Life Sciences 77 (2005) 3117–3126

www.elsevier.com/locate/lifescie

Extracellular inosine modulates ERK 1/2 and p38 phosphorylation

in cultured Sertoli cells: Possible participation in

TNF-alpha modulation of ERK 1/2

Luiz F. Souza a, Ana P. Horn a, Daniel P. Gelain a, Fernanda R. Jardim a,

Guido Lenz b, Elena A. Bernard a,TaDepartamento de Bioquımica, Instituto de Ciencias Basicas da Saude, Universidade Federal do Rio Grande do Sul,

(ICBS-UFRGS), Rua Ramiro Barcelos, 2600 anexo, CEP 90035-003, Porto Alegre, RS, BrazilbDepartamento de Biofısica, Instituto de Biociencias, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

Received 13 December 2004; accepted 25 May 2005

Abstract

Extracellular ATP and adenosine modulation of MAPKs is well described in different cells types, but few studies

have addressed the effects of extracellular inosine on these kinases. Previous results showed that hydrogen peroxide

and TNF-a increase extracellular inosine concentration in cultured Sertoli cells and this nucleoside protects Sertoli

cells against hydrogen peroxide induced damage and participates in TNF-a induced nitric oxide production. In view

of the fact that MAPKs are key mediators of the cellular response to a large variety of stimuli, we investigated the

effect of extracellular inosine on the phosphorylation of ERK 1/2 and p38 MAPKs in cultured Sertoli cells. The

involvement of this nucleoside in the activation of ERK 1/2 by TNF-a was also investigated. Inosine and the selective

A1 adenosine receptor agonist R-PIA increases the phosphorylation of ERK 1/2 and p38, and this was blocked by the

selective A1 adenosine receptors antagonists, CPT and DPCPX. These antagonists also inhibited TNF-a increase in

the phosphorylation of ERK 1/2. TNF-a also rapidly augmented extracellular inosine concentration in cultured

Sertoli cells. These results show that extracellular inosine modulates ERK 1/2 and p38 in cultured Sertoli cells,

possible troughA1 adenosine receptor activation. This nucleoside also participates in TNF-a modulation of ERK 1/2.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Extracellular inosine; TNF-a; MAPKs; ERK; p38; Sertoli cells

0024-3205/$ -

doi:10.1016/j.l

T Correspond

E-mail add

see front matter D 2005 Elsevier Inc. All rights reserved.

fs.2005.05.049

ing author. Fax: +55 51 3316 5535/5540.

ress: elenbern@ufrgs.br (E.A. Bernard).

L.F. Souza et al. / Life Sciences 77 (2005) 3117–31263118

Introduction

Extracellular purines are important regulators of many cellular functions. They activate P1 and P2

cell surface receptors. P1 receptors are mainly responsive to adenosine and P2 receptors to

extracellular nucleotides. P1 or adenosine receptors are divided into four subtypes: A1, A2A, A2B and

A3, which are all G protein-coupled receptors (Fredholm et al., 2001). P2 receptors are divided into

two distinct families: the P2X ligand-gated ionotropic channel family and the P2Y metabotropic G

protein-coupled family (Dubyak and El-Moatassim, 1993; Burnstock and Williams, 2000).

Extracellular inosine, the degradation product of adenosine deamination, was initially considered an

inactive metabolite of purine catabolism; however, recent publications have suggested its role in the

regulation of various processes. In mast cells, inosine stimulates degranulation via A3 adenosine

receptors, leading to increased vasopermeability in mice (Jin et al., 1997; Tilley et al., 2000). A

protective role has been assigned to inosine in many cell and animal models. In endotoxemia (Hasko

et al., 2000) and acute lung injury (Liaudet et al., 2002), inosine reduces the production of

inflammatory cytokines and increases the production of anti-inflammatory cytokines by macrophages,

improving the survival of mice. Extracellular inosine also prevents glial cell death during glucose

deprivation (Jurkowitz et al., 1998; Haun et al., 1996) and improves renal function in ischemia

(Fernando et al., 1976; De Rougemont et al., 1982). In addition, inosine protects RAW 264.7

macrophages from cell death induced by reactive oxygen species, by inhibiting poly (ADP-ribose)

polimerase activity (Virag and Szabo, 2001).

