisms by

which sorbitol may mediate phosphorylation of cPLA2, we

designed the following series of experiments.

I. Rodrıguez et al. / Cellular Signalling 14 (2002) 839–848 841

In HeLa cells, treatment with hyperosmolar concentra-

tions of sorbitol has been shown to increase EGFR tyrosine

phosphorylation [25], which is an indication for EGFR

activation. To analyse whether this effect was also present

in human keratinocytes, HaCaT cells were incubated for

different time periods with 600 mM sorbitol and then

tyrosine phosphorylation of the EGFR was analysed. Sorbi-

tol stimulated a strong tyrosine phosphorylation and thus

activation of the receptor in contrast to untreated cells. The

Western blot in Fig. 1B shows EGFR phosphorylation after

10 (Lane 1) and 30 min (Lane 3). This phosphorylation

holds for at least 120 min, the latest time point we analysed

in our experiments (data not shown). Thus, our results

demonstrate that in humans, keratinocytes treatment with

hyperosmolar concentrations of sorbitol produces a strong

tyrosine phosphorylation of EGFR and that this response is

very rapid.

3.3. Sorbitol-stimulated cPLA2 phosphorylation is depend-

ent on EGFR activation

Unstimulated HaCaT cells show a weak constitutive

phosphorylation of cPLA2 (Fig. 1A), which is concomitant

with a low basal EGFR phosphorylation (Fig. 1B, Lane 2).

Since a marked increase in EGFR activation was already

detectable after 10 min of sorbitol treatment (Fig. 1B, Lane

1), we postulated that cPLA2 in human keratinocytes is

activated under hyperosmotic stress through an EGFR

kinase-mediated signalling pathway.

To inhibit EGFR kinase activity, HaCaT cells were pre-

treated with tyrphostin AG1478 (1 AM), a selective inhibitor

of the EGFR kinase [26], for 30 min before addition of

sorbitol for 10 min, and then phosphorylation of cPLA2 by

band shift analysis was investigated.

In Fig. 1C, it is shown that treatment of the cells with

AG1478 alone had no effect on the constitutive phosphor-

ylation state of cPLA2. Furthermore, addition of sorbitol to

the cells resulted in a strong band shifting. When AG1478

was added to sorbitol-treated cells, a strong reduction in the

slow-migrating band was observed, indicating that the

tyrphostin was able to inhibit phospholipase phosphoryla-

tion (Fig. 1C). These results, therefore, demonstrate that

AG1478 treatment inhibits sorbitol-induced cPLA2 phos-

phorylation in HaCaT cells due to an inhibition of EGFR-

mediated phosphorylation. As a positive control, we inves-

tigated the effect of AG1478 on the EGF-stimulated cPLA2

phosphorylation. EGF increased the amount of phosphory-

lated cPLA2 and this increase was inhibited with AG1478

(Fig. 1C).

3.4. Effect of sorbitol on the activation of MAP kinases erk1/

2 and stress kinase p38 in HaCaT cells

The experiments shown above demonstrate that hyper-

osmotic stress with sorbitol increases EGFR activation.

Thus, we investigated whether this activation results in

switching on the ras–raf–erk kinase cascade, thereby even-

tually leading to the activation of cPLA2. It has been shown

earlier that cPLA2 can be phosphorylated by a MAP kinase-

dependent mechanism [3,27].

In a first step, HaCaT cells were treated for 10 min with

600 mM sorbitol and Western blot analysis of protein

extracts was performed with antibodies recognizing specif-

ically the dual phosphorylated form of erk1/2 kinases. As

shown in Fig. 2A (upper panel, Lane 1), a clear increase in

p42/44 phosphorylation was observed, whereas the p42

phosphorylation was much more pronounced in these cells.

These data indicate that sorbitol treatment activated the

erk1/2 cascade. We further analysed if the same treatment

was able to increase p38 phosphorylation, a stress kinase not

Fig. 1. (A) Effect of sorbitol on the phosphorylation of cPLA2 in HaCaT

cells. HaCaT cells were cultured as described in Materials and Methods,

serum-starved for 24 h and 600 mM sorbitol or 25 ng/ml EGF was added

for 10 min. An amount of 500 Ag of cellular protein extracts was

immunoprecipitated with a cPLA2 antibody. Immunoprecipitates were

separated on polyacrylamide gels, blotted onto PVDF membranes and

Western blot analysis was performed using the same antibody as for

immunoprecipitation. Blots were developed using the ECL system of

Amersham. The data are representative of six independent experiments with

comparable results. (B) Activation of the EGF receptor in sorbitol-treated

HaCaT cells. Cells were cultured as described above and treated with

sorbitol for 10 (Lane 1) or 30 min (Lane 3) or left untreated (Lane 2).

