Suppression of tumor necrosis factor α–induced matrix metalloproteinase 9 production in human salivary gland acinar cells by cepharanthine occurs via down-regulation of nuclear factor κB: A possible therapeutic agent for preventing the destruction of the acinar structure in the salivary glands of Sjögren's syndrome patients

Authors


Abstract

Objective

Our previous results suggested that suppression of tumor necrosis factor α (TNFα)–induced matrix metalloproteinase 9 (MMP-9) could prevent the destruction of acinar tissue in the salivary glands of patients with Sjögren's syndrome (SS). The present study was undertaken to investigate the effect of cepharanthine on the suppression of TNFα-induced MMP-9 production in NS-SV-AC, an SV40-immortalized normal human acinar cell clone.

Methods

After pretreatment with or without cepharanthine, NS-SV-AC cells were treated with TNFα alone or with a combination of TNFα and cepharanthine. The expression of MMP-9 was then examined at the protein and messenger RNA levels. In addition, the effect of cepharanthine on the morphogenetic behavior of NS-SV-AC cells cultured on type IV collagen–coated dishes in the presence of TNFα was examined.

Results

Although TNFα induced the production of MMP-9 in NS-SV-AC cells, this production was greatly suppressed when cells were pretreated with cepharanthine, followed by treatment with both TNFα and cepharanthine. In addition, cepharanthine suppressed the TNFα-stimulated NF-κB activity by partly preventing the degradation of IκBα protein in NS-SV-AC cells. When NS-SV-AC cells were seeded on type IV collagen–coated dishes in the presence of both TNFα and plasmin, type IV collagen interaction with the cells was lost and the cells entered apoptosis. However, pretreatment with cepharanthine restored the aberrant in vitro morphogenesis of the NS-SV-AC cells.

Conclusion

These results may indicate a molecular mechanism by which cepharanthine is able to protect against the destruction of the acinar structure in salivary glands from patients with SS.

Sjögren's syndrome (SS), one of the most common rheumatic diseases (1), is characterized by the eventual total replacement of the acinar structure by marked infiltration of lymphocytes into the salivary and lacrimal glands (2). The pathogenesis of this selective and progressive destruction of the acinar structure in salivary glands is not yet fully understood. However, accumulated evidence indicates a close relationship between cytokine expression in salivary gland tissue and the development and progression of this disease (3, 4). Expression of messenger RNA (mRNA) for various cytokines, such as tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), IL-2, and interferon-γ (IFNγ), has been detected in the salivary glands of humans as well as experimental animals during the development of SS.

Establishment of the normal acinar structure of the salivary glands is fully dependent on the integrity of extracellular matrices, including the basement membrane (5). The basement membrane consists mainly of type IV collagen and laminin, and its synthesis and degradation are tightly regulated by proteolytic enzymes and their inhibitors. However, disruption of acinar cell–basement membrane interactions by excessive production of proteolytic enzymes, such as matrix metalloproteinases (MMPs), could lead to the disruption of the acinar tissue. Because cytokines, including TNFα and IL-1β, have been shown to stimulate the production of collagenases (6, 7), it is conceivable that the cytokines contribute to the destruction of the basement membrane, which in turn, leads to the disruption of the acinar structure of the salivary gland. Moreover, structural changes in the basement membrane of salivary glands and increased levels of latent and active MMP-9 in saliva have recently been demonstrated in SS patients (8, 9). Taken together, these observations support the previous finding that MMP-9 is implicated in the pathogenesis of SS (10).

The type IV collagenase MMP-9 is an important determinant of the degradation of the basement membrane (11). The promoter region of the MMP-9 gene has been found to contain binding sites for nuclear factor κB (NF-κB) (12), and production of MMP-9 has been reported to be stimulated by TNFα (13). NF-κB, one of the major components induced by TNFα, is considered to be an important transcription factor in the regulation of the MMP-9 gene. Under nonstimulated conditions, NF-κB is retained in the cytoplasm by its inhibitory protein, IκBα. The binding of IκBα to NF-κB masks nuclear localization signals in NF-κB and prevents its translocation to the nucleus (14). Conversely, upon stimulation by external stimuli, such as TNFα, IκBα undergoes phosphorylation and is subsequently degraded through the ubiquitin/proteasome pathway. Degradation of IκBα leads to the nuclear translocation of NF-κB, which then stimulates the expression of its target genes (15).

Recently, we demonstrated that although NS-SV-AC cells (an SV40-immortalized normal human acinar cell clone) produced a large amount of MMP-9 in response to TNFα, a super-repressor form of IκBα (srIκBα) complementary DNA (cDNA)–transfected NS-SV-AC clone lost its responsiveness to TNFα in terms of MMP-9 production (16). In addition, suppression of TNFα-induced MMP-9 production restored the normal in vitro morphogenesis of acinar cells even when they were cultured on type IV collagen–coated plates in the presence of both TNFα and plasmin. Moreover, an immunohistochemical study using salivary gland tissue from SS patients indicated that acinar cells adjacent to the lymphocytic infiltrate exhibited enhanced expression of both MMP-9 and NF-κB compared with those distant from infiltrated lymphocytes as well as those in normal salivary glands (16, 17). It therefore seems likely that inhibition of TNFα-induced MMP-9 production in acinar cells may lead to the restored integrity of the acinar structure in SS salivary glands.

Based on these considerations, we postulated a working hypothesis concerning clinical therapy for patients with SS. We thought that the identification of drugs that suppress the TNFα-induced production of MMP-9 would be a promising strategy.

