Rapid effects of 17β-estradiol on cell signaling and function of Mytilus hemocytes

GENERAL AND COMPARATIVE

ENDOCRINOLOGY

www.elsevier.com/locate/ygcen

General and Comparative Endocrinology 136 (2004) 58–71

Rapid effects of 17b-estradiol on cell signaling and functionof Mytilus hemocytes

Laura Canesi,a,* Caterina Ciacci,a Michele Betti,a Lucia Cecilia Lorusso,a

Barbara Marchi,b Sabrina Burattini,c Elisabetta Falcieri,c and Gabriella Gallob

a Istituto di Scienze Fisiologiche, Universit�a di Urbino ‘‘Carlo Bo,’’ Loc. Crocicchia, 61029 Urbino (PU), Italyb DIBISAA, Universit�a di Genova, Genova, 16132 Italy

c Istituto di Scienze Morfologiche, Universit�a di Urbino ‘‘Carlo Bo,’’ Loc. Crocicchia, 61029 Urbino (PU), Italy

Received 14 August 2003; revised 2 December 2003; accepted 4 December 2003

Abstract

Estrogens affect the functioning of several non-reproductive tissues, the immune system in particular. In mammalian immu-

nocytes, 17b-estradiol (E2) has both dose- and cell-type specific effects and the responses to E2 seem to be mediated by rapid, non-

genomic mechanisms; these may be initiated at either membrane or cytosolic locations, and can result in both direct local effects,

such as modification of ion fluxes, and regulation of gene transcription secondary to activation of different kinase cascades, in-

cluding mitogen activated protein kinases (MAPKs). In this work, the short-term effects of E2 and the possible mechanisms of

estrogen-mediated cell signaling were investigated in the hemocytes, the immune cells of the bivalve mollusc, the mussel Mytilus

galloprovincialis Lam. The results show that E2 (25 nM) caused a rapid and significant increase in hemocyte cytosolic [Ca2þ]; lowerconcentrations (5 nM) showed a smaller, not significant effect. Both E2 concentrations affected the phosphorylation state of the

components of tyrosine kinase-mediated signal transduction MAPK- and STAT- (signal transducers and activators of transcription)

like proteins within 5–15min from E2 addition. A greater effect and clearer time course were observed with 25 nM E2: in particular,

E2 induced a transient increase in p-ERK2 MAPK and a persistent increase in p-p38 MAPK. Moreover, both STAT3 and STAT5

were tyrosine phosphorylated in response to E2. E2 (5 nM) induced both morphological (as evaluated by SEM) and functional

changes (such as extracellular release of hydrolytic enzymes, lysosomal membrane destabilisation, and stimulation of the bacteri-

cidal activity) within 10–30min from addition. Lysosomal membrane destabilisation induced by both E2 concentrations was

abolished by hemocyte preincubation with the p38 MAPK inhibitor SB203580, and significantly reduced by PD98059 and Wort-

mannin (inhibitors of ERK MAPK and PI3-K, respectively), this suggesting that rapid activation of kinase cascades is involved in

mediating the effects of E2 in mussel hemocytes. The antiestrogen Tamoxifen prevented or strongly reduced most, but not all, the

effects of E2. Western blotting with heterologous anti-ERa–anti-ERb-antibodies revealed the presence of immunoreactive ERa- andERb-like proteins in hemocyte protein extracts. Overall, our data support the hypothesis that the rapid effects and mechanisms of

action of 17b-estradiol are extremely conserved and that they may play a crucial role in endocrine–immune interactions in inver-

tebrates.

� 2003 Elsevier Inc. All rights reserved.

Keywords: 17b-Estradiol; Mytilus; Hemocytes; Cell signaling; Ca2þ; MAPK; STAT; Immunity

1. Introduction

Estrogens, with their most active 17b-estradiol (E2),

exert a broad spectrum of activities on a wide variety ofcells and tissues. The effects of E2 are known to be

* Corresponding author. Fax: +39-722-304-226.

E-mail address: [email protected] (L. Canesi).

0016-6480/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.ygcen.2003.12.003

mediated by intracellular estrogen receptors (ERa and

ERb) belonging to the steroid receptor superfamily;

these receptors act as ligand-inducible transcription

factors by binding to specific DNA target sequences andspecifically regulating expression of target genes (Hall

et al., 2001; Moggs and Orphanides, 2001; Nadal et al.,

2001). This is generally referred to as genomic or

‘‘classical pathway’’ of estrogen action; it involves direct

mail to: [email protected]

L. Canesi et al. / General and Comparative Endocrinology 136 (2004) 58–71 59

participation of the ERs as transcription factors withoutvirtually any other previous signaling step, and requires

hours to days for an increase in gene expression to oc-

cur. On the other hand, estrogens can also trigger rapid

(within seconds to minutes) effects through non-genomic

or ‘‘alternative pathways’’ (Guo et al., 2002; Kelly and

Levin, 2001; L€osel et al., 2003; Nadal et al., 2001; Segars

and Driggers, 2002a,b). These may be initiated at either

membrane or cytosolic locations and can result in bothdirect local effects (such as modification of ion fluxes)

and regulation of gene transcription secondary to acti-

vation of kinase cascades (involving cAMP, MAPKs,

PKC and PKA, PI-3K, etc.). Elicited responses depend

upon the cell type studied and the conditions used;

however, rapid changes in phosphorylation state of

mitogen activated protein kinases (MAPKs) and in cy-

tosolic [Ca2þ] are among the most common events ob-served in non-genomic effects of E2 (L€osel et al., 2003;Nadal et al., 2001; Segars and Driggers, 2002a,b). In

contrast to its genomic effects, the precise �alternative�signaling pathways involved in the rapid effects of E2 are

not yet completely understood in mammalian cells. Al-

though rapid actions of E2 have been attributed to ac-

tivation of putative estrogen receptors associated with

the cell surface (mER) (Benten et al., 2001; Hayneset al., 2002), the role, location, and molecular nature of

these receptors in different cell types is still under con-

troversial debate (L€osel et al., 2003; Nadal et al., 2001;

Segars and Driggers, 2002a,b).

Estrogens are not merely reproductive hormones,

and they affect the functioning of several non-repro-

ductive tissues (Haynes et al., 2002; Mendelsohn, 2000;

Morey et al., 1997; Razandi et al., 2000), among thesethe immune system (Guo et al., 2002; Stefano et al.,

2000; Stefano and Peter, 2001; Vegeto et al., 2001).

Estrogens have both dose- and cell-type specific effects

on immune cells and may act as pro-inflammatory and

anti-inflammatory stimuli depending on the setting

(Bruce-Keller et al., 2000; Guo et al., 2002; Vegeto

et al., 2001). E2 can modulate the function of neutro-

phil granulocytes, monocytes, and macrophages: thesecell types have been reported to express both nuclear

and membrane ERs and their responses to E2 seem to

be mediated by rapid non-genomic pathways (Benten

et al., 1998; Guo et al., 2002; Stefano et al., 2000;

Stefano and Peter, 2001).

In invertebrates, the possibility that estrogens may

modulate the immune function and the mechanisms

involved has not been investigated yet. In the bivalvemollusc, the mussel Mytilus, the presence of endogenous

17b-estradiol has been described in both tissues and

hemolymph (De Longcamp et al., 1974; Reis-Henriques

et al., 1990; Stefano et al., 2003a; Zhu et al., 2003). In

mussels, circulating hemocytes, resembling the mam-

malian monocyte/macrophage lineage, are responsible

for innate immunity (Renwrantz, 1990). The immune

function of Mytilus hemocytes involves activation ofcomponents of tyrosine kinase-mediated cell signaling;

in particular of MAPK- (Canesi et al., 2002a,b) and

STAT- (signal transducers and activators of transcrip-

tion) (Canesi et al., 2003a, in press) like proteins. In this

work, the possible short-term effects of 17b-estradiol onmussel hemocytes and the rapid mechanisms of estro-

gen-mediated signal transduction were investigated; in

particular, the effects of E2 on cytosolic [Ca2þ] and onthe phosphorylation state of MAPKs were evaluated.

Moreover, since tyrosine phosphorylation of STATs

plays a key role in activation of the hemocyte immune

response, the possible effect of E2 on STAT phosphor-

ylation was investigated.

