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Endocrinology Vol. 138, No. 3 1097-1105
Copyright © 1997 by The Endocrine Society

Articles

Tumor Necrosis Factor-{alpha} Regulates Plasminogen Activator Inhibitor-1 in Rat Testicular Peritubular Cells1

Brigitte Le Magueresse-Battistoni, Gilles Pernod, Lucien Kolodié, Anne-Marie Morera and Mohamed Benahmed

INSERM U-407, Bâtiment 3B, Centre Hospitalier Lyon-Sud, Pierre-Benite; and Laboratoire d’ Hématologie, Centre Hospitalo-Universitaire La Tronche (G.P., L.K.), Grenoble, France

Address all correspondence and requests for reprints to: Dr. Brigitte Le Magueresse-Battistoni, INSERM U-407, Bâtiment 3B, Centre Hospitalier Lyon-Sud, 69495 Pierre-Bénite Cedex, France.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We examined the regulation by tumor necrosis factor-{alpha} (TNF{alpha}) of plasminogen activator inhibitor-1 (PAI-1) in cultured peritubular cells recovered from 20-day-old rat testes. We demonstrated that TNF{alpha} in a nanomolar dose range stimulated PAI-1 messenger RNA (mRNA; Northern blots) as well as immunoreactive (Western blots) and bioactive (Stachrom) PAI-1 protein. Induction of PAI-1 mRNA started 4 h after the addition of TNF{alpha} (2.5-fold increase) and peaked (7-fold increase) after 24 h of treatment. Actinomycin D and cycloheximide inhibited the effects of TNF{alpha} on PAI-1 mRNA, suggesting that ongoing RNA and protein syntheses were required. The combined actions of transforming growth factor-{alpha} (TGF{alpha}), a potent inducer of PAI-1, and TNF{alpha} on PAI-1 were less than additive, suggesting the activation of some common pathway. TNF{alpha} action on PAI-1, like that of TGF{alpha} demonstrated previously, was masked by a preexposure to phorbol myristate acetate (a stimulator of protein kinase C) and strongly reduced by staurosporine (an inhibitor of the protein kinase C). Furthermore, using genistein to inhibit tyrosine kinase activity, we not only blocked the action of TGF{alpha} on PAI-1 [initiated upon binding to the tyrosine kinase epidermal growth factor/TGF{alpha} receptor (EGFR)], but also markedly reduced that of TNF{alpha}. Finally, TNF{alpha}, at a dose range that stimulated PAI-1, enhanced EGFR mRNA levels and EGF binding. Together, the present findings suggest that some of the biological effects of TNF{alpha} on PAI-1 might be secondary to de novo synthesis of EGFR. Because TNF{alpha} probably originates from testicular macrophages, such a regulation of PAI-1 by TNF{alpha} may occur in the context of physiological interactions between the testis and the immune system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PERITUBULAR cells are mesenchymal/stromal cells that surround the seminiferous tubules (1). They exist in multiple layers and are in contact through a complex extracellular matrix (basement membrane) with the basal surface of Sertoli cells. Sertoli cells are the other somatic component of the tubule. They are connected to each other by tight junctions. They extend up to the lumen of the tubule and are in close contact with germ cells throughout their development (2, 3). Peritubular cells are likely to be an important component of the testis. They are target cells for testosterone. They are located between the interstitium housing the steroidogenic Leydig cells and the interstitial macrophages, and the seminiferous tubules where meiosis proceeds. They provide structural integrity for the tubule. They cooperate with Sertoli cells in the formation of the tubule basement membrane (which constitutes a prefilter to help create the blood-testis barrier). They exhibit contractile properties to allow spermiation, and they regulate Sertoli cell function, growth, and differentiation, as reviewed recently (4).

The seminiferous tubule is a dynamic structure. It is characterized by a turnover of the extracellular matrix components (4), the migration of germ cells from the basal to the adluminal compartment with their subsequent entry into meiosis, and finally, the spermiation step (2). To allow these movements without perturbating the structural integrity of the seminiferous tubules and disturbing the blood-testis barrier, there must be a fine tuning of the different proteases and antiproteases present in the seminiferous tubules (see review in Ref.5). As the testis is under hormonal and local regulation (4, 6, 7, 8, 9), it is likely that the protease/antiprotease system is also under this interconnected control.

In this report, we focused our interest on plasminogen activator inhibitor-1 (PAI-1). It is secreted by peritubular cells (10) and inhibits the activity of the two plasminogen activators [namely the urokinase (u-PA) and the tissue (t-PA) types] (11, 12), which are produced by Sertoli cells (5, 13, 14). PAI-1 gene expression is induced by growth factors and cytokines (15). In testis, previous studies have reported that PAI-1 is up-regulated by transforming growth factor-ß1 (TGF-ß1) (16), basic fibroblast growth factor (bFGF) (17), and TGF{alpha} (17), factors for which peritubular cells exhibit the corresponding high affinity receptors (18, 19, 20). However, and although cytokines have been deeply implicated in testicular growth, development, and differentiation (8, 9), no report has yet identified whether cytokines could alter the plasminogen activation system.

