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Characterization of signaling pathways leading to Fas expression induced by TNF-: pivotal role of NF-B

Donatella Starace^*, Anna Riccioli^*, Alessio D’Alessio^*, Claudia Giampietri^*, Simonetta Petrungaro^*, Roberta Galli^*, Antonio Filippini^*, Elio Ziparo^* and Paola De Cesaris^,1

^* Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Histology and Medical Embryology, University of Rome "La Sapienza," Rome, Italy; and
Department of Experimental Medicine, University of L’Aquila, L’Aquila, Italy

¹Correspondence: Department of Histology and Medical Embryology, University of Rome "La Sapienza," 00161 Rome, Italy. E-mail: paola.decesaris{at}uniroma1.it

SPECIFIC AIMS

TNF- is known to induce strong up-regulation of Fas expression in mouse Sertoli cell cultures, leading to their apoptosis triggered by effector FasL-bearing cells. This mechanism may be involved in the pathogenesis of autoimmune diseases such as autoimmune orchitis. The aim of the study was to investigate the signal transduction mechanisms involved in Fas induction.

PRINCIPAL FINDINGS

1. p38 and ERKs are not involved in Fas up-regulation
p38, ERKs, and JNK are rapidly activated by TNF- in Sertoli cells. By Western blot analysis, we provide evidence that pretreatment of Sertoli cells with specific inhibitors of ERKs and p38 (namely, U0126 and SB203580) at all concentrations tested was ineffective in reducing Fas induction by TNF-. These data demonstrate that neither ERKs nor p38 is involved in regulating Fas expression.

2. JNK is not involved in Fas up-regulation
To inhibit JNK, we pretreated Sertoli cells with DMAP, which was effective in inhibiting JNK and Fas induction, as demonstrated by Western and Northern blot and by flow cytometric analysis. Since the mechanism of action of DMAP has not been defined, we used more specific JNK inhibitors to confirm these data. Cells were pretreated with SP600125 or D-JNKI1; both inhibitors inhibited JNK activation, but failed to inhibit Fas induction, showing that JNK is not involved in the TNF--induced Fas expression (Fig. 1 ). We therefore hypothesize that the inhibitory effect of DMAP on Fas up-regulation might depend on its action on other signaling pathways.

View larger version (30K): [in this window] [in a new window]   Figure 1. Inhibition of JNK by D-JNKI1 does not affect Fas expression induced by TNF-. Sertoli cells were pretreated with D-JNKI1 (2 µM) or control peptide D-TAT (2 µM) for 2 h before stimulation with 20 ng/mL TNF- for 30 min. The effect of D-JNKI1 on JNK activity was evaluated by JNK kinase assay. The Western blot was probed with a phospho-c-Jun antibody (A). After pretreatment with D-JNKI1 or D-TAT, Sertoli cells were stimulated with 20 ng/mL TNF- for 20 h and whole cell extracts (50 µg) were subjected to Western blot analysis using anti-Fas polyclonal Ab (M-20) (B). This Western blot is representative of 3 independent experiments.

3. Fas expression is regulated by NF-B
TNF- induces IB degradation and subsequent NF-B activation. Using the proteasome inhibitor lactacystin (LC) to inhibit IB degradation and NF-B activation, we show that NF-B is relevant for the regulation of Fas induction. In fact, LC inhibited Fas expression in a dose dependent fashion at both the mRNA and the protein level (Fig. 2 ). We show that DMAP pretreatment inhibits not only JNK but also IB phosphorylation, as assessed by Western blot analysis using a phospho-specific anti IB antibody. DMAP inhibits NF-B activation as demonstrated by a NF-B Luc reporter gene assay. We therefore hypothesize that the inhibitory effect of DMAP on Fas depends on its action on NF-B and not on JNK. Different cross-talk mechanisms between NF-B and JNK have been described. Some papers assess a synergic action between NF-B and JNK; others describe an antagonizing effect of NF-B on JNK. It has been demonstrated that once activated, NF-B is able to inhibit the JNK cascade. We investigated whether this mechanism might also be active in our model. Our data show that JNK and NF-B are persistently and simultaneously activated by TNF-. We can then hypothesize that activated NF-B is not able to inhibit JNK. On the other hand, in conditions of NF-B inhibition, increased JNK phosphorylation is detectable in cells treated with LC plus TNF-, but we believe this independent of NF-B down-regulation, since it is also induced by LC alone (Fig. 2) .