2. Materials and methods

2.1. Animals

Mussels (Mytilus galloprovincialis Lam.) 4–5 cm long,

were obtained from S.E.A. (Gabicce Mare, PU) and

kept for 1–3 days in static tanks containing artificial sea

water (ASW) (1L/mussel) at 16 �C. Sea water was

changed daily.

2.2. Hemolymph collection, preparation of hemocyte

monolayers, and hemocyte treatment

Hemolymph was extracted from the posterior ad-

ductor muscle of 8–20 mussels, depending on the ex-

periment, using a sterile 1ml syringe with a 18 G1/2 in.

needle. With the needle removed, hemolymph was fil-tered through a sterile gauze and pooled in 50ml Falcon

tubes at 4 �C. Hemolymph serum was obtained by cen-

trifugation of whole hemolymph at 200g for 10min and

the supernatant was sterilised through a 0.22 lm pore

size filter. Hemocyte monolayers were prepared as pre-

viously described (Canesi et al., 2002a). Briefly, aliquots

of 0.5ml of hemolymph, corresponding to about 1–

2� 106 cells, were seeded onto glass cover slips (40 by22mm) placed in plastic culture dishes and incubated at

16 �C for 30min to allow for cell attachment. Non-ad-

herent hemocytes were subsequently removed by gently

washing the preparations with sterilised ASW. Hemo-

cyte monolayers were added with 1.5ml of hemolymph

serum and kept before use at 16 �C.Hemocytes were incubated at 16 �C for different pe-

riods of times (from 3 to up to 60min, depending onthe experiment) with 17b-estradiol (from a 10mM

stock solution in ethanol) suitably diluted in either

ASW or hemolymph serum. Untreated and control

vehicle hemocyte samples were run in parallel. In ex-

periments with antiestrogens, Tamoxifen (from a

10mM stock solution in ethanol) was added 10min

before estradiol.

60 L. Canesi et al. / General and Comparative Endocrinology 136 (2004) 58–71

2.3. Single cell [Ca2þ]i measurements

Single cell [Ca2þ]i measurements were carried out as

previously described (Canesi et al., 1997). Aliquots of

40 ll of hemolymph were settled on glass cover slips for

20min at 16 �C, rinsed and loaded with 4 lM Fura2/AM

as previously described. [Ca2þ]i measurements were

carried out with an Olympus IMT-2 inverted micro-

scope equipped with an IMT2-RFL fluorescence at-tachment (Olympus Optical, Germany) and with a

MTI68 intensified camera (Oatencourt, England). Im-

ages were acquired at 15 s intervals using the CUE2

RMS 4.0 imaging system (Galai Production, Israel).

Background fluorescence was subtracted before analy-

sis. Free [Ca2þ] was calculated following Grynkiewitz et

al. (1985). Intracellular calibration was performed at the

end of each experiment adding the ionophore ionomycin(10 lM) and excess EGTA to obtain maximal and

minimal fluorescence ratios, respectively, as previously

described (Canesi et al., 1997). Data are means� SD of

at least five experiments, each involving at least 10–12

cells.

2.4. Electrophoresis and Western blotting

Levels of phosphorylated MAPKs and STATs in

whole cell extracts from hemocyte monolayers were

determined using phosphospecific antibodies as previ-

ously described (Canesi et al., 2002a, 2003a). Hemocyte

monolayers were incubated with E2 for 5, 15, 30, and

60min. Supernatants from each culture dish were dis-

carded and each hemocyte monolayer was lysed with

1ml of ice-cold lysis buffer [50mM Tris–HCl, pH 7.8,0.25M sucrose, 1% (w/v) SDS (sodium dodecyl sulfate),

1 lg/ml pepstatine, 10 lg/ml leupeptine, 2mM sodium

orthovanadate, 10mM NaF, 5mM EDTA, 5mM NEM

(N-ethylmaleimide), 40 lg/ml PMSF (phenylmethylsul-

phonyl fluoride), and 0.1% Nonidet-P40] and sonicated

for 45 s at 50W. Samples were boiled for 4min and then

centrifuged for 10min at 14,000g to remove insoluble

debris. Supernatants were mixed 1:1 (v:v) with samplebuffer (0.5M Tris–HCl, pH 6.8, 2% SDS, 10% glycerol,

4% of 2-mercaptoethanol, and 0.05% bromophenol

blue) and samples (normalised for protein content be-

fore loading to 30 lg of protein) were resolved either by

12% (for MAPKs) or by 7% (for STATs) SDS–poly-

acrylamide gel electrophoresis (Laemli, 1970). Pre-

stained molecular-mass markers were run on adjacent

lanes. The gels were electroblotted and stained withCoomassie blue according to Towbin et al. (1979). Blots

were probed with human recombinant specific anti-

phospho-ERK1/2, anti-phospho-p38, or anti-phospho-

SAPK/JNK as primary antibodies (1:1000) and

horseradish-peroxidase-conjugated goat anti-rabbit IgG

(1:3000) was used as a secondary antibody. Nitrocellu-

lose membranes were stripped for 30min at 50 �C with

stripping buffer (62.5mM Tris–HCl, pH 6.7, containing10mM bmercaptoethanol and 2% SDS) and reprobed

with anti-actin antibodies (1:1000) as a loading control.

Blots were also probed with the specific anti-phospho-

STAT3 and anti-phospho-STAT5 (1:500) as primary

antibodies, and horseradish-peroxidase-conjugated goat

anti-rabbit IgG (1:3000) was used as a secondary anti-

body. After probing with anti-phospho-STATs, mem-

branes were stripped and reprobed with anti-STAT3 oranti-STAT5 antibodies (1:500).

For detection of ER-like proteins, hemocyte protein

extracts were analysed by 10% SDS–PAGE and Western

blotting with human polyclonal anti-ERa or anti-ERbantibodies (1:500) as primary antibodies and horserad-

ish-peroxidase-conjugated rabbit anti-goat IgG (1:5000)

or goat anti-rabbit IgG (1:3000), respectively, as sec-

ondary antibodies.Immune complexes were visualised using an en-

hanced chemioluminescence Western blotting analysis

system (Amersham-Pharmacia) following the manufac-

turer�s specifications. Western blots films were digitised

(Chemidoc-Biorad) and band optical densities were

quantified using a computerised imaging system

(QuantityOne). Relative optical densities (arbitrary

units) were normalised for the control band in eachseries.

2.5. Scanning electron microscopy

Hemocyte monolayers were washed three times with

filtered ASW and then incubated with E2 for 10, 30, and

60min. Control cells were run in parallel. Cells were

fixed for 1 h in 2.5% glutaraldehyde in modified PBS(150mM phosphate buffer saline adjusted to 450mM

osmolarity by addition of NaCl). Fixed cells were rinsed

with the same buffer and post-fixed in 1% osmium te-

troxide in buffer for 1 h. Samples were then dehydrated

by graded ethanol solutions, critical point dried, and

gold coated as described by Falcieri et al. (2000). Cells

were then examined with a Philips 515 Scanning Elec-

tron Microscope at 15 kV.

2.6. Lysosomal membrane stability

Lysosomal membrane stability in control hemocytes

and hemocytes incubated with different concentrations

of E2 for 30min was evaluated by the Neutral red re-

tention assay as previously described (Canesi et al.,

2003b) essentially following the procedure of Lowe et al.(1995). Hemocyte monolayers on glass slides were in-

cubated with 30 ll of a NR solution (final concentration

40 lg/ml from a stock solution of NR 20mg/ml DMSO);

after 15min the excess dye was washed out, 30 ll of

artificial sea water were added, and slides were sealed

with a cover slip. Every 15min slides were examined

under an optical microscope and the percentage of cells


showing loss of dye from lysosomes in each field wasevaluated. For each time point 10 fields were randomly

observed. The endpoint of the assay was defined as

the time at which 50% of the cells showed sign of lyso-

somal leaking (the cytosol becoming red and the cells

rounded).

