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INTRODUCTION |
TNF-
1 has long been
known to be a potent mediator of inflammation (1). Even though much
information is known about the TNF-
receptors and how this cytokine
interacts with them, there is limited knowledge regarding the signal
transduction mechanisms involved (2).
We have previously demonstrated that cultured mouse Sertoli cells
treated with proinflammatory cytokines, including TNF-
, undergo an
increase in surface expression of adhesion molecules (ICAM-1 and
VCAM-1) and IL-6 production (3). IL-6 is one of the most potent
cytokines known to activate T and B lymphocytes (4, 5), and the
deregulation of IL-6 gene expression may be involved in the
pathogenesis of autoimmune diseases (6-8). The response of Sertoli
cells to inflammatory factors could therefore contribute to the
activation of lymphocytes and to their migration through the
blood-tubular barrier to reach autoantigenic germ cells, resulting in
the insurgence of autoimmune disorders (3, 9).
In the testis, TNF-
is known to be produced by germ cells (round
spermatids) (10) and to affect Sertoli cell activity (11) by binding to
the p55 receptor (12). However, little definitive data have come forth
delineating the post-receptor molecular events. TNF receptors do not
contain an intrinsic kinase domain, and recently, the involvement of
serine/threonine protein kinases, including the so-called
mitogen-activated protein kinases (MAPKs), has been proposed by a
number of investigators (13-17). MAPKs form a large family of
serine/threonine protein kinases activated by separate cascades and are
important mediators of signal transduction from the cell surface to the
nucleus. In mammalian cells, three distinct and parallel MAPK cascades
have been identified: p42/p44 MAPKs (18, 19), p38 (20-23), and
JNK/SAPK (24, 25). The activation of p42/p44 MAPKs constitutes a
crucial step in the pathway mediating cell proliferation in response to
growth factors (26-28), whereas p38 kinase and JNK/SAPK mediate
signals in response to cytokines and environmental stress such as heat
shock, osmotic stress, and UV light (20-22, 24, 25, 29). The fact that
these biological responses are distinct implies that the specificity of
external stimuli has to be maintained throughout each of the cascades. This specificity can result from selective phosphorylation (hence activation) operated by an upstream kinase (MEK1
(MAPK/extracellular signal-regulated kinase
kinase) or MEK2 for p42/p44 MAPKs, MKK3/MKK6 (MAPK kinase) for p38, and
MKK4/MKK7 for JNK/SAPK). To elucidate the biological function of the
different MAPKs and/or p38, PD98059 and SB203580 have been used as
specific inhibitors of MEK1 and of the activity of p38, respectively
(recently reviewed in Ref. 30). In this report, we demonstrate that, in
mouse Sertoli cells, TNF-
induces IL-6 production and ICAM-1/VCAM-1
expression by the simultaneous activation of two distinct signal
transduction pathways, p38 and JNK/SAPK, respectively.
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EXPERIMENTAL PROCEDURES |
Materials--
DNase, collagenase, recombinant murine TNF-
,
and murine IFN-
were purchased from Boehringer Mannheim (Mannheim,
Germany). Trypsin was from Difco. The pyridinylimidazole SB203580 was
from SmithKline Beecham and was dissolved in Me2SO. PD98059
was purchased from New England Biolabs Inc. and was dissolved in
Me2SO.
Sertoli Cell Cultures--
Sertoli cells were prepared from CD1
mice as described previously (31). Briefly, testes from 15-day-old
animals were sequentially digested for 20 min, first with Hanks'
solution containing 0.25% trypsin + 10 µg/ml DNase and then with
Hanks' solution supplemented with 0.1% collagenase + 10 µg/ml
DNase, to remove interstitial tissue and peritubular cells. Fragments
of seminiferous epithelium, mainly composed of Sertoli cells, were
cultured at 32 °C in 95% air and 5% CO2 in serum-free
minimum Eagle's medium (Gibco-BRL, Paisley, Scotland). After 3 days,
Sertoli cell monolayers were incubated at room temperature with 20 mM Tris-HCl buffer (pH 7.4) for 2 min to remove residual
germ cells present in the culture (32). Sertoli cell cultures were
routinely checked for possible contamination by macrophages and
peritubular myoid cells by indirect immunofluorescence with
anti-macrophage monoclonal antibody (Mac-1 antigen CD11/b, Boehringer
Mannheim) and by histochemical detection of alkaline phosphatase
activity (33).
