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Secretory leukocyte protease inhibitor inhibits expression of polymeric immunoglobulin receptor via the NF-κB signaling pathway
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Secretory leukocyte protease inhibitor inhibits expression of polymeric immunoglobulin receptor via the NF-B signaling pathway Yoshikazu Mikami a , Takashi Iwase a , Yusuke Komiyama b , Naoyuki Matsumoto a , Hidero Oki c , Kazuo Komiyama a,∗ a
Department of Pathology, Nihon University School of Dentistry, 1-8-13 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-8310, Japan Department of Oral and Maxillofacial Surgery, Dokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu-machi, Shimotsuga-gun, Tochigi, 321-0293, Japan c Department of Maxillofacial Surgery, Nihon University School of Dentistry, 1-8-13 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-8310, Japan b
a r t i c l e
i n f o
Article history: Received 9 June 2015 Received in revised form 16 July 2015 Accepted 17 July 2015 Available online xxx Keywords: SLPI pIgR Epithelial cells NF-B
a b s t r a c t Polymeric immunoglobulin receptor (pIgR) plays an important role in mucosal immune systems. Secretory immunoglobulin A, composed of secretory component of pIgR and a dimeric form of immunoglobulin A, is secreted on mucosal surfaces and serves as a biological defense factor. pIgR gene expression is reportedly induced by activation of the transcription factor nuclear factor (NF)-B. On the other hand, secretory leukocyte protease inhibitor (SLPI) is a glycoprotein that functions as a serine protease inhibitor. In alveolar epithelial cells, SLPI increases the level of IB, which indicates that it is an inhibitor of NF-B at the protein level. Taken together, SLPI may regulate pIgR expression; however, the specific mechanism by which this occurs is unclear. Therefore, the aim of this study was to elucidatethe influence of SLPI on pIgR expression.SLPI and pIgR localized in goblet cells and ciliated epithelial cells of the gastrointestinal tract, respectively. No cells were detected in which SLPI and pIgR were co-expressed. In addition, recombinant human SLPI stimulation of an epithelial cell line (HT-29) decreased the pIgR expression. The pIgR expression was also higher in SLPI-deficient Ca9-22 cells than in wild-type Ca9-22 cells. Furthermore, a luciferase assay using a NF-B reporter plasmid and real-time RT-PCR analysis indicated that when SLPI was present, the transcriptional activity of NF-B protein was suppressed, which was accompanied by anincrease in the protein, but not the mRNA,expression of IB. These results demonstrate that SLPI down-regulates pIgR expression through the NF-B signaling pathway by inhibiting degradation of IB protein. © 2015 Z. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction The mucosal membrane of the gastrointestinal tract is covered by ciliated non-keratinized epithelial cells. The gastrointestinal mucosa is constantly exposed to pathogenic exogenous antigens. Mucosa-associated lymphoid tissue (MALT) protects the host from pathogens. Polymeric immunoglobulin receptor (pIgR) plays an important role in MALT (Mestecky and McGhee, 1987; Brandtzaeg et al., 1994). pIgR is a single transmembrane protein that binds to dimeric immunoglobulin A (dIgA) and transports it through the polarized epithelium. The pIgR structure can be divided into the extracellular domain, the transmembrane domain, and the intracytoplasmic domain (Brandtzaeg et al., 1994; Mestecky and
∗ Corresponding author. Fax: +81 3 3219 8340. E-mail address: [email protected] (K. Komiyama).
McGhee, 1987). On the apical side of the epithelium, pIgR is cleaved and the extracellular domain that is bound to dIgA is released into the mucosal secretions. This cleaved extracellular domain of pIgR is known as the secretory component (SC), and molecules containing all three domains are called pIgR. Secreted dIgA in association with the SC is called secretory IgA (S-IgA). SIgA works as a biological defense factor (Brandtzaeg et al., 1994; Mestecky and McGhee, 1987; Rojas and Apodaca, 2002). The addition of lipopolysaccharide (LPS) or cytokines such as interferon-␥, interleukin-1, interleukin-4, or tumor necrosis factor-␣ to the culture supernatant of colon (HT-29) or bronchial (Calu-3) epithelial cell lines increases gene and protein expression of pIgR (Kvale et al., 1988; Phillips et al., 1990; Sollid et al., 1987). Furthermore, pIgR gene expression is induced by activation of the transcription factor nuclear factor (NF)-B (Bruno et al., 2011). However, several aspects of how pIgR expression is regulated remain unclear.
http://dx.doi.org/10.1016/j.molimm.2015.07.021 0161-5890/© 2015 Z. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Mikami, Y., et al., Secretory leukocyte protease inhibitor inhibits expression of polymeric immunoglobulin receptor via the NF-B signaling pathway. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.07.021
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Secretory leukocyte protease inhibitor (SLPI) is a glycoprotein with a molecular weight of approximately 11.7 kD that functions as a serine protease inhibitor (Seemüller et al., 1986). SLPI is produced by tracheal epithelial cells, salivary gland acinar cells, squamous cells, intestinal Paneth cells, and goblet cells (Bergenfeldt et al., 1996; Novak et al., 2007; Van Seuningen et al., 1995). It is found in secretions of the respiratory tract and the male and female genital tract, and in saliva (Bergenfeldt et al., 1996; Novak et al., 2007; Van Seuningen et al., 1995). Furthermore, the observation that SLPI gene expression increases during inflammation and inhibits the activities of enzymes such as neutrophil esterase, cathepsin G, trypsin, and chymotrypsin suggests that SLPI diminishes tissue destruction during inflammation (Fritz, 1988; Fryksmark et al., 1989; Vogelmeier et al., 1996). In alveolar epithelial cells, SLPI increases the IB protein level (Lentsch et al., 1999). By binding to NF-B, IB inhibits NF-B activation (Thompson et al., 1995). Accordingly, SLPI may play an important role in the suppression of pIgR gene expression. Therefore, the aim of this study was to elucidate the specific mechanism by which SLPI influences pIgR expression. Using immunohistochemistry, we investigated the localization of SLPI and pIgR in the human gastrointestinal tract and salivary glands. Furthermore, we determined the effect of SLPI on pIgR expression by adding recombinant human SLPI (rhSLPI) to colorectal cancerderived HT-29 cells that constitutively express pIgR (Johansen et al., 1998). In addition, we used oral squamous cell carcinoma-derived Ca9-22 cells, which express SLPI but scarcely express pIgR (Yang et al., 2014), to generate SLPI-deficient (SLPI) cells and investigated whether pIgR expression was induced in these cells.
in paraffin. Thereafter, 4 m thick sections were prepared. After deparaffinization, non-specific binding was blocked by incubating samples in 10% BSA prepared in PBS for 1 h. The specimens were incubated with an anti-human SLPI antibody (HyCult Biotech) or a horseradish peroxidase (HRP)-conjugated anti-human pIgR antibody (Chemicon International) for 1 h. Specimens incubated with the anti-human SLPI antibody were washed with PBS and then further incubated with a HRP-conjugated anti-mouse IgG antibody (Chemicon International) for 1 h. Specimens were subsequently incubated with 3,3 -diaminobenzidine (DAB). Nuclei were stained with hematoxylin. Before staining, HT-29 cells, wild-type Ca9-22 cells, and SLPI-1 cells were incubated with or without rhSLPI (10 g/ml) for 72 h. At the end of the incubation period, cells were spun onto microscope slides using a cytocentrifuge, fixed with 4% paraformaldehyde prepared in PBS for 15 min at room temperature, and immunostained with a HRP-conjugated anti-human pIgR antibody (DAKO Japan, Tokyo, Japan) as described above.