In experiments with kinase inhibitors, hemocyte

monolayers were pre-treated for 20min with PD98059

(for ERK MAPK), SB203580 (for p38 MAPK) orWortmannin (for PI3-kinase) suitably diluted in serum

from 10mM stock solutions in DMSO as previously

described (Canesi et al., 2002a,b). After pre-incubation,

supernatants were discarded and hemocytes were incu-

bated with E2 or serum for 30min and then tested for

the NR assay. Control hemocytes were maintained for

20min in serum in the presence of DMSO (0.1% final

concentration), as pre-treated cells, then washed andincubated with serum for 30min.

2.7. Lysosomal enzyme release

Lysosomal enzyme release by mussel hemocytes was

evaluated by measuring lysozyme activity in the extra-

cellular medium as previously described (Canesi et al.,

2003b). Lysozyme activity in aliquots of serum of con-trol hemocytes and hemocytes incubated with E2 for

different periods of time (15, 30, and 60min) was de-

termined spectrophotometrically as described by Chu

and La Peyre (1989). Briefly, lysozyme activity was de-

termined as the ability of aliquots of serum to lyse a

standard suspension of Micrococcus lysodeikticus

(15mg/100ml in 66mM phosphate buffer, pH 6.4) and

measured as decrease in absorbance at 450 nM. Henegg-white (HEW) lysozyme was used to construct a

standard curve and lysozyme activity was expressed as

HEW lysozyme equivalents/mg protein/ml.

2.8. Bacterial cultures, incubations of hemocyte monolay-

ers with Escherichia coli, and bactericidal assays

All these procedures were carried out as previouslydescribed (Canesi et al., 2001a, 2002a). The E. coli strain

MG1855 suspension in PBS (A650 ¼ 1, corresponding to

2–4� 109 colony forming units [CFU]/ml) was suitably

diluted in hemolymph serum and 1.5ml were added to

each hemocyte monolayer to yield a final bacteria/he-

mocyte ratio of approximately 50:1. Control cells were

added with 1.5ml of hemolymph serum. Incubations

were carried out at 16 �C for different periods of time.Hemocyte monolayers were pre-treated with E2 for

different periods of time (10 or 60min), then washed,

and incubated with E. coli in the presence of hemolymph

serum. Control hemocytes were run in parallel. Imme-

diately after the addition of bacteria (T ¼ 0) and at

different times of incubation (60 and 120min), super-

natants were collected from monolayers and hemocytes

were lysed by adding 5ml of cold distilled water and10min agitation. Supernatants and lysates from each

sample were pooled, 10-fold serial diluted, and plated

onto LB agar to enumerate culturable bacteria (CFU/

ml). Percentages of killing in control and E2-treated

hemocytes were determined in comparison to values

obtained at T ¼ 0. Samples were in triplicate.

2.9. Data analysis

The results are the mean of at least three experiments

in triplicate� SD. Data from densitometric analyses of

Western blots are means� SD of three independent

experiments. Statistical analysis was performed by using

the Mann–Whitney U test with significance at P 6 0:05.

2.10. Chemicals

All reagents were of analytical grade. 17b-Estradiol,E2–BSA, and Tamoxifen were from Sigma (St. Louis,

MO). Fura2/AM was from Molecular Probes (Eugene,

OR); ionomycin was from Calbiochem (La Jolla, CA).

Rabbit polyclonal anti-phospho-MAPK (ERK1/2)

(Thr202/Tyr204), anti-phospho-p38 MAPK (Thr180/

Tyr182), anti-phospho-SAPK/JNK (Thr183/Tyr185)(human recombinant) antibodies and polyclonal anti-

phospho-STAT3 (Tyr705), and anti-phospho-STAT5

(Tyr694) antibodies were obtained from New England

Biolabs (Beverly, USA). Mouse monoclonal anti-

STAT3 (F-2) and anti-STAT5 (G-2) antibodies, anti-

ERa (H-184), and anti-ERb (N-19) antibodies were

from Santa Cruz Biotechnology (CA). Anti-b-actin an-

tibodies (rabbit polyclonal) were from Sigma. Pre-stained LMW and HMW molecular-mass markers were

from Biorad (CA). All other reagents were purchased

from Sigma (St. Louis, MO).

3. Results

3.1. Effect of E2 on cytosolic [Ca2þ]

Fig. 1 shows the effect of E2 on cytosolic [Ca2þ] asdetermined by fluorescence microscopy in Fura2/AM

loaded hemocytes. E2 (5 nM) elicited a small and not

significant increase in [Ca2þ]. However, higher concen-

trations (25 nM) caused a sustained rise in cytosolic

[Ca2þ] (Fig. 1A); the [Ca2þ] level significantly increased

with respect to control values (+50%) about 75 s from E2

addition (C ¼ 74:5� 5:2 nM; E2: 112.8� 9.0 nM;

P 6 0:05) and reached an average 2-fold increase after

about 150 s (C ¼ 74:5� 5:2 nM; E2: 150.8� 12.0 nM;

P 6 0:05). The E2-mediated increase in cytosolic [Ca2þ]was abolished by lowering extracellular [Ca2þ] with

2mM EGTA, indicating that E2 induced an influx

of calcium from the extracellular medium (Fig. 1B).

Fig. 2. Effect of 5 nM E2 on MAPK phosphorylation in hemocyte

monolayers. Protein extracts from hemocytes were subjected to 12%

SDS–PAGE followed by Western blotting using polyclonal phospho-

specific antibodies to MAPKs. (A and B) The time course of ERK2

and p38 MAPK phosphorylation, respectively, in control hemocytes

and hemocytes incubated with E2 for different periods of time; (C) an

anti-actin blot is shown as a loading control. Blots are representative of

three independent experiments.

Fig. 1. Effects of E2 on cytosolic [Ca2þ] in Fura2/AM loaded hemo-

cytes. (A) Dose-dependence: Control (_); 5 nM E2 (rÞ; 25 nM E2 (�);

(B) effect of 25 nM E2 in the presence of extracellular EGTA (2mM) or

after pre-treatment with Tamoxifen (500 nM): Control (_); 25 nM E2

(�); EGTA/E2 (X); Tamoxifen (D); Tamoxifen/E2 (jÞ. Data are

means� SD of at least five experiments, each involving at least 10–12

cells.


The E2-mediated [Ca2þ] increase was not significantly

affected by hemocyte pretreatment (10min) with the

antiestrogen Tamoxifen at concentrations up to 500 nM

(Fig. 1B); at this concentration, Tamoxifen alone caused

a small, not significant increase in [Ca2þ]. In all sub-

sequent experiments, Tamoxifen was used at 100 nM.

3.2. Effect of E2 on phosphorylation of immunoreactive

MAPK- and STAT-like proteins

The effect of E2 on the phosphorylation state of

MAPKs was firstly evaluated in protein extracts from

hemocytes incubated with 5 nM E2 for different periods

of time by electrophoresis and Western blotting utilising

specific anti-phospho-MAPK antibodies as previouslydescribed (Canesi et al., 2002a). The results reported in

Fig. 2 show that this concentration of E2 rapidly affected

the phosphorylation state of different MAPKs. In par-

ticular (Fig. 2A) E2 did not apparently increase the level

of phosphorylated ERK2, the main ERK form detected

in mussel hemocytes utilising anti-phospho-ERKs anti-

bodies (Canesi et al., 2002a); however, a significant de-

crease in ERK2 phosphorylation was observed between15 and 60min (about )50% as evaluated by densito-

metric band analysis; P 6 0:05, not shown). On the other

hand, E2 significantly increased the level of phosphory-

lated stress activated p38 MAPK: a transient increase in

phosphorylated p38 was observed at 5min (about 1,6-

fold with respect to controls; P 6 0:05, not shown), fol-lowed by a decrease similar to that observed for ERK2

(Fig. 2B). A representative anti-actin blot is shown as aloading control (Fig. 2C).

