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Endocrinology, doi:10.1210/en.2006-0643
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Endocrinology Vol. 147, No. 11 5424-5430
Copyright © 2006 by The Endocrine Society

Interleukin-1ß Signals through a c-Jun N-Terminal Kinase-Dependent Inducible Nitric Oxide Synthase and Nitric Oxide Production Pathway in Sertoli Epithelial Cells

Tomomoto Ishikawa and Patricia L. Morris

Center for Biomedical Research, Population Council (T.I., 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, Population Council, The Rockefeller University, 1230 York Avenue, New York, New York 10021. E-mail: p-morris{at}popcbr.rockefeller.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Our recent Sertoli cell (SC) studies showed that the c-Jun N-terminal kinase (JNK) and inducible cyclooxygenase-2 (COX-2) pathways are key regulatory components of IL (IL-1{alpha}, IL-1ß, and IL-6) expression and START-domain containing StARD1 and StARD5 proteins. IL-1ß regulates SC autocrine/paracrine activities and subsequently influences developing germ cells and spermatogenesis. This study was designed to evaluate whether IL-1ß mediates high-output inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in these specialized epithelial cells and characterize gonadotropin and cytokine-regulation of NO. Purified SCs were maintained in serum-free cultures and treated with FSH (100 ng–1 µg/ml) or IL-1ß (10 ng/ml) in time-course studies. To determine obligatory intracellular pathways, treatments were conducted with or without activity inhibitors: COX-2 selective (NS-398, 10 µM) or JNK (SP600125, 10 µM) for 1, 3, 6, and 24 h. NOS mRNAs and proteins were evaluated by RT-PCR and Western analysis, respectively. NO and reactive oxygen species were measured by flow cytometry and ELISA. IL-1ß transiently induces intracellular NO (30 min) but not reactive oxygen species. Subsequently, iNOS mRNA and protein expression (3–6 h) significantly increased after IL-1ß but not FSH stimulation, and in time-dependent manner, markedly increased extracellular NO (24 h, 8-fold). No change in the constitutive endothelial NOS isoform was observed. Inhibition of JNK, but not COX-2, activity inhibits IL-1ß-induced iNOS expression and NO production. Such findings suggest that intra- and extracellular NO within the tubule may alert SCs monitoring the microenvironment to an aberrant cytokine, triggering antioxidant and antiinflammatory activities to avoid disruption of spermatogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THROUGHOUT SPERMATOGENESIS, significant interactions within the seminiferous tubule occur between Sertoli cells (SCs) and specific male germ cells (1, 2, 3, 4, 5, 6). For example, throughout spermiogenesis when haploid spermatids differentiate, cellular remodeling of elongating spermatids results in the shedding of their cytoplasm and contents therein as cytoplasts known as residual bodies (7, 8). Under physiological conditions, phagocytosis of residual bodies occurs by the surrounding SC and stimulates the release of multiple cytokines; induction of IL-6 and IL-1{alpha} expression and secretion was shown to be major components of this response (9). Our previous studies demonstrated that SC IL-6 and its receptor are regulated by FSH, consistent with physiological regulation of environmental cytokines (10, 11).

Recently, we showed in SCs that IL-1ß is a potent inducer of several ILs, cyclooxygenase-2 (prostaglandin G/H synthase, PGHS-2 or COX-2) expression and enzyme activity, and the phosphorylation of c-Jun N-terminal kinase (JNK). Activation of these signaling pathways resulted in changes in lipid binding transfer proteins levels such as steroidogenic acute regulatory protein (StAR)-1 and StARD5, and arachidonic acid use for prostaglandin production and secretion (12). Our subsequent studies showed that IL-1ß can temporally activate multiple kinases in an auto-amplifying circuit that regulates cytokine and prostaglandin expression and production (13). These IL-1ß-triggered events were not accompanied by reactive oxygen species (ROS) generation but rather by a lower basal ROS level than those in matched control (Ct) SCs, findings suggestive of an antiinflammatory response to IL-1ß (12). Studies from this laboratory and others indicate that several signal transduction pathways in the testis are activated in response to IL-1ß, and their components may interact in a cell-specific context that determines response.

JNK is activated by phosphorylation in response to numerous environmental stimuli, activated in settings of cell stress, mitogenesis, differentiation, and morphogenesis. For SCs, our studies suggest that JNK-mediated mechanisms may have relevance during Sertoli-germ cell associations and interactions. The physiological signaling molecule nitric oxide (NO) inhibits JNK by S-nitrosylation, preventing sustained activity of the kinase and stress-activated programmed cell death (14). Protein modifications by NO mediate physiological cellular and tissue responses (15). NO and S-nitrosylation of target proteins mediate IL-1ß action in steroidogenic cells such as testicular Leydig and ovarian granulosa cells (16, 17). Nitric oxide synthase (NOS) catalyzes the production of NO, a short-lived free radical with physiological or pathophysiological roles in nearly every organ system (18). There are two constitutive isoforms of NOS, neuronal NOS and endothelial (eNOS), and one high-output inducible isoform (iNOS); each are subject to phosphorylation (18). NO production by iNOS is generated after particular growth factors, cytokines, and increases in intracellular cAMP under physiological conditions and by certain cytokines such as TNF{alpha} during acute or chronic inflammation (19, 20). Increases in Sertoli NOS activity and iNOS expression are observed in response to a combination of several cytokines (interferon-{gamma}, TNF{alpha}, and IL-1{alpha}) or exposure to the potent inflammatory agent bacterial lipopolysaccharide (LPS) (21, 22). Our previous study indicates that overproduction of eNOS in transgenic mice may have additive deleterious effects on germ cells compromised by cryptorchidism (23). Round spermatid-produced factors in culture influence Sertoli iNOS mRNA expression and NO production (24). Such germ cell-to-epithelial cell interactions may represent an important mechanism for stage-specific physiological modulation of SC function during spermatogenesis.