Protein extracts were immunoprecipitated with an antibody to the EGF

receptor. Immunoprecipitates were separated, blotted and reacted with

antibody 4G10, specific for phosphotyrosine. (C) HaCaT cells were

pretreated with tyrphostin AG1478 (1 AM) for 30 min and then sorbitol was

added or not and incubation was continued for further 10 min. In parallel

experiments, cells were treated with EGF for 10 min in the absence or

presence of AG1478. Protein extracts were prepared and immunoprecipi-

tated with a cPLA2 antibody. Precipitates were separated on acrylamide gel

and Western blot analysis was performed with the same antibody. The data

are representative of four independent experiments with comparable results.

I. Rodrıguez et al. / Cellular Signalling 14 (2002) 839–848842

connected with the ras–raf cascade. Immunoblots with

antibodies specific for the phosphorylated form of p38

showed that sorbitol treatment resulted in a strong increase

in stress kinase activation (Fig. 2A, upper panel, Lane 3).

The bands in the lower panels represent the total amount of

p42/44 and p38 protein detected in the same Western blot.

These results were further confirmed by analysing the

enzymatic activity of immunoprecipitates of erk1/2 and p38

using myelin basic protein (MBP) or fusion proteins GST–

transcription factor ATF2 (GST–ATF2) as substrates (data

not shown).

We next analysed whether the time course of cPLA2

stimulation correlates with a stimulation of MAP kinase

erk1/2 after sorbitol treatment. Cells were incubated with

sorbitol for different periods of time and then protein

extracts were prepared and analysed for erk1/2 activation

and cPLA2 band shifting. As shown in Fig. 2B, phosphor-

ylation of erk1/2 increased continuously during the treat-

ment period, as revealed by immunoblotting with a specific

antibody for phospho-erk1/2. A similar stimulation kinetic

was observed for cPLA2 phosphorylation; the slow-migrat-

ing band, corresponding to the phosphorylated enzyme,

increased continuously during the time points analysed.

Thus, these results demonstrate that cPLA2 and erk1/2

phosphorylation show a similar kinetics, strongly suggesting

a relationship between both events.

3.5. Effect of MEK1 and p38 MAP kinase inhibition on

cPLA2 phosphorylation

Since we have shown that both the erk1/2 and the p38

MAP kinases were activated by sorbitol, we designed a

series of experiments to determine which kinase is involved

in activation of cPLA2 in HaCaT cells. To assess the effect

of stress kinase p38 or of MAP kinase erk1/2, we preincu-

bated HaCaT cells for 10 min with SB203580 (20 AM) or

PD98059 (10 AM), known to be selective inhibitors of p38

MAPK or MEK1 activation, respectively.

Cells were then treated for 10 min with sorbitol and

protein extracts were immunoprecipitated with the anti-

cPLA2 antibody. Western blot analysis was performed with

the same antibody. As shown in Fig. 2C, in untreated cells,

both forms of cPLA2, phosphorylated and nonphosphory-

lated, were observed. Treatment with SB203580 alone had

only a scarce effect on the constitutive enzyme phosphor-

ylation and was also not able to inhibit sorbitol-elicited

cPLA2 phosphorylation. In contrast, treatment with the

MEK1 inhibitor, PD98059, clearly inhibited the sorbitol-

mediated phosphorylation, demonstrating that sorbitol

increased cPLA2 phosphorylation mainly by a MEK-1-

driven mechanism. To demonstrate that this effect was

mediated by erk1/2 MAP kinases, we again treated HaCaT

cells with the inhibitor and protein extracts were analysed

Fig. 2. Effect of sorbitol on erk1/2 and p38 activation in HaCaT cells. (A) Cells were treated for 10 min with sorbitol (Lanes 1 and 3) or left untreated (Lanes

