Protective effects of lysophosphatidic acid (LPA) on chronic

ethanol-induced injuries to the cytoskeleton and on glucose uptake

in rat astrocytes

Monica Tomas,* Francisco Lazaro-Dieguez,� Juan M. Duran,� Pilar Marın,*

Jaime Renau-Piqueras* and Gustavo Egea�

*Centro de Investigacion, Hospital La Fe, Valencia

�Departament de Biologia Cellular i Anatomia Patologica, Facultat de Medicina, Institut d’Investigacions Biomediques August Pi

i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain

Abstract

Ethanol induces severe alterations in membrane trafficking in

hepatocytes and astrocytes, the molecular basis of which is

unclear. One of the main candidates is the cytoskeleton and the

molecular components that regulate its organization and

dynamics. Here, we examine the effect of chronic exposure to

ethanol on the organization and dynamics of actin and micro-

tubule cytoskeletons and glucose uptake in rat astrocytes.

Ethanol-treated cells cultured in either the presence or absence

of fetal calf serum showed a significant increase in 2-deoxy-

glucose uptake. Ethanol also caused alterations in actin

organization, consisting of the dissolution of stress fibres and

the appearance of circular filaments beneath the plasma

membrane. When lysophosphatidic acid (LPA), which is a

normal constituent of serum and a potent intercellular lipid

mediator with growth factor and actin rearrangement activities,

was added to ethanol-treated astrocytes cultured without fetal

calf serum, it induced the re-appearance of actin stress fibres

and the normalization of 2-deoxyglucose uptake. Furthermore,

ethanol also perturbed the microtubule dynamics, which

delayed the recovery of the normal microtubule organization

following removal of the microtubule-disrupting agent noco-

dazole. Again, pre-treatment with LPA prevented this alter-

ation. Ethanol-treated rodent fibroblast NIH3T3 cells that

constitutively express an activated Rho mutant protein (GTP-

bound form) were insensitive to ethanol, as they showed

no alteration either in actin stress-fibre organization or in

2-deoxyglucose uptake. We discuss the putative signalling

targets by which ethanol could alter the cytoskeleton and hex-

ose uptake and the cytoprotective effect of LPA against eth-

anol-induced damages. The latter opens the possibility that

LPA or a similar non-hydrolysable lipid derivative could be used

as a cytoprotective agent against the noxious effects of ethanol.

Keywords: actin, alcohol, glia, GLUT1, microtubules, Rho

GTPases.

J. Neurochem. (2003) 87, 220–229.

There is clinical and experimental evidence that ethanol

consumption disrupts development of the central nervous

system (CNS), leading to depression of neurogenesis and an

aberrant migration, which collectively contribute to the fetal

alcohol syndrome, an embryo pathology related to maternal

ethanol drinking that causes mental retardation in neonates

(Miller 1992; Streissguth et al. 1994; Eckardt et al. 1998).

As glial cells are also sensitive to ethanol, alterations in

neuron–glia interactions could account for the ethanol-

induced disruptions of brain development (Renau-Piqueras

et al. 1998; Guerri et al. 2001).

It has been clearly established that glycosylation machin-

ery is affected by chronic pre-natal ethanol exposure (Ghosh

et al. 1993; Lieber 1994; Renau-Piqueras et al. 1997; Ghosh

et al. 1998; Arndt 2001). Many glycosylated proteins and

lipids appear to mediate normal growth and tissue differen-

tiation during fetal development, and pre-natal ethanol

exposure severely alters glycosylation in hepatocytes and

astrocytes (Renau-Piqueras et al. 1987; Kim and Druse

Received April 24, 2003; revised manuscript received June 16, 2003;

accepted June 23, 2003.

Address correspondence and reprint requests to Gustavo Egea,

Departament de Biologia Cellular i Anatomia Patologica, Facultat de

Medicina, Universitat de Barcelona, C/Casanova 143, E-08036 Barce-

lona, Spain. E-mail: egea@medicina.ub.es

Abbreviations used: 2-DGlc, 2-deoxyglucose; FCS, fetal calf serum;

LPA, lysophosphatidic acid; LTB, latrunculin B; NZ, nocodazole; PKC,

protein kinase C; PKD, protein kinase D.

Journal of Neurochemistry, 2003, 87, 220–229 doi:10.1046/j.1471-4159.2003.01993.x

220 � 2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 220–229

1996; Renau-Piqueras et al. 1997; Minana et al. 2000;

Tomas et al. 2002). This alteration could impair the intra-

cellular transport of nascent glycoproteins and glycolipids.