2. Materials and methods
A 2,512 bp fragment containing the SLPI gene was amplified by long-range PCR using genomic DNA of wild-type Ca9-22 cells as the template and cloned into the EcoRV site of pBluescript II. The primers used for amplification were 5 - TTG GCC TCA TAG CCT TAC CTG GCA TAG GAA -3 and 5 - AAC GAA TCA CAG AAG CAG GAT GTG CAA AG -3 . For the SLPI disruption construct, the neomycin resistance gene under the control of the TK promoter was inserted between the NdeI sites. Ca9-22 cells (1 × 107 ) were transfected with 25 g of plasmid by electroporation using a Gene Pulser apparatus (Bio-Rad Laboratories, Richmond, CA) at 25 F and 500 V. Plasmids were linearized by digestion with BamHI. After transfection, cells were re-suspended in 30 ml of medium, plated into 96-well plates (100 l/well), and selected in medium containing G418 (Sigma Chemical Co.) at a final concentration of 150 g/ml. Resistant cell clones were picked and expanded, and the culture medium was collected for ELISA analysis.
2.1. Cells and reagents Ca9-22 and HT-29 cells were obtained from RIKEN BRC (Tsukuba, Japan) and American Type Culture Collection (Manassas, VA), respectively. All cells were maintained in ␣-Minimal Essential Medium (Wako, Osaka, Japan) containing 10% fetal bovine serum (Japan Bio Serum, Tokyo, Japan) and 1% penicillin-streptomycin (Wako) at 37 ◦ C in an atmosphere containing 5% CO2 . rhSLPI was purchased from R&D Systems (Minneapolis, MN). LPS and G418 (Geneticin® ) were purchased from Sigma Chemical Co. (St. Louis, MO). For rhSLPI and LPS treatment, cells were cultured in the presence or absence of rhSLPI (10 g/ml) and/or LPS (10 g/ml) for 24 h. Tissue blocks of the gastrointestinal tract were obtained from the Department of Pathology, Nihon University School of Dentistry. The studies were performed with the approval of the ethics committee of Nihon University School of Dentistry (2007–25).
2.3. Quantitation of SC by ELISA Culture medium was collected and stored at −80 ◦ C until use. The secretion levels of SC in the culture medium were measured using a Quantikine ELISA Kit (R&D Systems) according to the manufacturer’s instructions. Each experiment was performed in triplicate (n = 3).
2.4. Generation of SLPI cells
2.5. Southern blot hybridization 2.2. Immunostaining Gastrointestinal tract tissues were fixed immediately after excision with 5% acetic acid prepared in ethanol for 5 min. The tissues were embedded in paraffin, and microscope slides of 4 m thick sections were prepared. After deparaffinization, tissue sections were blocked with 10% bovine serum albumin (BSA) prepared in phosphate-buffered saline (PBS) for 1 h to avoid non-specific binding of antibodies. The specimens were incubated with a mouse monoclonal anti-human SLPI antibody (HyCult Biotech, Plymouth Meeting, PA) for 1 h, washed with PBS, and incubated with a TRITC-conjugated donkey anti-mouse IgG antibody (Chemicon International, Temecula, CA) for 1 h. The same specimens were subsequently incubated with a FITC-conjugated donkey anti-human pIgR antibody (Chemicon International). For salivary gland staining, tissues were fixed with 10% formaldehyde for 17 h and embedded
Genomic DNA was purified using a QIAGEN DNeasy Kit (QIAGEN, Gaithersburg, MD) according to the manufacturer’s instructions. Approximately 10 g of genomic DNA was digested with PstI or XbaI and resolved on a 0.7% agarose gel. Genomic DNA fragments were transferred from the gel to a nylon membrane (Amersham Pharmacia Biotech, Amersham, UK) using alkaline transfer. Hybridization with the probe was performed at 55 ◦ C overnight. Washing was then performed with washing buffer (2 M urea, 0.1% sodium dodecyl sulfate (SDS), 0.16 M NaCl, 0.01 M MgCl2 , 2% blocking reagent, and 0.05 M phosphate buffer, pH 7.0) at 60 ◦ C for 10 min and then with blocking reagent (0.05 M trishydroxyl aminomethane, 0.1 M NaCl, and 2 mM MgCl2 ) at room temperature for 5 min. The probe was produced via PCR of the neomycin resistance cassette and detected according to the manufacturer’s instructions using CDPStar (Amersham Pharmacia Biotech).
Please cite this article in press as: Mikami, Y., et al., Secretory leukocyte protease inhibitor inhibits expression of polymeric immunoglobulin receptor via the NF-B signaling pathway. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.07.021
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2.6. Analysis of mRNA Total RNA was isolated using RNAiso Plus (Takara Bio, Tokyo, Japan) according to the manufacturer’s instructions. First-strand cDNA was synthesized at 50 ◦ C for 1 h in 20 l of solution containing 1 g of total RNA, 1× first-strand buffer, 50 ng of random primers, 10 mM dNTP mixture, 1 mM dithiothreitol, and 0.5 U of SuperScript® III RNase H reverse transcriptase (Invitrogen, Carlsbad, CA). cDNA was then diluted 5-fold in sterile distilled water, and 2 l of cDNA was subjected to real-time RT-PCR using SYBR PreTM mix Ex Taq II (Takara Bio) on a CFX96 Real-Time System (Bio-Rad Laboratories). mRNA levels were normalized to that of -actin. For RT-PCR, first-strand cDNA was synthesized as described above. PCR was performed on a Smart Cycler using the following cycling conditions: 40 cycles of 95 ◦ C for 5 s and 68 ◦ C for 25 s. PCR products were subsequently separated by electrophoresis through a 2% agarose gel, which was stained with ethidium bromide, and photographed. The primer sets used are described in Supplemental Table 1. Each reaction was performed three times using cDNA prepared from different samples of RNA. Supplementry material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.molimm.2015.07.021
2.7. Luciferase assay The NBE × 6 Luc reporter plasmid, which contains the NF-B binding element (NBE) × 6 fused to the firefly luciferase gene, was purchased from Stratagene (La Jolla, CA). The Control Luc plasmid, which contains the SV40 promoter fused to the firefly luciferase gene (Promega, Madison, WI), was used as a control. Cells were seeded into 6-well plates at a confluency of 90% and incubated for 18 h. Two micrograms of NBE × 6 Luc or Control Luc was transfected into cells using Lipofectamine LTX (Invitrogen) along with 0.5 g of the Renilla luciferase plasmid (Promega). The luciferase assay was performed 48 h after transfection using a Dual Luciferase Assay Kit (Promega) according to the manufacturer’s instructions. The luciferase activities of NBE × 6 Luc and Control Luc were normalized to Renilla luciferase activity as an internal control.
2.8. Western blot analysis Cells were lysed in 1 × sample buffer (50 mM Tris-HCl, 2% SDS, 10% glycerol, and 6% 2-mercaptoethanol). Protein samples (10 g) were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were incubated with the indicated primary antibodies (diluted 1:500). Primary antibodies against -actin and IB were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and those against NF-B (p50, p52, and p65) were obtained from Cell Signaling Technology (Beverly, MA). After washing, membranes were incubated with secondary antibodies for 1 h, and immunoreactive bands were visualized using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech) according to the manufacturer’s instructions.
2.9. Statistical analysis Results are presented as the mean ± standard deviation (SD). Statistical differences were assessed using the Student’s t-test. Significant differences (p < 0.05) are indicated.
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Table 1 Localization of SLPI and pIgR in the gastrointestinal tract and the salivary glands. pIgR
SLPI
Co-localization
+++ –
– +++
N N
+++ +++ + –
– – + +++
N N O N
Gastrointestinal tract Ciliated epithelial cells Goblet cells Salivary glands Salivary gland demilune structures Proximal ductal epithelial cells Serous acinar cells Mucinous acinar cells
The number of ‘+’ indicates expression levels. O or N, co-localization of SLPI and pIgR was or was not observed, respectively.