On the fourth day of culture, Sertoli cell monolayers were treated with
recombinant murine TNF-
or IFN-
. At the indicated times, Sertoli
cell-conditioned media were collected and frozen (
20 °C) before
measurement of IL-6, while the cells were analyzed for ICAM-1 and
VCAM-1 expression by flow cytometric analysis or were lysed for Western
blotting and immunoprecipitation experiments.
Flow Cytometry--
Control and treated Sertoli cells were
detached with 0.02% EDTA and washed with cold phosphate-buffered
saline + 1% bovine serum albumin. For detection of adhesion molecules
on the cell surface of Sertoli cells, the following monoclonal
antibodies were used: fluorescein isothiocyanate-conjugated hamster IgG
anti-mouse CD54 (ICAM-1) or fluorescein isothiocyanate-conjugated rat
IgG2a anti-mouse VCAM-1 (INCAM-110) (Pharmingen, San Diego,
CA). Specific monoclonal antibodies or the appropriate isotypic control
monoclonal antibodies were used at 1 µg/106 cells for 30 min on ice. Cells were then washed twice with phosphate-buffered saline + 1% bovine serum albumin and analyzed with a FACSTAR flow cytometer
(Becton Dickinson Labware). Cells were gated using forward versus side scatter to exclude dead cells and debris. The
fluorescence of 104 cells/sample was acquired in
logarithmic mode for visual inspection of the distributions and in
linear mode for quantitating the expression of the relevant molecules
by calculating the mean fluorescence intensity.
Assay for IL-6 Activity--
Supernatants from mouse Sertoli
cells untreated or treated with TNF-
or SB203580 ± TNF-
were assayed for IL-6 activity. IL-6 was measured using a B9 cell
hybridoma growth factor assay (34) in which Sertoli cell-conditioned
media were used to supplement the IL-6-dependent B9 cell
line (kindly provided by Dr. Lucien Aarden, Central Laboratory of the
Netherlands Red Cross, Amsterdam). The proliferative response of B9
cells to IL-6 is expressed relative to a standard curve with known
amounts of IL-6. One unit of IL-6 is defined as the reciprocal of the
dilution giving 50% maximal stimulation of proliferation.
B9 proliferation was measured using the Biotrak cell proliferation
enzyme-linked immunosorbent assay system (Amersham Pharmacia Biotech),
which is based on the incorporation of 5-bromo-2'-deoxyuridine into DNA
of proliferating cells, followed by the addition of peroxidase-labeled anti-5-bromo-2'-deoxyuridine antibodies. Briefly, 5 × 103 B9 cells in 100 µl of complete RPMI 1640 medium
(Gibco-BRL) supplemented with 10% fetal calf serum were plated onto a
96-well microtiter plate and incubated at 37 °C in the presence of
serial dilutions of Sertoli cell supernatants or recombinant mouse IL-6
(Genzyme Corp., Cambridge, MA). 48 h later, treatment with
5-bromo-2'-deoxyuridine was performed for 16 h. After fixation,
peroxidase-labeled anti-5-bromo-2'-deoxyuridine antibodies were added.
The immunocomplexes were detected by incubation with a chromogenic
substrate (as indicated in the manufacturer's protocol), and the
resultant color was read at 450 nm in a microtiter plate
spectrophotometer. This assay was previously determined to be
insensitive to TNF-
. All data points illustrated are the average of
three wells and of three independent experiments.
Western Immunoblotting--
Total Sertoli cells lysates were
prepared by lysing and scraping the cells off the culture plate with 10 mM Tris-HCl (pH 6.8), 0.4 mM EDTA, 2% SDS, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml antipain, 1 mM phenylmethylsulfonyl fluoride (Sigma), and the following
phosphatase inhibitors: 10 mM sodium fluoride, 0.4 mM sodium orthovanadate, and 10 mM
pyrophosphate. The nuclear fraction was prepared by hypotonic lysis
with 10 mM Tris-HCl (pH 7.5) and protease and phosphatase
inhibitors. After 5 min on ice, cells were homogenized with 20 strokes
of a Dounce homogenizer and centrifuged for 7 min at 900 × g. The
pellet (nuclei) was resuspended in 2% SDS. The protein concentration
of each sample was determined using the micro-BCA method (Pierce).