Ethanol also reduces the expression and secretion of

neurotrophic-factor receptors, and the plasma membrane

levels of the highly sialylated form of the neural cell

adhesion molecule (PSA-NCAM) (Valles et al. 1994; Kim

and Druse 1996; Renau-Piqueras et al. 1997; Minana et al.

2000). Furthermore, chronic exposure to ethanol (30 mM)

enhances monosaccharide uptake and increases the protein

concentrations of GLUT1 (Tomas et al. 2002). However, the

mechanisms of these ethanol-induced changes in astrocytes

remain unknown.

In contrast to intermediate filaments (Renau-Piqueras et al.

1989), little is known about the effects of ethanol on

microtubule or actin cytoskeletons in glial cells. In fact, the

reported effects of ethanol in these cells could be thus

attributable to a primary alteration of the organization and

dynamics of the cytoskeleton. In this regard, chronic

exposure to ethanol alters actin cytoskeleton in astroglial

primary cultures from rat cerebral cortex (Allansson et al.

2001). This alteration might explain the ethanol-induced

increase in the uptake of 2-deoxyglucose (2-DGlc; Carver

et al. 1999; Tomas et al. 2002) as glucose uptake by

transport proteins (GLUTs) seems to be also dependent on

actin cytoskeleton (Bunn et al. 1999). It is important to

highlight that astrocytes are essential for neuronal glucose

metabolism as alterations to glucose transport in these cells

compromise neuronal function and brain development

(Wiesinger et al. 1997; Magistretti and Pellerin 1999).

Therefore, here we examine ethanol-induced alterations in

glucose uptake and cytoskeleton organization and dynamics.

Interestingly, these alterations are prevented by lysophos-

phatidic acid (LPA), which is a structurally simple phosp-

holipid and a potent intercellular lipid mediator present in

serum that elicits a broad spectrum of responses in diverse

cell types (Moolenaar 2000 for a review) including glial cell

survival and proliferation (Weiner and Chun 1999; Steiner

et al. 2002; Li et al. 2003) and actin rearrangements

(Ramakers and Moolenaar 1998).

Materials and methods

Reagents

Radiochemicals

2-Deoxy-D-[1–3H]glucose (specific activity 13.0 Ci/mmol) and

L-[35S]methionine (Met) (specific activity 40–500 Ci/mmol) were

from Amersham Pharmacia (Buckinghamshire, UK).

Antibodies

Anti-GLUT1 rabbit polyclonal antibody (Santa Cruz Biotechnology,

Santa Cruz. CA, USA), anti-actin mouse monoclonal antibody

(Sigma, St Louis, MO, USA), anti-b-tubulin and anti RhoA mouse

monoclonal antibodies (Santa Cruz), secondary Alexa-488 or Alexa-

546 F(ab¢)2 fragments were from Molecular Probes (Leyden, the

Netherlands).

Other reagents

Nocodazole and lysophosphatidic acid were from Sigma, latrun-

culin-B was purchased from Calbiochem (San Diego, CA, USA)

and TRITC-Phalloidin was from Molecular Probes. Culture reagents

were all from Invitrogen (Paisley, UK).

Primary cultures of rat astrocytes and NIH3T3 cells

Primary cultures of astrocytes from 21-day-old rat fetuses were

prepared from brain hemispheres as described elsewhere (Gomez-

Lechon et al. 1992). They were grown in a humidified atmosphere

of 5% CO2 and 95% air at 37�C. In these conditions the cells grew

rapidly for 7–10 days. The medium (Dulbecco’s modified Eagle’s

medium, DMEM) was changed every 2 days. We used astrocytes at

7 days because they release several neurotrophic factors and express

their receptor during proliferation (Lu et al. 1991; Valles et al.

1994). Cells were grown in the presence of ethanol, which was

added to the culture medium when the cells were settled (day 0).

Ethanol concentration in the medium was checked daily and

adjusted to a final concentration of 30 mM (ethanol evaporation after

24 h was 10–20%), which is similar to that reported in blood vessels

in pregnant chronic drinkers or when three to five alcoholic drinks

are consumed within 1 h by a woman weighing about 60 kg

(Eckardt et al. 1998). In some experiments, cells were exposed for

7 days to 50 or 100 mM ethanol. The purity of astrocyte cultures

was assessed by immunofluorescence using a mouse anti-glial

fibrillary acidic protein monoclonal antibody (Renau-Piqueras et al.