Sertoli cells are the somatic cells of seminiferous tubules, responsible for energy supply, mechanical

support and hormonal control of spermatogenesis. These cells express distinct purinoreceptors: A1

adenosine receptors, that are related to the inhibition of follicle stimulating hormone (FSH) increased

cAMP concentration (Monaco and Conti, 1986; Rivkees, 1994; Monaco et al., 1998); P2Y2 ATP

receptors, which regulate phosphatidyl-inositol turnover and calcium mobilization, inhibit cAMP

accumulation (Filippini et al., 1994) and increase c-glutamyl-transpeptidase and transferrin secretion

(Meroni et al., 1998); and ionotropic P2X(2,3 and 7) ATP receptors that are differentially present at

different stages of the seminiferous epithelium cycle (Glass et al., 2001). Previous works of our group

reported that Sertoli cells present different ectonucleotidases activities responsible for extracellular

purine metabolism (Casali et al., 2001) and that these cells secrete extracellular nucleotides and their

metabolites, including inosine (Gelain et al., 2003). This molecule has been shown to be involved in

TNF-a modulation of nitric oxide production (unpublished results) and protection against lipoperoxida-

tion and death induced by H2O2 (Gelain et al., 2004).

Mitogen-activated protein kinases (MAPKs) are serine/threonine kinases involved in the regulation

of cell cycle progression, proliferation, differentiation and death. The MAPK family consists of three

main subgroups: the extracellular signal-regulated kinases (ERKs), the stress-activated protein kinases

(SAPKs), also known as c-jun N terminal kinases (JNKs), and the p38 family. The modulation of

MAPKs by extracellular adenosine acting on any of the adenosine receptors (A1, A2A, A2B and A3)

through different mechanisms and in different cells is well described (revised in Schulte and

Fredholm, 2003). Extracellular ATP also modulates MAPKs through P2 receptors (Lenz et al., 2000;

Sellers et al., 2001; Shigemoto-Mogami et al., 2001; Bradford and Soltoff, 2002; Gendron et al.,

2003). However, the action of extracellular inosine on these kinases is not reported. In Sertoli cells,

ERK-dependent signalling is stage-specifically modulated by FSH during primary Sertoli cell

maturation (Crepieux et al., 2001) and transforming growth factor b 3 regulates various Sertoli

L.F. Souza et al. / Life Sciences 77 (2005) 3117–3126 3119

functions via MAPKs. (Lui et al., 2003). Furthermore, TNF-a activates ERK 1/2, p38 and JNK,

regulating interleukine-6 production, via p38, and intercellular adhesion molecule-1 expression, via

JNK (De Cesaris et al., 1998, 1999).

We investigated the modulation by extracellular inosine of ERK 1/2 and p38 MAPKs in cultured

Sertoli cells and studied the possible receptor involved. The role of extracellular inosine in the TNF-amodulation of ERK 1/2 was also investigated.

Materials and methods

Materials and animals

DMEM/F-12 medium was purchased from Gibco-BRL Life Technologies (Rockville, MD, USA).

Anti phospho-ERK 1/2 was purchased from Upstate (USA) and anti-phospho-p38 antibodies from

Promega (USA). TNF-a, R-PIA, CPT, DPCPX, MRS1220, inosine and all other drugs, reagents and

enzymes were purchased from Sigma Chemicals (St. Louis, MO, USA). Pregnant Wistar rats were

housed individually in plexiglass cages. Litters were restricted to eight pups each. The animals were

maintained on a 12 h light/dark cycle at a constant temperature of 23 8C, with free access to commercial

food and water. Male immature rats (18 days old) were killed by ether inhalation.

Isolation and culture of Sertoli cells

Sertoli cells were isolated as previously described (Rocha et al., 1997), following the method of Tung

and Fritz (Tung and Fritz, 1984) with modifications. Testes of immature 18 day-old Wistar rats were

removed, decapsulated and digested enzymatically with trypsin and deoxyribonuclease for 30 min at 34

8C, and then centrifuged at 750 g for 5 min. The pellet was washed with soybean trypsin inhibitor,

centrifuged and incubated with collagenase and hyaluronidase for 30 min at 34 8C followed by a

centrifugation for 10 min at 40 g. After this, the pellet was incubated with hyaluronidase for 30 min at 34

8C and then centrifuged at 40 g for 10 min. After counting, Sertoli cells were plated at a density of

3�105 cells/cm2 in DMEM:F12 (1 :1, low glucose) 1% FBS, supplemented with sodium bicarbonate,

HEPES and gentamicin, and maintained in a humidified atmosphere of 5% CO2 at 34 8C for 72 h. The

medium was changed every 24 h. Sertoli cell cultures were estimated to be 90–95% pure, as assessed by

alkaline phosphatase assay.

