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Molecular Endocrinology, doi:10.1210/me.2005-0423
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Molecular Endocrinology 20 (4): 904-915
Copyright © 2006 by The Endocrine Society

Spermatogonial Cell-Mediated Activation of an I{kappa}B{zeta}-Independent Nuclear Factor-{kappa}B Pathway in Sertoli Cells Induces Transcription of the Lipocalin-2 Gene

Ryu-Suke Fujino, Kiyoko Tanaka, Masami Morimatsu, Kazuhiro Tamura, Hiroshi Kogo and Takahiko Hara

Stem Cell Project Group (R.-S.F., K.Tan., T.H.), The Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, Bunkyo-ku, Tokyo 113-8613; Laboratory of Animal Experiment for Disease Model (M.M.), Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815; and Department of Endocrine Pharmacology (R.F., K.Tam., H.K.), Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan

Address all correspondence and requests for reprints to: Takahiko Hara, Stem Cell Project Group, The Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan. E-mail: thara{at}rinshoken.or.jp.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In spermatogenesis, Sertoli cells serve as supporting cells for the proliferation and differentiation of germ cells. However, it appears that Sertoli cell function is regulated by adjacent spermatogonial cells in the testis because expression of lipocalin-2 mRNA, which encodes an iron-siderophore-binding protein, is barely detectable in Sertoli cells of germ cell-deficient W/Wv mice, and more abundantly expressed in jsd/jsd mice. By employing a coculture system comprising immortalized Sertoli cells (designated as Sertoli-B) and c-Kit+ spermatogonial cells from 7-d-old mouse testis, we found that lipocalin-2 gene transcription in Sertoli cells is induced by a factor secreted from spermatogonial cells. Transfection of Sertoli-B cells with a series of reporter constructs encompassing an upstream region of the mouse lipocalin-2 gene revealed that a nuclear factor (NF)-{kappa}B binding consensus sequence in the proximal region of lipocalin-2 gene is responsible for transcriptional activation. A major NF-{kappa}B component, p65, bound to this region and translocated from the cytoplasm to the nucleus upon stimulation with spermatogonial cell-conditioned medium. Moreover, short interference RNA directed to p65 or a dominant-negative form of I{kappa}B{alpha} suppressed the spermatogonial cell factor-mediated transcription of lipocalin-2. However, NF-{kappa}B-activating inflammatory molecules, such as IL-1ß and lipopolysaccharide, did not induce lipocalin-2 mRNA in Sertoli-B cells and the expression of lipocalin-2 was unaffected in the testis of I{kappa}B{zeta}-deficient mice. These results demonstrate that spermatogonial cells regulate lipocalin-2 gene expression in Sertoli cells in a manner distinct from that employed by immune cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IN ADULT MOUSE testis, type-A spermatogonial stem cells proliferate and differentiate to form type-B spermatogonial cells and preleptotene primary spermatocytes before entering meiotic prophase for spermiogenesis (1). Self-renewal and faithful differentiation of spermatogonial stem cells are supported and regulated by various factors provided by Sertoli cells in the testis. Based on phenotypic characterization of infertile mutant male mice, a membrane-bound form of steel factor and glial cell line-derived neurotrophic factor have been identified as critical cytokines that are produced by Sertoli cells to act on spermatogonial stem cells (2, 3). Although these cytokines are produced by somatic cells to support germ cells, several past reports have shown that the genetic program of Sertoli cells is itself regulated by germ cell lineage (4, 5, 6, 7, 8). In addition, the phagocytic capacity or antibacterial activity of Sertoli cells is known to be enhanced by postmeiotic germ cells (9, 10). Thus, reciprocal intercellular communications between Sertoli cells and spermatogenic cells appear to be crucial for testis development and function.

We have previously reported that expression of many testicular genes, including prostaglandin D synthetase and lipocalin-2 (also called as 24p3, NGAL, siderocalin, or uterocalin), is not detected in the testis of germ cell-deficient W/Wv mice (11). In wild-type testes, expression of these genes occurred in somatic and germ cell fractions, but expression level was much higher in spermatogonia-rich jsd/jsd mutant mice than that in wild-type controls. These results implied that a reverse signal from spermatogonial cells to Sertoli cells may play an important role in testis development. However, it remains to be determined which factors and signaling molecules are involved in this intercellular regulation of Sertoli cells by spermatogonial cells.

In an effort to characterize a spermatogonial cell-derived factor acting on Sertoli cells, we here focused on the transcriptional control mechanism of the lipocalin-2 gene. Lipocalin-2 is an iron carrier protein, which binds to bacterial siderophores with high affinity (12, 13). In peritoneal macrophages, it was recently shown that stimulation by IL-1 or ligands for Toll-like receptors (TLR) rapidly induces transcription of lipocalin-2 via activation of a nuclear factor (NF)-{kappa}B pathway (14, 15). This cellular response was mediated by the inducible nuclear factor I{kappa}B{zeta} (also known as MAIL and INAP) (16, 17, 18). TLR/I{kappa}B{zeta}-dependent induction of lipocalin-2 mRNA is critical for innate immunity, as lipocalin-2-knockout mice exhibited an accelerated lethality after bacterial infection (15). In addition to its iron-sequestering function, lipocalin-2 has also been shown to induce apoptosis in hematopoietic cells (19) and to suppress malignant transformation (20). Although the physiological functions of lipocalin-2 protein in the testis have not been explored in detail, Lee et al. (21) recently reported that lipocalin-2 produced from epididymis enhances the motility of spermatozoa.