1989). All experiments using rats were approved by the appropriate

institutional review committee and performed in strict compliance

with the European Community Guide for the Care and Use of

Laboratory Animals.

NIH3T3 cells expressing either the GTP-bound form of Rho

(RhoAV14 mutant) or the empty vector (Mock cells) were generated

in the laboratory of J. C. Lacal (Jimenez et al. 1995). NIH3T3 cell

lines were all cultured in DMEM with 10% of FCS, but in the case

Mock and RhoV14 cell lines, geneticin (750 lg/mL) was present in

the culture medium.

Western blotting

NIH3T3 cells were washed in phosphate-buffered saline (PBS),

homogenized in extraction buffer (Tris-HCl 6 mM, EDTA 10 mM

and sodium dodecyl sulfate (SDS) 2%, pH 7.0) and incubated on ice

for 15 min. Cell lysates (20 lg per lane) were mixed with lithium

dodecyl sulfate (LDS sample buffer) and boiled for 3 min. Proteins

were separated in SDS polyacrylamide slab gels (15%). After

electrophoresis, the proteins were transferred to nitrocellulose paper,

which was incubated for 60 min with anti-RhoA monoclonal

antibody (1 : 1000). Membranes were washed in TBS-Tween and

then incubated with an alkaline phosphatase (AP)-conjugated goat

anti-mouse IgG (1 : 10 000). After 10–20 min of colour develop-

ment, the nitrocellulose sheets were washed and photographed.

Carbohydrate uptake

Astrocyte cultures were incubated with 2 mL of medium

containing [3H]2-DGlc and [35S]Met as previously reported

Effects of LPA on ethanol-induced injuries 221

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 220–229

(Tomas et al. 2002). The cells were maintained in this medium

for 5 min and incorporation was stopped by washing the cultures

three times in cold PBS. Supernatants and cells were collected

for radioactive counting. Cells were incubated overnight at 37�Cin the presence of 0.5 N NaOH and the amount of radioactivity

incorporated was measured in an automatic scintillation coun-

ter and, as the energy distribution spectra of the [3H] and [35S]

were superimposed, a two-channel counting program was used.

The dpm obtained in each channel was computed to obtain the

net dpm corresponding to each isotope. The results are expressed

as pmol/mg of protein. The protein concentration was deter-

mined by using the BCA protein assay kit (Pierce, Rockford, IL,

USA).

Fluorescence microscopy

Cultured cells were fixed with 4% formaldehyde in PBS for

30 min at room temperature, washed in PBS (3 · 5 min each)

and in PBS containing 50 mM ammonium chloride. Subsequently,

cells were permeabilized for 15 min with PBS containing 0.1%

saponin and 0.1% BSA. Actin stress fibres were visualized by

incubating cells for 45 min with 0.1 lg/mL of TRITC-labelled

phalloidin, as previously described (Valderrama et al. 1998). For

microtubules, cells were incubated first with a mouse anti-

b-tubulin monoclonal antibody (1 : 500) then with an anti-mouse

antibody conjugated with FITC. Finally, cells were rinsed several

times in PBS, mounted in Mowiol and observed in a fluorescence

microscope.

Statistical analysis

Data are presented as means ± SD. Differences between group

means were considered significant where P £ 0.05, as determined

by Student’s t-test.

Results

Ethanol increases the 2-deoxyglucose uptake

Recent data from our laboratories demonstrated that, upon

treatment with low concentrations of ethanol, the uptake of

labelled carbohydrates undergoes a significant increase

(Tomas et al. 2002). Here, we examined in detail the uptake

of [3H]2-deoxyglucose (2-DGlc), a derivative of glucose that

is not metabolized beyond phosphorylation, in untreated and

ethanol-treated astrocytes at growing stage (7 days in culture;

Renau-Piqueras et al. 1998). Astrocytes treated with ethanol

(30 mM) showed a significant increase in 2-DGlc uptake

(Fig. 1a). To determine whether this increase was dependent

on components contained in FCS, astrocytes were grown for

6 days in culture medium with FCS and one more day

without FCS (– FCS). Results showed that the basal 2-DGlc

uptake diminished both in control and ethanol-treated

astrocytes in comparison with cells cultured in the presence

of FCS (+ FCS) (Fig. 1a). Strikingly, the differences between

untreated and ethanol-treated cells remained significant, the

latter being more sensitive to FCS deprivation than the

former (Fig. 1a).