As peritubular cells face the interstitium, and macrophages are a potential source of tumor necrosis factor {alpha} (TNF{alpha}) within the testis (21), we sought to determine whether regulatory interactions could take place between the immune system and the peritubular cells that will modify the antiprotease activity of the tubule.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DMEM-Ham’s F-12 was obtained from Life Technologies (Grand Island, NY) and was supplemented with gentamicin (20 mg/L). Human recombinant TNF{alpha} was purchased from Prepro Tech (Canton, MA). Human recombinant TGF{alpha} was obtained from Sigma Chemical Co. (St. Louis, MO). Actinomycin D, cycloheximide, and staurosporine were of culture grade and purchased from Sigma. Genistein was purchased from Upstate Biotechnology (Lake Placid, NY).

Cell preparation
Peritubular and Sertoli cells were isolated from 20-day-old rats and cultured at 32 C in a humidified atmosphere of 5% CO2 as previously described (22), with certain modifications (19). A hyaluronidase treatment (0.1 mg/ml; 30 min; 34 C) was introduced after the first collagenase-dispase digestion, followed by collection of peritubular cells in the supernatant. After several washes in culture medium, cells were filtered through a 20-µm nylon screen. Cells were seeded in 60-cm2 petri dishes (Northern blots) or in 24-multiwell plates (binding studies) and cultured in 10% FCS until confluent. Four days later, cells were cultured in the absence of serum for 24–48 h. Alkaline phosphatase is a marker for peritubular myoid cells (23) and has been used routinely to detect peritubular myoid cells in the cell cultures. Alkaline phosphatase-positive cells were less than 5% of the total cell population in Sertoli cell cultures and greater than 85% in peritubular cell cultures.

Spermatogenic cells were isolated from 60- to 90-day-old rat testes by trypsinization. The resulting crude germ cell population (containing germ cells from all developmental steps) was submitted to a centrifugal elutriation using a rotor Beckman JE-6 (Fullerton, CA), as described previously (24, 25). Two fractions enriched at 80–85% with pachytene spermatocytes and early spermatids were harvested. After collection, the different cell populations were processed for RNA.

Treatment of peritubular cells in culture
On day 5 or 6 of culture, peritubular cells were treated for various lengths of time with different reagents (growth factors or drugs) as described in Results. Thereafter, culture media were collected, and cells were either scraped from the dishes and processed for RNA or used for binding studies. None of the treatments performed affected cell number or viability in our experimental conditions.

Isolation of RNA and Northern blot hybridization
Total RNA was isolated using acid-guanidinium thiocyanate-phenol-chloroform extraction in a single step procedure as reported previously (26). The polyadenylated [poly(A)+] RNA was isolated using the Promega (Charbonnieres, France) PolyA tract messenger RNA (mRNA) isolation system. The probes used for the hybridization were a 1.2-kilobases (kb) EcoRI-BglII fragment of the human PAI-1 complementary DNA (cDNA), a 2.2-kb EcoRI fragment encompassing the entire extracellular domain of the rat epidermal growth factor (EGF) receptor (ER1), a 0.8-kb EcoRI fragment encoding only part of the intracellular domain but not the extracellular domain (ER3), and a 1.3-kb rat glyceraldehyde-3-phosphate dehydrogenase cDNA (GAPDH). These cDNA probes were provided by Dr. L. R. Lund, Drs. S. H. Earp and J. M. Blanchard, respectively. Probes were labeled using the Promega random primed DNA labeling kit (SA, 109 dpm/µg DNA). Northern blot analysis was performed as previously described (19). A 0.24- to 9.5-kb Promega RNA ladder was used to determine the size of the transcripts.

Western blots
SDS-PAGE and Western blotting were carried out as described previously (27, 28). Primary antibody was a rabbit antirat PAI-1 IgG (dilution, 1:200) from American Diagnostics (1062, Greenwich, CT). Secondary antibody was a goat antirabbit IgG (dilution, 1:500) conjugated to alkaline phosphatase and was obtained from Dako (Trappes, France). Conditioned media from peritubular cells were concentrated 10 times using Centriprep (Mr cut-off, 10 kDa; Amicon, Beverly, MA). Proteins were separated on a 10% SDS-PAGE electrophoresis. A rat PAI-1 positive control (1060, American Diagnostics) and prestained mol wt standards were loaded in each SDS-PAGE gel. Under these conditions, PAI-1 migrated as a doublet band, a major band of 49 and a minor band of 46 kDa, as reported previously (28).