3. Results 3.1. Interstitial localization of SLPI and pIgR We first investigated the interstitial localization of pIgR and SLPI in the normal human gastrointestinal tract and salivary glands. The localization patterns of SLPI and pIgR are summarized in Table 1. In the gastrointestinal tract, pIgR was localized in ciliated epithelial cells. On the other hand, SLPI seemed to be localized in goblet cells (Fig. 1A). We did not detect cells in which SLPI and pIgR were co-expressed in the gastrointestinal tract (Fig. 1A). In the salivary glands, pIgR was strongly expressed in salivary gland demilune structures and proximal ductal epithelial cells and was weakly expressed in serous acinar cells (Fig. 1B). However, pIgR was not expressed in mucinous acinar cells. On the other hand, SLPI was strongly expressed in mucous acinar cells and weakly expressed in serous acinar cells (Fig. 1B). However, SLPI was not expressed in salivary gland demilune structures or proximal ductal epithelial cells (Fig. 1B). 3.2. The effect of rhSLPI treatment on SC secretion and pIgR expression in HT-29 cells We next investigated how SLPI affects pIgR expression in HT-29 cells. The basal concentration of SC in HT-29 cell culture supernatant after 3 days of culture in the absence of rhSLPI was 7.9 ± 0.3 g/ml (Fig. 2A). However, when HT-29 cells were cultured in the presence of rhSLPI for 3 days, the SC concentration in the culture supernatant was 6.1 ± 1.0 g/ml. Meanwhile, the addition of LPS (10 g/ml) significantly increased the amount of SC secreted by HT-29 cells into the culture supernatant (17.3 ± 2.2 g/ml). The LPS-induced increase in SC secretion by HT-29 cells was diminished by the addition of rhSLPI (7.8 ± 1.5 g/ml). We observed a similar pattern by immunostaining for pIgR when HT-29 cells were cultured in the presence or absence of rhSLPI (Fig. 2B). When rhSLPI was added to the HT-29 cell culture medium, the number of cells expressing pIgR greatly decreased (Fig. 2B). 3.3. The effect of SLPI knockout on SC secretion and pIgR expression in Ca9-22 cells To verify that pIgR exhibited an SLPI-dependent expression pattern in Ca9-22 cells, similar to HT-29 cells, we generated a cell line in which part of the SLPI gene was replaced with a neomycin resistance gene under the control of the TK promoter (TKpro +Neo). The replacement targeting strategy is illustrated in Fig. 3A. This construct was transfected into wild-type Ca9-22 cells, and neomycin-resistant clones were screened according to the secretion level of SC measured by an ELISA. The two clones that secreted the highest levels of SC into the culture supernatant (SLPI-1 and -2) were chosen for further experiments (Supplemental Fig. 1). We next confirmed that the SLPI gene was replaced with
Please cite this article in press as: Mikami, Y., et al., Secretory leukocyte protease inhibitor inhibits expression of polymeric immunoglobulin receptor via the NF-B signaling pathway. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.07.021
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Fig. 1. Interstitial localization of SLPI and pIgR. (A) Localizations of SLPI and pIgR in gastrointestinal tract tissue. Gastrointestinal tract tissue was co-stained with anti-SLPI and anti-pIgR antibodies, which were subsequently detected with TRITC-conjugated (red) and FITC-conjugated (green) secondary antibodies, respectively. Co-localization of SLPI and pIgR (yellow) was not observed. Hematoxylin and eosin (HE) staining was performed to label the tissue that was immunostained. (B) Localizations of SLPI and pIgR in salivary gland tissues. Salivary gland tissues were stained with an anti-SLPI (left) or anti-pIgR (right) antibody, which was subsequently labeled with a HRP-conjugated secondary antibody. The secondary antibody was detected using DAB. DNA was counterstained with hematoxylin. Black, gray, and white arrowheads indicate positively stained mucinous acinar cells, proximal ductal epithelial cells, and salivary gland demilune structures, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. The effect of rhSLPI on SC secretion by HT-29 cells. (A) Concentration of SC in the culture supernatant. Cells were cultured in the presence or absence of rhSLPI (10 g/ml) and/or LPS (10 g/ml), and then the basal concentration of SC in the culture supernatant was measured by an ELISA. * indicates a significant difference. Mean ± SD (n = 3, p < 0.05). (B) pIgR protein expression in HT-29 cells cultured in the presence or absence of rhSLPI. Cells were cultured in the presence or absence of rhSLPI (10 g/ml) and then stained with an anti-pIgR antibody as described in Section 2.
our targeting constructs in the two clones by hybridizing XbaI or PstI digests with a probe located on TKpro +Neo. Successful targeting was expected to produce a 4.8 kb (XbaI) fragment or 1.8 kb and 1.0 kb (PstI) fragments. The two clones were positive for 4.8 kb (XbaI) and 1.8/1.0 kb (PstI) fragments (Fig. 3B). Next, we performed RT-PCR to determine whether SLPI mRNA expression was suppressed in these two clones. Expression of SLPI was observed in wild-type Ca9-22 cells, but was completely suppressed in SLPI-1 and -2 cells (Fig. 3C). We further investigated whether mRNA expression of pIgR was increased in SLPI cells. Real-time RT-PCR showed that pIgR mRNA expression in SLPI-1 and -2 cells was higher than that in wild-type Ca9-22 cells and was similar to that in HT-29 cells (Fig. 4A). Immunostaining and ELISA analyses also showed remarkable pIgR protein expression in SLPI cells and secretion of SC into the culture supernatant, respectively. Contrastively, they were scarcely detected in wild-type Ca9-22 cells and in their culture supernatant (Fig. 4B and Supplemental Fig. 1).
Supplementry material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.molimm.2015.07.021 3.4. Signaling pathways involved in the suppression of SC secretion and pIgR expression by SLPI The results of these experiments demonstrated that SLPI suppresses SC secretion and pIgR expression. Therefore, we proceeded to investigate the molecular mechanism responsible for SLPImediated suppression of SC secretion and pIgR expression. Previous reports suggested that the NF-B signaling pathway is involved in SLPI-mediated suppression of pIgR expression (Lentsch et al., 1999). Therefore, we first analyzed the level of NF-B activation in cells used in the aforementioned experiment using a reporter plasmid in which six repeats of NBE were bound to the luciferase gene (see Section 2). Luciferase activity was higher in SLPI cells than in wild-type Ca9-22 cells (Fig. 5A). Luciferase activity was significantly
Please cite this article in press as: Mikami, Y., et al., Secretory leukocyte protease inhibitor inhibits expression of polymeric immunoglobulin receptor via the NF-B signaling pathway. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.07.021
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Fig. 3. Generation of SLPI cells. (A) Restriction maps of the SLPI locus, the gene disruption construct, and configuration of the targeted locus. The targeted construct is expected to disrupt exon 4. Relevant restriction enzyme sites shown are as follows: B, BamHI; N, NdeI; P, PstI; and X, XbaI. The position of the probe used for Southern hybridization is indicated. The new 4.8 kb XbaI and 1.0/1.8 kb PstI fragments hybridize with the probe if targeted integration of the construct occurs. (B) Restriction analysis of the targeted integration of the SLPI disruption construct. Genomic DNA from two SLPI clones (SLPI-1 and -2) was digested with XbaI or PstI, size-fractionated by 0.7% agarose gel electrophoresis, transferred to a nylon filter, and hybridized with the probe indicated in (A). (C) Suppression of SLPI expression from the transgene. Total RNA was isolated from the two SLPI clones (SLPI-1 and -2), and RT-PCR was performed. W.T.Ca9-22 indicates wild-type Ca9-22 cells.