Taken together, such findings suggest that intracellular and extracellular NO may play an active role in regulating SC function, with NO levels based on responses to distinct factors in the tubule’s microenvironment. Thus, it is likely that particular ILs and other cytokines, or combinations thereof, play multiple interactive roles, both hormone-dependent and independent, in the regulation and dysregulation of Sertoli function and male germ cell development. For this study, we sought to determine whether IL-1ß, gonadotropin signaling by FSH, or changes in intracellular cAMP induce SC iNOS and NO.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
SC preparations
SCs were purified from the testes of 18-d-old Sprague Dawley [Crl: CD (Sprague Dawley) BR-CD] rats purchased from Charles River Laboratories, Inc. (Kingston, NY). Animals were housed in standard lighting (12 h light, 12 h dark) with food and water allowed ad libitum in facilities approved by the American Association for the Accreditation of Laboratory Animal Care. Procedures involving the use of animals strictly followed the Guidelines for Care and Use of Laboratory Animals set forth by the National Institutes of Health and protocols received Institutional Animal Care and Use Committee approval. Experiments were repeated at least three times using individual SC purifications (40 testes pooled starting material) as described previously (25, 26). Primary cultures (≥95% pure) were maintained at a density of 1 x 107 cells per 100-mm polystyrene dish in phenol red-, serum-, and endotoxin-free DMEM/F-12 (Irvine Scientific, Santa Ana, CA) in a humidified incubator at 34 C, 5% CO2. The medium was supplemented with 2.5-µg/ml bovine insulin (Sigma, St. Louis, MO), 1 µg/ml transferrin (Calbiochem, La Jolla, CA), and 10 µg/ml bacitracin (Sigma). On d 3 ex vivo, SCs were rinsed twice with fresh serum- and phenol red-free culture medium and then treated with IL-1ß (10 ng/ml) in the absence or presence of COX-2 activity inhibitors NS-398 (10 µM) or the JNK inhibitor, SP600125 (10 µM). These experiments were conducted in a count-down manner, that is, a 24-h experimental clock began on d 3 with all matched SC harvested together at the end of the experiment on d 4 ex vivo. For the experiments reported herein, the time-specific vehicle Ct were harvested together with the experimental replicates on d 4; the vehicle Ct are represented as Ct (veh). At 1, 3, 6, and 24 h after either vehicle (Ct), IL-1ß, or addition of the inhibitor(s), whole cell extracts were isolated from each replicate for protein and total RNA analyses. Duplicate or triplicate culture dishes were used for each drug treatment and experiments were repeated at least twice. The mean (±SEM) of all the experiments was calculated for RNA analyses.

Drugs and reagents
Recombinant rat IL-1ß was purchased from R&D Systems (Minneapolis, MN). NS-398 (N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide) was purchased from Cayman Chemical (Ann Arbor, MI). SP600125 [(N1-methyl-substituted pyrazolanthrone (N1-methyl-1, 9-pyrazoloanthrone))] and forskolin (8, 13-Epoxy-7ß-(N-metylpiperazino-{gamma}-butyryloxy)-1{alpha}, 6ß, 9{alpha}-trihydroxy-labd-14-en-11-one) were purchased from Calbiochem (San Diego, CA). FSH (ovine FSH-19) was obtained from National Hormone and Pituitary Program [University of Maryland School of Medicine, Baltimore, MD, National Institute of Diabetes and Digestive and Kidney Diseases, and Dr. Parlow (Torrance, CA)]. Recombinant IL-1ß was dissolved in 0.1% BSA in PBS as a 100-fold (10 µg/ml) stock solution. Matched aliquots of 0.1% BSA were used in Ct cultures; the final BSA concentration was 0.0001%. COX-2 and JNK inhibitors were dissolved in dimethylsulfoxide (DMSO). Forskolin and FSH were dissolved in H2O and calcium- and magnesium-free PBS, respectively (10 µM and 1 µg/ml). Subsequent dilutions, as needed, were performed in serum- and phenol red-free medium on the day of the experiment. DMSO alone was used as matched vehicle Ct in all plates as required. DMSO concentration did not exceed 0.1%. Accutase was purchased from Innovative Cell Technologies, Inc. (San Diego, CA). The fluoroprobe, 4-amino-5-methylamino-2', 7'-difluorescein (DAF-FM) diacetate was purchased from Invitrogen (Carlsbad, CA) and used per manufacturer’s instructions. ROS was also determined in matched SCs using methods we previously described (12).

Protein extraction and Western analysis
Whole cell homogenates were extracted for protein lysates for 15 min on ice in buffer [10 mM Tris-HCl (pH 7.8) containing 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 mM dithiothreitol, 2 mM sodium orthovanadate, 2 µg/ml aprotinin, 2 µg/ml pepstatin, and 2 µg/ml leupeptin] (chemicals were from Sigma). Cellular debris was removed by centrifugation (12,000 x g, 15 min). The proteins in the supernatant were then subjected, under reducing conditions, to SDS-PAGE using 4–20% Tris-glycine gels (Novex, San Diego, CA), and were electrophoretically transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH). The membranes were probed with antibodies, as described below. The iNOS polyclonal antibody (1:1000) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA; sc-650). eNOS polyclonal antibody (1:1000) was obtained from BD Transduction Laboratories (Lexington, KY). Monoclonal anti-ß-actin antibody (1:2000) was purchased from Sigma. Blots were developed with the ECL Western blotting system (Amersham, Arlington Heights, IL) and exposed to x-ray film (Kodak, Rochester, NY). Densitometric analysis was performed using the PC version of NIH Image software (Scion Image; Scion Corp., Frederick, MD.) after scanning. For Western analyses, particular signal intensities in each lane were normalized with those for ß-actin on the same membranes, and data are expressed as arbitrary units relative to Ct, set as a value of one.

