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(IFN) Receptor Subunits: IFN Enhances Interferon Regulatory Factor-1 and Interleukin-1ß Converting Enzyme Expression1
Population Council (M.K., P.L.M.) and The Rockefeller University (P.L.M.), New York, New York 10021
Address all correspondence and requests for reprints to: Dr. Patricia L. Morris, Center for Biomedical Research, Population Council and The Rockefeller University, 1230 York Avenue, New York, New York 10021. E-mail: p-morris{at}popcbr.rockefeller.edu
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Abstract |
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(IFN) transmits its signal through a specific cell
surface receptor (IFNR), which consists of a primary ligand binding
-chain (IFNR) and a signaling ß-chain (IFNRß). Recent
studies identified the cytokines IFN, interleukin-6 (IL-6), IL-1,
and tumor necrosis factor- in testicular cells. Therefore, we: 1)
examined the expression of IFNR and IFNRß subunits in
freshly isolated and purified rat testicular cells; 2) examined the
differential regulation of receptor components by cytokines using
primary cultures of Sertoli cells; 3) identified the cell signaling
pathway components of testicular IFNR; and 4) characterized the
functional role of testicular IFN using primary Sertoli cells. We
demonstrated the messenger RNAs for both chains of IFNR in rat
testicular cells using Northern hybridization analysis. Western blot
analysis and immunocytochemistry showed that both specific IFNR
protein subunits were present in cultured primary Leydig and Sertoli
cells prepared from the testes of immature rats. The expression of both
IFNR component messenger RNAs in cultured Sertoli cells was
increased by its specific ligand (IFN), as well as IL-1 and tumor
necrosis factor-, in both a time- and dose-dependent manner.
IFN-activation of the Janus (JAK) tyrosine kinases, JAK1 and JAK2
proteins, indicate that IFNR, expressed in the Sertoli cell, is
functional. Moreover, IFN modulates the expression of interferon
regulatory factor (IRF)-1 and IL-1ß converting enzyme genes in
Sertoli cells. Thus, our data are suggestive of a role(s) for IFN-
in the regulation of distinct gene expression and cell-specific
sensitivity to apoptosis in the testis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IFNß (type I IFNs), initially described as the
product of virus-infected leukocytes and fibroblasts, and IFN (type
II IFN), produced when lymphocytes and natural killer cells are
stimulated by antigenic or mitogenic substances (1). In cultured cells,
IFN is produced by the somatic epithelial Sertoli cells and the
peritubular myoid cells, as well as germ cells. In contrast, IFN has
been shown to be produced by early spermatids but not somatic cells
(2). Targeted gene mutagenesis studies suggest that these IFNs can
alter the development of testicular germ cells. In transgenic mice
overexpressing either the IFN or IFNß gene, the process of normal
germ cell development (spermatogenesis) is disrupted with concomitant
destruction of germ cells (3, 4). Mice that lack the exon 9 of the
Fanconi anemia C gene have reduced numbers of male germ cells, and bone
marrow progenitor cells from these mice are hypersensitive to IFN
treatment (5). Such effects of IFNs are suggestive of specific receptor
molecules as targets interacting with cytokine ligands within the
seminiferous tubule (Sertoli and/or germ cells) or interstitium
(e.g. androgen-producing Leydig cells).
IFN receptor (IFNR) belongs to the class 2 cytokine receptor
family, which includes the two known chains of the IFN/ß receptor
(6) and is composed of at least two subunits: a 90-kDa -chain
(IFNR) that is required for ligand binding, ligand trafficking,
and signal transduction (7, 8); and a ß-chain (IFNRß) (also
known as accessory factor-1) that plays a critical role in signaling
and is species-matched to the extracellular domain of the -chain
(9). Although the expression of IFNR is sufficient for ligand
binding, its presence alone does not confer responsiveness to IFN
(9). Concomitant expression of IFNR and IFNRß is required
for transcriptional activation after IFN signaling (8, 9). Transient
transfections of a murine fibroblast cell line show that IFNRß
alone does not form a separate, independent binding site for IFN,
whereas the expression of the ß-chain increases the apparent affinity
for its ligand (10).