Western blots: ERK 1/2 and p38 phosphorylation

After 72 h, cultured Sertoli cells were incubated with or without inosine (0.01–1 mM), R-PIA(100

AM) or TNF-a 500 U/ml (25 ng/ml) for 5, 10 or 15 min. When present, adenosine receptor antagonists

were added 5 min before inosine, R-PIA or TNF-a. At the end of incubation, cultured Sertoli cells were

harvested with a cell scraper, centrifuged and lysed in 1% Triton/PBS. Equal amounts of soluble protein

(50 Ag) were run on SDS-PAGE 12% T, transferred to nitrocellulose and reacted with specific antibodies

for phosphorylated forms of ERK and p38. Peroxidase tagged secondary antibodies (anti immuno-

globulin) were used and the detection was performed using the enhanced chemiluminescence system

(ECL). Immunoblots were quantified by scanning of films followed by densitometric analysis. All

L.F. Souza et al. / Life Sciences 77 (2005) 3117–31263120

results were representative of three independent experiments and only statistically significant results are

reported in text.

Extracellular purine nucleotide assay

To investigate purine nucleotides and their metabolites in the incubation medium, we used the method

described by Cunha et al. (Cunha et al., 1989). Cultured Sertoli cells were gently washed three times to

remove remnants of medium and any dead or dying cells, and then incubated with HBSS (without

phenol-red, 15 mM HEPES) in the presence or absence of TNF-a 500 U/ml (25 ng/ml) for 5 min in 5%

CO2 at 34 8C. After incubation the medium was removed and centrifuged to eliminate debris. Samples

were evaporated in a vacuum centrifuge (�61 8C) and resuspended in 10% of the original volume.

Purines were determined by a reverse-phase HPLC system equipped with a C-18 column

(SupelcosilTM, SupelcoR, 25 cm�4.6 mm) and UV detector. The elution program consisted of: 10

min with 96% buffer A (KH2P04 100 mM, pH 6.5) and 4% buffer B (buffer A plus methanol 30%),

followed by a 5 min linear gradient up to 50% of buffer B, held for 10 min, at a flow rate of 1.25 ml/min

(UV absorption of 245 nm). Standard solutions were used to identify the substances detected.

Protein quantification

All the results were standardized against protein content, determined as described by Lowry et al.

(Lowry et al., 1951).

Results

We have previously described that Sertoli cell cultures secrete inosine, leading to the accumulation of

this purine in the extracellular medium (Gelain et al., 2003); this was induced by TNF-a (unpublished

results) and hydrogen peroxide (Gelain et al., 2004). MAPK cascades are involved in the response to

various stimuli, such as growth factors, cytokines and oxidative stress (Schulte and Fredholm, 2003;

Lander, 1997). To investigate whether extracellular inosine could modulate the activation of ERK 1/2 ,

the phosphorylation of this MAPK in cultured Sertoli cells treated with inosine was studied.

Extracellular inosine increased ERK 1/2 phosphorylation with maximal effect in 5 min, followed by

a slow dephosphorylation (Fig. 1A). The increase in phosphorylation of ERK 1/2 was seen at a

concentration of 0.1 mM inosine and was maximal at 1 mM (data not shown).

Fig. 1. Time dependence of extracellular inosine modulation of ERK 1/2 phosphorylation. After 72 h culture, Sertoli cells were

incubated for the indicated time periods in the presence of extracellular inosine 1 mM. Equal loading of cell lysates (50 Ag) wereresolved on SDS/PAGE and transferred to nytrocellulose before being probed with specific anti phospho-ERK 1/2.

Representative Western blot of three independent experiments is shown.

Fig. 2. Effect of selective A1 adenosine receptors antagonists on extracellular inosine and A1 adenosine receptor agonist

modulation of ERK 1/2 phosphorylation. After 72 h culture, Sertoli cells were incubated for 5 min in the presence or not of CPT

100 AM (A) or DPCPX 100 AM (B) (A1 antagonists) before incubation for 5 min in the presence or absence of extracellular

inosine 1mM or R-PIA 100 AM (A1 agonist). Cell lysates (50 Ag) were resolved on SDS/PAGE and transferred to

nytrocellulose before being probed with specific anti phospho-ERK 1/2. Representatives Western blots of three independent

experiments are shown.