The extent and time course of MAPK phosphoryla-

tion induced by E2 were more clearly observed increas-

ing the E2 concentration (Fig. 3); densitometric analysis

of phosphorylated protein bands are shown in inset

to Fig. 3. At 25 nM E2 a rapid increase in ERK2

phosphorylation was observed with a maximum (about2-fold at 5min; P 6 0:05), followed by a progressive

decrease similar to that observed with lower E2 con-

centrations (Fig. 3A). E2 also caused a large increase in

p38 MAPK phosphorylation (up to a 5-fold at 5min;

P 6 0:05) followed by a slow decrease; at 60min the level

of phosphorylated p38 was still twice as high as in

control cells (Fig. 3B). Moreover, both p54 and p46

JNK forms were progressively phosphorylated in re-sponse to estradiol, showing a maximum 2-fold increase

at 60min (P 6 0:05) (Fig. 3C). The same experiments

were carried out in hemocytes pre-treated with Tamox-

ifen (100 nM). The antiestrogen prevented the effect of

E2 on ERK2 phosphorylation (Fig. 3E). On the other

hand, the E2-induced phosphorylation of the stress-

activated MAPKs was not significantly affected by

Tamoxifen (Figs. 3F and G). Anti-actin blots are shownas loading controls (Figs. 3D and H).

The possible effect of E2 on other components of

tyrosine-kinase mediated cell signaling was also inves-

tigated by evaluating the phosphorylation state of

different STATs by Western blotting with anti-phospho-

STAT antibodies directed against the tyrosine phos-

phorylated sites of STAT3 and STAT5 isoforms as

previously described (Canesi et al., 2003a). E2 (5 nM)also caused a rapid and significant increase in the level

of both phosphorylated STAT3- and STAT5-like

immunoreactive proteins in mussel hemocytes (about

2-fold with respect to controls at 5min; P 6 0:05,not shown). Again, the effect was more evident with

25 nM E2, that induced a transient increase in the

level of phosphorylated STAT3 (up to 4-fold at

15min; P 6 0:05) (Fig. 4A), followed by a decrease tocontrol levels at 60min, whereas the level of total,

Fig. 4. Effect of 25 nM E2 on STAT tyrosine phosphorylation in hemocyte monolayers. Protein extracts from control hemocytes and hemocytes

incubated with E2 for different periods of time were subjected to 7% SDS–PAGE followed by Western blotting using polyclonal phosphospecific

antibodies to phosphorylated STAT3 (A) and STAT5 (B). Blots were stripped and reprobed with antibodies to the corresponding total, unphos-

phorylated STATs. (C and D) Represent the results of the same experiments carried out in Tamoxifen-pre-treated hemocytes. Bands were detected

using enhanced chemioluminescence reagents (see Section 2). Results are representative of three independent experiments. C, control. Inset: den-

sitometric analysis of blots from three independent experiments (mean�SD). Relative increases in band optical densities (arbitrary units) were

normalised for the control band in each series.

Fig. 3. Effect of 25 nM E2 on MAPK phosphorylation in hemocyte monolayers. Protein extracts from hemocytes were subjected to 12% SDS–PAGE

followed by Western blotting using polyclonal phosphospecific antibodies to MAPKs. (A–C) The time course of ERK2, p38, and JNK MAPK

phosphorylation, respectively, in control hemocytes and hemocytes incubated with E2 for different periods of time; (D) an anti-actin blot is shown as

a loading control. (E–H) Results of the same experiments carried out in hemocytes pre-treated with the antiestrogen Tamoxifen (100 nM, 10min).

Insets: densitometric analysis of blots from three independent experiments (mean� SD). Relative increases in band optical densities (arbitrary units)

were normalised for the control band in each series.



unphosphorylated STAT3-like protein did not change(Fig. 4B). E2 also caused a rapid and more persistent in-

crease in the level of p-STAT5, with a maximum at 5 and

15min (4-fold P 6 0:05) (Fig. 4C), without affecting the

level of unphosphorylated STAT5-like protein (Fig. 4D).

Tamoxifen pre-treatment abolished the effect of E2 on the

phosphorylation of STAT3 (Fig. 4E), but it was in-

effective on the E2-induced STAT5 phosphorylation

(Fig. 4F).The level of phosphorylated MAPKs and STATs did

not change in control hemocytes or in vehicle-treated

hemocytes incubated for different times in the absence of

estrogen (not shown).

3.3. Identification of estrogen-like receptors in mussel

hemocytes

In an attempt to identify the presence of ER-like

forms in mussel hemocytes, whole protein extracts were

also subjected to SDS–PAGE and Western blotting with

anti-ERa or anti-ERb antibodies. The results in Fig. 5

show that a single immunoreactive band of the MW of

approximately 70 kDa was identified by the anti-ERaantibody (Fig. 5A). Moreover, an immunoreactive band

of about 49 kDa was observed using the anti-ERb an-tibody (Fig. 5B). Immunoreactive ERa- and ERb-likeproteins were clearly expressed in hemocyte samples

obtained from different groups of mussels.

3.4. Effect of E2 on hemocyte morphology and function

The possible effects of E2 were investigated by scan-

ning electron microscopy (SEM) and the results areshown in Fig. 6. Control hemocytes were characterised

by a distinct shape, generally showing a rather smooth

margin on one side and several membrane extensions on

the others (Fig. 6A). Some extracellular material was

observed around the cells; this material could not be

eliminated by repeated washing of cells after adhesion,

Fig. 5. Identification of immunoreactive estrogen-like receptors in mussel hem

followed by Western blotting using polyclonal anti-ERa (A) and anti-ERb (B

extracts obtained from the hemolymph of different pools of mussels.

indicating that it was probably released by the cells andfixed during sample preparation. After the addition of

5 nM E2, progressive changes in hemocyte shape were

observed; these morphological changes were seen in

many cells at 10min of exposure (not shown), and in the

majority of cells at 30min (Figs. 6B and C). The most

evident effect of E2 treatment was a general decrease in

the number and length of membrane extensions; more-

over, an apparent increase in the release of extracellularmaterial was observed (Figs. 6B and C), this suggesting

that the degranulation process was stimulated by E2

addition. Such an effect was particularly evident around

some cells that became rounded or showed a charac-

teristic bipolar appearance (Fig. 6B). At longer incuba-

tion times (60min), many cells became rounded or

showed variable shapes, with emission of long pseudo-

podia or filopodia (Figs. 6D–F). The effects of E2 on cellmorphology were prevented by Tamoxifen pretreatment

(Fig. 7); Tamoxifen-treated hemocytes showed an ap-

pearance similar to that of untreated cells (Figs. 7A and

B) at both 30 and 60min of incubation with E2 (Figs. 7C

and D).

The effects of E2 on the hemocyte function were then

evaluated and the results are reported in Fig. 8. Fig. 8A

shows that 5 nM E2 caused a small but significant in-crease in the extracellular release of lysosomal hydro-

lytic enzymes, with a maximum (+30% respect to

control; P 6 0:05) at 30min incubation, this confirming

the microscopical observations. The effects of E2 on the

overall immune function was evaluated by determining

the microbicidal activity of hemocyte monolayers to-

wards E. coli as previously described (Canesi et al.,

2001a, 2002a,b) and the results are reported in Fig. 8B.In hemocytes pre-incubated with 5 nM E2 for 60min,

washed and then incubated with bacteria for different

periods of time (60 and 120min) no changes were ob-

served in the bactericidal activity towards E. coli. On the

other hand, addition of E2 10min before bacterial

challenge lead to a small but significant increase in the

ocytes. Hemocyte protein extracts were subjected to 10% SDS–PAGE

) antibodies. Samples loaded in different lanes correspond to hemocyte

Fig. 6. Effect of E2 (5 nM) on cell morphology of mussel hemocytes evaluated by scanning electron microscopy (SEM). (A) Control hemocyte; (B and

C) hemocytes incubated with E2 for 30min; (D–F) hemocytes incubated with E2 for 60min. Scale bars (A–D)¼ 10 lm; (E and F)¼ 1 lm.


percentage of bacterial killing (+17 and +20% withrespect to untreated hemocytes, at 60 and 120min

incubation, respectively; P 6 0:05).

Finally, the effect of E2 on the stability of lysosomalmembranes was evaluated. In hemocytes incubated for

30min with E2 and then tested for the NR assay, a clear

Fig. 8. Effects of 5 nM E2 on hemocyte function. Data are means�SD

of four experiments in triplicate. *P 6 0:05. (A) Effect of E2 on hemo-

cyte lysozyme release. Hemocytes were treated with E2 for different

periods of time and the lysozyme activity in the extracellular medium

was evaluated as described in Section 2. C (s); E2 (d). *P 6 0:05. (B)

Effect of E2 pretreatment on survival of E. coli to hemocyte bactericidal

activity. Hemocytes were pre-treated with E2 for either 10 or 60min,

washed, incubated with bacteria for different periods of time, and the

number of culturable bacteria per millilitre in the hemocyte monolayer

was evaluated as described in Section 2. Control (s) (untreated)

hemocyte samples were run in parallel; E2 10min (d); E2 60min (D).