Microfilaments and microtubules are involved

in the uptake of 2-DGlc

Most cell lines cultured in the absence of FCS show a

perturbed cytoskeleton. Therefore, to examine whether the

ethanol-produced increase in 2-DGlc uptake depends on

the integrity of the cytoskeleton, astrocytes were treated

with latrunculin B (LTB; 1 lM, final concentration) or

nocodazole (NZ; 30 lM, final concentration), disrupters of

actin filaments and microtubules, respectively. LTB

increased 2-DGlc uptake at 10 and 20 min, but the effect

was reversed after 40 min (Fig. 2), most likely as a result

of a reduction in cell viability. NZ treatment also increased

2DGlc uptake, which was sustained throughout the

experiment (Fig. 2).

Lysophosphatidic acid quickly increases the 2-DGlc

uptake in ethanol-treated astrocytes cultured in the

absence of FCS

We reasoned that the FCS must contain a component, or

components, that is directly involved in the increase of

2-DGlc uptake in ethanol-exposed cells. One of the most

abundant constituents of serum is lysophosphatidic acid

(LPA; 1-acyl-sn-glycerol-3-phosphate), which mediates

Fig. 1 LPA treatment normalizes the ethanol-induced increase of

2-DGlc uptake. Histogram showing the uptake of [3H]2-DGlc mono-

saccharide by control and ethanol (30 mM)-exposed astrocytes grown

for 7 days in the presence of FCS (+ FCS) or for 6 days with FCS and

one more day without FCS (– FCS). (a) In cells cultured in the pres-

ence of FCS, ethanol increased the 2-DGlc uptake. In the absence of

FCS, the levels of 2-DGlc uptake were lower but the differences

between untreated and ethanol-treated cells remained. (b) When LPA

(200 ng/mL) was added for 15, 30 or 60 min to astrocytes cultured

without FCS, the 2-DGlc uptake rose to the same levels observed in

astrocytes cultured with FCS, and it occurred faster in ethanol-treated

than in untreated cells. The data shown are representative of three

independent experiments and expressed as the mean ± SD. Asterisks

indicate significant differences (Student’s t-test, p £ 0.05) of ethanol-

treated versus untreated cells.

222 M. Tomas et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 220–229

multiple biological responses (actin reorganization, survival,

amongst many others) via G protein-coupled serpentine

receptors (Moolenaar et al. 1997). Taking into account that

ethanol increases the protein levels of the glucose transporter

GLUT1 in growing astrocytes (Tomas et al. 2002), which is

the main transporter in these cells (Maher et al. 1994;

Morgello et al. 1995), and GLUT1 activity seems to be

dependent on actin dynamics (Zhang and Ismail-Beigi 1998;

Bunn et al. 1999), we therefore examined the effects of LPA

on the 2-DGlc uptake in astrocytes deprived of FCS the last

24 h (– FCS). LPA treatment (200 ng/mL) normalized faster

the 2-DGlc uptake in ethanol-treated than in untreated cells

(Fig. 1b), similarly to ethanol-treated cells cultured in the

presence of FCS (Fig. 1a). This result indicates that LPA is

involved in the ethanol-induced increase in the 2-DGlc

uptake.

Ethanol alters the actin cytoskeleton organization

of astrocytes

We next examined whether actin cytoskeleton organiza-

tion was perturbed by ethanol (Fig. 3). For this, primary

cultures of rat astrocytes were treated for 7 days with

different concentrations of ethanol (30, 50 or 100 mM,

final concentrations). After fixation, actin was stained with

TRITC-phalloidin. Untreated astrocytes showed numerous

well-organized actin stress fibres (Fig. 3a). In contrast,

ethanol-treated cells showed fewer actin fibres, which, in

addition, were rearranged in a circular structure beneath

plasma membrane, leaving the central portion of cells devoid

of actin (Figs 3b and c). The severity of this alteration was

dependent on the ethanol concentration used.

LPA reverts the ethanol-induced alterations of the actin

cytoskeleton

We studied whether LPA could revert the ethanol-induced

alteration in the actin organization. For this, we used the

same experimental conditions described above for the

2-DGlc uptake experiments (Fig. 1b). Astrocytes cultured

in the absence of FCS (the last 24 h) showed few actin stress

fibres (not shown). When LPA was added to cells cultured

without FCS, both untreated (Fig. 3d) and ethanol-treated

cells (30 or 100 mM; Figs 3e and f, respectively) showed an

actin cytoskeleton organization indistinguishable from con-

trol (Fig. 3a).