Total RNA extraction
Total RNA was extracted from SCs using the TRIzol reagent (Life Technologies, Inc., Grand Island, NY) according to the manufacturer’s instructions. RNA was measured using 260/280 UV spectrophotometry.

RT-PCR analysis
Total RNA (2 µg) was reverse-transcribed for 15 min at 42 C. Reverse transcription was performed in a 20-µl mixture containing 5 mM MgCl2, 1x PCR buffer II, 4 mM each of deoxynucleotide triphosphate, 1 U/µl ribonuclease inhibitor, and 2.5 mM random hexamers. Samples were then denatured for 5 min at 99 C. A no-template Ct was performed for each experiment, establishing the absence of genomic contamination of the samples. PCR was performed using 3 µl of each reverse transcription product as a template. The following primers were used: iNOS sense primer, 5'-GCCTCCCTCTGGAAAGA-3'; antisense primer, 5'-TCCATGCAGACAACCTT-3' (500-bp product); and S16 ribosomal gene sense primer, 5'-TCCGCTGCAGTCCGTTCAAGTCTT-3'; antisense primer, 5'-GCCAAACTTCTTGGATTCGCAGCG-3' (385-bp product). AmpliTaq DNA polymerase (PE Applied Biosystems, Foster City, CA) was used at 25 mU/µl. The PCR mixture (25 µl) contained 2 mM MgCl2, 1x PCR buffer II, and each primer at 0.2 µM. Amplification was performed in a programmable thermal controller (model PTC-100; MJ Research, Inc., Watertown, MA). The samples were first denatured at 95 C for 2 min, followed by 30 PCR cycles; the temperature profile was 95 C (30 sec), 56 C (45 sec), and 72 C (1 min). After the last cycle, additional extension incubation at 72 C (4 min) was performed. After amplification, PCR products were sequenced to verify the products.

PCR products (5 µl of each sample) were subjected to size separation by polyacrylamide gel (4–20% Tris/boric acid/EDTA gels, Novex). The bands were visualized by UV fluorescence after staining with ethidium bromide (1 µg/ml) for 15 min. Densitometric analysis was performed using the PC version of NIH Image software (Scion Image) after photography with a computer-assisted camera (Kodak). For each experiment, iNOS levels were normalized with S16 values and were expressed in units relative to their matched Ct, set as a value of 1. Replicates from three to five primary experiments were subsequently analyzed.

ELISA
To determine the amount of total NO produced and released, matched replicates of Sertoli culture supernatants were assayed for the stable in vivo end products of NO oxidation, i.e. the sum of nitrite (NO2–) and nitrate (NO3–). SCs were treated with forskolin (10 µM), FSH (1 µg/ml), and IL-1ß (10 ng/ml), IL-1ß (10 ng/ml) with NS-398 (COX-2 activity-specific inhibitor; 10 µM), or SP600125 (JNK inhibitor; 10 µM) or matched vehicle blank Ct for 1, 3, 6, and 24 h. At indicated times, cell-free supernatants were transferred to sterile microcentrifuge tubes. Two 80-µl aliquots of conditioned medium from each sample were assayed using Nitrate/Nitrite ELISA kits according to the manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI). The total concentration of nitrate and nitrite was determined in duplicate samples of each experimental replicate by competitive binding enzyme immunoassay using the standard curve method. Fluorescence intensity was measured using a microplate reader (model MRX; Dynex Technologies, Inc., Chantilly, VA). Data were expressed as arbitrary units relative to Ct, set as a value of 1 for the duplicate samples from each individual experiment from a total of three.

NO and ROS flow cytometry analyses
The cell-permeant fluoroprobe DAF-FM passively diffuses across cellular membranes and was used to measure NO in SC after treatment with IL-1ß. ROS was measured as previously reported (12). On d 3 ex vivo, SCs were treated with IL-1ß (15 min, 30 min, 1 h, 3 h, or 6 h) or vehicle (negative Ct) at 34 C and then loaded with DAF-FM diacetate (at 5 µM final concentration) for 20 min at 34 C. SCs were rinsed once with serum-free media (2 min) to remove excess unincorporated probe and to allow complete de-esterification of the intracellular diacetates; cells were then quickly rinsed once again (1x PBS). SCs were harvested to a single cell suspension using Accutase (1.5 ml per well for 5 min at 34 C). Single SC-associated fluorescence was measured by flow cytometric analysis of 20,000 individual cell events in duplicate samples using a Becton Dickinson FACSCalibur cytometer. NO data were obtained from four to five separate SC isolations and subsequent experiments. ROS was measured by flow cytometry as previously reported (12).

Data analysis
Densitometric analyses are expressed in arbitrary units. All results are the mean ± SEM derived from the number of different experiments. Statistical analyses were performed using t test or paired t test, ANOVA. P ≤ 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Our recent studies showed that IL-1ß induces COX-2 and JNK activation, signaling that does not involve ROS generation but does subsequently regulate START (StAR-related lipid transfer protein or amino acid sequence) domain proteins and prostaglandin production The following experiments were conducted using primary SCs to determine whether IL-1ß regulates NO signaling.