Many recent studies demonstrate that the activation of the JAK/STAT
(Janus kinase/signal transducer and activator of transcription) pathway
is initiated when IFN is bound to its receptor complex. JAK1
associates with IFNR before ligand binding (11, 12), and IFN
treatment of cells results in the recruitment of JAK2 to IFNRß
(13, 14, 15). IFN-activated JAKs phospholylate STAT-1 protein, which is
translocated to the nucleus, resulting in transcriptional activation of
specific target genes (16). Several genes have been shown to be
activated by IFN, including interferon regulatory factor (IRF)-1
(17). IRF-1 mediates diverse functions, including tumor suppression,
myeloid differentiation, macrophage activation, antigen presentation,
and T- and B-cell differentiation (18). IRF-1 is also considered to be
the regulator of interleukin-1ß (IL-1ß) converting enzyme (ICE),
which can induce apoptosis in cells in conjunction with other apoptosis
pathway-related proteins (19). In several types of cells, the addition
of IFN induced IRF-1 gene expression, followed by increases in ICE
messenger RNA (mRNA), effects associated with a lowered cell threshold
to programmed cell death (20).
Recently, we demonstrated the differential activation of STATs by
IFN and IL-6, using primary rat Sertoli cells (21, 22). However, to
date, no direct evidence exists that the component chains of the
IFNR are expressed in the testis. Thus, we sought to examine the
expression of these two IFNR subunits in epithelial (Sertoli),
steroid-secreting Leydig cells, and male germ cells, using testicular
cells freshly isolated and purified from both immature and adult rats.
In the present study, we show: 1) the expression of both IFNR and
IFNRß in specific rat testicular cells; 2) that the IFNR mRNA
species expressed in primary Sertoli cells are regulated by various
testicular cytokines, including its cognate ligand; 3) that JAK1 and
JAK2 proteins are tyrosine phosphorylated rapidly, after IFN
treatment of Sertoli cells; and 4) that IFN regulates the expression
of IRF-1 and ICE gene and may contribute to testicular sensitivity to
apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxysteroid dehydrogenase (HSD)
positive; 42% 3ß-HSD positive] and Sertoli cells (93–95% inhibin,
desmin, vimentin, and enkephalin positive) were prepared from
18-day-old immature Sprague-Dawley (SD) rats (Charles River, Kingston,
NY) and cultured, as previously described (23). Briefly, the cells were
isolated by sequential collagenase digestions, plated onto 100-mm
polystyrene dishes, and cultured at 34 C in a humidified atmosphere of
5% CO2-95% air. Phenol red-free, serum-free, and
endotoxin-free DMEM/Ham’s F-12 medium (Irvine Scientific, Santa
Ana, CA) were supplemented with bovine insulin (Sigma, St. Louis, MO),
transferrin (Calbiochem, La Jolla, CA), and bacitracin (Sigma); and the
cells were cultured in serum-free conditions. The concentrations of
Leydig cells and Sertoli cells were 5 x 106 and
1 x 107 cells per 100-mm dish, respectively. On day
2, the medium was removed from the Leydig cell cultures, the cells were
washed with serum-free medium, and the protein was extracted. On day 3,
the medium was removed from the Sertoli cell cultures, the cells were
washed, and 4 ml serum-free medium was added. After the addition of
specific reagents in vitro, as indicated, RNA or protein was
extracted at the indicated times. Rat IFN (Genzyme, Cambridge, MA),
mouse IL-1, mouse IL-6, and mouse tumor necrosis factor- (TNF)
(R & D System, Minneapolis, MN) were dissolved in 0.1% BSA. Adult Leydig cells (>97% 3ß-HSD+) were prepared from SD rats (55–65 days old) and purified by Percoll (Pharmacia Biotech, Uppsala, Sweden) gradient and centrifugal elutriation using a Beckman JE-6B rotor (Beckman Instruments, Palo Alto, CA) (23). Adult Sertoli cells were isolated from SD rats (300–350 g) as previously described (24). Germ cells were isolated from adult SD rats (250–270 g) and purified by centrifugal elutriation using a Beckman JE5.0 system as previously reported; using somatic cell-specific markers, the elutriator-purified germ cell fractions were negative for Sertoli or Leydig cells (22).