LPA prevents the ethanol-induced alteration

in microtubule dynamics

It has been reported that LPA also stimulates the assembly of

stable microtubules independently of its effects on actin

filaments (Cook et al. 1998). Therefore, we studied whether

ethanol also alters microtubules. Ethanol (30 mM) did not

effect microtubule organization (Figs 4a and b). Nonetheless,

we reasoned that ethanol could impair the dynamics of

microtubules. Therefore, we examined the kinetics of

microtubule reassembly. For this, astrocytes were first treated

with nocodazole (NZ) to induce the complete disassembly

of microtubules (Figs 4c–e) and, subsequently, NZ was

removed from the culture medium. In ethanol-treated astro-

cytes, the kinetics of the microtubule reassembly (Figs 4g, j

and m) was slower than in untreated cells (Figs 4f, I and l).

In particular, the complete microtubule reassembly in

untreated astrocytes was observed at 45 min after NZ

removal (Fig. 4l), whereas in ethanol-treated cells reassem-

bly occurred only between 90 and 120 min after NZ washout

Fig. 2 Cytoskeleton disrupters alter the 2-deoxyglucose uptake in rat

astrocytes. Control (C) and ethanol-treated (E) astrocytes were cul-

tured with FCS for 7 days. Subsequently, control cells were incubated

with latrunculin B (LTB; 1 lM) or nocodazole (NZ; 30 lM) for several

periods, and the 2-DGlc uptake was measured as described in

Materials and methods. The disruption of actin and microtubule cyto-

skeletons produced different effects on the 2-DGlc uptake. The results

are the mean ± SD from three independent experiments. Asterisks

indicate significant differences (Student’s t-test, p £ 0.05) versus

control cells cultured with FCS.

Fig. 3 Ethanol induces a rearrangement of actin cytoskeleton, which

is reverted by LPA. Untreated and ethanol-treated astrocytes cultured

in the presence (+ FCS; a–c) of FCS, were stained with TRITC-

phalloidin to visualize the actin cytoskeleton organization. Ethanol

produced a significant rearrangement of actin stress fibres (b, c) in

comparison with untreated cells (a). When LPA was added to cells

cultured without FCS (+ LPA; d–f), numerous parallel actin stress

fibres re-appeared, both in control (d) and in ethanol-treated astro-

cytes (e, f). Bar, 10 lm.

Effects of LPA on ethanol-induced injuries 223

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 220–229

(not shown). In accordance with these observations, the

kinetics of the NZ-induced microtubule disassembly in

ethanol-treated cells was faster than in untreated cells (not

shown). As in 2-DGlc uptake and actin cytoskeleton

experiments, we next examined whether LPA also prevented

the alteration in the microtubule dynamics. Indeed, LPA

(200 ng/mL) normalized the kinetics of the reassembly of

microtubules in ethanol-treated cells (Figs 4h, k and n).

Together, these data indicate that ethanol treatment impairs

microtubule assembly and reassembly and that LPA prevents

these alterations.

NIH3T3 cells that constitutively express activated RhoA

are resistant to the ethanol-induced alterations on actin

and on 2-deoxyglucose uptake

Rho GTPase family governs the actin cytoskeleton organ-

ization, and in particular RhoA regulates the formation of

actin stress fibres (Hall 1998; for a review). As we have

observed that ethanol induced a rearrangement of actin

cytoskeleton, we examined whether the RhoA signalling

pathway could be involved in the mechanism by which

ethanol produces these actin cytoskeleton alterations. With

this aim, NIH3T3 cells that constitutively express a mutant

form of RhoA for its GTP-bound form (RhoAV14; mutated

Rho activated by Val14 substitution in the GTP binding site;

Jimenez et al. 1995) were treated with ethanol (100 mM).