In this study, we initially demonstrate that lipocalin-2 mRNA in Sertoli cells is induced by a factor secreted from c-Kit+ spermatogonial cells of juvenile mice. We further show that transcriptional activation of the lipocalin-2 gene in Sertoli cells is mediated by the spermatogonial cell factor in an NF-{kappa}B-dependent manner, which is distinct from the IL-1 receptor/TLR-mediated signaling process that occurs in inflammatory immune cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of Lipocalin-2 mRNA in Sertoli Cells Is Regulated by Spermatogonial Cells
Based on the observation that testicular expression of lipocalin-2 mRNA is much higher in jsd/jsd mice than W/WV mice, and expression of lipocalin-2 was detected in both somatic and germ cells of wild-type testes, we previously proposed that expression of the lipocalin-2 gene in Sertoli cells is spermatogonia-dependent (11). To demonstrate this phenomenon in vitro, we here established a coculture experimental system comprising Sertoli and spermatogonial cells. In testes of 7-d-old male mice, c-Kit is a reliable marker for separation of type A spermatogonia from somatic cells because spermiogenesis has not started at this stage (22). We cultured the c-Kit-negative testicular cell fraction from 7-d-old enhanced green fluorescent protein (EGFP) transgenic mice in the presence or absence of c-Kit+ spermatogonial cells from C57BL/6 mice for 24 h, after which EGFP+c-Kit– cells were sorted by fluorescence-activated cell sorting (FACS). Expression of sulfated glycoprotein-2 (SGP2) (23) in the c-Kit– cell fraction confirmed that it contains Sertoli cells (Fig. 1Go). On the other hand, mRNA for transketolase-like 1, a spermatogonia-specific marker gene (24), was only detectable in the c-Kit+ fraction (Fig. 1Go), confirming that separation of spermatogonial cells from Sertoli cells was successful. Before starting the primary culture, expression of lipocalin-2 mRNA was detected in the c-Kit– cells, but it was reduced to an undetectable level during 1 wk of culture (data not shown). However, after coculture with c-Kit+ cells, expression of Lipocalin-2 mRNA was reinitiated in the cultured c-Kit– cells (Fig. 1Go, arrow). The identity of PCR bands was confirmed by Southern blotting (Fig. 1Go, lower panels). These results indicated that expression of lipocalin-2 mRNA in the c-Kit– Sertoli cell population is triggered by spermatogonial cells in culture.


Figure 1
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  		Fig. 1. Spermatogonia-Dependent Expression of Lipocalin-2 mRNA in a Sertoli-Containing Cell Fraction

c-Kit– cells (mainly Sertoli cells) and c-Kit+ cells (containing spermatogonial cells) were prepared from testes of 7-d-old EGFP transgenic mice and C57BL/6 mice, respectively. They were cultured for 24 h singly or in combination. Total RNAs were extracted from EGFP+c-Kit– cells, EGFP–c-Kit+ cells, and EGFP+c-Kit– cells after coculture with EGFP–c-Kit+ cells, respectively. They were subjected to RT-PCR analysis with gene-specific primer sets as indicated. DNA bands after ethidium bromide staining are shown with ß-actin, which is the control for equal template cDNA. PCR cycle number and product size for each analysis are indicated. Lower panels (marked as *) are results of Southern blot analyses, conducted to confirm the identity of PCR products. An arrow highlights a band for lipocalin-2 mRNA, which was induced in c-Kit– cells by coculture with spermatogonial cells.

 
To eliminate somatic cells other than Sertoli cells in the c-Kit– fraction, we immortalized Sertoli cells by introducing the simian virus 40 large T antigen into the c-Kit– 7-d-old testicular cells. Among several stable cell lines, one line, referred to as Sertoli-B, was chosen as a representative Sertoli cell line because of its tightly interconnecting morphology (Fig. 2AGo) and expression of SGP2 and steel factor genes (Fig. 2BGo). Neither 3ß-hydroxysteroid dehydrogenase (3ß-HSD) gene, which is known to be specifically expressed in Leydig cells (25), nor spermatogonial marker genes (c-Kit and transketoloase-like 1) was detectable in Sertoli-B cells. Expression patterns of Sertoli-B cells were similar to the cultured c-Kit– cells derived from 7-d-old testes (Fig. 2BGo). When Sertoli-B cells were cocultured with spermatogonial cells, specific induction of lipocalin-2 mRNA was observed (Fig. 2CGo). Therefore, we successfully reproduced the spermatogonia-dependent expression of lipocalin-2 in an in vitro coculture system.


Figure 2
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  		Fig. 2. Induction of Lipocalin-2 in the Sertoli-B Cell Line by Coculture with Spermatogonial Cells

A, Morphological appearance of immortalized Sertoli-B cells under phase-contrast microscopy. B, Marker expression in the Sertoli-B cell line and c-Kit– testicular cells. Expression of marker genes for Sertoli cells (SGP2, steel factor), Leydig cells (3ß-HSD), and spermatogonial cells (c-Kit, transketolase-like 1) in Sertoli-B cells and c-Kit– cells from 7-d-old testes was examined by RT-PCR. RNA from 7-wk-old mouse testis was used as a positive control template. C, Induction of lipocalin-2 mRNA in Sertoli-B cells by coculture with spermatogonial cells. RNA samples of Sertoli-B cells before and after 24 h coculture with spermatogonial cells were subjected to RT-PCR analysis. In B and C, the ethidium bromide staining patterns after PCR are shown with cycle number and product size. ß-Actin was used to verify equal quantities of template cDNA.

 
Transcriptional Regulation of the Lipocalin-2 Gene by Spermatogonial Cells
To determine whether up-regulation of lipocalin-2 mRNA in Sertoli-B cells by spermatogonial cells occurs at a transcriptional or posttranscriptional level, we characterized a promoter region of the mouse lipocalin-2 gene using reporter assays. Initially, a genomic DNA fragment of approximately 3 kb upstream of an initiation codon was linked to a promoter-less luciferase cDNA vector. We transfected this construct into Sertoli-B cells and examined the reporter activity with or without addition of spermatogonial cells. Luciferase activity was markedly induced by coculture with spermatogonial cells (Fig. 3AGo). Similar analysis of a series of shorter DNA fragments ranging from 370 to 184 bp in a proximal promoter region of the lipocalin-2 gene, revealed that the DNA region between –231 and –214 contains a spermatogonial cell-responsive element (Fig. 3AGo). As shown in Fig. 3BGo, there is a sequence in this region 5'-GGGAATGTCCC-3', which is closely related to the NF-{kappa}B binding consensus sequence, 5'-GGG(A/G)NNT(C/T)CC-3' (26). When this NF-{kappa}B binding site was deleted from the –3124 to +53 reporter construct, spermatogonial cell-dependent transcriptional activation was abrogated (Fig. 3AGo), suggesting that NF-{kappa}B could be responsible for this transcriptional activation event.