All experiments were repeated 3–5 times, using different cell primary preparations, with comparable results obtained in the replicates of each. The results are presented as those typical for each experiment illustrated.
Procedures involving the use of animals strictly followed the Guidelines for the Care and Use of Laboratory Animals, set forth by the NIH.
Cell lines
EL4 (mouse lymphoma-derived) and P388D1 (mouse
macrophage-derived) cell lines were obtained from ATCC (Rockville, MD)
and were cultured as indicated.
Northern blot analysis
Total RNA was isolated using the Trizol reagent (Life
Technologies, Grand Island, NY). Poly (A)+ RNA was prepared
using the PolyATract mRNA Isolation System (Promega, Madison, WI). RNA
was subjected to electrophoresis, through a 1% agarose gel containing
formaldehyde, and was transferred to nylon membrane (MSI, Westboro,
MA). Complementary DNAs (cDNAs) of murine IFNR (full-length,
kindly provided by Dr. W. J. Murphy, University of Kansas), murine
IFNRß (full-length, obtained from ATCC), human IRF-1 (1.5-kb-long
insert, kindly provided by Dr. J. E. Darnell, Jr., Rockefeller
University), rat ICE (1035-bp-long insert, kindly provided by Dr. B.
Shivers, Parke-Davis, Ann Arbor, MI), or G3PDH (Clonetech, Palo Alto,
CA) were labeled with 32P-deoxycycidine triphosphate
(Amersham, Arlington Heights, IL) using random hexamers. The filters
were washed at high stringency (0.2 x saline sodium citrate/0.1%
SDS at 60 C) followed by exposure of the hybridized filters to Kodak
X-OMAT AR film (Eastman Kodak, Rochester, NY) and signals were analyzed
using the PhosphoImager with ImageQuant software (STORM system,
Molecular Dynamics, Sunnyvale, CA).
Protein extraction and immunoblotting
Cells were lysed for 30 min on ice in buffer (50 mM
Tris-HCl (pH 7.5) containing 1% NP-40, 0.1% SDS, 0.1% sodium
deoxycholate, 150 mM NaCl, 2 mM EDTA, 1
mM phenylmethlysulfonylflouride, 10 µg/ml aprotinin, 10
µg/ml pepstein, and 1 mM sodium orthovanadate). Cellular
debris was pelleted by centrifugation at 12,000 x g
for 10 min. Immunoprecipitations were carried out by standard methods.
The cell lysate or immunoprecipitate was mixed with an equal vol of
2 x Laemmli’s buffer containing 180 µM
ß-mercaptoethanol and was boiled for 3 min. The proteins in the
supernatant were then subjected, under reducing conditions, to SDS-PAGE
using 7.5% polyacrylamide gels, and were electrophoretically
transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene,
NH). The membranes were probed with antibodies, as described below.
Antimouse affinity-purified IFNR polyclonal antibody (pAb)
(sc-703, 1:1000) and antimouse affinity-purified IFNRß pAb
(sc-972, 1:1000) were obtained from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). Monoclonal antiphosphotyrosine antibody (4G10,
1:4000), antimouse JAK1 pAb (1:1000), and antihuman JAK2 pAb (1:1000)
were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY).
Blots were developed with the ECL Western blotting system
(Amersham).