Fig. 9. Effect of E2 on lysosomal membrane stability (NR retention

time) of mussel hemocytes. Data are means� SD of four experiments

in triplicate; *P 6 0:05. (A) Dose dependence in untreated (d) and

Tamoxifen-pre-treated (circ) hemocytes. (B) Effect of hemocyte pre-

treatment with different kinase inhibitors on the lysosomal membrane

destabili� induced by different concentrations of E2. Before E2 incu-

bation, hemocytes were pre-treated for 20min with SB203580 (20lM),

PD98059 (20 lM) or Wortmannin (0.1lM).

Fig. 7. Effect of E2 (5 nM) on cell morphology evaluated by (SEM) in tamoxifen-pre-treated hemocytes. (A) Control hemocyte; (B) hemocytes treated

with Tamoxifen for 10min; (C and D) hemocytes pre-treated with Tamoxifen (10min) and exposed to E2 for 30 and 60min, respectively. Scale bars

(A–D)¼ 10 lm.


concentration-dependent labilisation of lysosomal

membranes was observed (Fig. 9A). E2 caused a signifi-

cant decrease in lysosomal membrane stability (about

)30% and )56% at the concentrations of 5 and 25 nM,

respectively, with respect to control cells; P 6 0:05). At

higher concentrations (50 nM) hemocyte membranes

were almost completely destabilised. Tamoxifen pre-

treatment prevented the effect of E2 on lysosomal mem-

brane stability at all the concentrations tested (Fig. 9A).

In order to investigate whether the effects of E2 on

mussel hemocytes may be related to activation of kinase


cascades, the same experiments were carried out in he-mocytes pre-treated with different kinase inhibitors as

previously described (Canesi et al., 2002a) and the re-

sults are shown in Fig. 9B. Cell pre-treatment with

SB203580, a specific inhibitor of p38 MAPK activation,

completely abolished the lysosomal destabilisation in-

duced not only by 5 nM but also by 25 nM E2. The effect

of E2 was significantly reduced also by cell pre-treatment

with the ERK MAPK inhibitor PD98059 and withWortmannin, the inhibitor of PI3-kinase (phosphati-

dylinositol 3-kinase).

4. Discussion

Endogenous steroids and estrogen-like receptors have

been identified in the mollusc Octopus, indicating thatclassical estrogen signaling involved in the reproductive

functions is conserved in both vertebrates and inverte-

brates (Di Cosmo et al., 2002; Tosti et al., 2001). Steroid

hormones have been also identified in the mantle of the

mussel Mytilus edulis, showing no significant sex-related

differences in the concentration of 17b-estradiol (De

Longcamp et al., 1974; Reis-Henriques et al., 1990). In

mussel tissues and hemolymph nanomolar concentra-tions of 17b-estradiol have been recently measured

(Stefano et al., 2003a; Zhu et al., 2003). Both hemocytes

and ganglia have been shown to be responsive to E2 in

the high nanomolar range (Burlando et al., 2002; Stef-

ano et al., 2003a).

In this work, we investigated the possibility that 17b-estradiol may affect the function of mussel immune cells

through rapid signaling mechanisms. The results showthat 5 nM E2 induced cell shape changes, release of ex-

tracellular enzymes, and stimulation of bactericidal ac-

tivity; all these effects were observed from 10 to 30min

from estrogen addition. This E2 concentration did not

elicit significant changes in cytosolic [Ca2þ]; however, arapid increase in cytosolic [Ca2þ] was observed with

25 nM E2. Both concentrations significantly affected the

phosphorylation state of the components of tyrosinekinase-mediated signal transduction MAPK- and

STAT-like proteins. The effects of E2 on cell signaling

were rapid, occurring from seconds to minutes.

We have previously demonstrated that tyrosine ki-

nase mediated-cell signaling plays an important role in

the response of mussel cells to both bacterial challenge

and cytokines (Canesi et al., 2002a, 2003a), and to

growth factors (Canesi et al., 2001b). The results herereported show that both 5 and 25 nM E2 significantly

affected the phosphorylation state of different MAPKs

in mussel hemocytes. A greater response and clearer

time course were observed at 25 nM E2: a rapid and

transient phosphorylation of both the extracellularly

regulated ERK2 MAPK and the stress activated p38

MAPK and a progressive and persistent increase in

phosphorylated JNK MAPKs were observed. A signif-icant, although smaller increase in p38 MAPK phos-

phorylation and a similar time course were observed

also with 5 nM E2. Although with this concentration we

could not detect an early increase in p-ERK2, a decrease

in ERK2 phosphorylation from 15min similar to that

induced by 25 nM was observed; with lower concentra-

tions, such a decrease may follow a small and more

rapid increase that we were not able to detect in ourexperimental conditions. The time course of the phos-

phorylation/dephosphorylation of each MAPK may be

very rapid and depend on the hormone concentration

used.

The MAPK signal cascade has emerged as a non-

genomic pathway mediating the rapid response to

estrogens in many mammalian cells (Haynes et al.,

2002; L€osel et al., 2003; Nadal et al., 2001; Segars andDriggers, 2002b). Activation of the Src/Ras/MAPK

cascade represents one of the best studied examples of

rapid cytosolic actions of estrogens. Binding of estra-

diol to either cytosolic ERa and ERb determines the

interaction of ERs with Src, changing the conformation

of the kinase to an active state and activation of the

cascade (Nadal et al., 2001; Segars and Driggers,

2002b; Song et al., 2002; Wong et al., 2002). Both ERaand ERb are involved in the transmission of signals for

phosphorylation of the selective MAPK family mem-

bers, although each receptor differs in the kinetics of

MAPK activation depending on the cell type. ERaseems to preferentially lead to ERK activation, while

ERb is more closely linked to JNK/SAPK activation

(Wade et al., 2001; Haynes et al., 2002); moreover,

Tamoxifen is able to reduce MAPK activation medi-ated by ERa, whereas ERb-mediated MAPK activation

seems less sensitive to antiestrogen inhibition (Wade

et al., 2001). In mussel hemocytes, both ERK and p38

MAPK phosphorylation induced by E2 were transient,

and peaked at 5min; on the other hand, persistent JNK

MAPK activation (up to 1 h after estrogen treatment)

was observed. The antiestrogen Tamoxifen prevented

the effects of E2 on ERK phosphorylation, withoutaffecting the effects on both the stress activated p38 and

JNK MAPKs. These results suggest that, in analogy

with mammalian studies, in mussel hemocytes activa-

tion of different MAPKs by E2 may be related to

activation of different receptor isoforms and/or different

signaling pathways.

E2 also induced rapid and transient tyrosine phos-

phorylation of immunoreactive STAT-like componentsSTAT3 and STAT5 in mussel hemocytes. Phosphory-

lation of STAT1, STAT3, and STAT5 has been re-

cently demonstrated to play a key role in the hemocyte

activation (Canesi et al., 2003b). STAT proteins are

the only transcription factors known to be activated

by tyrosine phosphorylation: every STAT contains a

conserved tyrosine residue near the C terminus that is


specifically phosphorylated upon activation by receptorassociated Janus tyrosine kinases (Jaks); once phos-

phorylated, they dimerise and translocate from the

cytosol to the nucleus; serine phosphorylation is also

required for maximal transcriptional activity (Horvath,

2000). Each STAT family member shows a distinct

pattern of activation by growth factors and cytokines,

has a unique tissue distribution and can regulate the

transcription of particular genes (Horvath, 2000;Leaman et al., 1996). In mussel hemocytes, E2-induced

STAT3 phosphorylation was prevented by Tamoxifen,

but that of STAT5 was not. Like for MAPKs, different

pathways may lead to activation of different STATs by

estradiol. Our data are in line with those reported in

endothelial cells, where E2-activated ERa and ERbwere able to induce transcriptional activation of

STATs via rapid cytoplasmic transduction pathways(Bjornstrom and Sjoberg, 2002). In that study, the

effect of E2 was prevented by antiestrogens and

required at least three different signal transduction

pathways, including MAPK, Src-kinase, and PI-3K

activation, indicating a relationship between cytoplas-

mic and nuclear actions of the hormone in modulating

gene expression. Cross-talk between the Jak/STAT and

the Raf/MEK/MAPK has been demonstrated inmammalian cells in the action of growth factors and

cytokines (reviewed by Heinrich et al., 2003; Schwartz

et al., 2002) and it has been shown to play a role in

blood cell activation in Drosophila (Luo et al., 2002).