IL-1ß induces intracellular NO, JNK-dependent inducible NOS expression, and extracellular NO
Flow cytometry was employed for analysis of intracellular NO at specified times after IL-1ß using matched replicates in three separate Sertoli primary cell culture experiments. IL-1ß was added for 15 min, 30 min, 1 h, 3 h, 6 h, and 24 h before DAF-FM diacetate was added for an additional 20-min period at 34 C. Intracellular NO was significantly induced by IL-1ß treatment of 30 min (P ≤ 0.01; Fig. 1Go, inset). No significant changes were measured at earlier or later time points (not shown) (12). This change in intracellular NO was significant but transient and modest (15–20%), findings consistent with physiological signaling and a potential protective role. Consistent with our earlier study, IL-1ß treatment resulted in reduced basal levels of intracellular ROS at 30 min relative to matched Ct (data not shown).


Figure 1
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  		FIG. 1. IL-1ß, but not FSH or forskolin, significantly increases Sertoli JNK-mediated NO production. SCs were treated with IL-1ß (10 ng/ml; underscored lanes 1), forskolin (10 µM; unscored lanes 4; checkered), or FSH (1 µg/ml; unscored lanes 5; open bars) or matched vehicle Ct [Ct (veh); lanes 1–5] and total [nitrite + nitrate] concentrations in conditioned serum-free media replicates at 3, 6, and 24 h. measured by ELISA. Treatment with the JNK inhibitor SP600125 (underscored lanes 3; cross-hatched bars) and the COX-2 inhibitor NS-398 (underscored lanes 2; stippled bars) significantly inhibited IL-1ß (underscored lanes 1; closed bars) induced increases in extracellular [nitrite and nitrate]. Data were expressed as arbitrary units relative to Ct, set as a value of 1. Results are the mean ± SEM from three individual experiments. *, Significant difference (P ≤ 0.05) from the value for matched SCs treated only with the vehicle blank (Ct). Inset, Analysis of intracellular NO in living cells using flow cytometry. SCs were treated with either IL-1ß for 30 min or vehicle (negative Ct) and cells then loaded with DAF-FM diacetate for an additional 20 min at 34 C, i.e. a total of 50 min. The data shown represents the percentage of single SC-associated fluorescence, i.e. NO-positive cells, measured by flow cytometric analysis of 120,000 individual cell events in duplicate samples. *, Significant difference (P ≤ 0.05) from the value for matched SCs treated only with the vehicle blank (Ct (veh)). {dagger}, Significant difference (P ≤ 0.05) from the value for cells treated with IL-1ß + inhibitors compared with IL-1ß only (closed bars).

 
IL-1ß resulted in a significant and sustained elevation of extracellular nitrites and nitrates (Fig. 1Go, underscored lanes 1; closed bars). From 6–24 h after IL-1ß addition, a significant time-dependent stimulation of NO secretion was observed (1.7- and 8.1-fold, P ≤ 0.02 and P ≤ 0.001, respectively) compared with those of matched vehicle Ct (Ctveh; no underlining).

Endogeneous NO has been shown to affect the activities of both the JNK and COX-2 enzymes, and a physiological circuit of feedback has been suggested as a Ct mechanism in response to normal microenvironmental cues. Therefore, the following pharmacological experiments were performed to determine whether SC activation by IL-1ß results in iNOS-dependent NO induction. In the presence of the specific COX-2 activity inhibitor NS-398, IL-1ß induction of extracellular NO levels was not affected (Fig. 1Go; underscored lanes 2, lightly stippled bars). In the presence of the JNK activity inhibitor SP600125, IL-1ß induction of extracellular NO was significantly inhibited (Fig. 1Go; underscored lanes 3; cross-hatched bars).

In comparison, neither FSH nor forskolin stimulated extracellular NO secretion, findings indicating that increases in SC cAMP do not affect steady-state NO production (Fig. 1Go; unscored lanes 4 and 5, checkered and open bars, respectively). Our previous studies showed that IL-1ß significantly stimulates prostaglandin (PG) E2, F2{alpha}, and I2 production (13). Therefore, we next evaluated the effects of specific PGs directly on SC extracellular NO. No changes in extracellular NO level were observed after PG treatments (PGE2, PGF2{alpha}; 10 µM; data not shown).

Evaluation of IL-1ß effects on Sertoli iNOS expression
To determine the expression of iNOS mRNA in SC, total RNA extracted from SC treated with or without IL-1ß (10 ng/ml) was analyzed by RT-PCR. Figure 2AGo depicts the time course of IL-1ß stimulation at a concentration of 10 ng/ml for 0, 1, 3, 6, and 24 h. In vehicle-Ct SCs, the levels of iNOS mRNA were low, but began to increase as early as 3 h (1.9-fold over Ct, P < 0.01) after a single addition of IL-1ß and continued for up to 24 h (Fig. 2BGo). Neither FSH nor forskolin induced iNOS mRNA (data not shown).


Figure 2
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  		FIG. 2. IL-1ß induces steady-state iNOS mRNA levels in a time-dependent manner. A, An illustrative example of a semiquantitative RT-PCR analysis of iNOS mRNA in matched SCs treated with IL-1ß (10 ng/ml) or without (Ct) for the indicated times is shown. B, Densitometric analysis for all replicate sample cDNA product is shown. Results are the mean ± SEM. Data are normalized with 18S values and are expressed relative to Ct set as a value of 1. *, Significant difference (P ≤ 0.05) from the Ct value. Experiments were repeated three times.