Immunocytochemistry
Freshly isolated immature Leydig or Sertoli cells were placed on
chamber slides (Lab Tek, NUNC, Naperville, IL) and were cultured
without serum for 48 h at 34 C. The cells were then fixed, using
0.3% H2O2-methanol, for 10 min at room
temperature to quench the endogeneous peroxidase activity. The fixed
cells were then processed, using avidin-biotin complex (ABC) methods,
with the VECTASTAIN ABC kit (Vector, Burlingame, CA). The cells were
incubated overnight at 4 C with an anti-IFNR pAb (sc-703, 1:200)
or anti-IFNRß pAb (sc-972, 1:200). After washing with PBS, slides
were reacted with biotinylated antirabbit IgG antibody and ABC
reagents, and developed with DAB (DAKO, Carpinteria. CA). Neutralized
antibodies against each synthetic IFNR antigen (Santa Cruz
Biotechnology) or PBS were employed, in place of primary antisera, to
determine nonspecific immunoreactivity. Cells were counterstained with
Mayer’s hematoxylin solution (Sigma).
Data analysis
The significance of results was determined using Student’s
t test, ANOVA, and the Dunnett’s multiple-comparison test
for comparison of means, as needed. P values 
0.05
were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R mRNA species in isolated testicular cellsRs (IFNR and
IFNRß) are expressed in the testis, we performed Northern analysis
using RNA from freshly isolated rat testicular cells (Fig. 1A
). One microgram of poly
(A)+ RNA, prepared from EL4 cells, was used as positive
controls, because they express IFNR mRNA (25). Similarly, because
IFN regulates macrophage activation markers (26), 1 µg poly
(A)+ RNA from P388D1 cells and 5 µg rat spleen poly
(A)+ RNA were loaded on the same blot. Northern
hybridization, with a probe specific for murine IFNR or
IFNRß, detected the predicted transcripts (-chain, 2.3 kb;
ß-chain, 2.0 kb) in RNA from EL4 and P388D1 cells. Using RNA from rat
spleen, IFNR mRNA was almost the same size as that from mouse
cells, whereas IFNRß transcripts were found to be a rat-specific
shorter length (about 1.8 kb). Freshly isolated immature and adult
Leydig cells expressed both chains of IFNR mRNA species. IFNR
mRNAs were also detected in Sertoli cells prepared from the testis of
immature and adult rats. In these somatic cells, the level of IFNR
mRNAs did not depend on maturational status or age. Although only
IFNRß transcripts were consistently detected at low levels in rat
germ cells, despite longer exposure times, we were unable to detect a
signal for IFNR mRNA using this murine cDNA as a hybridization
probe (data not shown). In P388D1 cells, the accumulation of IFNR
and IFNRß mRNA are shown to be almost the same levels, whereas in
EL4 cells, the amount of -chain gene was significantly more abundant
than that of the ß-chain. The level of IFNRß mRNA seemed to be
expressed at higher levels than that of IFNR in testicular
cells.
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R and IFNRß immunoreactivities in
cultured immature rat Sertoli and Leydig cellsR protein in Sertoli and Leydig cells from
immature rats, Western blot analysis and immunocytochemistry were
performed. For Western analyses, proteins were isolated from total cell
lysates of primary Leydig cells or Sertoli cells cultured in serum-free
medium (day 2 or day 3, respectively); simultaneously, proteins from
EL4 cell lysates were used as a positive control. The 90-kDa chain of
IFNR (-chain) was detected in the immunoprecipitates from Sertoli
or Leydig proteins (Fig. 1B
, lanes 2 and 3). In addition, total cell
lysates (prepared from EL4, Sertoli, or Leydig cells) showed a doublet
located about 66 kDa, when probed with anti-IFNRß pAb (Fig. 1B, lanes 4–6). This double-band pattern was similar to that reported
previously (14). The expression of IFNR and IFNRß proteins
in cultured primary Sertoli and Leydig cells was then confirmed by
immunocytochemical studies. Positive staining for both IFNR and
IFNRß proteins was visualized in the form of small granules
distributed over the cytoplasm of each cell type. Using these
immunological reagents, the reactivities of IFNR protein, in both
Sertoli and Leydig cells, were clearly detectable (although they were
faint), whereas IFNRß protein was strongly positive in these cell
types (Fig. 2).