2 and 4). Protein extracts were separated by acrylamide gel electrophoresis and immunoblotted with antibodies recognizing the activated, phosphorylated

forms of MAP kinases p42/44 or p38 (upper panels). For showing equal protein loading on the gels, blots were stripped and were immunoblotted with

antibodies recognizing total p42/44 or p38 protein (lower panels). The data are representative of four independent experiments with comparable results. (B)

Time course of erk1/2 and cPLA2 phosphorylation. Cells were treated with sorbitol or EGF for the indicated time points; Western blot analyses for phospho-

erk1/2, unphosphorylated erk1/2 or cPLA2 were performed as described in the legends for Fig. 1A and (A). (C) Effect of inhibitors of erk1/2 and p38

activation on cPLA2 phosphorylation. HaCaT cells were treated for 10 min with SB203580 (20 AM), PD98059 (10 AM) with or without sorbitol (600 mM).

Protein extracts were immunoprecipitated and Western blot analysis was performed with the antibody to cPLA2. The data are representative of three

independent experiments with comparable results. (D) Effect of PD98059 on erk1/2 phosphorylation. Cells were preincubated for 10 min with PD98059 (10

AM) and then treated for 10 min with or without sorbitol. Western blot analyses for detection of phospho-erk1/2 or unphosphorylated erk1/2 were performed

as described in the legend for (A).

I. Rodrıguez et al. / Cellular Signalling 14 (2002) 839–848 843

for p42/44 activation. As shown in Fig. 2D, PD98059 had

no effect on erk1/2 activation, but was able almost com-

pletely to inhibit sorbitol-mediated erk1/2 phosphorylation.

In summary, these results show that cPLA2 phosphorylation

is driven by a MEK-1/erk1/2-mediated mechanism.

3.6. Effect of sorbitol on arachidonic acid release and PGE2

formation

We also analysed the effects of sorbitol on arachidonic

acid release and PGE2 formation by prelabelling HaCaT

cells for 24 h with 1-[14C]arachidonic acid and subsequent

treatment for different periods with sorbitol.

Sorbitol stimulated a rapid and significant release of

arachidonic acid, reaching a maximum at 10 min (Fig.

3A). This increase correlates with the activation of erk1/2

after 10 min of sorbitol treatment (Fig. 2A). The release of

arachidonic acid was followed by a significant increase in

PGE2 levels starting after 30 min (Fig. 3B). Together, these

data show that sorbitol treatment of HaCaT cells leads to an

activation of cPLA2 as was shown by arachidonic acid

release and PGE2 formation.

In parallel experiments with a time course of 4 h, we did

not observe a sorbitol-mediated increase in extracellular

phospholipase A2 activity, which was determined using 1-

[14C]-labelled Escherichia coli membranes [28], indicating

that sorbitol has no effect on secreted phospholipases A2 in

HaCaT cells (data not shown).

3.7. Effect of calcium depletion on cPLA2 phosphorylation

Activation of cPLA2 has been described to be the result

of an increased calcium concentration and phosphorylation

with concomitant membrane translocation of the enzyme.

Since a fraction of the enzyme is already constitutively

phosphorylated in HaCaT cells (see Fig. 1A), we decided

to analyse the role of calcium on cPLA2 phosphorylation

in sorbitol-treated cells. For this, cells were incubated in

calcium-containing medium and were treated with sorbitol.

As shown in Fig. 4A, sorbitol stimulated a rapid increase

in calcium followed by a delayed decrease to the basal

levels.

The next question was whether this calcium was of

extracellular origin or derives from intracellular stores. For

this, HaCaT cells were incubated for 15 min in medium

without calcium in the presence of 0.5 mM EGTA. Under

these conditions, HaCaT cells are depleted of calcium of

extracellular origin. In parallel, cells were treated with

sorbitol for 15 min. As is shown in Fig. 4B, sorbitol still

increases intracellular calcium in the absence of extracellular

calcium ions.

As a positive control for detection of intracellular cal-

cium, HaCaT cells were treated with ionomycin, which is

known to release calcium from intracellular stores. As shown

in Fig. 4D, in calcium-free and EGTA-containing medium,

ionomycin treatment produced a clear intracellular calcium

increase, which indicates that under the conditions used, the

measurement of intracellular calcium works properly.