Colorimetric assay of PAI-1
Stachrom PAI-1 (Diagnostica Stago, Asnieres-Sur-Seine, France) is a colorimetric assay based on the ability of PAI-1 to form a complex with urokinase and inhibit the generation of plasmin (29). The reaction mixture contains either PAI-1 calibrators or the test sample plus a cocktail of urokinase, plasminogen, and inhibitors of {alpha}2-antiplasmin and {alpha}2-macroglobulin. The amount of PAI-1 is inversely proportional to the amount of plasmin generated. Results are expressed as amidolytic units (AU) per ml and correspond to the amount of bioactive PAI-1 present in the concentrated samples. The sensitivity of the assay ranged from 1–2 AU/ml. Inter- and intravariations were 8.6% and 7.9%, respectively.

125I labeling of EGF and binding assays
Purified EGF (1 µg) was labeled using 125I (Amersham, Les Ulis, France) by the chloramine-T procedure (30). Preparations of [125I]EGF with a specific activity of 200–300 µCi/µg were obtained by this method. For measurements of [125I]EGF binding, cells were seeded to a confluence of 500,000 cells in 24-multiwell plates. Cells were washed once with the culture medium and then incubated with [125I]EGF (0.13 nM) at 4 C in the absence of unlabeled EGF (total binding) or in the presence of an excess (300-fold) of unlabeled EGF (nonspecific binding). The cells were then washed with PBS containing BSA (1 mg/ml) at 4 C and solubilized in 0.5 N sodium hydroxide-0.4% deoxycholate. The radioactivity was measured in a {gamma}-counter. For all experiments, specific binding is the difference between total binding and nonspecific binding. Binding data were evaluated by the method of Scatchard with the aid of EBDA and Ligand computer programs (31), which were converted to BIOSOFT by G. A. McPherson and distributed by Elsevier Biosoft (Cambridge, UK).

Data analysis
The band densities were determined by scanning densitometric analysis using the Bioprofil scanner (Vilber Lourmat, Marnes-la-Vallee, France). The amount of RNA in each lane of each blot was internally standardized within a blot by assessing the amount of GAPDH mRNA per lane. All experiments were repeated at least three times, with independent cell preparations. A representative experiment of each series is presented. The significance of the results was determined by ANOVA, linear regression analysis, or Student’s t test when comparing data from two groups. Differences are accepted as significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Regulation of PAI-1 mRNA levels
The addition of TNF{alpha} in doses ranging from 0.125–25 ng/ml induced in a dose-dependent manner (P < 0.01) the 3-kb signal, corresponding to the PAI-1 mRNA, after 8 h of treatment (Fig. 1Go). A maximal increase (3.7-fold) occurred with 12.5 ng/ml (0.7 nM) of TNF{alpha}, and the ED50 was 1 ng/ml (0.06 nM).



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  		Figure 1. TNF{alpha}-induced enhancement of PAI-1 mRNA levels. Peritubular cells were treated for 8 h with increasing doses of TNF{alpha}, ranging from 0.125–25 ng/ml, after which total RNA was extracted and analyzed by Northern blots. The membranes were successively hybridized with PAI-1 and GAPDH cDNA probes. A Northern blot is shown in the upper panel (lane 1, 0; lane 2, 0.125; lane 3, 1.25; lane 4, 12.5; lane 5, 25 ng/ml TNF{alpha}). Lower panel, Data on PAI-1 mRNA levels yielded by scanning the autoradiographs were normalized to the GAPDH signal, expressed as a percentage of the control value, and are the mean ± SEM of three dishes.

 
As shown in Fig. 2Go, the effect of TNF{alpha} was time dependent. Results are expressed as a percentage of the time-matched controls (from 2–48 h). The first significant effects were noted after 4-h exposure to TNF{alpha} (a 2.46-fold increase; P < 0.01). The stimulation reached a plateau at 24 h (a 6.72-fold increase; P < 0.001) and thereafter was maintained for additional 24 h.



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  		Figure 2. Kinetics of the TNF{alpha}-induced increase in PAI-1 mRNA levels. Peritubular cells were treated or not with 12.5 ng/ml TNF{alpha} for up to 48 h, after which total RNA was extracted and analyzed by Northern blots. Membranes were sequentially hybridized with the PAI-1 and GAPDH cDNA probes. Autoradiographs were scanned, and results were normalized to the GAPDH signal. Values are the mean ± SEM of three dishes and are expressed as a percentage of the time-matched control value. a, P < 0.01; b, P < 0.001 (compared to the respective time-matched control).

 
Regulation of PAI-1 secretion
By Western blotting, two proteins that migrated at 49 and 46 kDa were observed in peritubular cell-conditioned medium. This doublet comigrated with purified rat HTC PAI-1. Addition of TNF{alpha} (12.5 ng/ml) increased approximately 2.7-fold (P < 0.02; n = 4) the levels of immunoreactive PAI-1 after 24 h (Fig. 3Go). In addition, we found that TNF{alpha} increased 2.7-fold (P < 0.01) the secretion of bioactive PAI-1 measured after 48 h of treatment. Indeed, control cells secreted 10.42 ± 2.52 AU/ml (n = 3), and TNF{alpha}-treated cells secreted 27.60 ± 2.22 AU/ml (n = 3).