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R and IFNRß mRNA species
expression in cultured immature Sertoli cellsR genes of testicular cells, we used primary
Sertoli cells to initially investigate the kinetics of their effect(s)
on both IFNR mRNA species, using Northern analysis. The same blot
was sequentially hybridized with specific cDNA probes for both chains.
Changes in IFNR and IFNRß mRNA levels were examined at 3, 6,
and 18 h after the addition of 10 ng/ml IFN, TNF, IL-1,
or IL-6. IFNR - and ß-chain mRNAs were detected at time zero
(primary culture, day 3), and their levels increased slightly when
cultured in serum-free medium. The addition of their homologous ligand,
IFN, resulted in a small (but significant) increase in both - and
ß-chain mRNA levels at 18 h. Similarly, TNF and IL-1
increased IFNR mRNAs. However, there were differences observed in
the kinetics of their effects on IFNR and IFNRß mRNAs. There
was a more rapid onset of increases in the mRNA for the IFNRß
subunit than that of IFNR, after the addition of either TNF or
IL-1. IFNRß mRNA levels increased up to 6 h, after which
the level decreased by 18 h. In contrast, IL-6 had no effect on
the expression of IFNR mRNAs within 18 h (Fig. 3).
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R and IFNRß mRNA levels were determined.
Because the kinetic studies shown above revealed that IL-1 increased
IFNR mRNAs earlier than IFN or TNF, as appropriate, we used
different lengths of treatment time among these reagents. The addition
of IFN (18 h), TNF (18 h), or IL-1 (6 h) at increasing doses
resulted in a dose-dependent increase in IFNR gene levels. A
significant elevation of both IFNR mRNA species occurred after
treatment with 1 ng/ml IFN, 10 ng/ml TNF, or 1 ng/ml IL-1
(Fig. 4).
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R treatment of Sertoli cells for 10 min. Data in Fig. 5 (upper panels) demonstrate
that both JAK1 (lane 2) and JAK2 (lane 4) were tyrosine phosphorylated
after the addition of IFN, as shown by sequential
immunoprecipitation with the respective antiserum of the JAKs and
Western blotting with 4G10. When blots were stripped of
antiphosphotyrosine antibody and reprobed with antiserum to the two
respective kinases (lower panels), similar levels of protein
were observed.
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treatment of primary Sertoli cells.
Significant increases in IRF-1 mRNA level were first detected at 1
h, with consistent increases up to 18 h. Because IRF-1 has been
shown to be a regulator of ICE gene expression, we next examined the
levels of ICE mRNA by rehybridizing the same membrane with a rat ICE
cDNA probe. The apparent induction of ICE transcript was first seen at
6 h after IFN addition, with increases in mRNA to 18 h
(Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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treatment affects the testis, we provide here the first
demonstration that IFNR is expressed in the rat testis and that
IFN induces the expression of the apoptotic pathway-related genes,
IRF-1 and ICE, in primary Sertoli cells (21, 22, 27, 28, 29, 30, 31).
Intratesticular administration of human IFN decreased the number of
Sertoli and germ cells in adult mice (27), suggesting that
inappropriate IFN exposure could increase the programmed cell death
of Sertoli cells and/or decrease the proliferation of germ cells,
either directly or indirectly. Chronic administration of IFN to mice
results in reduced testicular weights, decreased sperm count and
concentration, and abnormalities in sperm morphology (28), consistent
with IFN-induced disruption of spermatogenesis. In addition, early
studies showed that IFN treatment inhibits gonadotropin-stimulated
testosterone production, as well as the level of mRNAs for
steroidogenic enzymes in cultured porcine and mouse Leydig cells (29, 30). IFN increases the expression of integrin ligands, adhesion to
lymphocytes, and secretion of IL-6 in cultured mouse Sertoli cells
(31). Together, these data suggest that IFN action in the testis may
be associated with multiple cell types; however, to our knowledge there
are no data available regarding the cell-specific localization of
testicular IFNR components.