Fig. 4. pIgR expression in SLPI cells. (A) mRNA expression of pIgR. mRNA expression was measured by real-time RT-PCR. * indicates significantly different from wild-type Ca9-22 cells (W.T.Ca9-22). HT-29 cells were used as a positive control. Mean ± SD (n = 3, p < 0.05). (B) Protein expression of pIgR. Cells were stained with an anti-pIgR antibody as described in Section 2.
lower in HT-29 cells treated with rhSLPI than in HT-29 cells not treated with rhSLPI (Fig. 5B). Next, we investigated whether these changes in luciferase activity were caused by changes in the level of NF-B expression. Western blot analysis found no difference in the level of NF-B protein expression between SLPI and wildtype Ca9-22 cells (Fig. 5C) or between HT-29 cells treated with and without rhSLPI (Fig. 5D). Similar results were obtained by RT-PCR; no differences were observed in the level of NF-B mRNA between SLPI and wild-type Ca9-22 cells or between HT-29 cells treated with and without rhSLPI (data not shown). Expression of IB, a component of the NF-B signaling pathway, was analyzed in the same cells and culture conditions. IB protein expression was lower in SLPI cells than in wild-type Ca9-22 cells and was higher in HT-29 cells treated with rhSLPI than in HT-29 cells not treated with rhSLPI (Fig. 5C and D). Meanwhile, there was no difference in the mRNA level of IB between these cells (Fig. 5E and F). 4. Discussion The localization of SLPI has been demonstrated in many types of tissues, including salivary glands, the gastrointestinal tract,
mammary glands, skin, respiratory organs, kidneys, pancreas, and reproductive organs, using immunohistochemistry and in situ hybridization (Bergenfeldt et al., 1996; Novak et al., 2007; Van Seuningen et al., 1995). SLPI is expressed in the stratum granulosum in normal skin, tracheal Clara cells (non-ciliated bronchiolar secretory cells), serous cells in the respiratory tract, serous acinar cells in the parotid gland and submandibular glands, uterine cervix mucous cells, and Paneth and goblet cells in the gastrointestinal tract (Bergenfeldt et al., 1996; Franken et al., 1989; Into et al., 2006). Similar to the previous reports, we detected SLPI expression in goblet cells in the gastrointestinal tract. In addition, we observed SLPI expression in mucus acinar cells in salivary glands. However, there was only a minimal amount of expression in serous acini. The discrepancies between our results and the previous studies may be due to the use of in situ hybridization to investigate mRNA expression in previous reports, whereas we used immunohistochemistry to detect proteins. However, differences were noted in the tissues that contained SLPI-expressing and pIgR-expressing cells. This suggests that SLPI expression suppresses that of pIgR and/or vice versa. Previous studies reported strong SLPI expression in lung squamous cell carcinoma-derived HS-24 cells, lung epithelium-derived A594
Please cite this article in press as: Mikami, Y., et al., Secretory leukocyte protease inhibitor inhibits expression of polymeric immunoglobulin receptor via the NF-B signaling pathway. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.07.021
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Fig. 5. The NF-B signaling pathway in SLPI-mediated suppression of pIgR expression. (A and B) Transcriptional activity of NF-B in wild-type Ca9-22 cells (W.T.Ca9-22) and SLPI clones. Cells were transfected with the NF-B Luc or Control Luc plasmid, and then luciferase activity was measured. * indicates significantly different from wild-type Ca9-22 cells. Mean ± SD (n = 3, p < 0.05). (B) Transcriptional activity of NF-B in HT-29 cells. Cells transfected with the NF-B Luc or Control Luc plasmid were cultured in the presence or absence of rhSLPI (10 g/ml), and then luciferase activity was measured. * indicates significantly different from cells not treated with rhSLPI. Mean ± SD (n = 3, p < 0.05). (C) Protein expression of NF-B (p50, p52, and p60) and IB in wild-type Ca9-22 cells and SLPI clones. Western blot analysis was performed as described in Section 2. -actin was used as the internal control. (D) Protein expression of NF-B (p50, p52, and p60) and IB in HT-29 cells. Cells were cultured in the presence or absence of rhSLPI (10 g/ml), and then Western blot analysis was performed. -actin was used as the internal control. (E) mRNA expression of IB in wild-type Ca9-22 cells and SLPI clones. Total RNA was isolated from each type of cell, and then real-time RT-PCR was performed. Mean ± SD (n = 3). (F) mRNA expression of IB in HT-29 cells. Cells were cultured under the same conditions as described in (D), and then real-time RT-PCR was performed. Mean ± SD (n = 3).
cells, and gingival squamous cell carcinoma-derived Ca9-22 cells (Yang et al., 2014). pIgR was not expressed in these cells. Furthermore, SLPI is expressed at low levels or is not expressed in HT-29, Caco2, and T84 cells. This is in contrast to pIgR, which is expressed in all of these colon carcinoma cell lines (Si-Tahar et al., 2000). The present study found that LPS stimulation augmented the SC secretion level in HT-29 cell culture supernatant. However, the addition of rhSLPI suppressed the LPS-mediated increase in the level of secreted SC. The secretion level of SC was higher in SLPI Ca9-22 cells than in wild-type Ca9-22 cells (supplemental Fig. 1). These results demonstrate that SLPI suppresses SC secretion into the culture supernatant. A complex of pIgR bound to dIgA passes through the cytoplasm, and S-IgA is secreted in the lumen when part of the pIgR on the epithelial cell apical surface is cleaved by a serine protease. SLPI suppresses this cleavage of pIgR by inhibiting the activity of the serine protease (Pilette et al., 2003). Accordingly, suppression of pIgR cleavage by a serine protease may be one reason why the addition of rhSLPI decreased the amount of SC in the culture supernatant. Real-time RT-PCR data indicated that pIgR mRNA expression was strongly increased in SLPI Ca9-22 cells (approximately 30-fold in comparison to the level in wild-type Ca922 cells). Decreased protein expression of pIgR was demonstrated by a reduction in the number of HT-29 cells that were positively immunostained for pIgR following rhSPLI treatment. Thus, we infer that the main reason for the SLPI-mediated decrease in SC secretion into the culture supernatant is suppression of pIgR gene expression. SLPI suppresses NF-B activation by preventing IB degradation (Baeuerle, 1998; Bruno et al., 2011). Specifically, IB is phosphorylated by IB kinase, and then phosphorylated IB is degraded by the proteasome (Baeuerle, 1998; Bruno et al., 2011). This results in NF-B activation and increased pIgR expression (Bruno et al., 2011). Based on these reports, we hypothesize that suppression of pIgR expression by SLPI involves the NF-B signaling pathway through IB protein degradation, rather than inhibition of IB gene transcription. The luciferase assay performed using the NF-B reporter plasmid indicated that the transcriptional activity of NF-B protein was suppressed when SLPI was present. However, no changes in the mRNA or protein level of
NF-B were observed in these cells. These results suggest that post-translational modification (e.g., phosphorylation or binding of inhibitory factors) suppresses the transcriptional activity of NF-B protein. In support of this hypothesis, the level of IB protein, but not of mRNA, changed in parallel with the level of NF-B activity. pIgR is expressed in epithelial cells of various tissues, although certain epithelial cells do not express pIgR. The mechanism of suppression that leads to these differences was unclear. The results of this study demonstrate that pIgR expression was regulated in epithelial cells, at least in part, by SLPI through the NF-B signaling pathway. Acknowledgments This work was supported in part by JSPS KAKENHI (#24593066), grants from the Dental Research Center, and the Sato Fund of the Nihon University School of Dentistry. References Baeuerle, P.A., 1998. IkappaB-NF-kappaB structures: at the interface of inflammation control. Cell 95, 729–731. Bergenfeldt, M., Nyström, M., Bohe, M., Lindström, C., Polling, A., Ohlsson, K., 1996. Localization of immunoreactive secretory leukocyte protease inhibitor (SLPI) in intestinal mucosa. J. Gastroenterol. 31, 18–23. Brandtzaeg, P., Krajci, P., Lamm, M.E., Kaezel, C.S., 1994. Epihelial and hepatobiliary transport of polymeric Immunoglobulin. In: Ogra, P.L., Mestecky, J., Lamm, M.E., Strober, W., McGhee, J.R., Bienenstock, J. (Eds.), Handbook of Mucosal Immunology. Academic Press, San Diego, pp. 113–126. Bruno, M.E., Frantz, A.L., Rogier, E.W., Johansen, F.E., Kaetzel, C.S., 2011. Regulation of the polymeric immunoglobulin receptor by the classical and alternative NF-(B pathways in intestinal epithelial cells. Mucosal Immunol. 4, 468–478. Franken, C., Meijer, C.J., Dijkman, J.H., 1989. Tissue distribution of antileukoprotease and lysozyme in humans. J. Histochem. Cytochem. 37, 493–498. Fritz, H., 1988. Human mucus proteinase inhibitor (human MPI). Human seminal inhibitor I (HUSI-I), antileukoprotease (ALP), secretory leukocyte protease inhibitor (SLPI). Biol. Chem. Hoppe Seyler 369, 79–82. Fryksmark, U., Jannert, M., Ohlsson, K., Tegner, H., Wihl, J.A., 1989. Secretory leukocyte protease inhibitor in normal, allergic and virus induced nasal secretions. Rhinology 27, 97–103. Into, T., Inomata, M., Kanno, Y., Matsuyama, T., Machigashira, M., Izumi, Y., Imamura, T., Nakashima, M., Noguchi, T., Matsushita, K., 2006.