Figure 3
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  		Fig. 3. Search for a Spermatogonial Cell-Responsive Promoter Region of the Lipocalin-2 Gene in Sertoli-B Cells

A, Sertoli-B cells were transfected with a series of reporter constructs which carry various lengths of genomic DNA fragments for upstream region of mouse lipocalin-2 gene. The longest reporter construct with a deletion in the NF-{kappa}B binding site between –231 and –214 (–3124 del) was also generated and introduced into Sertoli-B cells. One day after transfection, cells were cocultured with spermatogonial cells for 24 h and were lyzed to measure the luciferase activities. Each value represents a mean ratio of firefly vs. Renilla luciferase activities (F/R) ± SD [n = 4; *, P < 0.001; **, statistically no significance vs. (–) spermatogonial cells]. B, Primary nucleotide sequence of the –235 to +56 region of mouse lipocalin-2 gene. Positions for putative binding sites of transcription factors (NF-{kappa}B, C/EBPß, and HSF2) and TATA box are shown.

 
Spermatogonial Cells Secrete a Lipocalin-2-Inducing Factor
We next sought to determine whether the putative lipocalin-2-inducer of spermatogonial origin is a secreted factor or a membrane-associated molecule. Sertoli-B cells were stimulated with spermatogonial cell conditioned medium (SGC-CM) or conditioned medium from the c-Kit– somatic cells (somatic cell CM), and subjected to RT-PCR, Western blotting, and the reporter assay with the –231/luciferase construct. We first observed lipocalin-2 mRNA induction by SGC-CM, but not by somatic cell CM (Fig. 4AGo, upper). In addition, lipocalin-2 protein was secreted in the media only after the SGS-CM stimulation (Fig. 4AGo, lower). Induction of lipocalin-2 mRNA in Sertoli-B cells by SGC-CM occurred at the transcriptional level, as the –231/luciferase reporter was trans-activated by SGC-CM, but not by somatic cell CM (Fig. 4BGo). Moreover, this biological activity of SGC-CM was destroyed by incubation at 98 C for 10 min (Fig. 4BGo). These results indicated that spermatogonial cells secrete a lipocalin-2-inducing protein.


Figure 4
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  		Fig. 4. Induction of Lipocalin-2 by a Secreted Factor from Spermatogonial Cells

A, Sertoli-B cells were stimulated with medium, SGC-CM or somatic cell CM for 24 h and subjected to RT-PCR and immunoprecipitation analyses. Upper panels, Ethidium bromide staining patterns of PCRs with cycle number and product size. ß-Actin was used for the loading control. In a lower panel, 35S-methionine labeled lipocalin-2 protein in a culture supernatant of SGC-CM-stimulated Sertoli-B cells was visualized by SDS-PAGE followed by autoradiography. B, Trans-activation of a proximal promoter of lipocalin-2 gene by SGC-CM. Sertoli-B cells transfected with the –231/luciferase construct were incubated with medium, SGC-CM, conditioned medium of the c-Kit– (somatic) cells, and heat-inactivated SGC-CM. One day after culture, luciferase reporter activity of each sample was measured and F/R values were calculated. Each value represents the average ± SD (n = 4; *, P < 0.001; **, statistically no significance vs. medium control).

 
Binding of NF-{kappa}B p65 and p50 to the Lipocalin-2 Promoter in Sertoli-B Cells
Having identified an NF-{kappa}B binding consensus sequence as a critical element in the proximal promoter region of the lipocalin-2 gene, we examined whether representative components of the NF-{kappa}B complex bind to this element. First, a nuclear extract from SGC-CM-stimulated Sertoli-B cells was subjected to EMSA. We detected a slow migrating protein complex with the lipocalin-2 NF-{kappa}B binding consensus sequence (Fig. 5AGo, open arrowhead). This band was competed away by an excess of matched oligonucleotide, but not by a mutated oligonucleotide (Fig. 5AGo, left). Addition of anti-p65 or anti-p50 antibody supershifted the band position substantially (Fig. 5AGo, closed arrowheads). Consistent with this observation, chromatin immunoprecipitation (ChiP) assays also revealed specific complex formation of the lipocalin-2 promoter with NF-{kappa}B p65 and p50 (Fig. 5BGo). A control antibody did not bring down chromatin containing the lipocalin-2 promoter. These results suggested that NF-{kappa}B p65 and p50 are binding proteins to the proximal promoter region of mouse lipocalin-2 gene. To further support this possibility, we observed translocation of NF-{kappa}B p65 from the cytoplasm to the nucleus in Sertoli-B cells upon stimulation with SGC-CM (Fig. 5Go, C and D). Taken together, it is apparent that a spermatogonial cell-derived factor activates the NF-{kappa}B pathway in Sertoli-B cells and that NF-{kappa}B p65 and p50 are intimately involved in regulating the lipocalin-2 proximal promoter.