STAT members are involved in the indirect effects

of ERs on non-ERE (estrogen responsive elements)

containing promoters (Segars and Driggers, 2002a).

Cross-talk between steroid- and growth factor-mediated signaling pathways may explain the conver-

gence of the effects of E2 on MAPK and STAT

phosphorylation also in invertebrate cells.

E2 (5 nM) induced progressive changes in hemocyte

morphology, as evaluated by SEM; the main changes

observed in adherent hemocytes were a reduction in the

number and length of cell extensions, degranulation,

progressive rounding, and formation of long filopodia.These effects were prevented by Tamoxifen. In mam-

malian cells, E2-mediated cell shape changes can be

mediated by signaling pathways involving both Ca2þ-and kinase-dependent cascades. In immunocytes, rapid

estrogen-mediated Ca2þ-signaling coupled to nitric ox-

ide release initiates cell rounding and reduction of cell

adherence; these effects are apparently mediated by

membrane-associated estrogen receptors (Stefano andPeter, 2001). In bovine aortic endothelial cells, E2-in-

duced cytoskeletal and morphological changes were

mediated by p38 MAPK activation (Razandi et al.,

2000). In the MCF-7-derived LTED (long term estrogen

deprived) breast cancer cell line, E2 rapidly induced

cell shape changes, with formation of membrane

ruffles, pseudopodia, and ERa membrane translocation.

These effects were mediated by stimulation of Shc–MAPK pathways, and were prevented by antiestrogens

(Song et al., 2002).

The apparent induction of hemocyte degranulation

by E2 suggested by microscopical observations was

confirmed by the increase in extracellular lysozyme ac-

tivity as a marker of lysosomal enzyme release. A similar

rapid, non-genomic action of E2 on the release of pro-

lactin has been described in a subset of lactotrophs inthe rat pituitary (Christian and Morris, 2002). The effect

of E2 on lysosomal function was confirmed by data

obtained on lysosomal membrane stability. The results

demonstrate that a significant concentration-dependent

destabilisation of lysosomal membranes was induced by

E2 at all concentrations tested; such an effect was pre-

vented by Tamoxifen. Another evidence for the rapid

effect of E2 on hemocyte function is given by the resultsof bacterial killing. When E2 (5 nM) was added shortly

(5–10min) before bacterial challenge, the overall im-

mune function of mussel hemocytes (i.e., their bacteri-

cidal activity) was significantly stimulated; however,

prolonged pre-treatment with E2 (60min) was appar-

ently uneffective.

Overall, our results show that E2 affects the function

of mussel hemocytes leading to cell shape changes, re-lease of extracellular hydrolytic enzymes, activation of

the lysosomal machinery, and stimulation of bacterici-

dal activity. All the effects of E2 on the hemocyte func-

tion were rapid, since they were observed from 10 to

30min from estrogen addition. This time-course is

consistent with those of the rapid changes in signaling

components induced by the hormone, and suggests the

possibility that, like in mammalian cells, both Ca2þ- andkinase-mediated signaling may play a role in mediating

the action of E2 on the hemocyte.

Burlando et al. (2002) demonstrated that the hemo-

cyte lysosomal system is a target for the action of E2;

activation of a Ca2þ-dependent PLA2 was shown to be

the main pathway responsible for the effects of E2 on

different lysosomal functions. These effects were ob-

served in hemocytes exposed to 50 nM E2 for up to25min, and a corresponding slow, large, and persistent

increase in [Ca2þ] was observed. However, the [Ca2þ]rise did not fully account for the effect E2 on lysosomal

membrane destabilisation, suggesting that other, Ca2þ-independent mechanisms, may be involved in mediating

this specific effect of estradiol. PLA2 activation plays a

role in mediating the hemocyte bactericidal activity

(Canesi et al., 2002b); therefore, a Ca2þ-dependentPLA2 might be also involved in mediating some of the

effects of E2 observed in our study. However, in our

experimental conditions, the rise in [Ca2þ] observed with

25 nM E2 was much smaller and more rapid than that

reported by Burlando et al. (2002). What is more, lower

E2 concentrations (5 nM) that did not significantly in-

crease cytosolic [Ca2þ], were able to affect both cell


morphology and functions as well as MAPK and STATphosphorylation. This suggests that signaling pathways

other than those involving Ca2þ may play a major role

in mediating the effects of E2 in mussel hemocytes in our

experimental conditions. In particular, activation of ki-

nase cascades may be involved in mediating the rapid

effects of lower E2 concentrations.

This possibility was therefore investigated utilising

specific kinase inhibitors. In mussel hemocytes, p38MAPK activation induced by E. coli and bactericidal

activity were inhibited by the p38 MAPK inhibitor

SB203580 and by the PI3-kinase inhibitor Wortmannin,

whereas the ERK inhibitor PD98059 had no significant

effects (Canesi et al., 2002a,b); these data also suggested

that in mussel hemocytes, like in mammalian cells, PI3-

kinase would lie upstream of p38 MAPK activation.

The effects of kinase inhibitors were evaluated on thelysosomal membrane destabilisation induced by E2.

Lysosomal membrane stability, that is generally con-

sidered as representative of both hemocyte function in

general (Lowe et al., 1995) and of the immune response

in particular (Hauton et al., 2001), was also chosen since

it was significantly affected by E2 at all the concentra-

tions tested. The results clearly show that SB20358

abolished the effect of both 5 and 25 nM E2; PD98059and Wortmannin also had a significant inhibitory effect.

These data indicate that activation of kinase cascades,

p38 MAPK in particular, but also ERK MAPK and

PI3-kinase, may be involved in mediating the effect of E2

in mussel hemocytes, like in mammalian cells (L€osel etal., 2003; Pedram et al., 2002; Segars and Driggers,

2002b). However, it is clear that the transduction path-

ways involved in modulating the hemocyte function maybe very complex and that each effect elicited by different

concentrations of E2 may result from cross-talk between

multiple signaling components. The problem of how the

rapid signaling pathways involving Ca2þ- and kinase-

mediated cascades are integrated in eliciting an array of

cellular responses to E2 is still largely unresolved in

mammalian cells (Hall et al., 2001; L€osel et al., 2003;

Nadal et al., 2001).Overall, our data support the hypothesis that the

rapid signaling used by estradiol is extremely conserved

and may play a crucial role in endocrine–immune in-

teractions in invertebrates. However, we do not have at

present direct evidence for the location and nature of the

receptors responsible for the effects of E2 in mussel he-

mocytes. In Mytilus ganglia, an E2-mediated signaling

pathway at the cell surface coupled to NO productionwas suggested by the fact that membrane impermeant

E2–BSA was as effective as free E2 (Stefano et al.,

2003a). In hemocytes, E2–BSA induced lysosomal

membrane destabilisation and phosphorylation of both

MAPK and STAT members similar to those elicited by

free E2 (data not shown). However, significant effects

were observed at concentrations higher (100 nM) than

those of free E2, even when the E2–BSA preparation wastreated with charcoal/dextran to rule out the effect due

to the presence of free E2 associated to commercial E2–

BSA preparations (Nadal et al., 2001; Pappas et al.,

1995). The lower potency of this compound in binding

the membrane ER (90% of that of estrogen) can be

partly due to the fact that the bulky BSA protein limits

access of E2 to the receptor sites (Morey et al., 1997;

Pappas et al., 1995). Moreover, in mussel hemocytes,part of E2–BSA seems to be rapidly internalised through

rapid phagocytosis (as indicated by preliminary ob-

servations by fluorescence microscopy with E2–BSA-

FITC). Therefore, this approach seems unsuitable to

clearly demonstrate an extracellular action of the hor-

mone in our system.