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  		Figure 3. Western blot analysis of the TNF{alpha}-induced enhancement of PAI-1 antigen. Peritubular cells were treated (lanes 2 and 4) or not (lane 3) for 24 h with 12.5 ng/ml TNF{alpha}. Lane 1, Rat positive PAI-1; Culture media were collected, concentrated, and analyzed by Western blot.

 
Actions of actinomycin D and cycloheximide
To determine whether ongoing RNA and protein synthesis were required for TNF{alpha} action on PAI-1 mRNA levels, peritubular cells were treated with actinomycin D (5 µg/ml) or cycloheximide (50 µg/ml). After 30 min and without changing the culture medium, cells were treated with or without TNF{alpha} (12.5 ng/ml). Incubations were stopped 8 h later. The results presented in Fig. 4Go indicate that actinomycin D inhibited significantly by 4-fold (P < 0.001), and cycloheximide by 65% (P < 0.05) the TNF{alpha}-induced enhancement of PAI-1 mRNA levels. Of note and as previously described (12, 17), cycloheximide by itself enhanced PAI-1 mRNA levels.



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  		Figure 4. Effects of actinomycin D (AD) and cycloheximide (CX) on the TNF{alpha}-induced increase in PAI-1 mRNA levels. Peritubular cells were pretreated with either AD (+ AD) or CX (+ CX) for 30 min (basal, no addition during pretreatment), after which cells were treated with (+ TNF alpha; 12.5 ng/ml) or without (control) TNF{alpha}. The incubations were continued for 8 h, after which total RNA was extracted and analyzed by Northern blots. Membranes were successively hybridized with the PAI-1 and GAPDH cDNA probes. A Northern blot is shown in the upper panel (lanes 1–3, control; lanes 4–6, + TNF{alpha}; lanes 1 and 4, basal; lanes 2 and 5, + AD; lanes 3 and 6, + CX). Lower panel, Data on PAI-1 mRNA levels were normalized to the GAPDH signal, expressed as a percentage of the basal control value (no TNF{alpha}, no AD, no CX) and are the mean ± SEM of three dishes. a, P < 0.001; d, P < 0.01 (compared to untreated cells). b, P < 0.001; c, P < 0.05 (compared to TNF{alpha}-treated cells).

 
Combined actions of TNF{alpha} and TGF{alpha}
TGF{alpha} is synthesized by peritubular cells (20). It is a potent and fast acting inducer of PAI-1 in our culture model (17). We thus examined the combined actions of TGF{alpha} and TNF{alpha} on PAI-1 mRNA levels. Peritubular cells were treated for 8 h with TNF{alpha} and/or TGF{alpha} at doses eliciting the maximal increase in PAI-1 mRNA levels. The data presented in Fig. 5Go show that PAI-1 mRNA levels in TGF{alpha}- and TNF{alpha}-treated cells were not significantly different (P = 0.2, using ANOVA) from PAI-1 mRNA levels measured in TGF{alpha}-treated cells, suggesting that the two factors may activate some common pathway.



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  		Figure 5. Combined effects of TGF{alpha} and TNF{alpha}. Peritubular cells were treated for 8 h with or without TGF{alpha} (10 ng/ml) or TNF{alpha} (12.5 ng/ml). Total RNA was extracted and analyzed by Northern blots. Membranes were successively hybridized with the PAI-1 and GAPDH cDNA probes. A Northern blot is shown in the upper panel (lane 1, control; lane 2, + TGF{alpha}; lane 3, + TNF{alpha}; lane 4, + TGF{alpha} + TNF{alpha}). Lower panel, PAI-1 mRNA data yielded by scanning the autoradiographs were normalized to the GAPDH signal and are the mean ± SEM of three dishes. ns, Not significant (P = 0.2, using ANOVA).

 
Effects of phorbol 12-myristate 13-acetate (PMA) and staurosporine
As we previously demonstrated that TGF{alpha}-induced PAI-1 may involve the protein kinase C (PKC) pathway (17), we analyzed the PKC dependency of TNF{alpha} regulation of PAI-1 mRNA levels. Addition of either TNF{alpha} or PMA (100 nM), a stimulator of PKC and PAI-1 mRNA levels (17) (Fig. 6Go), did not further significantly increase PAI-1 mRNA levels in cells pretreated for 24 h with 100 nM PMA (Fig. 6Go). We also employed staurosporine to inhibit PKC. The dose of 100 nM was selected because this dose did not affect PAI-1 mRNA levels in control cells (Fig. 7Go), whereas it inhibited 100% of the PMA-induced response (not shown). We observed that the stimulation exerted by TNF{alpha} was +339% (P < 0.01) in cells not exposed to staurosporine and +102% (P < 0.04) in cells exposed to staurosporine. Thus, exposure to staurosporine resulted in a 70% loss of responsiveness to TNF{alpha} (Fig. 7Go).