Equal amounts of proteins (70 µg) were subjected to
SDS-polyacrylamide gel electrophoresis and then transferred onto a
nitrocellulose (Hybond C, Amersham Pharmacia Biotech) or polyvinylidene
difluoride (Immobilon-P, Millipore Corp.) membrane depending on the
primary antibody used. When anti-phosphotyrosine monoclonal antibodies (Upstate Biotechnology, Inc.) were used, the nitrocellulose was saturated with 3% gelatin in Tris-buffered saline; otherwise, nonfat
dry milk was used.
Phospho-specific anti-p38 and phospho-specific anti-ATF-2 antibodies
were purchased from New England Biolabs Inc. After the first and second
antibodies, the membranes were washed three times for 15 min with
Tris-buffered saline + 0.05% Tween. The secondary antibodies were
horseradish peroxidase conjugates (Zymed Laboratories, Inc.), and
detection was performed using the chemiluminescence system (ECL,
Amersham Pharmacia Biotech).
Immunoprecipitation--
For the immunoprecipitation
experiments, the lysates were prepared in 20 mM Tris-HCl
(pH 7.5), 0.15 M NaCl, 0.5% Triton X-100, 1 mM
MgCl2, and 1 mM CaCl2 supplemented
with protease and phosphatase inhibitors. 4 µg of
anti-phosphotyrosine monoclonal antibody were added to 1 mg of cell
lysates and incubated overnight at +4 °C. The samples were
challenged with 50 µl of protein G-Sepharose beads (Amersham
Pharmacia Biotech) for 2 h at +4 °C and subsequently microcentrifuged. The beads were washed three times with ice-cold lysis
buffer and then resuspended in Laemmli sample buffer. The samples were
boiled for 5 min and run on a 12% SDS-polyacrylamide gel. The proteins
were transferred onto nitrocellulose, and the membrane was incubated
with anti-p38 antibody (C-20, Santa Cruz Biotechnology). Detection was
performed using the chemiluminescence system.
JNK/SAPK Kinase Assay--
This assay was performed using the
JNK/SAPK assay kit purchased from New England Biolabs Inc. and
following the manufacturer's instructions. The kit employs an
N-terminal c-Jun bound to Sepharose to selectively "pull down"
JNK/SAPK from the cell lysate, after which the kinase reaction is
carried out in the presence of unlabeled ATP (24, 25). c-Jun
phosphorylation is selectively measured using phospho-specific c-Jun
antibodies.
The cell lysates were prepared from Sertoli cells treated for 10, 16, and 25 min with TNF-
. The kinase reaction was terminated by the
addition of 3× SDS sample buffer, and the samples were run on a 12%
SDS-polyacrylamide gel, followed by transfer onto a polyvinylidene
difluoride membrane. The membrane was subsequently incubated with
phospho-c-Jun antibodies and then with secondary horseradish
peroxidase-conjugated anti-rabbit antibody, and finally, chemiluminescence detection was performed with LumiGLO provided with
the kit.
MAPK Assay--
This assay was performed using the MAPK assay
kit purchased from New England Biolabs Inc. Briefly, phospho-specific
antibodies to MAPKs were utilized to selectively immunoprecipitate
active MAPKs from Sertoli cell lysates under control conditions after treatment for 25 min with TNF-
or with TNF-
+ PD98059. PD98059 at
75 and 25 µM was added 1 h before TNF-
treatment.
The resulting immunoprecipitate was then incubated with an Elk-1 fusion
protein in the presence of ATP, which allows immunoprecipitated active MAPK to phosphorylate Elk-1 (35, 36). The kinase reaction was
terminated by the addition of 3× SDS sample buffer, and the samples
were run on a 12% SDS-polyacrylamide gel, followed by transfer onto a
polyvinylidene difluoride membrane. The membrane was subsequently
incubated with phospho-Elk-1 antibodies and then with secondary
horseradish peroxidase-conjugated anti-rabbit antibody, and finally,
chemiluminescence detection was performed with LumiGLO provided with
the kit.
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RESULTS |
TNF-
Induces Phosphorylation of p38 and ATF-2--
To
investigate the possible involvement of tyrosine phosphorylation in the
signal transduction mechanisms utilized by TNF-
in Sertoli cells, we
assayed the effect produced by the protein-tyrosine kinase inhibitors
herbimycin A (HMA) and genistein on TNF-
-induced ICAM-1 and VCAM-1
expression.