The response of mussel hemocytes to E2 may be

ascribed to the presence of ER-like forms. Tamoxifenpretreatment prevented some, but not all, the effects of

E2. The antiestrogen ICI 182,780 is generally utilised

together with Tamoxifen to discriminate between the

two receptor forms ERa and ERb. In Mytilus ganglia,

both compounds prevented E2-induced NO production

(Stefano et al., 2003a). In mussel hemocytes the effect

of ICI 182,780 is similar to that of Tamoxifen (data

not shown). However, the molecular mechanisms ofaction of these antiestrogens closely depend on the

receptor type are not completely understood even in

mammalian cells (Barkhem et al., 1998; Hall et al.,

2001; Haynes et al., 2002; Wade et al., 2001). The re-

sults obtained with Tamoxifen in hemocytes are of

difficult interpretation since its specificity towards the

putative ER-like forms present in mussel hemocytes is

unknown.The results of Western blotting with heterologous

anti-ERa–anti-ERb-antibodies revealed the presence of

immunoreactive ERa- and ERb-like proteins in hemo-

cyte protein extracts. Since each of the two antibodies

used is directed against a specific region in the amino-

terminal domain of the human receptor forms ERa and

ERb, respectively, which is the highly divergent domain

between the two mammalian forms, the results indicatethat both ERa- and ERb-like forms are expressed in

mussel hemocytes. On the other hand, only an ERb-likeform was previously detected with the same technique in

mussel ganglionic tissue (Stefano et al., 2003a); more-

over, a 266 bp sequence showing 100% homology with

the human ERb was found to be expressed by this tissue

(Stefano et al., 2003b). ER-like forms highly homolo-

gous to human ERs are also present in the opistho-branch mollusc Aplysia (Thornton et al., 2003);

however, the transcriptional activity of the Aplysia ER

seems to be constitutive, and independent of estrogen

activation. An ancient steroid receptor (AncSR1) which

is shared by other invertebrate groups, may have re-

tained this capacity, that seems to be lost in the Aplysia

lineage (Thornton et al., 2003).


The identification and localisation of the receptor(s)and signaling pathways involved in mediating the effects

of steroids, and estrogens in particular, in different in-

vertebrate groups still represents a largely unexplored

field of research. As underlined by Thornton et al.

(2003), this subject is of particular importance when

considering that most environmental regulations and

testing programs for endocrine disrupters are focused on

vertebrates, and that a much broader range of animaltaxa could be subjected to endocrine disruption by ste-

roid-mimicking chemicals or steroid-blocking com-

pounds in the environment.

References

Barkhem, T., Carlsson, B., Nilsson, Y., Enmark, E., Gustafsson, J.,

Nilsson, S., 1998. Differential response of estrogen receptor a and

estrogen receptor b to partial estrogen agonists/antagonists. Mol.

Pharmacol. 54, 105–112.

Benten, W.P.M., Lieberherr, M., Giese, G., Wunderlich, F., 1998.

Estradiol binding to cell surface raises cytosolic free calcium in T

cells. FEBS Lett. 422, 349–353.

Benten, W.P.M., Stephan, C., Lieberherr, M., Wunderlich, F., 2001.

Estradiol signaling via sequestrable surface receptors. Endocrinol-

ogy 142, 1669–1677.

Bjornstrom, L., Sjoberg, M., 2002. Signal transducers and activators of

transcription as downstream targets on nongenomic estrogen

receptor actions. Mol. Endocrinol. 16, 2202–2214.

Bruce-Keller, A.J., Keeling, J.L., Keller, J.N., Huang, F.F., Camon-

dola, S., Mattson, M.P., 2000. Antiinflammatory effects of estrogen

on microglia activation. Endocrinology 141, 3646–3656.

Burlando, B., Marchi, B., Panfoli, I., Viarengo, A., 2002. Essential role

of Ca2þ-dependent phospholipase A2 in estradiol-induced lyso-

somal activation. Am. J. Physiol. Cell Physiol., C1461–C1468.

Canesi, L., Ciacci, C., Orunesu, M., Gallo, G., 1997. Effects of

epidermal growth factor on isolated digestive gland cells from

mussels (Mytilus galloprovincialis Lam.). Gen. Comp. Endocrinol.

107, 221–228.

Canesi, L., Pruzzo, C., Tarsi, R., Gallo, G., 2001a. Surface interactions

between Escherichia coli and hemocytes of the Mediterranean

mussel Mytilus galloprovincialis Lam. leading to efficient bacterial

clearance. Appl. Environ. Microbiol. 67, 464–468.

Canesi, L., Betti, M., Ciacci, C., Gallo, G., 2001b. Insulin-like effect of

Zinc in Mytilus digestive gland cells: modulation of tyrosine kinase

mediated cell signaling. Gen. Comp. Endocrinol. 122, 60–66.

Canesi, L., Betti, M., Ciacci, C., Scarpato, A., Citterio, B., Pruzzo, C.,

Gallo, G., 2002a. Signaling pathways involved in the physiological

response of mussel hemocytes to bacterial challenge: the role of

stress-activated p38 MAPK. Dev. Comp. Immunol. 26, 325–334.

Canesi, L., Betti, M., Ciacci, C., Scarpato, A., Citterio, B., Pruzzo, C.,

Gallo, G., 2002b. Bacterial killing byMytilus hemocytes as a model

for investigating the signaling pathways involved in mussel immune

defence. Mar. Environ. Res. 54, 547–551.

Canesi, L., Betti, M., Ciacci, C., Citterio, B., Pruzzo, C., Gallo, G.,

2003. Tyrosine kinase-mediated cell signaling in the activation of

Mytilus hemocytes: possible role of STAT-like proteins. Biol. Cell,

in press.

Canesi, L., Ciacci, C., Betti, M., Scarpato, A., Citterio, B., Pruzzo, C.,

Gallo, G., 2003b. Effects of PCB congeners on the immune

function of Mytilus hemocytes: alterations of tyrosine kinase-

mediated cell signaling. Aquat. Toxicol. 63, 293–306.

Christian, H.C., Morris, J.F., 2002. Rapid actions of 17b-oestradiol ona subset of lactotrophs in the rat pituitary. J. Physiol. 539, 557–566.

Chu, F.L.E., La Peyre, J.F., 1989. Effect of environmental factors and

parasitism on hemolymph lysozyme and protein of American

oysters (Crassostrea virginica). J. Invert. Pathol. 54, 224–

232.

De Longcamp, D., Lubet, P., Drosdowsky, M., 1974. The in vitro

biosynthesis of steroids by the gonad of the mussel (Mytilus edulis).

Gen. Comp. Endocrinol. 22, 116–127.

Di Cosmo, A., Di Cristo, C., Paolucci, M., 2002. A estradiol-17beta

receptor in the reproductive system of the female of Octopus

vulgaris: characterization and immunolocalization. Mol. Reprod.

Dev. 61, 367–375.

Falcieri, E., Bassini, A., Pierpaoli, S., Luchetti, F., Zamai, L., Vitale,

M., Guidotti, L., Zauli, G., 2000. Ultrastructural characterization

of maturation, platelet release, and senescence of human cultured

megacaryocytes. Anat. Rec. 253, 90–99.

Grynkiewitz, G., Poenie, M., Tsien, R.Y., 1985. A new generation of

Ca2þ indicators with greatly improved fluorescence properties. J.

Biol. Chem. 260, 3440–3450.

Guo, Z., Krucken, J., Benten, P.M., Wuderlich, F., 2002. Estradiol-

induced nongenomic calcium signaling regulates genotropic signal-

ing in macrophages. J. Biol. Chem. 277, 7044–7050.

Hall, J.M., Couse, J.F., Korach, K.S., 2001. The multifaceted

mechanisms of estradiol and estrogen receptor signaling. J. Biol.

Chem. 276, 36869–36872.

Hauton, C., Hawkins, L.E., Hutchinson, S., 2001. Response of

hemocyte lysosomes to bacterial inoculation in the oysters Ostrea

edulis L. and Crassostrea gigas (Thunberg) and the scallop Pecten

maximus (L.). Fish Shellfish Immunol. 11, 143–153.