GTP-bound RhoA-expressing cells were insensitive to the

expected actin dissolution produced by the absence of FCS

(Figs 5d and e), and they showed an actin stress fibres pattern

(Fig. 5f) that was indistinguishable from that shown by cells

cultured in the presence of FCS (Fig. 5c). Unlike wild-type

(Fig. 5g) and Mock cells (Fig. 5h), RhoAV14-expressing

cells treated with 30 mM (not shown) or 100 mM ethanol

(Fig. 5i) showed a virtually unaltered actin stress fibre

Fig. 4 LPA prevents the ethanol-induced

impairment in the reassembly of micro-

tubules. Control (a) and ethanol (30 mM)-

treated (b) astrocytes were fixed and

stained to a-tubulin to visualize microtubule

cytoskeleton. Next, cells were first treated

with nocodazole (NZ; 30 lM, 60 min), which

produced the disassembly of microtubules

(c–e). Subsequently, NZ was removed from

the medium and the kinetics of the reas-

sembly of microtubules was examined

(f–n). In control cells, the microtubule

reassembly was complete after 30–45 min

(i, l) whereas in ethanol-treated cells at

these times (j, m) microtubules were still

disassembled although the characteristic

microtubule aster emerging from centrioles

(j) and some microtubules tracks were

evident (m). When LPA (200 ng/mL;

60 min) was added to ethanol-treated cells

(h, k, n), the kinetics of the reassembly of

microtubules was indistinguishable from

control (f, i, l). Bar, 10 lm.

224 M. Tomas et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 220–229

organization (compare Figs 5a, c and i). Furthermore, these

cells did not show any increase in 2-DGlc uptake (Fig. 5j).

Discussion

Ethanol perturbs glucose uptake and the organization

and dynamics of the cytoskeleton

It has been postulated that GLUT1 is a stress-response

protein (Wertheimer et al. 1991; Mueckler 1994; Olson

and Pessin 1996). Ethanol increases glucose transport,

like other stressful stimuli such as hypoxia, inhibition of

oxidative phosphorylation, hyperosmolarity, incubation in a

low-glucose medium, or at high pH (Ismail-Beigi 1993;

Hwang and Ismail-Beigi 2001, and references herein).

However, unlike all these stimuli, the ethanol-induced

increase in 2-deoxyglucose uptake is concomitant to an

increase in the GLUT1 protein content (Tomas et al. 2002),

which may be an adaptive response to long-term ethanol

exposure. Although, how ethanol treatment increases GLUT1

and glucose uptake is unclear, the cytoskeleton could be

involved. This hypothesis is supported by the observation

that GLUT1 activity is regulated by actin cytoskeleton

(Zhang and Ismail-Beigi 1998). The astrocyte contains a

cytosolic GLU1-binding protein named Glut1CBP, which

tethers GLUT1 to the actin-binding protein a-actinin-1

(j)

Fig. 5 NIH3T3 cells that constitutively express a GTP-bound Rho

mutant protein are resistant to ethanol-induced actin disassembly.

NIH3T3 cell lines (wild-type, WT; transfected with a empty plasmid,

Mock NIH3T3; and constitutively expressing activated RhoA protein

(RhoAV14) were fixed and stained to actin with TRITC-phalloidin. All

three cell lines showed a well-organized actin cytoskeleton with den-

sely packed actin stress fibres (a–c). When these cell lines were cul-

tured overnight in the absence of FCS (– FCS), there was a

dissolution of stress fibres in WT and Mock NIH3T3 cells (d and e,

respectively) but, in contrast, the activated RhoA-expressing cells

showed an actin cytoskeleton organization indistinguishable from

control NIH3T3 cells (f and a, respectively), which is indicative of the

continuous expression of activated RhoA protein in these cells. In

addition, these cells also overexpress larger amounts of RhoA protein

as shown by western blot (inset in c). Ethanol treatment (100 mM;

5 days) induced the dissolution of actin stress fibres in WT (g) and

Mock (h), but not in RhoAV14-expressing cells (i), which showed a

virtually unaltered actin cytoskeleton organization in comparison with

untreated control cells (c). (j) Measurement of the 2-DGlc uptake in

wild type, Mock and RhoAV14-expressing NIH3T3 cells cultured in the

absence or presence of ethanol (100 mM) for 5 days. Unlike wild type

and Mock cells, those expressing the mutant GTP-bound form of

RhoA showed no increase in the 2-DGlc uptake. These results are the

mean of two independent experiments. Bar, 10 lm.

Effects of LPA on ethanol-induced injuries 225

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 220–229

(Bunn et al. 1999). GLUT1 is thus restricted to some plasma

membrane domains and regulated in its activity. Therefore,

GLUT1 activity could be affected by actin rearrangements,

which is indeed what we observe when astrocytes were

treated either with latrunculin B (Fig. 2) or ethanol (Fig. 1a).