Figure 5
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  		Fig. 5. Interaction of NF-{kappa}B Components with the Promoter Region of the Lipocalin-2 Gene

A, EMSA. Nuclear extracts from SGC-CM-stimulated Sertoli-B cells were incubated with 32P-labeled oligonucleotide corresponding to the NF-{kappa}B consensus sequence in the lipocalin-2 promoter and applied to the gel electrophresis and autoradiography. Effects of excess amount of cold competitors and antibodies against NF-{kappa}B p65 and p50 were tested. A specific DNA-protein complex (indicated by NF-{kappa}B) and supershifted bands by anti-p65 or p50 (marked by SS-p65 or SS-p50) are shown. B, ChiP assay. Anti-p65, anti-p50, or control antibody was added to equal volume of chromatin fraction of SGC-CM-stimulated Sertoli-B cells. Immunoprecipitated complexes were analyzed by PCR with primers that detect a proximal promoter region of lipocalin-2 gene. Ethidium bromide staining patterns of PCRs and loaded chromatin fractions are shown with PCR cycle number and product size. C, Nuclear tanslocation of NF-{kappa}B p65 in Sertoli-B cells by stimulation with SGC-CM. Cytoplasmic and nuclear protein fractions of Sertoli-B cells with or without SGC-CM stimulation were subjected to Western blot analysis using anti-p65 antibody. D, Sertoli-B cells were cultured in control medium or SGC-CM for 1 h and fixed. Localization of p65 was visualized by immunofluorescent staining (original magnification, x400). Microscopically identified location of nucleus in the SGC-CM-stimulated cells is marked in the bottom panel.

 
NF-{kappa}B Is Critical for Lipocalin-2 Gene Transcription in Sertoli-B Cells
We next explored the importance of the NF-{kappa}B pathway in the SGC-CM-mediated induction of lipocalin-2 in Sertoli-B cells, by using short interference RNA (siRNA) to specifically suppress NF-{kappa}B p65 or p50 mRNA. Knockdown of p65 in Sertoli-B cells abolished lipocalin-2 mRNA, whereas transfection of p50 siRNA slightly lowered the level of lipocalin-2 mRNA (Fig. 6AGo, upper). Quantitative measurement of the PCR bands by National Institutes of Health Image software revealed that a 75% knockdown of p65 mRNA resulted in 80% suppression of lipocalin-2 mRNA, whereas a 70% reduction of p50 mRNA caused only 19% suppression of lipocalin-2 mRNA. Specific reduction of p65 and p50 proteins by siRNAs was confirmed by immunoblotting (Fig. 6AGo, lower). Because repression of p65 mRNA or p50 mRNA by corresponding mutated siRNAs did not occur (Fig. 6AGo, upper), nonspecific or cross-interference by siRNA was not an issue in these experiments. EMSA and ChiP assays indicated that both p65 and p50 interact with the lipocalin-2 proximal promoter (Fig. 5Go, A and B). Therefore, the lesser effect of p50 knockdown is likely due to the p50 being a weaker activator on the NF-{kappa}B-mediated gene stimulation. Removal of p50 could permit the formation of p65/p65 homodimers that enhances the transcription. We concluded that NF-{kappa}B p65 is critically required for the expression of lipocalin-2 mRNA in SGC-CM-stimulated Sertoli-B cells.


Figure 6
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  		Fig. 6. Requirement of the NF-{kappa}B Pathway for Lipocalin-2 Induction in Sertoli-B Cells

A, Sertoli-B cells were infected with retrovirus expression vector for siRNAs directed to p65 or p50 and their mutant forms. In upper panels, expression of mRNAs for lipocalin-2, p65, p50, and ß-actin (template control) after 24 h stimulation with SGC-CM was examined by RT-PCR. The ethidium bromide staining patterns are shown with PCR cycle number and product size. In lower panels, quantity of p65 or p50 protein in the SGC-CM-stimulated Sertoli-B cells was analyzed by Western blotting. B, Sertoli-B cells were transfected with lipocalin-2 promoter reporter (–231/luciferase construct) (left panel) or NF-{kappa}B consensus reporter (right panel) in the presence of I{kappa}B{alpha}M or lacZ (control) expression vector. At 24 h after stimulation with SGC-CM, luciferase reporter activities of cell lysates were measured and ratios of firefly vs. Renilla luciferase activities (F/R) were calculated. Each value represents the average ± SD (n = 4; *, P < 0.001 vs. SGC-CM-stimulated lacZ control).

 
To further confirm the dependency of lipocalin-2 transcription on the NF-{kappa}B pathway, we transfected an expression vector for the dominant-negative form of I{kappa}B{alpha} (I{kappa}B{alpha}M), which is resistant to proteolytic degradation by virtue of its mutations in two serine residues (27). I{kappa}B{alpha}M is known to block translocation of the NF-{kappa}B complex to the nucleus. Upon cotransfection of I{kappa}B{alpha}M cDNA with the –231/luciferase construct, transactivation of the reporter gene by SGC-CM was inhibited in Sertoli-B cells when compared with the cells transfected with the control vector (Fig. 6BGo, left). Similar suppression pattern of the reporter activity was demonstrated when the NF-{kappa}B consensus reporter was tested (Fig. 6BGo, right). These results provide further evidence that induction of lipocalin-2 in Sertoli-B cells is NF-{kappa}B dependent.

The NF-{kappa}B-Activating Molecule in SGC-CM Is Distinct from Inflammatory Cytokines
To characterize a lipocalin-2-inducing substance in SGC-CM, we compared its biological activity with inflammatory cytokines that are known to activate the NF-{kappa}B signaling pathway. Sertoli-B cells were transfected with the –231/luciferase construct and stimulated for 24 h with IL-1ß, TNF{alpha} or lipopolysaccharide (LPS). IL-1ß and LPS enhanced the reporter activity in Sertoli-B cells, but only 17.7% and 23.0% of the level of SGC-CM stimulated cells, respectively, whereas TNF{alpha} was inactive (Fig. 7AGo, left). Higher concentrations of IL-1ß and LPS did not increase the reporter activity (data not shown). Furthermore, induction of lipocalin-2 mRNA in Sertoli-B cells was detectable after stimulation with SGC-CM, but not after exposure to IL-1ß and LPS (Fig. 7BGo). Because even the NF-{kappa}B consensus reporter gene was not significantly activated in Sertoli-B cells by IL-1ß, TNF{alpha}, or LPS (Fig. 7AGo, right), these cells presumably lack receptors or signal transducing molecules of the NF-{kappa}B pathway for those cytokines. Loss of FSH receptor expression has been reported in previously established immortalized Sertoli cells (28, 29). Our data clearly indicate that the NF-{kappa}B-activating molecule in SGC-CM is distinct from these inflammatory mediators.