Heinrich, P.C., Berhmann, I., Hoam, S., Hermanns, H.M., Muller-

Newen, G., Schaper, R., 2003. Principles of interleukin (IL)-6-type

cytokine signaling and ist regulation. Biochem. J. 374, 1–20.

Haynes, M.P., Li, L., Russel, K.S., Bender, J.R., 2002. Rapid vascular

cell responses to estrogen and membrane receptors. Vasc. Phar-

macol. 38, 99–108.

Horvath, C.M., 2000. STAT proteins and transcriptional responses to

extracellular signals. Trends Biochem. Sci. 25, 496–502.

Kelly, M.J., Levin, E.R., 2001. Rapid actions of plasma membrane

estrogen receptors. Trends Endocrinol. Metab. 12, 152–156.

Leaman, D.W., Leung, S., Li, X., Stark, G.R., 1996. Regulation of

STAT-dependent pathways by growth factors and cytokines.

FASEB J. 10, 1578–1588.

Laemli, U.K., 1970. Cleavage of structural proteins during assembly of

the head bacteriophage T4. Nature 227, 680–685.

L€osel, R.F., Falkestein, E., Feuring, M., Schultz, A., Tillmann, H.-C.,

Rossol-Haseroth, K., Wehling, M., 2003. Non genomic steroid

action: controversies, questions, and answers. Physiol. Rev. 1, 965–

1016.

Lowe, D.M., Fossato, V.U., Depledge, M.H., 1995. Contaminant-

induced lysosomal membrane damage in blood cells of mussels

Mytilus galloprovincialis from the Venice Lagoon: an in vitro study.

Mar. Ecol. Prog. Ser. 129, 189–196.

Luo, H., Rose, P.E., Roberts, T.M., Dearolf, C.R., 2002. The

hopscotch Jak kinase requires the Raf pathway to promote cell

blood differentiation in Drosophila. Mol. Genet. Genom. 267, 57–

63.

Mendelsohn, M.E., 2000. Nongenomic, estrogen receptor-mediated

activation of endothelial nitric oxide synthase. Circ. Res. 87, 956–

960.

Moggs, J.G., Orphanides, G., 2001. Estrogen receptors: orchestrators

of pleiotropic cellular responses. EMBO Rep. 2, 775–781.

Morey, A.K., Pedram, A., Razandi, M., Prins, B.A., Hu, R.-M.,

Bieasiada, E., Levin, E.R., 1997. Estrogen and progesterone inhibit

vascular smooth muscle proliferation. Endocrinology 138, 3330–

3339.

Nadal, A., D�ıaz, M., Valverde, M.A., 2001. The estrogen trinity:

membrane, cytosolic and nuclear effects. News Physiol. Sci. 16,

251–255.


Pappas, T.C., Gametchu, B., Watson, C.S., 1995. Membrane estrogen

receptors identified by multiple antibody labeling and impeded-

ligand binding. FASEB J. 9, 404–410.

Pedram, A., Razandi, M., Aitkenhead, M., Hughes, C.C., Levin, E.R.,

2002. Integration of non genomic and genomic actions of estro-

gens. Membrane-initiated signaling by steroid to transcription and

biology. J. Biol. Chem. 277, 50768–50775.

Razandi, M., Pedram, A., Levin, E.R., 2000. Estrogen signals to the

preservation of endothelial cell form and function. J. Biol. Chem.

276, 38540–38546.

Reis-Henriques, M.A., Le Guellec, D., Remy-Martin, J.P., Adessi,

G.L., 1990. Studies of endogenous steroids from the marine

molluscs Mytilus edulis L. by gas chromatography and mass

spectrometry. Comp. Biochem. Physiol. B 95, 303–309.

Renwrantz, L., 1990. Internal defence system of Mytilus edulis. In:

Stefano, G.B. (Ed.), Neurobiology of Mytilus edulis. Manchester

University, Manchester, pp. 256–275.

Segars, J.H., Driggers, P.H., 2002a. Estrogen action and cytoplasmic

signaling cascades. Part I: membrane-associated signaling com-

plexes. Trends Endocrinol. Metab. 13, 349–354.

Segars, J.H., Driggers, P.H., 2002b. Estrogen action and cytoplasmic

signaling cascades. Part II: the role of growth factors and

phosphorylation in estrogen signaling. Trends Endocrinol. Metab.

13, 422–427.

Song, R.X.D., Mcpherson, R.A., Adam, L., Bao, Y., Shupnik, M.,

Kumar, R., Santen, R.J., 2002. Linkage of rapid estrogen action to

MAPK activation by ERa-Shc association and Shc pathway

activation. Mol. Endocrinol. 16, 116–127.

Stefano, G.B., Cadet, P., Breton, C., Goumon, Y., Prevot, V.,

Dessaint, J.P., Beauvillain, J.-C., Roumier, A.S., Welters, I.,

Salzet, M., 2000. Estradiol-stimulated nitric oxide release in human

granulocytes is dependent on intracellular calcium transients:

evidence of a cell surface estrogen receptor. Blood 95, 3951–3958.

Stefano, G.B., Peter, D., 2001. Cell surface receptors coupled to cNOS

mediate immune and vascular tissue regulation: therapeuthic

implications. Med. Sci. Monit. 7, 1066–1074.

Stefano, G.B., Cadet, P., Mantione, K., Cho, J.J., Jones, D., Zhu, W.,

2003a. Estrogen signaling at the cell surface coupled to nitric oxide

release in Mytilus edulis nervous system. Endocrinology 144, 1234–

1240.

Stefano, G.B., Zhu, W., Mantione, K., Jones, D., Salamon, E., Cho,

J.J., Cadet, P., 2003b. 17-b estradiol downregulates ganglionic

microglial cells via nitric oxide release: presence of an estrogen

receptor b transcript. Neuroendocrinol. Lett. 24, 130–136.

Schwartz, J., Huo, J.S., Piwien-Pilipuk, G., 2002. Growth hormone

regulated gene expression. Minerva Endocrinol. 27, 231–241.

Thornton, J.W., Need, E., Crews, D., 2003. Resurrecting the ancestral

steroid receptor: ancient origin of estrogen signaling. Science 301,

1714–1717.

Tosti, E., Di Cosmo, A., Cuomo, A., Di Cristo, C., Gragnaniello, G.,

2001. Progesterone induces activation in Octopus vulgaris sperma-

tozoa. Mol. Reprod. Dev. 59, 97–105.

Towbin, H., Stahelin, T., Gordon, J., 1979. Electrophoretic transfer of

protein from polyacrylamide gels to nitrocellulose sheets: proce-

dure and some applications. Proc. Natl. Acad. Sci. USA. 76, 4350–

4354.

Vegeto, E., Bonincontro, C., Pollio, G., Sala, A., Viappiani, S., Nardi,

F., Brusarelli, A., Viviani, B.., Ciana, P., Maggi, A., 2001. Estrogen

prevents the lipolysaccharide-induced inflammatory response in

microglia. J. Neurosci. 21, 1809–1818.

Wade, C.B., Robinson, S., Shapiro, R.A., Dorsa, D.M., 2001.

Estrogen receptor ERa and ERb exhibit unique pharmacologic

properties when coupled to activation of the mitogen-activated

protein kinase pathway. Endocrinology 142, 2336–2342.

Wong, C.-W., McNally, C., Nickbrag, E., Komm, B.S., Cheskis, B.J.,

2002. Estrogen receptor-interacting protein that modulates its

nongenomic activity-crosstalk with Src/Erk phosphorylation cas-

cade. Proc. Natl. Acad. Sci. USA 99, 14783–14788.

Zhu, W., Mantione, K., Jones, D., Salamon, E., Cho, J.J., Cadet, P.,

Stefano, G.B., 2003. The presence of 17-b estradiol in Mytilus

edulis gonadal tissues: evidence for estradiol isoforms. Neuroen-

docrinol. Lett. 24, 136–140.

Rapid effects of 17β-estradiol on cell signaling and function of Mytilus hemocytes

Documents

Transcript of Rapid effects of 17β-estradiol on cell signaling and function of Mytilus hemocytes