As shown in Fig. 1A, HMA
reduced, in a dose-dependent manner, the expression of both
adhesion molecules, whereas genistein was ineffective on VCAM-1
expression and positively up-regulated ICAM-1 (Fig. 1B). The
latter result is in agreement with the data reported by Tiisala
et al. (37) that show that pretreatment of endothelial cells
with genistein, followed by TNF-
stimulation, induces ICAM-1
expression.

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Fig. 1.
Flow cytometric analysis of the expression of
ICAM-1 and VCAM-1 in mouse Sertoli cells treated with TNF- after
pretreatment with tyrosine kinase inhibitors. Cells were
preincubated for 24 h with the indicated concentrations of HMA
(A) and genistein (B), and then TNF- (20 ng/ml) was added for 16 h. The results are expressed as percent of
mean fluorescence intensity of TNF- -treated cells.
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Such results prompted us to investigate the pattern of tyrosine
phosphorylation in Sertoli cells stimulated for different times with
TNF-
with or without HMA and genistein pretreatment. Using
anti-phosphotyrosine antibodies in Western blotting, we demonstrated
that TNF-
treatment was responsible for a rapid and
dose-dependent tyrosine phosphorylation of a protein of
~38-40 kDa (Fig. 2, A and
B). Immunoprecipitation with anti-Tyr(P) antibodies and
subsequent blotting with anti-p38 antibodies demonstrated that TNF-
indeed induced the phosphorylation of p38 (Fig.
3). The definitive confirmation of this
result was obtained using antibodies that specifically recognize p38 in
the phosphorylated form. Fig. 4 shows
that, under control conditions, Sertoli cells contained the
phosphorylated form of p38 and that, after 10 min of TNF-
stimulation, the phosphorylation (hence the activation) of p38
significantly increased. Moreover, as shown in Fig. 4, HMA treatment
greatly reduced the phosphorylation of p38.

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Fig. 2.
Anti-phosphotyrosine Western blot of Sertoli
cell whole extracts. Cells were treated with TNF- for the
indicated times (minutes) (A) and at the indicated doses
(B). C, control.
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Fig. 3.
TNF- up-regulates tyrosine-phosphorylated
p38. Sertoli cells lysates (1 mg) were immunoprecipitated with
anti-Tyr(P) monoclonal antibodies. After SDS-polyacrylamide gel
electrophoresis and Western blotting, the filter was incubated with
specific antibodies against p38.
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Fig. 4.
Effect of HMA on the phosphorylation of
p38. Shown are Western blots of Sertoli cell whole extracts
treated for the indicated times (minutes) with 250 ng/ml TNF- ± 0.5 µM HMA and immunoblotted with specific antibodies to the
phosphorylated form of p38. Densitometric analysis revealed that,
considering control (C) = 1, the relative phosphorylation
was as follows: 10 min + HMA = 0.56, 10 min = 1.86, 16 min + HMA = 1.09, 16 min = 2.26, 25 min + HMA = 0.49, and 25 min = 1.14. p38+ represents the phosphorylated protein
from C6 glioma cells treated with anisomycin.
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Since the transcription factor ATF-2 is the substrate that is
phosphorylated with great avidity by activated p38 and by JNK/SAPK (38), we analyzed the effect of TNF-
on ATF-2. Using specific anti-phospho-ATF-2 antibodies, we demonstrated that TNF-
induces the
rapid phosphorylation of ATF-2 from Sertoli cell nuclear fractions and
that the maximal effect is reached after 16 min (Fig.
5).

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Fig. 5.
Time course of ATF-2 phosphorylation induced
by TNF- . Sertoli cells were treated for the indicated times
(minutes) with 250 ng/ml TNF- . The antibody used is specific for the
phosphorylated form of ATF-2. ATF-2+ is the phosphorylated
protein from NIH 3T3 cells treated with anisomycin.
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Activation of p38 Mediates IL-6 Production, but Not ICAM-1 and
VCAM-1 Induction--
Both TNF-
and IFN-
induce ICAM-1 and
VCAM-1 expression in Sertoli cells, whereas only TNF-
treatment
increases the production of IL-6 (3). These data suggest that
differential signal transduction pathways are involved in the different
response of Sertoli cells to TNF-
. To clarify the role of p38 in the
biological activity of TNF-
on Sertoli cells, we treated the cells
with SB203580, a specific inhibitor of p38 kinase activity (20, 30,
39).
Fluorescence-activated cell sorting analysis demonstrated that neither
ICAM-1 (Fig. 6) nor VCAM-1 (not shown)
expression, induced by TNF-
, was affected by increasing doses of
SB203580 (up to 20 µM). Conversely, under the same
experimental conditions, the secretion of biologically active IL-6 was
completely inhibited by SB203580 at 20 µM (Fig.