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  		Figure 6. Effect of long term PMA pretreatment on the TNF{alpha}-induced increase in PAI-1 mRNA levels. After a 24-h exposure to 100 nM PMA (+ PMA), cells were stimulated with or without TNF{alpha} (12.5 ng/ml) or PMA (100 nM) for 8 h. Total RNA was extracted and analyzed by Northern blots. Membranes were successively hybridized with the PAI-1 and GAPDH cDNA probes. A Northern blot is shown in the upper panel (lanes 1–3, no PMA pretreatment; lanes 4–6, + PMA pretreatment; lanes 1 and 4, control; lanes 2 and 5, + TNF{alpha}; lanes 3 and 6, + PMA). Lower panel, Data on PAI-1 mRNA levels were normalized to the GAPDH signal and are the mean ± SEM of three dishes. a, P < 0.001; b, P < 0.02 (compared to cells not pretreated with PMA). ns, Not significant compared to PMA-pretreated cells.

 


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  		Figure 7. Effect of staurosporine (ST) on the TNF{alpha}-induced increase in PAI-1 mRNA levels. Peritubular cells were pretreated with (+ ST) or without (- ST) staurosporine (100 nM) for 30 min, after which cells were treated with (+ TNF alpha; 12.5 ng/ml) or without (control) TNF{alpha}. The incubations were continued for 8 h, after which total RNA was extracted and analyzed by Northern blots. Membranes were successively hybridized with the PAI-1 and GAPDH cDNA probes. A Northern blot is shown in the upper panel (lanes 1 and 2, control; lanes 3 and 4, + TNF{alpha}; lanes 1 and 3, - ST; lanes 2 and 4, + ST). Lower panel, Data on PAI-1 mRNA levels were normalized to the GAPDH signal and are the mean ± SEM of three dishes. a, P < 0.01 (compared to untreated cells); b, P < 0.04 (compared to ST-treated cells); c, P < 0.02 (compared to TNF{alpha}-treated cells).

 
Action of genistein
TGF{alpha} acts upon binding to its high affinity tyrosine kinase receptor, the EGF/TGF{alpha} receptor (EGFR). To further investigate the interference in the action of TNF{alpha} and of TGF{alpha} on PAI-1, we analyzed the action of genistein, an inhibitor of tyrosine kinases (32). The protocol included pretreatment with 10 µg/ml genistein, followed by 8-h exposure to either TGF{alpha} (10 ng/ml) or TNF{alpha} (25 ng/ml). Doses higher than 10 µg/ml were found to affect the basal steady state levels of PAI-1 mRNA levels (not shown). The results presented in Fig. 8Go indicate that genistein abrogated the TGF{alpha}-induced enhancement of PAI-1 mRNA levels. The stimulation exerted by TNF{alpha} was +125% (P < 0.05) in cells exposed to genistein and +767% (P < 0.001) in cells not exposed to genistein. Thus, exposure to genistein resulted in an 84% loss of responsiveness to TNF{alpha}.



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  		Figure 8. Effect of genistein on the TGF{alpha}- and TNF{alpha}-induced increase in PAI-1 mRNA levels. Peritubular cells were pretreated with (+ genistein) or without (- genistein) genistein for 30 min, after which cells were treated with or without (control) 10 ng/ml TGF{alpha} or 25 ng/ml TNF{alpha}. The incubations were continued for 8 h, after which total RNA was extracted and analyzed by Northern blots. Membranes were successively hybridized with the PAI-1 and GAPDH cDNA probes. A Northern blot is shown in the upper panel (lanes 1–3, - genistein; lanes 4–6, + genistein; lanes 1 and 4, control; lanes 2 and 5, + TGF{alpha}; lanes 3 and 6, + TNF{alpha}). Lower panel, Data on PAI-1 mRNA levels were normalized to the GAPDH signal and are the mean ± SEM of three dishes. a, P < 0.005; b, P < 0.001 (compared to untreated cells); ns, not significant; c, P < 0.05 (compared to genistein-treated cells).

 
TNF{alpha} up-regulated EGF binding and EGFR mRNA
Finally, we analyzed whether TNF{alpha} could regulate EGFR. As previously described (20), we found that peritubular cells exhibited high affinity receptors for EGF, with a Kd value of 100 pM and 15,000 binding sites/cell (not shown). Addition of TNF{alpha} (20 ng/ml) to cultured peritubular cells resulted in a time-dependent increase in EGF binding (Fig. 9Go). The increase first became apparent by 6 h after the addition of TNF{alpha} 20 ng/ml (+19%; P < 0.02). A plateau was observed after 26 h (+55%; P < 0.001).