Figure 7
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  		Fig. 7. Effect of Known NF-{kappa}B Activating Substances on the Transactivation of Lipocalin-2 Gene

A, Sertoli-B cells were transfected with lipocalin-2 promoter reporter (–231/luciferase construct) (left panel) or NF-{kappa}B consensus reporter (right panel). After stimulation with SGC-CM, IL-1ß, LPS, or TNF{alpha} for 24 h, luciferase activities of cell lysates were measured and ratios of firefly vs. Renilla luciferase activities (F/R) were calculated. Each value represents the average ± SD (n = 4; *, P < 0.001; **, statistically no significance vs. medium control). B, Sertoli-B cells after 24 h stimulation with SGC-CM, IL-1ß, TNF{alpha}, or LPS were subjected to total RNA extraction followed by RT-PCR. DNA bands after ethidium bromide staining are shown with PCR cycle number and product size. ß-Actin is the control for equal amounts of template cDNA.

 
Recent reports demonstrated that IL-1ß or TLR ligands rapidly up-regulate lipocalin-2 expression in immune cells by inducing I{kappa}B{zeta} mRNA (14). We therefore tested the possibility that I{kappa}B{zeta} is involved in the SGC-CM-mediated induction of lipocalin-2 in Sertoli-B cells. However, expression of I{kappa}B{zeta} mRNA was detected in Sertoli-B cells without any stimulation and its amount was not augmented by the SGC-CM stimulation (Fig. 8AGo, left). As expected, IL-1ß and LPS did not affect the lipocalin-2 mRNA level (Fig. 8AGo, right). Moreover, levels of lipocalin-2 mRNA in the testis and epididymis were similar in I{kappa}B{zeta} null and heterozygote mice (Fig. 8BGo). These data clearly demonstrate that machinery of the lipocalin-2 induction in Sertoli-B cells by SGC-CM is distinct from the I{kappa}B{zeta}-dependent IL-1/TLR signaling pathway employed by innate immune cells.


Figure 8
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  		Fig. 8. Lipocalin-2 Expression in Testis Is Independent from I{kappa}B{zeta}

A, Total RNAs were extracted from Sertoli-B cells before and after stimulation with SGC-CM, IL-1ß, or LPS for 24 h. Expression of I{kappa}B{zeta} mRNA was examined by RT-PCR. B, Expression of lipocalin-2 mRNA in testis and epididymis of I{kappa}B{zeta} knockout mice was examined by RT-PCR [(–/–): homozygous mutant; (+/–): heterozygous mutant]. The ethidium bromide staining patterns are shown with PCR cycle number and product size. ß-Actin was used as the template control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In our previous studies, we observed that 24 testicular genes, including lipocalin-2, are not expressed in testes of W/Wv mice when compared with those of jsd/jsd mice (11). Because Sertoli cells are present in the W/Wv mouse and the jsd/jsd mouse testis contains spermatogonial cells at a higher frequency, we speculated that spermatogonial cells positively regulate a genetic program in Sertoli cells. In this study, we obtained direct evidence of such spermatogonia-dependent gene expression in Sertoli cells. Lipocalin-2 mRNA is normally produced in both spermatogonial cells and Sertoli cells in the testis. When primary Sertoli cells were cultured in the absence of spermatogonial cells, lipocalin-2 expression was shut down. Upon coculture with spermatogonial cells or in their conditioned medium, lipocalin-2 expression in Sertoli cells was reinitiated, suggesting that spermatogonial cell-derived factors positively regulate expression of this gene in Sertoli cells. We further investigated the molecular basis for this phenomenon and proved that the NF-{kappa}B pathway is involved in the transcriptional activation of lipocalin-2 gene. A series of in vitro experiments provided an explanation for why lipocalin-2 mRNA is almost undetectable in the testes of W/Wv mice, which lack spermatogonial cells but possess Sertoli cells. Interestingly, among other suppressed genes in W/Wv mice, expression of major histocompatibility complex region class IQ and spi proteinase inhibitor genes was induced in a similar manner in primary Sertoli cells by coculture with spermatogonial cells, although mRNA expression of these genes were not induced by SGC-CM (Fujino, R.-S., K. Tanaka, and T. Hara, unpublished data). Thus, there must be other, as yet unidentified, interactions between spermatogonial cells and Sertoli cells in vivo.

Since Syed and Hecht first reported that Sertoli cell gene expression can be up- or down-regulated by germ cells in a coculture system (4), several lines of evidence have supported the contention that germ cell-mediated regulation of Sertoli cell function influences testicular development. For example, it has been shown that pachytene spermatocytes and round spermatids induce the serotonin receptor and fra-1/c-fos genes in Sertoli cells, respectively (5, 6). It has also been shown that germ cells negatively regulate transcription of the cathepsin L gene (7). More recently, Delfino et al. (8) showed that in Sertoli cells, the androgen receptor gene is transcriptionally activated by TNF{alpha} produced from round spermatids. In that situation, NF-{kappa}B p65 and p50 bind to the enhancer/promoter region of the androgen receptor gene to activate its transcription in rat Sertoli cells (30). In contrast to such postmeiotic germ cell-mediated regulation of Sertoli cell function, in this study we provide the first evidence that a spermatogonial cell-derived factor induces transcription of the lipocalin-2 gene. Underlying mechanism of this phenomenon appears to be different from the TNF{alpha}-mediated NF-{kappa}B activation in the case of the androgen receptor gene control because Sertoli-B cells did not respond to TNF{alpha}.