View larger version (46K): [in this window] [in a new window]   Figure 2. Lactacystin inhibits TNF--induced Fas expression. Sertoli cells were pretreated for 2 h with lactacystin (LC) and treated with 20 ng/mL TNF- for 20 h. Whole extracts (50 µg) were subjected to Western blot analysis using anti-Fas, anti-phospho-specific JNK, and anti-JNK1 polyclonal antibodies. The blot was incubated with anti--tubulin Ab as control of equal amount of protein loaded. Representative of 3 independent experiments (A). Sertoli cells were pretreated for 2 h with lactacystin (LC), then treated with 20 ng/mL TNF- for 20 h. Total RNA (20 µg/lane) was analyzed by Northern blotting. Nylon filter was hybridized with 32P-labeled mouse Fas cDNA probe. The integrity and equal loading of RNA were ascertained by ethidium bromide staining of the gel before transfer (B, lower panel). A representative blotting of 3 independent experiments is shown.

CONCLUSIONS AND SIGNIFICANCE

The Fas/FasL system is considered one of the central mechanisms in the homeostasis of immune response. Fas expression requires strict regulation to avoid indiscriminate cell death, since elevated Fas levels are associated with pathological complications in autoimmune diseases. The testis is known to be a major source of FasL in the body but, until now, the role of the Fas system in this organ has not been clearly elucidated. We have demonstrated that TNF- induces a strong up-regulation of Fas expression in mouse Sertoli cell cultures, leading to their apoptosis triggered by effector FasL-bearing cells. We have also shown that TNF- increases the surface expression of the adhesion molecules ICAM-1 and VCAM-1 on Sertoli cells, enhancing the binding of lymphocytes to Sertoli cells. A strong proinflammatory role of TNF- in the testis is demonstrated by its ability to increase IL-6 production by Sertoli cells. It has been demonstrated that TNF- plays a central role in the pathogenesis of experimental autoimmune orchitis. All together, these data suggest that increased Fas expression on the cell surface might be a key event in the pathogenesis of autoimmune orchitis by inducing leakage of the blood-tubular barrier as a consequence of Sertoli cell apoptosis.

The present paper sheds new insight on signal transduction mechanisms involved in the up-regulation of Fas induced by TNF- in mouse primary cultured Sertoli cells. We show that NF-B plays a central role in Fas induction whereas p38, ERKs, and JNK are not involved. It has been demonstrated that the murine Fas promoter contains AP-1 and NF-B binding sites and that AP-1 sites are not sufficient for TNF--induced Fas expression, because deletion of AP-1 sites in the Fas promoter did not affect reporter activity in NIH-3T3 fibroblasts. This might agree with our data showing that JNK, the upstream kinase of c-Jun, a member of the AP-1 complex, is not involved in the regulation of TNF--induced Fas expression. A collaborative action between NF-B and JNK has been demonstrated on the transcriptional induction of several genes involved in inflammation and apoptosis. Although in our model JNK and NF-B are both activated, our findings provideevidence that only NF-B is involved in the regulation of Fas expression; JNK seems not to be essential. In different models the IB/NF-B system plays an important role in regulating several key molecules involved in inflammation and in apoptosis. Our findings obtained in primary cultured of differentiated Sertoli cells corroborate data from other groups demonstrating that NF-B plays a pivotal role in the regulation of Fas expression. Also in agreement with a major role played by NF-B vs. JNK in regulating Fas expression is that, in the presence of LC (i.e., in conditions of NF-B inhibition and Fas down-regulation), JNK phosphorylation is high. That proteasome inhibitors activate JNK has been described, but the mechanisms involved are still under investigation.

Understanding the molecular mechanisms activated by cytokines could be relevant to the pathogenesis of autoimmune disorders in immune privileged districts of the body and may contribute to the discovery of new targets for a pharmacological strategy aimed to down-regulate key molecules involved in inflammatory process as well as in apoptosis.

View larger version (13K): [in this window] [in a new window]   Figure 3. Schematic diagram. TNF- leads to the activation of ERKs, p38, JNK, and NF-B. By using specific inhibitors for each pathway (U0126, SB203580, DJNKI-1 and lactacystin), we demonstrate that NF-B is involved in Fas up-regulation. More studies are needed to clarify the molecular mechanism of DMAP that inhibits JNK/NF-B and Fas expression. Dotted arrows: hypothetical step of inhibition.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2726fje;

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