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  		Figure 9. Kinetics of the TNF{alpha}-induced increase in EGF binding. Peritubular cells were treated or not with 20 ng/ml TNF{alpha} for up to 50 h. Cells were washed and then incubated with [125I]EGF in the absence (total binding) or presence of an excess of unlabeled EGF (nonspecific binding). Data represent the specific binding (total binding minus nonspecific binding) of EGF in control and TNF{alpha}-treated cells. Values are the mean ± SEM of nine (control) or three (+ TNF{alpha}) dishes. a, P < 0.02; b, P < 0.001 (vs. control cells).

 
We next investigated whether the increase in EGF binding was secondary to the increase in EGFR mRNA levels in cultured peritubular cells. We first analyzed the size of the EGFR transcripts. As shown in Fig. 10aGo using the ER3 cDNA probe, three major bands, migrating at 9.6, 6.5, and 5 kb, and a diffuse and minor band of 1.3 kb were observed in cultured peritubular cells (lane 4) and, to a lesser extent, in 20-day-old rat cultured Sertoli cells (lane 3). A very weak hybridization pattern was detected in early spermatids (lane 2), but not in pachytene spermatocytes (lane 1). We next stripped this blot and hybridized it with the ER1 cDNA probe, which extends 5' to the ER3 cDNA probe and encompasses the extracellular domain. A hybridization pattern similar to that yielded with the ER3 cDNA probe, was observed except that a 2.7-kb band appeared with an intensity similar to the other transcripts (Fig. 10bGo).



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  		Figure 10. EGFR transcripts in testicular cells. Poly(A)+ RNA (5 µg) prepared from freshly isolated pachytene spermatocytes (lane 1), early spermatids (lane 2), 5-day cultured 20-day-old rat Sertoli cells (lane 3), and 5-day cultured 20-day-old rat peritubular cells (lane 4) was analyzed by Northern blots. Membranes were successively hybridized with the ER3 (a) and then with the ER1 (b) cDNA probes. The sizes of the RNA ladder are indicated on the left. The arrowheads point to the EGFR transcripts.

 
Addition of TNF{alpha} (12.5 ng/ml) for 6 h resulted in a significant stimulation of about 2.2-fold (P < 0.01; n = 3) of each transcript (Fig. 11Go). In routine analysis, we worked with total RNA rather than poly(A+). Under these conditions, we could only quantify the 9.6-kb transcript; the other hybridizing bands were too diffuse (Fig. 11Go). Maximal and half-maximal increases in the 9.6-kb transcript were obtained with 25 ng/ml (1.4 nM) and 2 ng/ml (0.12 nM) TNF{alpha} (Fig. 11Go). The first significant effects (1.63-fold increase; P < 0.02) were noted after 2-h exposure to TNF{alpha} (25 ng/ml). The plateau of stimulation (a 7.6-fold increase; P < 0.001) was reached after 24 h of treatment (Fig. 12Go).



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  		Figure 11. TNF{alpha}-induced increase in EGFR mRNA levels. a, Peritubular cells were treated for 6 h with 12.5 ng/ml TNF{alpha}, after which poly(A)+ was prepared and loaded onto gels (1 µg/ml) for Northern blot analysis. Membranes were hybridized successively with the ER1 and GAPDH cDNA probes. Lanes 1 and 3, Control; lane 2, + TNF{alpha}. The arrowheads point to the four major EGFR transcripts. b, Peritubular cells were treated for 8 h with increasing doses of TNF{alpha}, after which total RNA was prepared and analyzed. Membranes were hybridized sequentially with the ER1 and GAPDH cDNA probes. Lane 1, 0; lane 2, 0.125; lane 3, 1.25; lane 4, 12.5; lane 5, 25 ng/ml TNF{alpha}. The arrowhead points to the 9.6-kb transcript. c, Data for the 9.6-kb mRNA levels yielded by scanning the autoradiographs were normalized to the GAPDH signal, expressed as a percentage of the control value, and are the mean ± SEM of three dishes.

 


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  		Figure 12. Kinetics of the TNF{alpha}-induced increase in EGFR mRNA levels. Peritubular cells were treated or not with TNF{alpha} (25 ng/ml) for up to 24 h, after which total RNA was extracted and analyzed by Northern blots. Blots were successively hybridized with the ER1 and GAPDH cDNA probes. Autoradiographs were scanned, and data for the 9.6-kb mRNA levels were normalized to the GAPDH signal. Values are the mean ± SEM of triplicate dishes. a, P < 0.02; b, P < 0.01, c, P < 0.001 (compared to the time-matched control).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrated that PAI-1 gene expression in cultured peritubular cells recovered from 20-day-old rat testes was up-regulated by TNF{alpha}. We also presented evidence suggesting that part of the TNF{alpha}-induced enhancement of PAI-1 might be secondary to a de novo synthesis of EGFR.