Next, we performed a preincubation of HaCaT cells for

30 min with EGTA in calcium-free medium. By this

method, cells are triggered to empty their intracellular

stores, and all the released calcium ions are chelated in

the calcium-free medium by EGTA. This procedure induces

a depletion of the cells from intracellular calcium. Indeed,

under these conditions, an increase in intracellular calcium

by sorbitol was no longer observed (Fig. 4C). Similar data

were obtained with ionomycin treatment under the same

conditions (Fig. 4E).

Fig. 3. Effect of sorbitol on arachidonic acid release (A) and PGE2

formation (B) in HaCaT cells. (A) HaCaT cells were prelabelled for 24 h

with 1-[14C]arachidonic acid, and after medium change cells were treated

with 600 mM sorbitol or vehicle. Lipids were extracted from the

supernatants and separated by thin layer chromatography as described in

Materials and Methods. Arachidonic acid was quantified with a

phosphorimager. The data represent the means of two independent

experiments F S.D. (n= 6). The values are expressed as percent of controls

of the respective time points. * * *P < .001; *P < .05; Student’s t test. (B)

Cells were treated with 600 mM sorbitol or vehicle for the indicated

periods. Cell culture supernatants were collected and PGE2 was analysed as

described in Materials and Methods. Data represent the meansF S.D.

(n= 6) of a representative experiment. This experiment was repeated twice

with comparable results.

I. Rodrıguez et al. / Cellular Signalling 14 (2002) 839–848844

Together, these results clearly show that the sorbitol-

mediated increase in cytoplasmic calcium mainly derived

from intracellular stores.

We, therefore, took advantage of these results and

analysed cPLA2 phosphorylation in HaCaT cells in cal-

cium-free medium in the absence or presence of EGTA. As

shown in Fig. 5, a 30-min preincubation of the cells with

EGTA in calcium-free medium, which depletes the cells

from intracellular calcium (Fig. 4C), did not result in an

inhibition of sorbitol-mediated cPLA2 phosphorylation, i.e.,

the classical mobility shift for the phosphorylated form of

the enzyme was still detectable.

These results, therefore, strongly suggest that in HaCaT

cells, phosphorylation of the enzyme by sorbitol was not

dependent on an increase in intracellular calcium.

3.8. Subcellular localization of cPLA2 in cells treated with

sorbitol

It has been suggested that activation of the cPLA2 is the

result of phosphorylation and subsequent translocation of

cPLA2 to the cellular membranes. We, therefore, analysed

the distribution of the enzyme in subcellular fractions after

Fig. 4. Effect of sorbitol on calcium release in HaCaT cells. (A) Cells were incubated in calcium-containing HBSS and treated with 600 mM sorbitol. (B, D)

Cells were incubated in calcium-free HBSS containing 0.5 mM EGTA and, in parallel, they were treated with 600 mM sorbitol (B) or 300 nM ionomycin (D).

(C, E) Cells were preincubated for 30 min in calcium-free HBSS containing 0.5 mM EGTA to deplete the cells from intracellular calcium and to chelate

released extracellular calcium. After this incubation period, they were treated without medium change for 10 min with 600 mM sorbitol (C) or 300 nM

ionomycin (E). Intracellular calcium was monitored as described in Materials and Methods. The data are representative of four independent experiments with

comparable results.

Fig. 5. Analysis of cPLA2 phosphorylation by sorbitol under calcium-free

conditions. Protein extracts from cells incubated in calcium-free HBSS with

10-min sorbitol treatment or without sorbitol, and extracts from cells

incubated in calcium-free medium containing EGTA with or without 30-

min preincubation and 10-min sorbitol treatment were prepared, immuno-

precipitated with the cPLA2 antibody and immunoblotted with the same

antibody to demonstrate band shifting.

I. Rodrıguez et al. / Cellular Signalling 14 (2002) 839–848 845

sorbitol treatment by Western blot analysis. As shown in

Fig. 6A, in nontreated cells, most of the enzymes were

detected in the cytosol, with only small amounts in the

membranes and in the nuclear fraction. Treatment with

sorbitol resulted in a remarkable increase of the enzyme in

the fraction containing the nuclei, whereas no major changes

were observed in the cytosol and membrane fractions.

Maximal effects were observed after 30 min. This increase

must be due to a translocation of cPLA2 from the cytosol to

the nuclear fraction, since no major differences could be

observed in the membrane fractions before and after sorbitol

treatment (Fig. 6A).