In addition to our previous report of the IL-6 receptor expression in
rat Sertoli and Leydig cells, the expression of several cytokine
receptor subtypes (e.g. those receptors for IL-1, IL-3,
TNF, LIF and gp130) has been demonstrated in the mammalian testis
(32, 33, 34, 35, 36). Several of these receptors show testis-specific patterns of
expression of their components. The IL-3 receptor -chain (but not
the ß-chain) is expressed in mouse Leydig cells (34). Although the
55-kDa chain of TNF receptor is present and is under hormonal
control, the 75-kDa chain is not detected in isolated porcine Sertoli
cells (35). Our present study showed that both chains of IFNR were
expressed in freshly isolated testicular cells. In contrast to the
comparatively strong signals for IFNR components in rat somatic
cells of the testis, their expression in the germ cells was apparently
less abundant; using Northern analyses with the murine cDNA, we could
detect only faint bands for IFNRß mRNA. Further studies are
necessary to delineate the functional status of the IFNR components
in germ cells. The cell-specific expression of both the ligand and its
receptor will, no doubt, play an important role in determining the
testicular targets for IFN. Likewise, the presence of the respective
downstream signal components will be additional determinants in
mediating IFN-induced effects on gene expression in testicular
cells.
To date, our studies have focused on the role of cytokines in the local
regulatory interactions between Sertoli and Leydig cells (21, 22, 23, 32, 36). Because cooperation with other cytokines and growth factors
(produced in an autocrine or paracrine fashion) is often required to
achieve full functional responses, cell-to-cell interactions likely
play an important role during the spermatogenic process (37). Several
cytokines and growth factors are produced in the testis. TNF mRNA
and proteins have been detected in the haploid round spermatids (38).
Both IL-1 and IL-6 are produced by Leydig and Sertoli cells (23, 39). Recently, both IFN mRNA and protein were found in
PHA-stimulated early spermatids, but, in contrast, were not produced by
peritubular cells, Sertoli cells, or pachytene spermatocytes (2). Our
present findings are consistent with the role of the cytokines as
regulatory molecules involved in cell-to-cell interactions in the
testis. Somatic cells (Leydig and Sertoli) are the predominant cell
populations in the 18-day-old rat testis (after this age in the rat,
more mature germ cells are present); overall, the relative cellular
contributions of the germ cell compartment expands, and they become the
most numerous cellular components of the adult testis. Our study
indicates that there are no differences in the levels of IFNR mRNAs
between freshly isolated immature and adult Sertoli cells. Thus, under
physiologic conditions, the presence of germ cells may not affect the
expression of IFNR mRNA in somatic cells.