 
To more fully evaluate the effects of IL-1ß on iNOS induction at the level of its translation and steady-state protein levels in SC, proteins were isolated from whole cell lysates of SC treated with IL-1ß as indicated. Western analyses showed a significant time-dependent induction of iNOS but not eNOS (Fig. 3AGo). Significant induction of iNOS protein by IL-1ß (Fig. 3BGo; closed bars) was first evident within 3 h (2.3-fold; P < 0.001) and was maximally elevated by 6 h (6-fold; P < 0.001) compared with Ct. In contrast, constitutive levels of eNOS protein were not affected at any time after IL-1ß (Fig. 3BGo, open bar).


Figure 3
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  		FIG. 3. IL-1ß induces SC iNOS, but not eNOS, expression. A, SCs were cultured in the presence or absence (Ct) of IL-1ß (10 ng/ml) for various periods of time. From three separate primary experiments, whole cell protein extracts were prepared from SC replicates. A representative experiment is shown. Sequential Western analyses demonstrated induction of iNOS protein (130 kDa) but not eNOS protein (140 kDa). The same membranes were reprobed for ß-actin (42 kDa) levels for normalization and to ensure equal protein loading in each lane. B, Densitometric analysis is shown. iNOS (closed bar) and eNOS (open bar) protein levels were normalized to matched ß-actin values and results expressed in arbitrary units with the matched Ct set as a value of 1. Results are the mean ± SEM. *, Significant difference (P ≤ 0.05) from the Ct value.

 
Inhibition of JNK but not COX-2 significantly reduces IL-1ß induction of iNOS
Pretreatment with the JNK-specific inhibitor (SP600125, 10 µM) significantly inhibited IL-1ß-induced iNOS protein levels (Fig. 4Go, B and C, cross-hatched) compared with Ct (Fig. 4Go, B and C, open bar). In contrast, the specific COX-2 activity inhibitor (NS-398, 10 µM) did not affect IL-1ß induction of iNOS (Fig. 4Go, A and C, stippled). No change in the level of eNOS was observed after either IL-1ß alone or together with either the JNK or COX-2 inhibitor (data not shown). Induction of iNOS was significantly attenuated by JNK inhibition as seen at 6 h after IL-1ß addition (Fig. 4CGo; ≥ 50% decrease, P < 0.001).


Figure 4
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  		FIG. 4. Inhibition of JNK, but not COX-2, reduces IL-1ß induction of iNOS. A and B, Western analyses performed from whole cell lysates from SC treated with IL-1ß (10 ng/ml) alone or IL-1ß for 1, 3, and 6 h after a pretreatment with NS-398 (A; 10 µM) or SP600125 (B; 10 µM) and compared with their matched vehicle Ct (Ct; A and B, respectively). C, Densitometric analysis is shown from data of three experiments. iNOS protein levels are normalized to ß-actin values and are expressed in arbitrary units with Ct set as a value of 1. Results are the mean ± SEM. *, Significant difference (P ≤ 0.001) from the IL-1ß- and vehicle-treated value.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To our knowledge, this is the first study to demonstrate in SCs that IL-1ß directly activates JNK, which in turn, induces the iNOS-NO cascade. The data demonstrate that IL-1ß exposure significantly induces intracellular NO generation resulting in extracellular NO secretion by SCs.

In the mammalian testis, the process of spermatogenesis is regulated by a complex interplay of endocrine and paracrine signals. SCs provide a specialized, antiinflammatory microenvironment within the seminiferous tubules for germ cell development. We recently showed that IL-1ß is a potent inducer of several ILs and PG production in SCs, effects due to the phosphorylation of JNK, and the induction of COX-2 expression and activity. Hence, both pathways are involved in a SC cytokine regulatory loop (12). Other studies showed that either IL-1ß alone or in a combination with interferon-{gamma} and TNF{alpha} induction after LPS stimulation increase SC iNOS expression and NO release (21, 22, 27). The precise molecular signals involved in such proinflammatory mechanisms remain unknown.

The MAPKs are important intracellular signaling molecules that regulate cell function and survival in the testis. Three subfamilies of MAPKs have been identified in mammals: ERK, JNK, and p38 MAPK kinases. MAPKs are involved in the induction of high-output iNOS-NO signaling in multiple cell types. SCs express each of these kinases. Recent studies showed that IL{alpha} activates p38 MAPK but not ERK and that p38 mediates the mitogenic effects of IL-1{alpha} on proliferating rat SCs from postnatal d 8 and 9 testes (28). In differentiated, nonproliferating SCs, our studies show that IL-1ß increases time-dependent phosphorylation of JNK without affecting phosphorylation of p38 or p42/p44 ERK (12, 13). JNK phosphorylation occurs within 30 min of IL-1ß, coincident with the first detectable increase in intracellular NO. The subsequent phase of IL-1ß stimulation of NO occurs by iNOS induction. NO generation and extracellular secretion could be prevented by pretreatment with a JNK inhibitor.