To substantiate this point further, we performed indirect

immunofluorescence with the cPLA2 antibody to detect the

Fig. 6. Cellular localization of cPLA2 in HaCaT cells after sorbitol treatment. (A) Control and sorbitol-treated cells were homogenized and centrifuged at

5000� g for 15 min. The sediment was taken as the nuclear fraction. The supernatant was further centrifuged for 120 min at 100,000� g and proteins in

sediment (membranes) and supernatant (cytosol) were precipitated with the cPLA2 antibody as described in Materials and Methods. Reaction products were

detected with the ECL system. The data are representative of three independent experiments with comparable results. (B, C) Translocation of cPLA2 in HaCaT

cells treated with sorbitol as revealed by immunofluorescence. Cells were grown on coverslips and, after treatment with vehicle or sorbitol for 30 min, they

were fixed for 30 min as described in Materials and Methods. After incubation with the cPLA2 antibody (1:250 dilution in PBS), cells were washed and

incubated with a second antibody labelled with Alexa-418. (B) Control. (C) Sorbitol.

I. Rodrıguez et al. / Cellular Signalling 14 (2002) 839–848846

localization of the enzyme after sorbitol treatment. In

untreated cells, most of the fluorescence is observed as

regularly distributed in the cell (Fig. 6B), as already

described [13]. After sorbitol treatment, a large increase in

perinuclear fluorescence can be observed (Fig. 6C), thus

demonstrating that treatment with sorbitol results in enzyme

translocation to the membrane fraction located around the

nucleus. A similar translocation to the perinuclear mem-

branes has been already observed in basophilic leukemia

cells [29] or in CHO cells (see Refs. [30,31] for a review)

upon phospholipase activation.

4. Discussion

cPLA2 has been shown to be activated under different

conditions, like growth factors, phorbol esters, LPA, G-

protein-coupled receptors and a series of other different

stimuli. The main effect of cPLA2 is thought to be related to

the release of arachidonic acid, the rate-limiting step for the

biosynthesis of eicosanoids, which may be involved in

proinflammatory processes in the skin (for reviews, see

Refs. [2,32]). This study demonstrates that exposure of

HaCaT cells to hyperosmotic stress activates signalling

pathways, leading to upregulation of cPLA2 followed by

arachidonic acid release and PGE2 formation.

The sorbitol-stimulated increase in arachidonic acid

release exactly follows the time course of cPLA2 phosphor-

ylation, both reaching a maximum at 10 min and remaining

at a lower but elevated level, when compared to the control.

The formation of PGE2 occurred with a slight delay starting

at 30–60 min and increased continuously during the time

course of the experiment. We assume that this delay is due

to an induction of cyclooxygenase-2 (COX-2) at the tran-

scriptional level because the HaCaT cells used in our studies

do not express COX-2 under unstimulated conditions

(unpublished observations). It was already shown by other

stress stimuli, such as UV-B irradiation [33] or platelet-

activating factor treatment [34], that in HaCaT cells, COX-2

mRNA expression starts with a delay of 30–60 min

compared to arachidonic acid release.

Activation of cPLA2 has been postulated to be dependent

on two different steps: serine-505 phosphorylation, and

translocation of the enzyme to cellular membranes in a

calcium-dependent process [3,12,27]. However, it seems

that these conditions are not always necessary for activation

and that, depending on cell type and stimulus, the conditions

are different concerning calcium increase or serine phos-

phorylation. In P388D1 macrophages, phosphorylation

seems to be necessary but not sufficient [15,35], and in

liver macrophages, phosphorylation is not necessary at all

for activation [36].

Our results show that in HaCaT cells, a part of cPLA2 is

constitutively phosphorylated under unstimulated condi-

tions. Treatment with hyperosmolar concentrations of sor-

bitol resulted in an increased phosphorylation of the

enzyme, concomitant with its translocation to perinuclear

membranes. These results, therefore, suggest that in HaCaT

cells, both membrane translocation and phosphorylation are

necessary for enzyme activation.