Ethanol-induced actin rearrangement may also perturb the

receptor-mediated endocytosis of GLUT1. In fact, receptor-

mediated endocytosis is actin-dependent (Qualmann and

Kessels 2002) and significantly inhibited by ethanol (Megıas

et al. 2000; Nagy et al. 2002). Therefore, the here-reported

increase in 2-DGlc uptake could be the result of an (partial)

inhibition of the GLUT1 endocytosis, which thereafter would

stay functionally longer in plasma membrane (Fig. 6).

In contrast, in hepatocytes, colonic cells and neurons,

ethanol alters the organization and dynamics of microtubules

(Banan et al. 1998; Yoon et al. 1998; Rovasio and Battiato

2002). We here observed that the steady state microtubule

cytoskeleton organization of ethanol-treated astrocytes is

virtually unaltered but, in contrast, its dynamics is impaired.

In particular, microtubule reassembly after nocodazole

treatment was significantly delayed. This indicates that the

functionality of cellular processes that depend on dynamic

microtubules will be perturbed, as is the case of membrane

trafficking (Yoon et al. 1998; Guash et al. 1992; Larkin

et al. 1996; Renau-Piqueras et al. 1997). It has been

suggested that in hepatocytes the microtubule disruption

produced by ethanol is a consequence of the high-affinity

interaction of acetaldehyde with a-tubulin, which becomes

useless for microtubule polymerization (Sorrell and Tuma

1987; Smith et al. 1992; Hamm-Alvarez and Sheetz 1998;

Yoon et al. 1998). Similar results have also been reported

with purified rabbit skeletal-muscle actin in vitro (Xu et al.

1989). Although the production of acetaldehyde in astrocytes

is much lower than in hepatocytes (Eyserric et al. 1997), this

mechanism may explain the ethanol-induced alterations in

microtubule dynamics. Like ethanol, the disruption of

microtubules by nocodazole also increases 2-DGlc uptake

(Fig. 2). Interestingly, LPA treatment normalizes the 2-Dglc

uptake levels in ethanol-treated cells cultured in the absence

of FCS (Fig. 1b). LPA is a component of serum that

stimulates RhoA signalling pathway, which not only governs

actin cytoskeleton organization (Nobes and Hall 1995) but

also stabilizes microtubules (Cook et al. 1998). With regard

to the latter, LPA could be considered as a cytoprotectant as it

has been previously reported with prostaglandin in ethanol-

treated Caco-2 cells (Banan et al. 1999). Collectively, our

results on LPA show a role of the cytoskeleton in glucose

uptake, suggesting that ethanol could interfere in this

association. Nonetheless, other molecular mechanisms could

also explain the alterations induced by ethanol, particularly

those involving the dynamics and organization of actin

cytoskeleton (see below).

Signalling molecules that regulate actin dynamics

and organization as putative targets of ethanol

Actin organization is regulated by the Rho family members

Rho, Rac and Cdc42, whose downstream signalling

pathways usually induce the appearance of stress fibres,

membrane ruffles/lamelipodia, and filopodia, respectively

(Hall 1998; Hall and Nobes 2000; for reviews). LPA is an

intercellular lipid mediator with growth factor-like activity,

which activates plasma membrane-specific G protein

coupled receptor, leading to Rho activation and, subse-

quently, to stress fibre formation in fibroblasts (Nobes

et al. 1995; Moolenaar et al. 1997). Thus, the finding that

the effect of ethanol on cell cytoskeleton and glucose

uptake is reverted by LPA suggests that ethanol interferes

in the Rho signalling pathway. Consistent with this are the

results in NIH3T3 fibroblasts that constitutively overex-

press the GTP-bound form of RhoA: neither actin cyto-

skeleton organization nor glucose uptake was significantly

altered by ethanol treatment. However, the effect of LPA

treatment indicates the partial involvement of Rho signal-

ling pathway, but not in coupling LPA receptor and hetero-

trimeric G proteins to RhoA otherwise LPA treatment

Fig. 6 Potential signalling pathways through which ethanol could

mediate actin and glucose uptake injuries and LPA its protective

action. Ethanol inhibits PKC and/or PLD activities, which reduces the

PIP2 levels and alters actin polymerization and actin cytoskeleton

organization. Concomitantly to this effect, ethanol also inhibits

receptor-mediated endocytosis of GLUT1 receptors located in plasma

membrane, a process that is also dependent on actin filaments. We

postulate that the cytoprotective effect of LPA is mediated by the

activation of the RhoA signalling pathway. In particular, RhoA acti-

vates Rho kinase, which, on the one hand, stimulates PI(4)P5-kinase,

raising the levels of PIP2, which in turn leads to net actin polymer-

ization, and on the other hand, this increase in filamentous actin

induces the formation of stress fibres. Importantly, this net actin

polymerization also leads to the assembly of a submembranous

actin filament that regulates GLUT1 activity.