JNK activates multiple transcription factors such as c-Jun, activating protein-1, activating transcription factor-2, nuclear transcription factor-{kappa}B (NF-{kappa}B), and histone modifications, initiating complex transcriptional regulation in response to extracellular stimuli (29, 30, 31, 32, 33). IL-1ß has been shown to regulate NF-{kappa}B nuclear translocation and binding to DNA. Recent studies in transformed and transfected immortalized cell lines recently demonstrated that tyrosine nitration of the NF-{kappa}B p65 subunit inactivates its activity (34). Endogeneous nitric oxide has been shown to regulate the extent of NF{kappa}B by cytokines through nitrosylation and subsequent repression of inhibitory {kappa}B kinase in transformed lung epithelial cells and Jurkat cells (35). To date, cell-specific regulation has not been clearly defined using nontransformed or nontransfected normal epithelial cells.

SCs are hormonally regulated primarily by the gonadotropin FSH mediated by ligand binding and activation of its G protein-coupled receptor and the catalytic unit of adenylate cyclase, rapid phosphorylation of cAMP regulatory binding protein, and increases in cAMP transcriptional activation of cAMP response element-containing genes (36, 37, 38, 39, 40, 41). Neither FSH nor forskolin induction of cAMP resulted in a change in iNOS expression or NO production using these methods of detection.

The presence of iNOS in the normal testis suggests that NO is generated under physiological conditions. Given the highly complex and coordinated series of events that regulate the conversion of a stem cell to a spermatozoa, transient NO production by iNOS activity could mediate rapid cytokine or growth factor regulatory signaling during spermatogenesis (42). In the seminiferous tubule, pathological levels of iNOS and NO expression follows treatment of rodents with bacterial LPS, contributing, in part, to the observed germ cell damage, sloughing, and apoptosis (43, 44). Consistent with these findings, our previous studies demonstrated that cryptorchidism-induced apoptosis of germ cells is accelerated in the testes of transgenic mice with overexpression of endothelial NO synthase and NO production (23).

Data obtained from studies with cells derived from other tissues suggest that it may be the balance between NO and ROS that determines whether NO acts in a protective or a destructive capacity (44). Using flow cytometry, we demonstrated that basal ROS is reduced after IL-1ß exposure but, when challenged, SCs can mount a robust burst of ROS in response to a known oxidative stress (12). Taken together, our studies are consistent with an antioxidant role for IL-1ß in SC by concomitant regulation of NO and ROS generation.

In summary, these studies have identified for the first time an IL-1ß-JNK-mediated regulatory mechanism for inducible NOS and NO production in the SC. Several studies in cells as diverse as pancreatic islet cells, cardiac myocytes, sperm cells, and microglia cells suggest that NO is involved in the regulation of COX-2 activity, mobilization and use of arachidonic acid, and prostaglandin production (45, 46, 47, 48). Taken together, our data indicate that NO signaling plays a role in IL-1ß regulation of autocrine and paracrine functions of the SC. Based on the findings of our experimental studies to date, a model is proposed (Fig. 5Go), which illustrates potential mechanisms for the effects of IL-1ß-JNK-NO-signaling on SC activities (12, 13). Specific components of these pathways may provide novel therapeutic targets for interventional use to prevent testicular dysfunction during chronic inflammatory or oxidative stresses.


Figure 5
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  		FIG. 5. JNK as a SC molecular switch for intracellular and extracellular NO signaling. A model illustrates potential IL-1ß-JNK-NO signaling involved in SC physiological autocrine and paracrine activities. Two phases of intracellular NO production were demonstrated in the present study. The first phase (≤60 min) is a rapid and transient increase (#1) likely due to the activation of basal NOS (either or both iNOS and eNOS) present constitutively in the SC. The second phase is JNK-dependent and due to an induction (#2) of iNOS mRNA expression and subsequent increases in iNOS protein (≤3 h), NO production and secretion of nitrites and nitrates (<6 h). The current findings are put in the context of our previous studies (12 13 ). See text for details.

 


   Acknowledgments
 
The authors express their appreciation for expertise in cell handling by Lyann Mitchell, technical assistance by KeumSil Hwang, and flow cytometry by Catherine Rapelje. The use of the Population Council’s Cell Biology and Flow Cytometry Facility is gratefully acknowledged.


   Footnotes
 
This study was supported by National Institutes of Health Grants HD29428 and HD39024 (to P.L.M.).

T. Ishikawa, M.D., conducted these studies in fulfillment, in part, of his Ph.D. degree requirements as well as postdoctoral training.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 3, 2006

Abbreviations: COX, Cyclooxygenase; Ct, control; DAF-FM, 4-amino-5-methylamino-2', 7'-difluorescein; DMSO, dimethylsulfoxide; eNOS, endothelial NOS; NO, nitric oxide; NOS, nitric oxide synthase prostaglandin G/H synthase (PGHS-2 or COX-2); JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; PG, prostaglandin; ROS, reactive oxygen species; SC, Sertoli cell; StAR, steroidogenic acute regulatory protein.

Received May 15, 2006.