Activation of cPLA2 has been shown to be dependent on

calcium, although in some cell types, it was reported that the

enzyme could be activated in the absence of calcium [15]. In

HaCaT cells, sorbitol treatment generates a rapid, transient

increase in the concentration of cytosolic calcium. These

data are in line with recently published results also showing

an increase in cytoplasmic calcium in HaCaT cells after

sorbitol shock [1]. Whether this rise in calcium was neces-

sary for cPLA2 phosphorylation was analysed by depleting

HaCaT cells of intra- and extracellular calcium ions and

analysing cPLA2 mobility shift by gel electrophoresis. Our

results demonstrated that protein mobility shift was not

dependent on the presence of calcium, indicating that

preincubation of the cells in medium devoid of calcium

and containing EGTA does not affect sorbitol-mediated

cPLA2 phosphorylation. An alternative explanation for our

results might be that cPLA2 is activated in a calcium-

independent manner through phosphatidylinositol bisphos-

phate (PIP2), as was recently described in P388D1 macro-

phages [37,38]. Whether PIP2 levels in sorbitol-treated

HaCaT cells are increased is so far unknown.

What kind of MAP kinase is necessary to phosphorylate

cPLA2 seems to be cell type- and stimulus-dependent. Our

experiments using inhibitors of MAP kinases erk1/2

(PD98059) or stress kinase p38 (SB203580) show that

human keratinocytes phosphorylate cPLA2 mainly by an

erk1/2-mediated mechanism. This is in contrast to other

cells types and other treatments, in which either both

enzymes or preferentially p38 MAP kinase is responsible

for serine-505 phosphorylation. In human neutrophils,

cPLA2 activation by FcgRIIA/IIIB is exclusively dependent

on erk1/2 activation [20,39], but in macrophages, it seems

that both p38 and erk1/2 are involved in cPLA2 phosphor-

ylation [40]. In human platelets, it has been described that

cPLA2 is activated by p38 stress kinase after sorbitol treat-

ment [41], which is in contrast to the results found in our

experiments. Thus, activation of cPLA2 seems to be depend-

ent on the cell type in addition to the stimulus used.

However, it should be taken into consideration that

SB203580 only inhibits p38a and p38h, thus making it

possible that the other two isoforms of the stress kinase may

also be involved in cPLA2 activation. In our case, this seems

rather unlikely, since treatment with the MEK1 inhibitor,

PD98058, efficiently inhibited cPLA2 phosphorylation.

Our experiments using the specific EGFR kinase inhib-

itor, AG1478 [26], show that this inhibitor was able to

impair sorbitol-stimulated cPLA2 phosphorylation (see Fig.

1C). These results, therefore, imply that the EGFR must be

involved in the sorbitol-mediated activation of the cPLA2.

We have shown that treatment of HaCaT cells with sorbitol

activates the EGFR, which indicates that this receptor might

be the main signal mediator of hyperosmotic stress. That

I. Rodrıguez et al. / Cellular Signalling 14 (2002) 839–848 847

these effects were not restricted to sorbitol was substantiated

by experiments showing that hyperosmolar concentrations

of mannitol elicited the same effects as described for

sorbitol (results not shown).

Thus, our results suggest a scenario where treatment of

human keratinocytes with hyperosmolar concentrations of

sorbitol results in increased activation of the EGFR. The

activated receptor switches on the ras–raf–MEK kinase

cascade and activates MAP kinases erk1/2. This activation

produces an increased cPLA2 phosphorylation, which can

be inhibited by PD98059, an inhibitor of MEK1 activation.

Interestingly, hyperosmotic stress also increases p38 activa-

tion, known to be responsible in several cell types for

phosphorylation of serine-505 of cPLA2 [40]. However,

treatment with SB28049, an inhibitor of p38a and p38hactivation, has only a weak effect on the phosphorylation

state of the enzyme. Thus, HaCaT cells under osmotic stress

modulate cPLA2 activation through an EGFR-mediated

mechanism and mainly independent of stress kinase p38

activation.

In summary, we present pieces of evidence that hyper-

osmotic stress by nonionic compounds, which are constit-

uents of drug preparations for the treatment of skin diseases

or which are used in transdermal therapeutic systems, may

per se have effects on metabolic processes in keratinocytes,

thereby changing the proliferation behaviour and mediator

production in the epidermis.

Acknowledgements

We thank J. Fischer for excellent technical work.

Margarita M. Marques has been supported by an

UNESCO/L’OREAL fellowship. We also thank Dr. H.

Spring for help with confocal microscopy.

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