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Secretory leukocyte protease inhibitor inhibits expression of polymeric immunoglobulin receptor via the NF-B signaling pathway Yoshikazu Mikami a , Takashi Iwase a , Yusuke Komiyama b , Naoyuki Matsumoto a , Hidero Oki c , Kazuo Komiyama a,∗ a
Department of Pathology, Nihon University School of Dentistry, 1-8-13 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-8310, Japan Department of Oral and Maxillofacial Surgery, Dokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu-machi, Shimotsuga-gun, Tochigi, 321-0293, Japan c Department of Maxillofacial Surgery, Nihon University School of Dentistry, 1-8-13 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-8310, Japan b
a r t i c l e
i n f o
Article history: Received 9 June 2015 Received in revised form 16 July 2015 Accepted 17 July 2015 Available online xxx Keywords: SLPI pIgR Epithelial cells NF-B
a b s t r a c t Polymeric immunoglobulin receptor (pIgR) plays an important role in mucosal immune systems. Secretory immunoglobulin A, composed of secretory component of pIgR and a dimeric form of immunoglobulin A, is secreted on mucosal surfaces and serves as a biological defense factor. pIgR gene expression is reportedly induced by activation of the transcription factor nuclear factor (NF)-B. On the other hand, secretory leukocyte protease inhibitor (SLPI) is a glycoprotein that functions as a serine protease inhibitor. In alveolar epithelial cells, SLPI increases the level of IB, which indicates that it is an inhibitor of NF-B at the protein level. Taken together, SLPI may regulate pIgR expression; however, the specific mechanism by which this occurs is unclear. Therefore, the aim of this study was to elucidatethe influence of SLPI on pIgR expression.SLPI and pIgR localized in goblet cells and ciliated epithelial cells of the gastrointestinal tract, respectively. No cells were detected in which SLPI and pIgR were co-expressed. In addition, recombinant human SLPI stimulation of an epithelial cell line (HT-29) decreased the pIgR expression. The pIgR expression was also higher in SLPI-deficient Ca9-22 cells than in wild-type Ca9-22 cells. Furthermore, a luciferase assay using a NF-B reporter plasmid and real-time RT-PCR analysis indicated that when SLPI was present, the transcriptional activity of NF-B protein was suppressed, which was accompanied by anincrease in the protein, but not the mRNA,expression of IB. These results demonstrate that SLPI down-regulates pIgR expression through the NF-B signaling pathway by inhibiting degradation of IB protein. © 2015 Z. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction The mucosal membrane of the gastrointestinal tract is covered by ciliated non-keratinized epithelial cells. The gastrointestinal mucosa is constantly exposed to pathogenic exogenous antigens. Mucosa-associated lymphoid tissue (MALT) protects the host from pathogens. Polymeric immunoglobulin receptor (pIgR) plays an important role in MALT (Mestecky and McGhee, 1987; Brandtzaeg et al., 1994). pIgR is a single transmembrane protein that binds to dimeric immunoglobulin A (dIgA) and transports it through the polarized epithelium. The pIgR structure can be divided into the extracellular domain, the transmembrane domain, and the intracytoplasmic domain (Brandtzaeg et al., 1994; Mestecky and
∗ Corresponding author. Fax: +81 3 3219 8340. E-mail address: [email protected] (K. Komiyama).
McGhee, 1987). On the apical side of the epithelium, pIgR is cleaved and the extracellular domain that is bound to dIgA is released into the mucosal secretions. This cleaved extracellular domain of pIgR is known as the secretory component (SC), and molecules containing all three domains are called pIgR. Secreted dIgA in association with the SC is called secretory IgA (S-IgA). SIgA works as a biological defense factor (Brandtzaeg et al., 1994; Mestecky and McGhee, 1987; Rojas and Apodaca, 2002). The addition of lipopolysaccharide (LPS) or cytokines such as interferon-␥, interleukin-1, interleukin-4, or tumor necrosis factor-␣ to the culture supernatant of colon (HT-29) or bronchial (Calu-3) epithelial cell lines increases gene and protein expression of pIgR (Kvale et al., 1988; Phillips et al., 1990; Sollid et al., 1987). Furthermore, pIgR gene expression is induced by activation of the transcription factor nuclear factor (NF)-B (Bruno et al., 2011). However, several aspects of how pIgR expression is regulated remain unclear.
http://dx.doi.org/10.1016/j.molimm.2015.07.021 0161-5890/© 2015 Z. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Secretory leukocyte protease inhibitor (SLPI) is a glycoprotein with a molecular weight of approximately 11.7 kD that functions as a serine protease inhibitor (Seemüller et al., 1986). SLPI is produced by tracheal epithelial cells, salivary gland acinar cells, squamous cells, intestinal Paneth cells, and goblet cells (Bergenfeldt et al., 1996; Novak et al., 2007; Van Seuningen et al., 1995). It is found in secretions of the respiratory tract and the male and female genital tract, and in saliva (Bergenfeldt et al., 1996; Novak et al., 2007; Van Seuningen et al., 1995). Furthermore, the observation that SLPI gene expression increases during inflammation and inhibits the activities of enzymes such as neutrophil esterase, cathepsin G, trypsin, and chymotrypsin suggests that SLPI diminishes tissue destruction during inflammation (Fritz, 1988; Fryksmark et al., 1989; Vogelmeier et al., 1996). In alveolar epithelial cells, SLPI increases the IB protein level (Lentsch et al., 1999). By binding to NF-B, IB inhibits NF-B activation (Thompson et al., 1995). Accordingly, SLPI may play an important role in the suppression of pIgR gene expression. Therefore, the aim of this study was to elucidate the specific mechanism by which SLPI influences pIgR expression. Using immunohistochemistry, we investigated the localization of SLPI and pIgR in the human gastrointestinal tract and salivary glands. Furthermore, we determined the effect of SLPI on pIgR expression by adding recombinant human SLPI (rhSLPI) to colorectal cancerderived HT-29 cells that constitutively express pIgR (Johansen et al., 1998). In addition, we used oral squamous cell carcinoma-derived Ca9-22 cells, which express SLPI but scarcely express pIgR (Yang et al., 2014), to generate SLPI-deficient (SLPI) cells and investigated whether pIgR expression was induced in these cells.
in paraffin. Thereafter, 4 m thick sections were prepared. After deparaffinization, non-specific binding was blocked by incubating samples in 10% BSA prepared in PBS for 1 h. The specimens were incubated with an anti-human SLPI antibody (HyCult Biotech) or a horseradish peroxidase (HRP)-conjugated anti-human pIgR antibody (Chemicon International) for 1 h. Specimens incubated with the anti-human SLPI antibody were washed with PBS and then further incubated with a HRP-conjugated anti-mouse IgG antibody (Chemicon International) for 1 h. Specimens were subsequently incubated with 3,3 -diaminobenzidine (DAB). Nuclei were stained with hematoxylin. Before staining, HT-29 cells, wild-type Ca9-22 cells, and SLPI-1 cells were incubated with or without rhSLPI (10 g/ml) for 72 h. At the end of the incubation period, cells were spun onto microscope slides using a cytocentrifuge, fixed with 4% paraformaldehyde prepared in PBS for 15 min at room temperature, and immunostained with a HRP-conjugated anti-human pIgR antibody (DAKO Japan, Tokyo, Japan) as described above.