Here, we report that IFN-, TNF-, and IL-1-treatment
enhances the expression of IFNR mRNA species in a dose- and
time-related manner using a model system of cultured primary Sertoli
cells. The effect of IFN on its receptor has been shown to vary in
several cell types. IFN augments the expression of both IFNR
subunits in L929 fibroblasts. IFN induces the expression of
IFNR on CD4+ T helper cell type 1 but results in a
loss of receptor ß-chain expression on T helper type 2 cells (40). In
THP-1 cells, IFN does not modify IFNR mRNA (41, 42). TNF
and IL-1 exert a growth inhibitory effect, mediated by an increase
in IFNR expression; this enhancement was caused by
transcriptional activation of the IFNR gene in human colorectal
carcinoma cells (43). Also, in THP-1 cells, TNF and IL-6 induce
IFNR gene expression by transcriptional and posttranscriptional
mechanisms (41). A wide range of biological activities of TNF and
IL-1 overlap and are indistinguishable, even though these cytokines
are neither structurally related nor do they react with related
cellular receptors. Both TNF and IL-1 have been shown to activate
nuclear factor-
B (NFB), a pleiotropic transcriptional factor that
is recently reported to be tightly associated with cellular viability
through the mechanisms of apoptosis (44, 45, 46). It is possible that
IL-1 and TNF might act by increasing IFNR gene expression
through the induction of proteins that interact with NFB-like
enhancer elements in the Sertoli cell. In contrast, IL-6 did not
modulate the IFNR mRNA species in Sertoli cells within 18 h,
despite the presence of the IL-6R. In human monocytic THP-1 cells,
TNF and IL-6 up-regulate the IFNR gene through different
mechanisms, and IL-6 seems to exert its effect by increasing the
half-life of IFNR mRNA (41). A difference in the stability of
testicular IFNR mRNA with other cell types may abrogate the IL-6
effect without affecting that of TNF in Sertoli cells. We also
provide evidence that the induction of IFNRß mRNA is more rapid
than that of -chain, after treatment of Sertoli cells with TNF or
IL1. Transfection studies show that expression of ß-chain
increases the affinity of IFN binding to IFNR, whereas IFN
does not bind the ß-chain (10).
In the past few years, the identification of the JAK-STAT pathway has
helped to clarify the intracellular components involved in the action
of certain cytokines on specific genes (47). To examine whether IFNR
modulates this JAK-STAT pathway in Sertoli cells, we studied the
phosphorylation of JAK1 and JAK2 proteins in IFN-treated Sertoli
cells. Binding of IFN to IFNR induces the activation of the
tyrosine kinases JAK1 and JAK2 in Sertoli cells, results consistent
with the presence of both chains of IFNR in Sertoli cells, because
JAK1 is constitutively associated with the IFNR and JAK2 with the
IFNRß. We recently showed that, in contrast to IL-6, IFN
regulates c-fos mRNA through the activation of STAT1 and
that genistein treatment abolished the recruitment of STAT1 protein
into the nucleus, showing that tyrosine phosphorylation is required for
such IFN signaling in rat Sertoli cells (21, 22).
Furthermore, we show here the IFN-induced increased expression
of the apoptotic pathway-related genes, IRF-1 and ICE, in Sertoli
cells. IRF-1 and IRF-2 are structurally similar DNA-binding factors
that were originally identified as regulators of the type I IFN system.
IRF-1 expression is necessary for the antiviral action of IFNs against
some viruses (17). Recently, IRF-1 has been shown to play an essential
role in apoptosis, including DNA damage-induced apoptosis (19, 48). A
study using IRF-1 null mice showed that IRF-1 is a transcriptional
activator of the ICE gene, the first member of mammalian homologues of
the Caenorhabditis elegans cell death gene ced-3
(19). Overexpression of ICE in some cell lines induces apoptosis (49).
Most recently, it has been suggested that IFN causes an increase in
ICE gene expression through the induction of IRF-1; significant
increases in the levels of ICE in cells are thought to be important in
programmed cell death (20). In most cell types, IRF-1 induction by
cytokine stimulation is rapid and transient. In contrast (unlike other
cell types), in Sertoli cells, the expression of IRF-1 continuously
increases up to 18 h, with the first significant increase in ICE
mRNA being observed at 6 h. As for c-fos, another
immediately early gene, we recently demonstrated that the level of its
mRNA showed a consistent pattern of rapid and transient induction in
IFN-treated Sertoli cells, in contrast to that found after IL-6
addition (21, 22). There may be a cell-specific mechanism involved in
regulating IRF-1 gene in the Sertoli cell. Further studies are required
to establish the role of IRF-1/ICE signaling pathway in IFN-mediated
apoptosis in the testis.