L.F. Souza et al. / Life Sciences 77 (2005) 3117–3126 3121

A1 adenosine receptor is present in Sertoli cells (Monaco and Conti, 1986; Rivkees, 1994). The

phosphorylation of ERK 1/2 induced by inosine or a selective A1 adenosine receptor agonist, R-PIA

((R)-N6-(2-phenylisopropyl)-adenosine), was inhibited by the specific A1 adenosine receptor

antagonists, CPT (cyclopentil-teophyline) and DPCPX (8-cyclopentil-1,3-dipropylxanthine) (Fig. 2).

Extracellular inosine activates A3 adenosine receptors in mast cells (Jin et al., 1997; Tilley et al., 2000).

Rivkees (1994) reported a low expression of this receptor mRNA in Sertoli cells and suggested that this

was due to contamination with germ cells. To avoid any effect of inosine on A3 adenosine receptors,

Sertoli cells were incubated with inosine or R-PIA in the presence of an A3 adenosine receptor

antagonist (MRS 1220 100 AM), and this did not alter inosine or R-PIA induced ERK phosphorylation

(data not shown).

TNF-a is said to activate ERK 1/2 in cultured Sertoli cells (De Cesaris et al., 1998) and our

unpublished results show that this cytokine increases extracellular inosine concentrations in these cells

after prolonged incubation. In order to show whether the secretion of inosine plays a role in the

activation of ERK 1/2 by TNF-a, we measured the concentration of extracellular inosine; this was

Fig. 3. TNF-a modulation of extracellular inosine concentration. After 72 hours culture, Sertoli cells were incubated for 5 min

in the presence of TNF-a 500 U/ml (25 ng/ml). Extracellular inosine in the incubation medium was measured by HPLC. Results

are presented as meanFS.E.M for at least three dishes and are representative of three independent experiments. *p b0.05

respectively to control, ANOVA/Student–Newman Keuls post-hoc.

Fig. 4. Effect of selective A1 adenosine receptors antagonists on TNF-a modulation of ERK 1/2 phosphorylation. After 72

h culture, Sertoli cells were incubated for 5 min in the presence or absence of CPT 100 AM or DPCPX 100 AM (A1 antagonists)

before incubation for 15 min in the presence of TNF-a 500 U/ml (25 ng/ml). Cell lysates (50 Ag) were resolved on SDS/PAGE

and transferred to nytrocellulose before being probed with specific anti phospho-ERK 1/2. Representative Western blot of three

independent experiments is shown.

L.F. Souza et al. / Life Sciences 77 (2005) 3117–31263122

increased after only 5 min of incubation (Fig. 3). To demonstrate the participation of this nucleoside in

cytokine induced phosphorylation of ERK 1/2, Sertoli cells were incubated with TNF-a in the presence

of the selective A1 antagonists CPT and DPCPX, which completely abbrogated the effect of the cytokine

on ERK 1/2 phosphorylation (Fig. 4).

As well as ERK 1/2, inosine also activated p38 kinase in a time dependent way, with maximal effect

at 5 min (Fig. 5A). The selective antagonist CPT inhibited the extracellular inosine effect on p38

phosphorylation (Fig. 5B).

Discussion

ERK 1/2 and p38 phosphorylation in cultured Sertoli cells are modulated by extracellular inosine.

Adenosine modulation of MAPKs via all four adenosine receptors is demonstrated. These effects have

been studied in cells that usually express adenosine receptors and in cells stably transfected with

adenosine receptors (Schulte and Fredholm, 2003). It has also been shown that extracellular inosine

Fig. 5. Extracellular inosine modulation of p38 phosphorylation and effect of selective A1 adenosine receptors antagonist. (A)

After 72 h culture, Sertoli cells were incubated for the indicated time periods in the presence of extracellular inosine 1 mM. (B)

After 72 h culture, Sertoli cells were incubated for 5 min in the presence or not of CPT 100 AM (A1 antagonist) before

incubation for 5 min in the presence of extracellular inosine 1 mM. Cell lysates (50 Ag) were resolved on SDS/PAGE and

transferred to nytrocellulose before being probed with specific anti phospho-p38. Representatives Western blots of three

independent experiments are shown.