The central location of peritubular cells within the testis, between the interstitial tissue and the inner of the seminiferous tubules, suggests that these cells may not only establish a dialogue with Sertoli cells and Leydig cells as reviewed previously (4), but also with testicular macrophages through diffusible factors. Recently, TNF{alpha} has been shown to be produced by testicular macrophages (21). In addition, increasing evidence accumulated over the past years suggests that TNF{alpha} may have a profound influence on Sertoli cell and Leydig cell function and differentiation (see review in Refs. 8 and 9). Therefore, in the present study we investigated whether TNF{alpha} could also regulate peritubular cells by assessing the action of this cytokine on PAI-1, a marker for peritubular cells (10). Our interest in studying PAI-1 stems from the fact that net protease activity within the gonad is likely to insure the structural cohesion of the tubule and to be involved in the formation of seminiferous cord structures during fetal life, in the remarkable restructuring occurring at specific stages of spermatogenesis, and in the turnover of the extracellular matrix (see review in Ref.5).

We first showed that TNF{alpha} enhanced both PAI-1 mRNA and protein levels. The concentrations of TNF{alpha} used in this study are well within the Kd of the two high TNF{alpha} affinity receptors described previously (33). Our data are also consistent with previous studies showing induced PAI-1 activity in cultured human endothelial, carcinoma, or fibrosarcoma cells treated with TNF{alpha} (see review in Ref.12). We observed that the action of TNF{alpha} on PAI-1 required ongoing RNA synthesis. Additionally, posttranscriptional events such as TNF{alpha}-induced stabilization of PAI-1 mRNA might explain the difference in the amplitude of the effects exerted by TNF{alpha} on PAI-1 mRNA and on PAI-1 protein (approximately a 7-fold vs. a 3-fold increase). Interestingly, induction of PAI-1 mRNA levels by TNF{alpha} started 4 h after the addition of the cytokine, and mRNA levels continued to increase with time. The stimulation reached a plateau at 24 h and thereafter was maintained for an additional 24 h. These data contrast with the kinetics of enhancement of PAI-1 mRNA induced with either bFGF or TGF{alpha} (17). Indeed, the effects of bFGF and TGF{alpha} on PAI-1 mRNA peaked by 4 h and reached a nadir by 24 h. In addition, cycloheximide abrogated TNF{alpha} action on PAI-1, suggesting a role of protein mediators in the signal transduction pathway. By contrast, the inhibitor of protein synthesis augmented in an additive manner bFGF- and TGF{alpha}-induced enhancement of PAI-1 (17). Thus, our results suggested that upon stimulation by TNF{alpha}, a cascade of events was involved, leading to a sustained stimulation of PAI-1. A similar delayed action of TNF{alpha} has been previously demonstrated on Sertoli cell inhibin mRNA levels (34).

In an attempt to elucidate the cascade of events by which TNF{alpha} could enhance PAI-1, we examined the combined actions of TGF{alpha} and TNF{alpha}. Indeed, it is known that TNF{alpha} can enhance TGF{alpha} and EGFR in human fibroblasts and pancreatic cancer cells (35, 36, 37). EGFR is the high affinity tyrosine kinase receptor for TGF{alpha}. It is up-regulated by TGF{alpha} (38). In addition, TGF{alpha} is synthesized by peritubular cells (20), and it stimulates PAI-1 (17). We found that TNF{alpha} did not significantly enhance PAI-1 mRNA levels in cells treated with a maximal dose of TGF{alpha}. This finding suggested the activation of some common pathway in response to TGF{alpha} and TNF{alpha}.

It is known that PMA augments PAI-1 gene expression (17, 39), that TGF{alpha}-induced enhancement of PAI-1 may involve PKC activation (17), and that TNF{alpha} activates multiple intracellular signaling mechanisms, including PKC (40). We thus examined the PKC dependency of TNF{alpha} regulation of PAI-1. We observed that down-regulation of PKC (provoked by long term pretreatment with PMA) rendered cells refractory to further TNF{alpha} treatment, and that staurosporine significantly decreased the effects of TNF{alpha}. However, the actions of TNF{alpha} could simply be masked by the baseline increase in PAI-1 mRNA levels observed in PMA-pretreated cells. In addition, staurosporine is not a specific inhibitor of PKC. Therefore, more experiments will be required to firmly establish that activation of PKC may be a common mechanism through which TGF{alpha} and TNF{alpha} induce PAI-1.