2. Materials and methods
A 2,512 bp fragment containing the SLPI gene was amplified by long-range PCR using genomic DNA of wild-type Ca9-22 cells as the template and cloned into the EcoRV site of pBluescript II. The primers used for amplification were 5 - TTG GCC TCA TAG CCT TAC CTG GCA TAG GAA -3 and 5 - AAC GAA TCA CAG AAG CAG GAT GTG CAA AG -3 . For the SLPI disruption construct, the neomycin resistance gene under the control of the TK promoter was inserted between the NdeI sites. Ca9-22 cells (1 × 107 ) were transfected with 25 g of plasmid by electroporation using a Gene Pulser apparatus (Bio-Rad Laboratories, Richmond, CA) at 25 F and 500 V. Plasmids were linearized by digestion with BamHI. After transfection, cells were re-suspended in 30 ml of medium, plated into 96-well plates (100 l/well), and selected in medium containing G418 (Sigma Chemical Co.) at a final concentration of 150 g/ml. Resistant cell clones were picked and expanded, and the culture medium was collected for ELISA analysis.
2.1. Cells and reagents Ca9-22 and HT-29 cells were obtained from RIKEN BRC (Tsukuba, Japan) and American Type Culture Collection (Manassas, VA), respectively. All cells were maintained in ␣-Minimal Essential Medium (Wako, Osaka, Japan) containing 10% fetal bovine serum (Japan Bio Serum, Tokyo, Japan) and 1% penicillin-streptomycin (Wako) at 37 ◦ C in an atmosphere containing 5% CO2 . rhSLPI was purchased from R&D Systems (Minneapolis, MN). LPS and G418 (Geneticin® ) were purchased from Sigma Chemical Co. (St. Louis, MO). For rhSLPI and LPS treatment, cells were cultured in the presence or absence of rhSLPI (10 g/ml) and/or LPS (10 g/ml) for 24 h. Tissue blocks of the gastrointestinal tract were obtained from the Department of Pathology, Nihon University School of Dentistry. The studies were performed with the approval of the ethics committee of Nihon University School of Dentistry (2007–25).
2.3. Quantitation of SC by ELISA Culture medium was collected and stored at −80 ◦ C until use. The secretion levels of SC in the culture medium were measured using a Quantikine ELISA Kit (R&D Systems) according to the manufacturer’s instructions. Each experiment was performed in triplicate (n = 3).
2.4. Generation of SLPI cells
2.5. Southern blot hybridization 2.2. Immunostaining Gastrointestinal tract tissues were fixed immediately after excision with 5% acetic acid prepared in ethanol for 5 min. The tissues were embedded in paraffin, and microscope slides of 4 m thick sections were prepared. After deparaffinization, tissue sections were blocked with 10% bovine serum albumin (BSA) prepared in phosphate-buffered saline (PBS) for 1 h to avoid non-specific binding of antibodies. The specimens were incubated with a mouse monoclonal anti-human SLPI antibody (HyCult Biotech, Plymouth Meeting, PA) for 1 h, washed with PBS, and incubated with a TRITC-conjugated donkey anti-mouse IgG antibody (Chemicon International, Temecula, CA) for 1 h. The same specimens were subsequently incubated with a FITC-conjugated donkey anti-human pIgR antibody (Chemicon International). For salivary gland staining, tissues were fixed with 10% formaldehyde for 17 h and embedded
Genomic DNA was purified using a QIAGEN DNeasy Kit (QIAGEN, Gaithersburg, MD) according to the manufacturer’s instructions. Approximately 10 g of genomic DNA was digested with PstI or XbaI and resolved on a 0.7% agarose gel. Genomic DNA fragments were transferred from the gel to a nylon membrane (Amersham Pharmacia Biotech, Amersham, UK) using alkaline transfer. Hybridization with the probe was performed at 55 ◦ C overnight. Washing was then performed with washing buffer (2 M urea, 0.1% sodium dodecyl sulfate (SDS), 0.16 M NaCl, 0.01 M MgCl2 , 2% blocking reagent, and 0.05 M phosphate buffer, pH 7.0) at 60 ◦ C for 10 min and then with blocking reagent (0.05 M trishydroxyl aminomethane, 0.1 M NaCl, and 2 mM MgCl2 ) at room temperature for 5 min. The probe was produced via PCR of the neomycin resistance cassette and detected according to the manufacturer’s instructions using CDPStar (Amersham Pharmacia Biotech).
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2.6. Analysis of mRNA Total RNA was isolated using RNAiso Plus (Takara Bio, Tokyo, Japan) according to the manufacturer’s instructions. First-strand cDNA was synthesized at 50 ◦ C for 1 h in 20 l of solution containing 1 g of total RNA, 1× first-strand buffer, 50 ng of random primers, 10 mM dNTP mixture, 1 mM dithiothreitol, and 0.5 U of SuperScript® III RNase H reverse transcriptase (Invitrogen, Carlsbad, CA). cDNA was then diluted 5-fold in sterile distilled water, and 2 l of cDNA was subjected to real-time RT-PCR using SYBR PreTM mix Ex Taq II (Takara Bio) on a CFX96 Real-Time System (Bio-Rad Laboratories). mRNA levels were normalized to that of -actin. For RT-PCR, first-strand cDNA was synthesized as described above. PCR was performed on a Smart Cycler using the following cycling conditions: 40 cycles of 95 ◦ C for 5 s and 68 ◦ C for 25 s. PCR products were subsequently separated by electrophoresis through a 2% agarose gel, which was stained with ethidium bromide, and photographed. The primer sets used are described in Supplemental Table 1. Each reaction was performed three times using cDNA prepared from different samples of RNA. Supplementry material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.molimm.2015.07.021
2.7. Luciferase assay The NBE × 6 Luc reporter plasmid, which contains the NF-B binding element (NBE) × 6 fused to the firefly luciferase gene, was purchased from Stratagene (La Jolla, CA). The Control Luc plasmid, which contains the SV40 promoter fused to the firefly luciferase gene (Promega, Madison, WI), was used as a control. Cells were seeded into 6-well plates at a confluency of 90% and incubated for 18 h. Two micrograms of NBE × 6 Luc or Control Luc was transfected into cells using Lipofectamine LTX (Invitrogen) along with 0.5 g of the Renilla luciferase plasmid (Promega). The luciferase assay was performed 48 h after transfection using a Dual Luciferase Assay Kit (Promega) according to the manufacturer’s instructions. The luciferase activities of NBE × 6 Luc and Control Luc were normalized to Renilla luciferase activity as an internal control.
2.8. Western blot analysis Cells were lysed in 1 × sample buffer (50 mM Tris-HCl, 2% SDS, 10% glycerol, and 6% 2-mercaptoethanol). Protein samples (10 g) were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were incubated with the indicated primary antibodies (diluted 1:500). Primary antibodies against -actin and IB were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and those against NF-B (p50, p52, and p65) were obtained from Cell Signaling Technology (Beverly, MA). After washing, membranes were incubated with secondary antibodies for 1 h, and immunoreactive bands were visualized using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech) according to the manufacturer’s instructions.
2.9. Statistical analysis Results are presented as the mean ± standard deviation (SD). Statistical differences were assessed using the Student’s t-test. Significant differences (p < 0.05) are indicated.
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Table 1 Localization of SLPI and pIgR in the gastrointestinal tract and the salivary glands. pIgR
SLPI
Co-localization
+++ –
– +++
N N
+++ +++ + –
– – + +++
N N O N
Gastrointestinal tract Ciliated epithelial cells Goblet cells Salivary glands Salivary gland demilune structures Proximal ductal epithelial cells Serous acinar cells Mucinous acinar cells
The number of ‘+’ indicates expression levels. O or N, co-localization of SLPI and pIgR was or was not observed, respectively.