L.F. Souza et al. / Life Sciences 77 (2005) 3117–3126 3123

activates A3 adenosine receptors in mast cells (Jin et al., 1997; Tilley et al., 2000); however, in Sertoli

cells, inosine activated ERK 1/2 phosphorylation was not prevented by an A3 adenosine receptor

antagonist (MRS1220). The inhibititory effect of selective A1 antagonists, CPT and DPCPX, on

extracellular inosine modulation of ERK 1/2 and p38 phosphorylation, in addition to the activation of

these kinases by the A1 selective agonist R-PIA, suggests that extracellular inosine could be activating

the A1 adenosine receptor in Sertoli cells. Taking into account that in mast cells the binding of

extracellular inosine to this receptor is negligible (Jin et al., 1997), this could suggest that the A1

adenosine receptor expressed by Sertoli cells has a different affinity for extracellular inosine than the

receptor present on mast cells.

We have previously demonstrated the increase of extracellular inosine by hydrogen peroxide and

shown that this nucleoside protects Sertoli cells against oxidative stress-induced damage (Gelain et al.,

2004). The activation of ERK 1/2 is involved in the protection against oxidative induced cell death in

cAMP potentiation of noradrenaline neuroprotection on dopaminergic neurons (Troadec et al., 2002),

estrogen neuroprotection (Mize et al., 2003) and calcitonin gene-related peptide protection in cultured

smooth muscle cells (Schaeffer et al., 2003). In Sertoli cells, the inosine-stimulated phosphorylation of

ERK 1/2, described in this paper, could be involved in the protection against hydrogen peroxide

previously cited. Oxidative stress modulates MAPKs (Lander, 1997), suggesting that extracellular

inosine could participate in hydrogen peroxide modulation of Sertoli cell functions.

In the testis, TNF-a is produced, under physiological conditions, mainly by round spermatids (De et

al., 1993), and exerts a regulatory function on spermatogenesis. However, in inflammatory responses,

interstitial macrophages can account for considerable production of this cytokine (Xiong and Hales,

1993; Moore and Hutson, 1994). Sertoli cells express both TNF-a receptors (Mauduit et al., 1996; De

Cesaris et al., 1999) and this cytokine modulates diverse functions of these cells related to the

maintenance of spermatogenesis, including lactate production (Nehar et al., 1997) and interleukin-6

(Riccioli et al., 1995), inhibin (Magueresse-Battistoni et al., 1995), transferrin (Sigillo et al., 1999) and

insulin-like growth factor binding protein 3 expression (Besset et al., 1996). TNF-a also modulates

various FSH actions (Mauduit et al., 1993), the Fas-ligand system (Pentikainen et al., 2001) and tight

junction dynamics (Siu et al., 2003) in Sertoli cells.

Our unpublished results show that prolonged TNF-a treatment increases extracellular inosine

content in cultured Sertoli cells. It was also shown that this increase is related to increased nitric oxide

production. In the current article, we show that the TNF-a-induced increase in extracellular inosine

content is very rapid, indicating that this nucleoside is acting as a mediator in ERK 1/2 modulation.

This is supported by the results with selective A1 adenosine receptor antagonists CPT and DPCPX,

which inhibited cytokine stimulated ERK1/2 phosphorylation. Miki and Eddy (2002) recently reported

that TNF-R1 exhibits ATPase activity that is involved in the regulation of the aggregation of this

receptor when activated by TNF-a. This ATPase activity could lead to alteration in the intracellular

purine content when TNF-a binds to TNF-R1, which, via equilibrative nucleotide/nucleoside

transporters, could alter the extracellular purine level. No alteration of extracellular adenosine

concentration was observed in TNF-a treated Sertoli cells, which indicates that this nucleoside is not

involved.

In the present work, we have shown that extracellular inosine increases ERK 1/2 and p38

phosphorylation in Sertoli cells, possibly through A1 adenosine receptor activation. TNF-a also rapidly

increases the extracellular inosine concentration and A1 antagonists block TNF-a-induced phosphor-

ylation of ERK 1/2, suggesting that extracellular inosine plays a role in cytokine signalling.

L.F. Souza et al. / Life Sciences 77 (2005) 3117–31263124

Acknowledgements

This work was supported by CAPES, CNPq, FAPERGS and PROPESQ/UFRGS.

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