Accepted for publication July 26, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Steinberger E, Steinberger A, Ficher M 1970 Study of spermatogenesis and steroid metabolism in cultures of mammalian testes. Recent Prog Horm Res 26:547–588[Medline]
  2. Russell LD, Peterson RN 1985 Sertoli cell junctions: morphological and functional correlates. Int Rev Cytol 94:177–211[Medline]
  3. Skinner MK 1991 Cell-cell interactions in the testis. Endocr Rev 12:45–77[CrossRef][Medline]
  4. de Kretser DM, Loveland KL, Meinhardt A, Simorangkir D, Wreford N 1998 spermatogenesis. Hum Reprod 13(Suppl 1):1–8
  5. Griswold MD 1998 The central role of Sertoli cells in spermatogenesis. Semin Cell Dev Biol 9:411–416[CrossRef][Medline]
  6. Parvinen M 1982 Regulation of the seminiferous epithelium. Endocr Rev 3:404–417[CrossRef][Medline]
  7. Toppari J, Kangasniemi M, Kaipia A, Mali P, Huhtaniemi I, Parvinen M 1991 Stage- and cell-specific gene expression and hormone regulation of the seminiferous epithelium. J Electron Microsc Tech 19:203–214[CrossRef][Medline]
  8. Hecht NB 1998 Molecular mechanisms of male germ cell differentiation. Bioessays 20:555–561[CrossRef][Medline]
  9. Syed V, Stephan J-P, Gerard N, Legrand A, Parvinen M, Bardin CW, Jegou B 1995 Residual bodies activate Sertoli cell interleukin-1a (IL-1a) release, which triggers IL-6 production by an autocrine mechanism, through the lipoxygenase pathway. Endocrinology 136:3070–3078[Abstract]
  10. Okuda Y, Sun X-R, Morris PL 1994 Interleukin-6 (IL-6) mRNAs expressed in Leydig and Sertoli cells are regulated by cytokines, gonadotropins and neuropeptides. Endocrine 2:617–624
  11. Okuda Y, Morris PL 1994 Identification of interleukin-6 receptor (IL-6R) mRNA in isolated Sertoli and Leydig cells: regulation by gonadotropin and interleukins in vitro. Endocrine 2:1163–1168
  12. Ishikawa T, Hwang K, Lazzarino D, Morris PL 2005 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-1ß regulated by activation of c-Jun N-terminal kinase and cyclooxygenase-2 and cytokine induction. Endocrinology 146:5100–5111[Abstract/Free Full Text]
  13. Ishikawa T, Morris PL 2006 A multi-step kinase-based Sertoli cell autocrine-amplifying loop regulates prostaglandins, their receptors, and cytokines. Endocrinology 147:1706–1716[Abstract/Free Full Text]
  14. So HS, Park RK, Kim MS, Lee SR, Jung BH, Chung SY, Jun CD, Chung HT 1998 Nitric oxide inhibits c-Jun N-terminal kinase 2 (JNK2) via S-nitrosylation. Biochem Biophys Res Commun 247:809–813[CrossRef][Medline]
  15. Bruckdorfer R 2005 The basics about nitric oxide. Mol Aspects Med 26:3–31[CrossRef][Medline]
  16. Chun SY, Eisenhauer KM, Kubo M, Hsueh AJ 1995 Interleukin-1ß suppresses apoptosis in rat ovarian follicles by increasing nitric oxide production. Endocrinology 136:3120–3127[Abstract]
  17. Tatsumi N, Fujisawa M, Kanzaki M, Okuda Y, Okada H, Arakawa S, Kamidono S 1997 Nitric oxide production by cultured rat Leydig cells. Endocrinology 138:994–998[Abstract/Free Full Text]
  18. Xie Q, Nathan C 1994 The high-output nitric oxide pathway: role and regulation. J Leukoc Biol 56:576–582[Abstract]
  19. Nathan C, Xie QW 1994 Regulation of biosynthesis of nitric oxide. J Biol Chem 269:13725–13728[Free Full Text]
  20. Sparrow JR, Nathan C, Vodovotz Y 1994 Cytokine regulation of nitric oxide synthase in mouse retinal pigment epithelial cells in culture. Exp Eye Res 59:129–139[CrossRef][Medline]
  21. Bauche F, Stephan JP, Touzalin AM, Jegou B 1998 In vitro regulation of an inducible-type NO synthase in the rat seminiferous tubule cells. Biol Reprod 58:431–438[Abstract/Free Full Text]
  22. Stephan JP, Guillemois C, Jegou B, Bauche F 1995 Nitric oxide production by Sertoli cells in response to cytokines and lipopolysaccharide. Biochem Biophys Res Commun 213:218–224[CrossRef][Medline]
  23. Ishikawa T, Kondo Y, Goda K, Fujisawa M 2005 Overexpression of endothelial nitric oxide synthase in transgenic mice accelerates testicular germ cell apoptosis induced by experimental cryptorchidism. J Androl 26:281–288[Abstract/Free Full Text]
  24. Fujisawa M, Tatsumi N, Fujioka H, Kanzaki M, Okuda Y, Arakawa S, Kamidono S 2000 Nitric oxide production of rat Leydig and Sertoli cells is stimulated by round spermatid factor(s). Mol Cell Endocrinol 160:99–105[CrossRef][Medline]
  25. Jenab S, Morris PL 1998 Testicular leukemia inhibitory factor (LIF) and LIF receptor mediate phosphorylation of signal transducers and activators of transcription (STAT)-3 and STAT-1 and induce c-fos transcription and activator protein-1 activation in rat Sertoli but not germ cells. Endocrinology 139:1883–1890[Abstract/Free Full Text]
  26. Kanzaki M, Morris PL 1998 Identification and regulation of testicular interferon {gamma} (IFN-{gamma}) receptor subunits: IFN-{gamma} enhances interferon regulatory factor-1 (IRF-1) and interleukin-1ß converting enzyme (ICE) expression. Endocrinology 139:2636–2644[Abstract/Free Full Text]