3. Results 3.1. Interstitial localization of SLPI and pIgR We first investigated the interstitial localization of pIgR and SLPI in the normal human gastrointestinal tract and salivary glands. The localization patterns of SLPI and pIgR are summarized in Table 1. In the gastrointestinal tract, pIgR was localized in ciliated epithelial cells. On the other hand, SLPI seemed to be localized in goblet cells (Fig. 1A). We did not detect cells in which SLPI and pIgR were co-expressed in the gastrointestinal tract (Fig. 1A). In the salivary glands, pIgR was strongly expressed in salivary gland demilune structures and proximal ductal epithelial cells and was weakly expressed in serous acinar cells (Fig. 1B). However, pIgR was not expressed in mucinous acinar cells. On the other hand, SLPI was strongly expressed in mucous acinar cells and weakly expressed in serous acinar cells (Fig. 1B). However, SLPI was not expressed in salivary gland demilune structures or proximal ductal epithelial cells (Fig. 1B). 3.2. The effect of rhSLPI treatment on SC secretion and pIgR expression in HT-29 cells We next investigated how SLPI affects pIgR expression in HT-29 cells. The basal concentration of SC in HT-29 cell culture supernatant after 3 days of culture in the absence of rhSLPI was 7.9 ± 0.3 g/ml (Fig. 2A). However, when HT-29 cells were cultured in the presence of rhSLPI for 3 days, the SC concentration in the culture supernatant was 6.1 ± 1.0 g/ml. Meanwhile, the addition of LPS (10 g/ml) significantly increased the amount of SC secreted by HT-29 cells into the culture supernatant (17.3 ± 2.2 g/ml). The LPS-induced increase in SC secretion by HT-29 cells was diminished by the addition of rhSLPI (7.8 ± 1.5 g/ml). We observed a similar pattern by immunostaining for pIgR when HT-29 cells were cultured in the presence or absence of rhSLPI (Fig. 2B). When rhSLPI was added to the HT-29 cell culture medium, the number of cells expressing pIgR greatly decreased (Fig. 2B). 3.3. The effect of SLPI knockout on SC secretion and pIgR expression in Ca9-22 cells To verify that pIgR exhibited an SLPI-dependent expression pattern in Ca9-22 cells, similar to HT-29 cells, we generated a cell line in which part of the SLPI gene was replaced with a neomycin resistance gene under the control of the TK promoter (TKpro +Neo). The replacement targeting strategy is illustrated in Fig. 3A. This construct was transfected into wild-type Ca9-22 cells, and neomycin-resistant clones were screened according to the secretion level of SC measured by an ELISA. The two clones that secreted the highest levels of SC into the culture supernatant (SLPI-1 and -2) were chosen for further experiments (Supplemental Fig. 1). We next confirmed that the SLPI gene was replaced with
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Fig. 1. Interstitial localization of SLPI and pIgR. (A) Localizations of SLPI and pIgR in gastrointestinal tract tissue. Gastrointestinal tract tissue was co-stained with anti-SLPI and anti-pIgR antibodies, which were subsequently detected with TRITC-conjugated (red) and FITC-conjugated (green) secondary antibodies, respectively. Co-localization of SLPI and pIgR (yellow) was not observed. Hematoxylin and eosin (HE) staining was performed to label the tissue that was immunostained. (B) Localizations of SLPI and pIgR in salivary gland tissues. Salivary gland tissues were stained with an anti-SLPI (left) or anti-pIgR (right) antibody, which was subsequently labeled with a HRP-conjugated secondary antibody. The secondary antibody was detected using DAB. DNA was counterstained with hematoxylin. Black, gray, and white arrowheads indicate positively stained mucinous acinar cells, proximal ductal epithelial cells, and salivary gland demilune structures, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. The effect of rhSLPI on SC secretion by HT-29 cells. (A) Concentration of SC in the culture supernatant. Cells were cultured in the presence or absence of rhSLPI (10 g/ml) and/or LPS (10 g/ml), and then the basal concentration of SC in the culture supernatant was measured by an ELISA. * indicates a significant difference. Mean ± SD (n = 3, p < 0.05). (B) pIgR protein expression in HT-29 cells cultured in the presence or absence of rhSLPI. Cells were cultured in the presence or absence of rhSLPI (10 g/ml) and then stained with an anti-pIgR antibody as described in Section 2.
our targeting constructs in the two clones by hybridizing XbaI or PstI digests with a probe located on TKpro +Neo. Successful targeting was expected to produce a 4.8 kb (XbaI) fragment or 1.8 kb and 1.0 kb (PstI) fragments. The two clones were positive for 4.8 kb (XbaI) and 1.8/1.0 kb (PstI) fragments (Fig. 3B). Next, we performed RT-PCR to determine whether SLPI mRNA expression was suppressed in these two clones. Expression of SLPI was observed in wild-type Ca9-22 cells, but was completely suppressed in SLPI-1 and -2 cells (Fig. 3C). We further investigated whether mRNA expression of pIgR was increased in SLPI cells. Real-time RT-PCR showed that pIgR mRNA expression in SLPI-1 and -2 cells was higher than that in wild-type Ca9-22 cells and was similar to that in HT-29 cells (Fig. 4A). Immunostaining and ELISA analyses also showed remarkable pIgR protein expression in SLPI cells and secretion of SC into the culture supernatant, respectively. Contrastively, they were scarcely detected in wild-type Ca9-22 cells and in their culture supernatant (Fig. 4B and Supplemental Fig. 1).
Supplementry material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.molimm.2015.07.021 3.4. Signaling pathways involved in the suppression of SC secretion and pIgR expression by SLPI The results of these experiments demonstrated that SLPI suppresses SC secretion and pIgR expression. Therefore, we proceeded to investigate the molecular mechanism responsible for SLPImediated suppression of SC secretion and pIgR expression. Previous reports suggested that the NF-B signaling pathway is involved in SLPI-mediated suppression of pIgR expression (Lentsch et al., 1999). Therefore, we first analyzed the level of NF-B activation in cells used in the aforementioned experiment using a reporter plasmid in which six repeats of NBE were bound to the luciferase gene (see Section 2). Luciferase activity was higher in SLPI cells than in wild-type Ca9-22 cells (Fig. 5A). Luciferase activity was significantly
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Fig. 3. Generation of SLPI cells. (A) Restriction maps of the SLPI locus, the gene disruption construct, and configuration of the targeted locus. The targeted construct is expected to disrupt exon 4. Relevant restriction enzyme sites shown are as follows: B, BamHI; N, NdeI; P, PstI; and X, XbaI. The position of the probe used for Southern hybridization is indicated. The new 4.8 kb XbaI and 1.0/1.8 kb PstI fragments hybridize with the probe if targeted integration of the construct occurs. (B) Restriction analysis of the targeted integration of the SLPI disruption construct. Genomic DNA from two SLPI clones (SLPI-1 and -2) was digested with XbaI or PstI, size-fractionated by 0.7% agarose gel electrophoresis, transferred to a nylon filter, and hybridized with the probe indicated in (A). (C) Suppression of SLPI expression from the transgene. Total RNA was isolated from the two SLPI clones (SLPI-1 and -2), and RT-PCR was performed. W.T.Ca9-22 indicates wild-type Ca9-22 cells.
Fig. 4. pIgR expression in SLPI cells. (A) mRNA expression of pIgR. mRNA expression was measured by real-time RT-PCR. * indicates significantly different from wild-type Ca9-22 cells (W.T.Ca9-22). HT-29 cells were used as a positive control. Mean ± SD (n = 3, p < 0.05). (B) Protein expression of pIgR. Cells were stained with an anti-pIgR antibody as described in Section 2.