Several studies imply that in the testis, IFNs play a negative
role in the spermatogenic process. A histological study of the testes
from IFN gene transgenic mice showed a marked vacuolization of some
of the seminiferous tubules. Such observations are consistent with the
response of the Sertoli cells to a wide variety of injuries, often
resulting in sterility (3). In the testes of transgenic mice carrying
an exogenous IFNß gene, abnormal pachytene spermatocytes and
spermatids were observed. In contrast, germ cells in the early stages
of meiotic prophase and Sertoli cells seemed normal (4). Mice without
the IFNR are fertile; but when these mice were infected by
vaccinia virus, viral replication reached titers 102- to
103-fold greater in the testes of deficient mice, compared
with those found in wild-type mice, implying disruption of normal
testicular defense functions (50). The testes of men with acquired
immunodeficiency syndrome exhibit altered spermatogenesis, often with
oligozoospermia or azoospermia, with human immunodeficiency
virus-associated proteins and particles found in testicular germ cells
(51, 52). Therefore, it will be critical to ascertain the components of
the testicular antiviral defense system, to understand the mechanisms
of virus-induced testicular damage.
To our knowledge, the present study is the first to demonstrate IFNR
expression in rat testicular cells and to further characterize the
effects of IFN in cultured Sertoli cells (21, 22). Our data are
supportive of a physiologic role for IFNR and its ligand during
spermatogenesis.
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Acknowledgments |
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Footnotes |
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Received November 3, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunol 149:1671–1675[Abstract]
receptor expression in RAJI and IM9
lymphoblastoid cells vs. THP-1 monocytic cells by IFN-
and phorbol myristate acetate. J Immunol 144:4688–4696[Abstract]
-interferon receptors. Cancer Res 53:636–640This article has been cited by other articles:
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  T. Ishikawa and P. L. Morris Interleukin-1{beta} Signals through a c-Jun N-Terminal Kinase-Dependent Inducible Nitric Oxide Synthase and Nitric Oxide Production Pathway in Sertoli Epithelial Cells Endocrinology, November 1, 2006; 147(11): 5424 - 5430. [Abstract] [Full Text] [PDF]
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 M. Vigodner, T. Ishikawa, P. N. Schlegel, and P. L. Morris SUMO-1, human male germ cell development, and the androgen receptor in the testis of men with normal and abnormal spermatogenesis Am J Physiol Endocrinol Metab, May 1, 2006; 290(5): E1022 - E1033. [Abstract] [Full Text] [PDF]
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T. Ishikawa and P. L. Morris A Multistep Kinase-Based Sertoli Cell Autocrine-Amplifying Loop Regulates Prostaglandins, Their Receptors, and Cytokines Endocrinology, April 1, 2006; 147(4): 1706 - 1716. [Abstract] [Full Text] [PDF]
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T. Ishikawa, K. Hwang, D. Lazzarino, and P. L. Morris Sertoli Cell Expression of Steroidogenic Acute Regulatory Protein-Related Lipid Transfer 1 and 5 Domain-Containing Proteins and Sterol Regulatory Element Binding Protein-1 Are Interleukin-1{beta} Regulated by Activation of c-Jun N-Terminal Kinase and Cyclooxygenase-2 and Cytokine Induction Endocrinology, December 1, 2005; 146(12): 5100 - 5111. [Abstract] [Full Text] [PDF]
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 K M Detjen, K Farwig, M Welzel, B Wiedenmann, and S Rosewicz Interferon {gamma} inhibits growth of human pancreatic carcinoma cells via caspase-1 dependent induction of apoptosis Gut, August 1, 2001; 49(2): 251 - 262. [Abstract] [Full Text] [PDF]
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M. Kanzaki and P. L. Morris Growth Hormone Regulates Steroidogenic Acute Regulatory Protein Expression and Steroidogenesis in Leydig Cell Progenitors Endocrinology, April 1, 1999; 140(4): 1681 - 1686. [Abstract] [Full Text]
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