We also challenged cells with genistein, an inhibitor of the tyrosine kinases (32). As expected, TGF{alpha} action on PAI-1 was totally inhibited by genistein. In addition, we found that genistein markedly reduced TNF{alpha}-induced enhancement of PAI-1 mRNA levels. However, it cannot be excluded that TNF{alpha}-induced PAI-1 activates tyrosine kinases different from the EGFR in our culture model. For example, TNF{alpha} has been shown in different human cell lines to activate the JNK protein kinase signal transduction pathway, and JNK protein kinases are activated by dual phosphorylation on tyrosine and threonine (41, 42). In addition, TNF{alpha} has been shown to induce the expression of bFGF (and bFGF induces PAI-1) (17) and nerve growth factor (42). These factors act upon binding to high affinity tyrosine kinase receptors, which have been identified in peritubular cells (19, 43).

We finally investigated the action of TNF{alpha} on EGFR mRNA levels and EGF binding. We found that TNF{alpha} at a dose range that stimulated PAI-1 enhanced EGFR mRNA levels and EGF binding, following a kinetics and with an amplitude comparable to those reported earlier for human fibroblasts (35, 36, 37). Together with our previous findings, this study raised the possibility that some of the TNF{alpha} effects on PAI-1 may result from de novo synthesis of EGFR. However, the kinetics of the TNF{alpha} enhancement of PAI-1 mRNA levels (first significant effects at 4 h) and of EGF binding (first significant effects at 6 h) suggest that other mechanisms are triggered by TNF{alpha} to enhance PAI-1. Previous studies have reported that TNF{alpha} could activate nuclear factor-{kappa}B and stimulate fos and jun genes in the first hour after the addition of the cytokine (44, 45, 46). As the products of the fos and jun genes interact to enhance transcription of the AP-1-responsive genes, it would be of interest to determine in our culture model whether TNF{alpha} action on PAI-1, which is an AP-1-responsive gene (39), also involved an induction of the fos and jun genes.

Our study also revealed that EGFR mRNA was present as four major transcripts of 9.6, 6.5, 5, and 2.7 kb in peritubular cells, to a lesser extent in Sertoli cells, and very faintly in early spermatids. These data extend the findings of a previous study using freshly isolated testicular cells and a S1 nuclease procedure (20). All transcripts except the 2.7-kb one are sufficient in size to code for the full-length EGFR whose coding region alone spans 3.6 kb (47). It would be of interest to determine whether the 2.7-kb transcript in testicular cells, like that in liver cells (47), is generated by alternative splicing of the primary receptor mRNA and encodes a truncated and secreted form of the EGFR.

Whether the in vitro data presented here reflect a potential physiological (or physiopathological) role of TNF{alpha} in the male gonad function requires further studies. In this context, it should be remembered that whereas TNF{alpha} produced by germ cells (the other testicular source for TNF{alpha} identified to date) (48) cannot cross the blood-testis barrier and thus cannot reach peritubular cells, TNF{alpha} may also originate apart from testicular macrophages, from the circulatory system. This would be the case particularly in patients with critical illness, burns, and sepsis. Interestingly, those patients have depressed gonadal function. Consistently, administration of TNF{alpha} to rats induced a dramatic decline in testosterone levels and severe epithelium damage. Furthermore, TNF{alpha} has been shown in vitro to reduce testosterone production by Leydig cells and to antagonize the actions of FSH and insulin-like growth factor I in Sertoli cells (9). As male infertility is associated in some cases with abnormal thickening of the seminiferous tubule lamina propria or boundary tissue (49, 50), it would be of interest to determine whether an excess of TNF{alpha} after an infection or an inflammation leads to an increase in PAI-1, thickening the tubule basement membrane and reducing fertility. Nonetheless, it should be noted that mice with single deficiencies of lymphotoxin (51), 55-kDa (52) or 75-kDa (53) TNF receptors, or PAI-1 (54) were apparently healthy and fertile. Thus, this would suggest that other cytokines or growth factors (8, 9) and other antiproteases (5) present in the testis exert redundant or overlapping functions, bypassing the knocked out gene.

In summary, by use of a model of cultured peritubular cells, this study has demonstrated that besides being regulated by cAMP analogs (16), TGFß (16), bFGF (17), and TGF{alpha} (17), PAI-1 gene expression is also under the control of TNF{alpha}. Given that PAI-1 inhibits Sertoli cell plasminogen activator activity, TNF{alpha} might be involved in the dialogue between Sertoli cells and peritubular cells by participating in the regulation of proteolytic activity within the seminiferous tubules.


   Acknowledgments
 
We are indebted to Dr. H. S. Earp (University of North Carolina, Chapel Hill, NC), Dr. R. L. Lund (The Finsen Laboratory, Copenhagen, Denmark), and Dr. J. M. Blanchard (Faculté des Sciences, Montpellier, France) for providing, respectively, the rat ER1 and ER3, the human PAI-1, and the rat GAPDH cDNA probes. We thank Annick Simon for technical assistance. We are grateful to Dr. Indrani Bagchi (Population Council, New York, NY) for her careful reading of the manuscript.


   Footnotes
 
1 This work was supported by INSERM and Ministère de la Recherche et de l’Enseignement Supérieur. Back

Received July 22, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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