lower in HT-29 cells treated with rhSLPI than in HT-29 cells not treated with rhSLPI (Fig. 5B). Next, we investigated whether these changes in luciferase activity were caused by changes in the level of NF-B expression. Western blot analysis found no difference in the level of NF-B protein expression between SLPI and wildtype Ca9-22 cells (Fig. 5C) or between HT-29 cells treated with and without rhSLPI (Fig. 5D). Similar results were obtained by RT-PCR; no differences were observed in the level of NF-B mRNA between SLPI and wild-type Ca9-22 cells or between HT-29 cells treated with and without rhSLPI (data not shown). Expression of IB, a component of the NF-B signaling pathway, was analyzed in the same cells and culture conditions. IB protein expression was lower in SLPI cells than in wild-type Ca9-22 cells and was higher in HT-29 cells treated with rhSLPI than in HT-29 cells not treated with rhSLPI (Fig. 5C and D). Meanwhile, there was no difference in the mRNA level of IB between these cells (Fig. 5E and F). 4. Discussion The localization of SLPI has been demonstrated in many types of tissues, including salivary glands, the gastrointestinal tract,
mammary glands, skin, respiratory organs, kidneys, pancreas, and reproductive organs, using immunohistochemistry and in situ hybridization (Bergenfeldt et al., 1996; Novak et al., 2007; Van Seuningen et al., 1995). SLPI is expressed in the stratum granulosum in normal skin, tracheal Clara cells (non-ciliated bronchiolar secretory cells), serous cells in the respiratory tract, serous acinar cells in the parotid gland and submandibular glands, uterine cervix mucous cells, and Paneth and goblet cells in the gastrointestinal tract (Bergenfeldt et al., 1996; Franken et al., 1989; Into et al., 2006). Similar to the previous reports, we detected SLPI expression in goblet cells in the gastrointestinal tract. In addition, we observed SLPI expression in mucus acinar cells in salivary glands. However, there was only a minimal amount of expression in serous acini. The discrepancies between our results and the previous studies may be due to the use of in situ hybridization to investigate mRNA expression in previous reports, whereas we used immunohistochemistry to detect proteins. However, differences were noted in the tissues that contained SLPI-expressing and pIgR-expressing cells. This suggests that SLPI expression suppresses that of pIgR and/or vice versa. Previous studies reported strong SLPI expression in lung squamous cell carcinoma-derived HS-24 cells, lung epithelium-derived A594
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Fig. 5. The NF-B signaling pathway in SLPI-mediated suppression of pIgR expression. (A and B) Transcriptional activity of NF-B in wild-type Ca9-22 cells (W.T.Ca9-22) and SLPI clones. Cells were transfected with the NF-B Luc or Control Luc plasmid, and then luciferase activity was measured. * indicates significantly different from wild-type Ca9-22 cells. Mean ± SD (n = 3, p < 0.05). (B) Transcriptional activity of NF-B in HT-29 cells. Cells transfected with the NF-B Luc or Control Luc plasmid were cultured in the presence or absence of rhSLPI (10 g/ml), and then luciferase activity was measured. * indicates significantly different from cells not treated with rhSLPI. Mean ± SD (n = 3, p < 0.05). (C) Protein expression of NF-B (p50, p52, and p60) and IB in wild-type Ca9-22 cells and SLPI clones. Western blot analysis was performed as described in Section 2. -actin was used as the internal control. (D) Protein expression of NF-B (p50, p52, and p60) and IB in HT-29 cells. Cells were cultured in the presence or absence of rhSLPI (10 g/ml), and then Western blot analysis was performed. -actin was used as the internal control. (E) mRNA expression of IB in wild-type Ca9-22 cells and SLPI clones. Total RNA was isolated from each type of cell, and then real-time RT-PCR was performed. Mean ± SD (n = 3). (F) mRNA expression of IB in HT-29 cells. Cells were cultured under the same conditions as described in (D), and then real-time RT-PCR was performed. Mean ± SD (n = 3).
cells, and gingival squamous cell carcinoma-derived Ca9-22 cells (Yang et al., 2014). pIgR was not expressed in these cells. Furthermore, SLPI is expressed at low levels or is not expressed in HT-29, Caco2, and T84 cells. This is in contrast to pIgR, which is expressed in all of these colon carcinoma cell lines (Si-Tahar et al., 2000). The present study found that LPS stimulation augmented the SC secretion level in HT-29 cell culture supernatant. However, the addition of rhSLPI suppressed the LPS-mediated increase in the level of secreted SC. The secretion level of SC was higher in SLPI Ca9-22 cells than in wild-type Ca9-22 cells (supplemental Fig. 1). These results demonstrate that SLPI suppresses SC secretion into the culture supernatant. A complex of pIgR bound to dIgA passes through the cytoplasm, and S-IgA is secreted in the lumen when part of the pIgR on the epithelial cell apical surface is cleaved by a serine protease. SLPI suppresses this cleavage of pIgR by inhibiting the activity of the serine protease (Pilette et al., 2003). Accordingly, suppression of pIgR cleavage by a serine protease may be one reason why the addition of rhSLPI decreased the amount of SC in the culture supernatant. Real-time RT-PCR data indicated that pIgR mRNA expression was strongly increased in SLPI Ca9-22 cells (approximately 30-fold in comparison to the level in wild-type Ca922 cells). Decreased protein expression of pIgR was demonstrated by a reduction in the number of HT-29 cells that were positively immunostained for pIgR following rhSPLI treatment. Thus, we infer that the main reason for the SLPI-mediated decrease in SC secretion into the culture supernatant is suppression of pIgR gene expression. SLPI suppresses NF-B activation by preventing IB degradation (Baeuerle, 1998; Bruno et al., 2011). Specifically, IB is phosphorylated by IB kinase, and then phosphorylated IB is degraded by the proteasome (Baeuerle, 1998; Bruno et al., 2011). This results in NF-B activation and increased pIgR expression (Bruno et al., 2011). Based on these reports, we hypothesize that suppression of pIgR expression by SLPI involves the NF-B signaling pathway through IB protein degradation, rather than inhibition of IB gene transcription. The luciferase assay performed using the NF-B reporter plasmid indicated that the transcriptional activity of NF-B protein was suppressed when SLPI was present. However, no changes in the mRNA or protein level of
NF-B were observed in these cells. These results suggest that post-translational modification (e.g., phosphorylation or binding of inhibitory factors) suppresses the transcriptional activity of NF-B protein. In support of this hypothesis, the level of IB protein, but not of mRNA, changed in parallel with the level of NF-B activity. pIgR is expressed in epithelial cells of various tissues, although certain epithelial cells do not express pIgR. The mechanism of suppression that leads to these differences was unclear. The results of this study demonstrate that pIgR expression was regulated in epithelial cells, at least in part, by SLPI through the NF-B signaling pathway. Acknowledgments This work was supported in part by JSPS KAKENHI (#24593066), grants from the Dental Research Center, and the Sato Fund of the Nihon University School of Dentistry. References Baeuerle, P.A., 1998. IkappaB-NF-kappaB structures: at the interface of inflammation control. Cell 95, 729–731. Bergenfeldt, M., Nyström, M., Bohe, M., Lindström, C., Polling, A., Ohlsson, K., 1996. Localization of immunoreactive secretory leukocyte protease inhibitor (SLPI) in intestinal mucosa. J. Gastroenterol. 31, 18–23. Brandtzaeg, P., Krajci, P., Lamm, M.E., Kaezel, C.S., 1994. Epihelial and hepatobiliary transport of polymeric Immunoglobulin. In: Ogra, P.L., Mestecky, J., Lamm, M.E., Strober, W., McGhee, J.R., Bienenstock, J. (Eds.), Handbook of Mucosal Immunology. Academic Press, San Diego, pp. 113–126. Bruno, M.E., Frantz, A.L., Rogier, E.W., Johansen, F.E., Kaetzel, C.S., 2011. Regulation of the polymeric immunoglobulin receptor by the classical and alternative NF-(B pathways in intestinal epithelial cells. Mucosal Immunol. 4, 468–478. Franken, C., Meijer, C.J., Dijkman, J.H., 1989. Tissue distribution of antileukoprotease and lysozyme in humans. J. Histochem. Cytochem. 37, 493–498. Fritz, H., 1988. Human mucus proteinase inhibitor (human MPI). Human seminal inhibitor I (HUSI-I), antileukoprotease (ALP), secretory leukocyte protease inhibitor (SLPI). Biol. Chem. Hoppe Seyler 369, 79–82. Fryksmark, U., Jannert, M., Ohlsson, K., Tegner, H., Wihl, J.A., 1989. Secretory leukocyte protease inhibitor in normal, allergic and virus induced nasal secretions. Rhinology 27, 97–103. Into, T., Inomata, M., Kanno, Y., Matsuyama, T., Machigashira, M., Izumi, Y., Imamura, T., Nakashima, M., Noguchi, T., Matsushita, K., 2006.
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