226 M. Tomas et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 220–229

could not prevent the ethanol-induced alterations. There-

fore, another molecular target(s) may be affected, as in the

case of protein kinase C (PKC) and phospholipase D

(PLD). PKC activates various phosphatidylinositol kinases,

whose coupled enzymatic activity leads to the formation of

PIP2, which in turns stimulates actin polymerization (Yin

and Janmey 2003). Importantly, this polymerized actin can

be used for stress fibre assembly, which is governed by

GTP-RhoA and the activation of Rho kinase, ROCK

(Fig. 6). Interestingly, ethanol inhibits some PKC isoforms,

in particular a, b1 and c (Slater et al. 2001). We therefore

postulate that ethanol in astrocytes also perturbs some PKC

activities, which may induce the disruption or rearrange-

ment of actin cytoskeleton and, consequently, the alteration

of physiological processes that depend on actin such as

glucose uptake and endocytosis (Bunn et al. 1999; Qual-

mann and Kessels 2002). The involvement of PKC in the

noxious effects of ethanol is also supported by the fact

that, in CaCo-2 cells, treatment with PKC activators/

stimulators, prior to exposure to ethanol, significantly

reduced cell injury on cell viability and microtubule

disruption (Banan et al. 1999). In contrast, primary alcoh-

ols (mainly 1-butanol and ethanol) are specific inhibitors of

PLDs. Their activity results in the hydrolysis of phosphat-

idylcholine to form phosphatidic acid, which regulates

intracellular vesicular transport, particularly in distal steps

of the secretory pathway (Siddhanta et al. 2000, and

references therein). Furthermore, a distinctive feature of

mammalian PLDs is their functional association with actin

cytoskeleton, based on their ability to directly bind both

G-actin (inhibiting its activity; Lee et al. 2001; Kusner

et al. 2002) and actin-binding proteins (Cross et al. 1996;

Steed et al. 1996; Park et al. 2000), and also on

phosphatidic acid stimulates type I phosphatydilinositol-4-

phosphate 5 kinases (PI(4,5)P-kinases; Jenkins et al. 1994;

Kam and Exton 2001). How then does LPA or overex-

pressed GTP-bound RhoA prevent the ethanol-induced

impairments in actin cytoskeleton organization and 2-DGlc

uptake? The mechanism may be the stimulation of PI(4,5)P

kinase through Rho kinase (Oude et al. 2000), which is

also a downstream effector of both PKC and PLD. The

PIP2 thus generated produces net actin polymerization,

which then leads to LPA-induced formation of stress fibres

and the assembly of a submembranous actin cytoskeleton,

which is directly or indirectly coupled to GLUT1 activity.

All these relationships are depicted in Fig. 6.

In conclusion, the findings reported here support the

hypothesis that the cytoskeleton is a major target for ethanol-

induced damage in astrocytes. The protective effects of LPA

on the chronic ethanol-induced impairments on actin and

microtubule cytoskeletons and on glucose uptake in rat

astrocytes open the possibility that LPA or a similar non-

hydrolysable lipid derivative could be used as a cytoprotec-

tant against the noxious effects of ethanol.

Acknowledgements

The authors thank Juan Carlos Lacal (IIB CSIC-UAM, Madrid) for

NIH3T3 cell lines, Jesus Avila (CBMSO, CSIC-UAM, Madrid) and

Miguel Morales (IDIBAPS, Barcelona) for helpful suggestions, and

Robin Rycroft for editorial assistance. This work is supported by

grants from MCyT (SAF2000-0042 to GE and BFI2001-0123-CO2-

02 to GE and JR-P) and FIS (PI020073 to JR-P). MT and JMD are

recipients of pre-doctoral fellowships from FIS, FL-D from

IDIBAPS, and PM from CIEN (FIS).

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