Transcriptional regulation of lipocalin-2 gene has been studied in detail by Cowland et al. (31) using human lung epithelial cells. In these cells, lipocalin-2 (NGAL in their report) mRNA is induced by IL-1 or LPS stimulation via activation of NF-{kappa}B. The responsible NF-{kappa}B binding sequence, 5'-GGGAATGTCCC-3' (–180 to –170), found in the human lipocalin-2 gene, is perfectly conserved in the murine lipocalin-2 gene at positions –220 to –230. Similar to human lung epithelial cells, mouse Sertoli-B cells responded to IL-1ß and LPS weakly, but not to TNF{alpha}, in terms of the transcriptional activation of lipocalin-2. However, the extent of activation by IL-1ß and LPS was much less than that induced by SGC-CM. Moreover, lipocalin-2 mRNA was undetectable in Sertoli-B cells after stimulation with IL-1ß or LPS. These results may suggest that signal transducers and/or transcriptional regulators for lipocalin-2 gene are different between lung epithelial cells and Sertoli cells, even though they share the similar NF-{kappa}B pathway for stimulus-dependent transcriptional activation.

More recently, Yamamoto et al. (14) reported that I{kappa}B{zeta}, an inducible I{kappa}B, mediates the induction of lipocalin-2 in LPS-stimulated peritoneal macrophages. Because the lipocalin-2-siderophore complex protects mice from bacterial propagation (15), the TLR-I{kappa}B{zeta}-lipocalin-2 signaling cascade is an important cellular system for innate immunity. In contrast to immune cells, however, expression of I{kappa}B{zeta} in Sertoli-B cells was not changed by stimulation with SGC-CM or IL-1ß. Furthermore, expression of lipocalin-2 mRNA in testis and epididymis was not affected by disruption of the I{kappa}B{zeta} gene. These data support the notion that transcriptional regulation of lipocalin-2 gene in Sertoli cells is independent of I{kappa}B{zeta}. In testis, an alternatively spliced I{kappa}B{zeta} mRNA is produced (16). Therefore, it is possible that distinct regulation and physiological roles of I{kappa}B{zeta} exist in male reproductive organs.

Lipocalin-2 serves as an inducible antimicrobial protein in airway epithelial cells, hepatocytes, and peritoneal macrophages (14, 31, 32). In the uterus of female mice, this protein is secreted from the epithelium during the proestrous phase of the estrous cycle and around parturition (33, 34). In males, lipocalin-2 is produced from the caput region of the epididymis and the ferric ion-lipocalin-2 complex is incorporated by spermatozoa into the caudal epididymis (35). Interestingly, previous reports revealed that incubation of spermatozoa with lipocalin-2 in vitro increased intracellular cAMP concentration and enhanced sperm motility (21, 36). Therefore, the regulation of spermatozoa by lipocalin-2 may depend, in part, on its iron-transporting activity. As mice deficient in the lipocalin-2 gene are reported to be fertile (15), there could be another iron transporter that functionally compensates for lipocalin-2 in vivo. In the epididymis, lipocalin-2 may also play a role in protecting gametes against bacterial infection. In this study, we demonstrated that steady-state expression of lipocalin-2 mRNA from juvenile Sertoli cells is influenced by adjacent spermatogonial cells. This might relate to a previous report by Grandjean et al. (10) showing that an antimicrobial substance is produced from the 15P-1 Sertoli cell line and that its activity is augmented by the addition of round spermatids in culture. Further investigation will clarify whether a similar lipocalin-2-inducing activity is produced from germ cells at other stages of differentiation.

In this work, we established a new, unique Sertoli cell line, Sertoli-B, to provide evidence for the spermatogonia-dependent expression of lipocalin-2. Because lipocalin-2 mRNA was induced in primary Sertoli cells by coculture with spermatogonial cells, it is likely that the Sertoli-B cell line retains this intercellular communication capacity. Some other Sertoli cell lines established from c-Kit– testicular cells in our laboratory and 15P-1 Sertoli cell line exhibited constitutive expression of lipocalin-2 (data not shown). These results indicate that resetting of gene expression by preculture in the absence of germ cells is important to reveal the spermatogonia-responsive machinery of the lipocalin-2 gene. By taking advantage of the Sertoli-B cell line and the –231/luciferase reporter assay, we are currently attempting to identify a lipocalin-2-inducing molecule in SGC-CM.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mice
C57BL/6, ICR (Nihon SLC, Hamamatsu, Japan) or transgenic mice expressing EGFP (37) were used for cell culture. Male mice (12 month old) deficient in the I{kappa}B{zeta} (we previously reported as MAIL) gene and heterozygous littermates were prepared as previously described (38). All animal experiments were performed according to institutionally approved protocols.

Preparation of Spermatogonial Cells and Sertoli Cells
Testes of 7-d-old male mice were isolated and decapsulated under a microscope. Seminiferous tubules were digested with type I collagenase (1 mg/ml) (Worthington, Lakewood, NJ), hyarulonidase (1 mg/ml) (Sigma, St. Louis, MO) and deoxyribonuclease I (100 U/ml) (Roche Diagnostics, Indianapolis, IN) in PBS at 37 C for 30 min with gentle agitation. An equal volume of 0.05% trypsin-0.5 mM EDTA solution (Sigma) was added and further incubated at 37 C for 5 min. Dissociated testicular cells primarily containing Sertoli cells and spermatogonial cells were washed in DMEM and filtered through a 70-µm cell strainer (BD Biosciences, San Jose, CA).

Spermatogonial cells were isolated from somatic cells by using magnetic beads. In brief, cells were incubated on ice for 30 min with a 1:50 dilution of biotin-conjugated rat antimouse c-Kit monoclonal antibody (2B8; BD Biosciences) in 5% fetal calf serum (FCS)-PBS. The cell suspension was washed once with labeling buffer (0.5% BSA and 2 mM EDTA in PBS) and incubated at 4 C for 30 min with a 1:5 dilution of antibiotin MACS beads (Miltenyi Biotec, Bergisch Gladbach, Germany) in the labeling buffer. c-Kit-positive cells were separated by two rounds of magnetic sorting on auto-MACS (Miltenyi Biotec). Simultaneously, the c-Kit-negative fraction was saved from the two rounds of magnetic sorting.