  27. Meroni SB, Suburo AM, Cigorraga SB 2000 Interleukin-1ß regulates nitric oxide production and {gamma}-glutamyl transpeptidase activity in Sertoli cells. J Androl 21:855–861[Abstract]
  28. Petersen C, Svechnikov K, Froysa B, Soder O 2005 The p38 MAPK pathway mediates interleukin-1-induced Sertoli cell proliferation. Cytokine 32:51–59[CrossRef][Medline]
  29. Bonizzi G, Piette J, Merville MP, Bours V 1997 Distinct signal transduction pathways mediate nuclear factor-{kappa}B induction by IL-1ß in epithelial and lymphoid cells. J Immunol 159:5264–5272[Abstract]
  30. Bleich D, Chen S, Wen Y, Nadler JL 1997 The stress-activated c-Jun protein kinase (JNK) is stimulated by lipoxygenase pathway product 12-HETE in RIN m5F cells. Biochem Biophys Res Commun 230:448–451[CrossRef][Medline]
  31. Madamanchi NR, Bukoski RD, Runge MS, Rao GN 1998 Arachidonic acid activates Jun N-terminal kinase in vascular smooth muscle cells. Oncogene 16:417–422[CrossRef][Medline]
  32. Whitmarsh AJ, Yang SH, Su MS, Sharrocks AD, Davis RJ 1997 Role of p38 and JNK mitogen-activated protein kinases in the activation of ternary complex factors. Mol Cell Biol 17:2360–2371[Abstract]
  33. Yang SH, Sharrocks AD, Whitmarsh AJ 2003 Transcriptional regulation by the MAP kinase signaling cascades. Gene 320:3–21[CrossRef][Medline]
  34. Park SW, Huq MD, Hu X, Wei LN 2005 Tyrosine nitration on p65: a novel mechanism to rapidly inactivate nuclear factor-{kappa}B. Mol Cell Proteomics 4:300–309[Abstract/Free Full Text]
  35. Reynaert NL, Ckless K, Korn SH, Vos N, Guala AS, Wouters EF, van der Vliet A, Janssen-Heininger YM 2004 Nitric oxide represses inhibitory {kappa}B kinase through S-nitrosylation. Proc Natl Acad Sci USA 101:8945–8950[Abstract/Free Full Text]
  36. Griswold MD, Heckert L, Linder C 1995 The molecular biology of the FSH receptor. J Steroid Biochem Mol Biol 53:215–218[CrossRef][Medline]
  37. Steinberger A, Hintz M, Heindel JJ 1978 Changes in cyclic AMP responses to FSH in isolated rat Sertoli cells during sexual maturation. Biol Reprod 19:566–572[Abstract]
  38. Steinberger A, Heindel JJ, Lindsey JN, Elkington JS, Sanborn BM, Steinberger E 1975 Isolation and culture of FSH responsive Sertoli cells. Endocr Res Commun 2:261–272[Medline]
  39. Meyer TE, Waeber G, Lin J, Beckmann W, Habener JF 1993 The promoter of the gene encoding 3',5'-cyclic adenosine monophosphate (cAMP) response element binding protein contains cAMP response elements: evidence for positive autoregulation of gene transcription. Endocrinology 132:770–780[Abstract]
  40. Waeber G, Meyer TE, LeSieur M, Hermann HL, Gerard N, Habener JF 1991 Developmental stage-specific expression of cyclic adenosine 3',5'-monophosphate response element-binding protein CREB during spermatogenesis involves alternative exon splicing [published erratum appears in Mol Endocrinol 1993 Nov;7(11):1501]. Mol Endocrinol 5:1418–1430[CrossRef][Medline]
  41. Walker WH, Cheng J 2005 FSH and testosterone signaling in Sertoli cells. Reproduction 130:15–28[Abstract/Free Full Text]
  42. de Kretser DM, Loveland KL, Meinhardt A, Simorangkir D, Wreford N 1998 Spermatogenesis. Hum Reprod 1:1–8[Medline]
  43. O’Bryan MK, Gerdprasert O, Nikolic-Paterson DJ, Meinhardt A, Muir JA, Foulds LM, Phillips DJ, de Kretser DM, Hedger MP 2005 Cytokine profiles in the testes of rats treated with lipopolysaccharide reveal localized suppression of inflammatory responses. Am J Physiol Regul Integr Comp Physiol 288:R1744–R1755
  44. O’Bryan MK, Schlatt S, Phillips DJ, de Kretser DM, Hedger MP 2000 Bacterial lipopolysaccharide-induced inflammation compromises testicular function at multiple levels in vivo. Endocrinology 141:238–246[Abstract/Free Full Text]
  45. Corbett JA, Kwon G, Turk J, McDaniel ML 1993 IL-1ß induces the coexpression of both nitric oxide synthase and cyclooxygenase by islets of Langerhans: activation of cyclooxygenase by nitric oxide. Biochemistry 32:13767–13770[CrossRef][Medline]
  46. Herrero MB, Franchi AM, Gimeno MA 1995 Mouse spermatozoa can synthesize PGE2 and 5-HETE in vitro: stimulatory action of nitric oxide. Prostaglandins Leukot Essent Fatty Acids 53:347–350[CrossRef][Medline]
  47. Goodwin DC, Gunther MR, Hsi LC, Crews BC, Eling TE, Mason RP, Marnett LJ 1998 Nitric oxide trapping of tyrosyl radicals generated during prostaglandin endoperoxide synthase turnover. Detection of the radical derivative of tyrosine 385. J Biol Chem 273:8903–8909[Abstract/Free Full Text]
  48. LaPointe MC, Isenovic E 1999 Interleukin-1ß regulation of inducible nitric oxide synthase and cyclooxygenase-2 involves the p42/44 and p38 MAPK signaling pathways in cardiac myocytes. Hypertension 33:276–282[Abstract/Free Full Text]



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