7). Moreover, IFN-
treatment, which increased ICAM-1 expression (Fig.
8A), failed to activate the p38 pathway (Fig. 8B). We can therefore conclude that
activation of the p38 pathway is involved in IL-6 production, but not
in ICAM-1 and VCAM-1 expression.

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Fig. 6.
Flow cytometric analysis of the expression of
ICAM-1. Sertoli cells were treated with 50 ng/ml TNF- for
18 h (A). 20 µM SB203580 was added 2 h before TNF- treatment (B). CTR, control.
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Fig. 7.
Effect of SB203580 on IL-6 production induced
by TNF- . Sertoli cells were preincubated for 2 h with the
indicated concentrations of SB203580 (SB) before the
addition of 50 ng/ml TNF- for 18 h. Conditioned media were
assayed for IL-6 activity using the proliferative response of B9
hybridoma cells. Each point represents the mean of triplicate samples
of at least three experiments.
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Fig. 8.
IFN- up-regulates ICAM-1 expression, but
does not activate p38. A, flow cytometric analysis of the
expression of ICAM-1 in mouse Sertoli cells treated for 24 h with
500 units/ml IFN- ; B, Western blot of Sertoli cell whole
extracts treated for the indicated times (minutes) with 500 units/ml
IFN- . The antibody used is specific for the phosphorylated form of
p38. p38+ is the phosphorylated protein from C6 glioma cells
treated with anisomycin. CTR and C,
control.
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TNF-
Activates p42/p44 MAPK and JNK/SAPK Signal Transduction
Pathways--
We analyzed whether, in Sertoli cells, TNF-
treatment
induces the activation of the p42/p44 MAPK pathway using a MAPK assay. As shown in Fig. 9, TNF-
activated
p42/p44 MAPKs, as demonstrated by the increased phosphorylation of its
substrate, Elk-1. Pretreatment of Sertoli cells with 75 µM PD98059, a specific inhibitor of MEK1 (30, 40, 41),
the upstream kinase that activates p42/p44 MAPKs, completely abolished
the phosphorylation of Elk-1.

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Fig. 9.
PD98059 inhibits p42/p44 MAPKs activated by
TNF- . Sertoli cells were preincubated for 1 h with PD98059
at the indicated doses and then stimulated with TNF- for 25 min. The
activation of p42/p44 MAPKs was analyzed by a specific
immunoprecipitation/kinase assay of its substrate (Elk-1) as described
under "Experimental Procedures." Phosphorylation of Elk-1 was
visualized by Western blotting using phospho-Elk-1 antibody.
C, control.
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We therefore tested the effects of PD98059 on ICAM-1 and VCAM-1
expression and on IL-6 production. This treatment was without effect
and failed to modify either adhesion molecule expression, as revealed
by fluorescence-activated cell sorting analysis, or basal and
stimulated IL-6 production. These data indicate that, although p42/p44
MAPKs are fully activated, they are not involved in the biological
response of Sertoli cells to TNF-
.
We further investigated if, in Sertoli cells, TNF-
was able to
activate the JNK/SAPK MAPK subtype, the activation of which leads to
c-Jun phosphorylation. Using a JNK/SAPK assay, we measured the
phosphorylation of c-Jun. Fig. 10 shows
that SAPK-induced phosphorylation of c-Jun appeared after 16 min and
increased strongly after 25 min of treatment with TNF-
.

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Fig. 10.
Activation of JNK/SAPK activity by
TNF- . JNK/SAPK activity was evaluated by Western blot analysis
using phospho-c-Jun antibody. TNF- was added to Sertoli cells at 250 ng/ml for the indicated times (minutes).
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We can therefore conclude that the p38 cascade is specifically involved
in IL-6 production by Sertoli cells, as demonstrated by the fact that,
following TNF-
treatment, stimulation of IL-6 secretion, but not of
ICAM-1 and VCAM-1 expression, is prevented by the p38 kinase inhibitor
SB203580. Moreover, treatment with IFN-
, which induces ICAM-1 and
VCAM-1 but not IL-6, does not affect p38 phosphorylation. In addition,
the phosphorylation of c-Jun by TNF-
suggests the involvement of the
JNK/SAPK pathway in the regulation of ICAM-1 and VCAM-1 in Sertoli
cells.