Primary culture of Sertoli cells and all coculture experiments were performed in DMEM supplemented with 10% FCS and 0.5% penicillin-streptomycin (Sigma). Sertoli-B cell line was established by introduction of an expression vector for simian virus 40 large T antigen [pSVLT (OD) neo, provided by Dr. S. Imai] (39) into c-Kit-negative cells of 7-d-old C57BL/6 mice via electroporation at 400 V, 500 µF (Gene pulser II; Bio-Rad, Hercules, CA). Transfected cells were selected by subculture in the presence of G418 (1 mg/ml) (Invitrogen, Carlsbad, CA) and subjected to isolation of single cell-derived colonies. All coculture and transfection experiments were carried out within tenth passages of Sertoli-B cell line. The supernatant from 1-d culture of c-Kit+ spermatogonial cells was used as SGC-CM.

FACS
EGFP transgenic primary Sertoli cells and spermatogonial cells were cocultured, and EGFP+c-Kit– cells were isolated by FACS. Cocultured cells (107/ml) were stained with biotinylated anti-c-Kit antibody on ice for 30 min and then incubated with allophycocyanin-conjugated streptavidin (10 µg/ml) (Molecular Probes, Eugene, OR). Cells were resuspended in PBS containing propidium iodide (2 µg/ml) (Sigma) and subjected to cell sorting on FACSVantage (BD Biosciences).

RT-PCR and Southern blotting
Total RNAs were isolated by using Trizol (Invitrogen) and subjected to first-strand cDNA synthesis with SuperScript II reverse transcriptase and random hexamer (Invitrogen). RT-PCRs were carried out as previously described (11). We used 5'-AAGGTGTACCACTCCTGTCT-3' and 5'-TCCTCGACAACCTTCCATTG-3' for detection of c-Kit (PCR product: 330 bp), 5'-TAACCCTCAACTATGTCGCC-3' and 5'-TGAAGAGAGCACACAGTCAC-3' for Steel factor (PCR product: 344 bp), 5'-AGGAGGAATTCTCCAAGCTG-3' and 5'-AGTACTGCCTTCTCAGCCAT-3' for 3ß-HSD (PCR product: 370 bp), 5'-CGGGGACTATGACTTGAATG-3' and 5'-GACTCGAACAGGAGCCTGGA-3' for NF-{kappa}B p65 (PCR product: 358 bp), 5'-CGGAACTGGGCAAATGTTTC-3' and 5'-GGCCCCACATAGTTGCAAAT-3' for NF-{kappa}B p50 (PCR product: 249 bp), and 5'-AGTCTCCCAAGTATGATTCC-3' and 5'-TTTCTTCCTGCTCTACCTGC-3' for I{kappa}B{zeta} (PCR product: 333 bp), respectively. Primer sequences for lipocalin-2, SGP2, transketolase-like 1, and ß-actin were described in a previous report (11). In initial experiments, PCR products were separated on 1% agarose gel electrophoresis and subjected to Southern blotting with a 32P-end-labeled gene specific internal oligonucleotide (21 oligomers) as a probe, according to standard procedures. Sequence information of these oligonucleotides will be provided upon request.

Transfection of siRNA
For the introduction of siRNA into Sertoli-B cells, we newly constructed a retroviral siRNA expression vector carrying puromycin resistant gene (designated as pRePS vector), which is basically the same as pRETRO-SUPER vector (40). Sequences of sense oligonucleotides for the expression of siRNAs were as follows: 5'-GATCAATGGCTACACAGGA-3' for p65 siRNA; 5'-GATCAATTGCTGCACAGGA-3' for mutated p65 siRNA; 5'-GGCCCATCACACGGAGGGC-3' for p50 siRNA; and 5'-GGCCCATTACATGGAGGGC-3' for mutated p50 siRNA, respectively. Sertoli-B cells were infected with each siRNA retroviral vector as previously described (41) and subjected to a drug selection in the presence of puromycin (2 µg/ml; Sigma). Resistant cells in bulk culture were stimulated with SGC-CM for 24 h and subjected to total RNA extraction followed by RT-PCR and Western blot analyses.

Reporter Assay
A genomic DNA sequence containing the mouse lipocalin-2 gene was obtained from a public database (Ensembl Gene ID: ENSMUSG00000026822). DNA fragments (–3124 to +53, –370 to +53, –285 to +53, –231 to +53, –214 to +53, –184 to +53) covering a region upstream from a transcriptional initiation site of mouse lipocalin 2 gene were PCR-amplified using splenic DNA of the C57BL/6 mouse as a template and cloned into an MluI/SmaI site of the pGL3 basic vector (Promega, Madison, WI). A –3124/+53 reporter construct carrying a deletion in the NF-{kappa}B recognition motif and an NF-{kappa}B consensus reporter possessing 5'-GGGGACTTTCCC-3' upstream of the –214/+53 construct were prepared by PCR. Sertoli-B cells were transfected with each reporter construct in combination with the pRL-CMV vector (Promega) using Effectene transfection reagent (QIAGEN, Hilden, Germany). In dominant-negative-I{kappa}B{alpha} transfection experiments, Sertoli-B cells were transfected with pCMV-I{kappa}B{alpha}M (BD Biosciences) or pcDNA-lacZ (Invitrogen) in combination with a reporter construct carrying the –231 to +53 region (hereafter called the –231/luciferase construct) and the pRL-CMV vector (Promega). At 24 h after transfection, SGC-CM was added to the culture medium. As controls, Sertoli-B cells were stimulated with IL-1ß (10 ng/ml) (R&D Systems, Minneapolis, MN), TNF{alpha} (10 ng/ml) (Peprotech, London, UK), or LPS (100 ng/ml) (Sigma). After an additional 24 h in culture, enzymatic activities of reporter genes were analyzed by Dual-Glo Luciferase reporter assay (Promega) with a luminometer Mithras LB940 (Berthold Technologies, Bad Wildbad, Germany).

EMSA
Nuclear extracts of Sertoli-B cells were prepared after a 24-h culture period in the presence of SGC-CM by as previously described (42). Double-stranded oligonucleotides were end-labeled with [{gamma}32P]-ATP (Amersham Pharmacia Biotech, Piscataway, NJ) using T4 polynucleotide kinase (Takara, Ohtsu, Japan). Sequences of oligonucleotides used in this study were 5'-CCCTGGGAATGTCCCTCTGG-3' and 5'-CCAGAGGGACATTCCCAGGG-3' for an NF-{kappa}B consensus site in the mouse lipocalin-2 gene promoter, and 5'-CCCTGGCAATGTCGCTCTGG-3' and 5'-CCAGAGCGACATTGCCAGGG-3' for a mutated competitor. Binding reactions of 32P-labeled DNA probe with nuclear proteins were carried out as previously described (43) at room temperature for 30 min in the presence or absence of an excess amount of competitor unlabeled oligonucleotide. In some samples, 1 µl of anti-NF-{kappa}B p65 antibody (sc-372; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-NF-{kappa}B p50 antibody (sc-1190; Santa Cruz) was added and incubated at room temperature for 30 min. Reacted samples were separated on a 4% acrylamide gel and exposed to a BAS cassette 2040 (Fujifilm, Tokyo, Japan) followed by analysis with a BAS-2500 phosphor imager and Image Gauge version 3.2 (Fujifilm).

ChiP Assay
The chromatin fraction was harvested from Sertoli-B cells after stimulation with SGC-CM for 1 h according to a previously published method (44). Cell extracts were sonicated to yield DNA fragments of 0.1–1.0 kb in size and preincubated with 100 µl of 10% Protein G PLUS-agarose (Santa Cruz) at 4 C for 2 h. After centrifugation, 2 µg of anti-NF-{kappa}B p65 (sc-372), anti-p50 (sc-1190), or control antibody (sc-7426, Santa Cruz) was added to an equally divided supernatant in the presence of skim milk (5%) (Difco, Detroit, MI), and tubes were gently rotated at 4 C overnight. Protein G PLUS-agarose (50 µl) was added to each tube and incubated at 4 C for 3 h. DNA was eluted from beads and subjected to PCR with a primer set (5'-TGGGAATGTCCCTCTGGTCC-3' and 5'-GGTTTCCACAGCTACTAGGT-3'), which amplifies 284 bp of the upstream region (–231 to +53) of mouse lipocalin-2 gene.

Immunoprecipitation and Western blot analysis
First, SGC-CM and somatic cell conditioned medium (CM) were individually dialyzed against methionine-free DMEM (Invitrogen) for 48 h. Sertoli-B cells in a 30-mm plate were cultured in 0.5 ml of the methinine-free SGC-CM or somatic cell-conditioned medium for 30 min and 50 µCi of 35S-labeled methionine (Amersham) was added. After 6 h in culture, media were harvested and incubated with goat antimouse lipocalin-2 antibody (AF1857; R&D Systems) at 4 C for 16 h. Protein A-Sepharose (Amersham) was added and gently agitated at 4 C for 1 h. After washing the resin several times, each immunocomplex was analyzed by SDS-PAGE followed by autoradiography. Western blot analysis was carried out as previously described (43). In brief, total cellular proteins (20 µg per lane) were electrophoretically separated, blotted on a polyvinylidene difluoride membrane (Bio-Rad), and incubated with anti-p65 (sc-109; Santa Cruz) or anti-p50 antibody (KAP-TF112; Stressgen, Victoria, Canada) at 2 µg/ml. Cytoplasmic and nuclear fraction of Sertoli-B cells was prepared according to the previous report (45).

Immunofluorescence Staining
To determine NF-{kappa}B localization, Sertoli-B cells were plated at 1 x 103 cells per well in four-well dishes (Nunc, Roskilde, Denmark). After 24 h, cells were stimulated with SGC-CM and fixed in 100% methanol (Wako, Osaka, Japan) at –20 C for 5 min. Fixed cells were washed thrice with PBS, blocked with 5% skim milk in PBS, and incubated with anti-p65 antibody (sc-372) diluted 1:100 in PBS at room temperature for 60 min. After washing thrice with PBS, samples were incubated with Cy3-conjugated donkey antirabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), diluted 1:200 in PBS for 60 min. Fluorescence signals were analyzed using an LSM510 confocal microscopy system (Carl Zeiss, Oberkochen, Germany).


   ACKNOWLEDGMENTS
 
We thank Yoshiki Futamata (Nippon Becton Dickinson) for help in the initial cell sorting experiments, Dr. Shin-ichiro Imai (Washington University School of Medicine) for pSVLT (OD) neo vector, and Dr. Michinori Kohara (The Tokyo Metropolitan Institute of Medical Science) for kind help in the luciferase reporter assay.


   FOOTNOTES
 
This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

The authors declare that they have no competing financial interests.

First Published Online December 1, 2005

Abbreviations: ChiP, Chromatin immunoprecipitation; EGFP, enhanced green fluorescent protein; FACS, fluorescence-activated cell sorting; FCS, fetal calf serum; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase; LPS, lipopolysaccharide; NF, nuclear factor; SGC-CM, spermatogonial cell conditioned medium; SGP2, sulfated glycoprotein-2; siRNA, short interference RNA; TLR, Toll-like receptor.

Received for publication October 24, 2005. Accepted for publication November 22, 2005.


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P J O'Shaughnessy, L Hu, and P J Baker
Effect of germ cell depletion on levels of specific mRNA transcripts in mouse Sertoli cells and Leydig cells
Reproduction, June 1, 2008; 135(6): 839 - 850.
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