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DISCUSSION |
Sertoli cells play an important role in the regulation of
spermatogenesis, including its hormonal control (42, 43). Moreover, this cell type is implicated in the immune tolerance of testicular autoantigens, both by establishing the blood-testis barrier, which segregates autoantigenic germ cells, and by secreting immunosuppressive factor(s) into the interstitial environment (44, 45).
The insurgence of autoimmune disorders in the testis has been reported
(46, 47). In these pathologic conditions, a massive intratubular
leukocyte infiltration has been described that allows leukocytes to
reach immunogenic germ cells after crossing the blood-testis barrier
and thus overwhelming the immune tolerance system (48).
The inflammatory process implies a series of different steps:
"rolling" of leukocytes on the endothelial wall, adhesion, and finally migration to the adjacent tissue. Leukocyte extravasation requires the progressive expression of various adhesion molecules on
the surface of endothelial cells (49). Among these molecules, ICAM-1
and VCAM-1 are inducible by inflammatory stimuli also in a set of
non-vascular cells (50, 51).
TNF-
is a proinflammatory cytokine, and we previously demonstrated
that it is a strong inducer of surface expression of ICAM-1 and VCAM-1
and of IL-6 production by Sertoli cells (3). These responses lead to
increased adhesion between lymphocytes and Sertoli cells. Our data
suggest that the biological effects of TNF-
on Sertoli cells may
play a pathogenic role in the outbreak of autoimmune disorders in the
testis. However, the intracellular signaling pathways that bring about
these responses are largely unknown.
In this work, we have reported the early biochemical events triggered
by TNF-
in mouse cultured Sertoli cell. We have shown that TNF-
induces a rapid and time- and dose-dependent tyrosine phosphorylation of p38 kinase. Moreover, pretreatment with HMA, a
powerful inhibitor of tyrosine kinases, significantly decreases both
the expression of ICAM-1 and VCAM-1 and the phosphorylation of p38.
However, treatment of Sertoli cells with SB203580, a specific inhibitor
of p38 kinase, does not affect the expression of ICAM-1 and VCAM-1
induced by TNF-
, indicating that p38 is not involved in the signal
transduction pathway that leads to this response. On the other hand,
the same treatment completely inhibited IL-6 production induced by
TNF-
, providing strong evidence that TNF-
regulates IL-6
production through the activation of p38, as already demonstrated in
the L929 cell line (52).
Using the p38-specific inhibitor SB203580, we further demonstrated
that, in Sertoli cells, TNF-
regulates the expression of surface
adhesion molecules and the secretion of IL-6 through two different
transduction pathways. Such conclusions are further confirmed by the
fact that IFN-
, an inducer of ICAM-1 and VCAM-1 but not of IL-6,
fails to activate p38 in these cells.
We then performed experiments to investigate the involvement of p42/p44
MAPKs in the biological responses of Sertoli cells to TNF-
. TNF-
activates this pathway as demonstrated by the phosphorylation of Elk-1,
a substrate of p42/p44 MAPKs. However, treatment with PD98059, a
specific inhibitor of MEK1 (the upstream kinase that activates p42/p44
MAPKs), failed to interfere with adhesion molecule expression and with
IL-6 production.
In conclusion, we have demonstrated that the biological responses of
Sertoli cells to TNF-
are under dual control: the activation of p38
leads to IL-6 production, but neither p38 nor p42/p44 MAPKs regulate
the induction of ICAM-1 and VCAM-1. In Sertoli cells, TNF-
also
activates JNK/SAPK as demonstrated by the increased c-Jun
phosphorylation. The activation of this pathway could be responsible
for the up-regulation of ICAM-1 and VCAM-1 in Sertoli cells. The
different regulation of the biological responses induced by TNF-
could be due to the differential recruitment of distinct signal
transducer proteins such as TRADD (TNF
receptor-1-associated death
domain protein), TRAF2 (TNF
receptor-associated factor), and
RIP (receptor-interacting protein)
(53). In this regard, it might be of interest to analyze the role of
the kinase RIP, a proximal intermediate of TNF-
signaling, and its
involvement in JNK/SAPK activation (54). The dual control of the
effects induced by TNF-
in Sertoli cells could represent a useful
experimental model to further dissect the different signal transduction
pathways mediated by TNF receptors.
We thank Dr. Fioretta Palombi (University of
Rome) and Dr. Antony Galione (University of Oxford) for critical
comments that improved this manuscript.