Cepharanthine, a bisbenzylisoquinoline (biscoclaurine) alkaloid extracted from Stephania cephalantha Hayata, has been used widely for the treatment of patients with leukopenia (18), nasal allergy (19), and venomous snake bites (20). Although the exact mechanism has not been elucidated, cepharanthine exerts immunomodulatory effects by enhancing the cytotoxic effect of natural killer cells and macrophages (21, 22), suggesting that it plays a possible role in the regulation of signaling pathways of cytokines.

In the present study, we examined the effect of cepharanthine on the TNFα-induced MMP-9 production in NS-SV-AC cells. We found that TNFα-induced MMP-9 production was effectively suppressed by cepharanthine through the down-regulation of NF-κB activity.

MATERIALS AND METHODS

Cells and media.

The characteristics of NS-SV-AC cells have been described in detail elsewhere (23). This cell clone was cultured at 37°C in serum-free keratinocyte medium (Gibco BRL, Grand Island, NY) in an incubator with an atmosphere containing 5% CO2.

Growth assay.

Cells (1 × 104/well) were grown in 96-well plates (Falcon, Oxnard, CA) in serum-free keratinocyte medium in the presence of cepharanthine (Kaken Syouyaku, Tokyo, Japan) at concentrations of 0, 5, 10, and 20 μg/ml. After the appropriate incubation periods, 10 μl of a 5-mg/ml preparation of MTT was added to each well and incubation was continued for 4 hours. The absorbance was measured with a Titertek spectrophotometer (Flow, Irvine, UK) at 570 nm with a reference wavelength of 630 nm. All assays were run in triplicate.

Enzyme-linked immunosorbent assay (ELISA) for TNFα.

For quantitative determination of TNFα produced by cepharanthine-treated NS-SV-AC cells, a microtiter-based sandwich ELISA was used. A commercially available TNFα assay kit was purchased from Genzyme Diagnostics (Cambridge, MA). Briefly, NS-SV-AC cells were treated with 10 μg/ml of cepharanthine for 4 days, and conditioned medium was concentrated by the method previously described (24). Biotinylated anti-human TNFα antibody and 100 μg of total protein from conditioned medium were added to each well of a microtiter plate coated with monoclonal anti-human TNFα antibody. Plates were then incubated for 1 hour at 37°C.

After 5 washes, 100 μl of working substrate consisting of tetramethylbenzidine and hydrogen peroxide was added, and the plates were incubated for another 10 minutes at room temperature. Optimal absorbance was then read at 450 nm using a Titertek spectrophotometer. All assays were performed in triplicate.

Substrate gel analysis.

NS-SV-AC cells were pretreated for 24 hours with or without cepharanthine (10 μg/ml) and then treated for 24 hours with either TNFα (10 ng/ml) or a combination of TNFα (10 ng/ml) and cepharanthine (10 μg/ml). MMP-9 activity was assayed according to the method described previously (25). Aliquots of conditioned medium (20 μg) were mixed with sample buffer without β-mercaptoethanol, applied directly to 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel containing 1 mg/ml of gelatin (Wako, Osaka, Japan), and electrophoresed.

After removal of SDS from the gel by incubation in 2.5% Triton X-100 for 30 minutes, the gel was incubated at 37°C for 14 hours in reaction buffer consisting of 50 mM Tris HCl, pH 7.6, 0.2M NaCl, 5 mM CaCl2, and 0.02% Brij-35. MMP-9 activity was identified by its ability to clear the substrate at its characteristic molecular weight, and was visualized after staining with Coomassie blue R250.

Preparation of nuclear and cytosolic extracts.

Cells were seeded on 100-mm plastic petri dishes (Falcon). Twenty-four hours after seeding, cells were pretreated with or without cepharanthine (10 μg/ml) for 24 hours, and nuclear extracts were obtained after treatment with either TNFα (10 ng/ml) alone or a combination of TNFα and cepharanthine according to previously described methods (26, 27). Cells were washed twice with ice-cold phosphate buffered saline, resuspended for 15 minutes in 400 μl of ice-cold lysis buffer, consisting of 10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT), 0.5 mg/ml benzamidine, and 2 mg/ml aprotinin. Nonidet P-40 was added to achieve a final concentration of 0.3%, and the lysates were vortexed before being pelleted in a microfuge. The supernatants from this centrifugation were designated cytosolic extracts.

Each nuclear pellet was resuspended in 50 μl of extraction buffer, consisting of 10 mM HEPES, pH 7.9, 400 mM NaCl, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, and 2 mg/ml of aprotinin, then placed on ice for 30 minutes. The nuclear extracts were pelleted, and the supernatants from this step were designated nuclear extracts.

Oligonucleotide labeling and electrophoretic mobility shift assay (EMSA).

The probe consisted of NF-κB–specific double-stranded oligonucleotides with the sequence 5′-AGTTGAGGGGACTTTCCCAGGC-3′ containing the κB site from the κ light chain enhancer in B cells. Oligonucleotides were end-labeled with γ32P-ATP with the use of polynucleotide kinase, and unincorporated γ32P-ATP was removed with Sephadex G-25–packed spin columns (Pharmacia, Uppsala, Sweden).

EMSA was carried out as previously described (27). Briefly, 1 μg of nuclear extract was mixed with the labeled probes in a 20-μl volume in buffer (10 mM HEPES, pH 7.9, 50 mM KCl, 0.2 mM EDTA, 2.5 mM DTT, 10% glycerol, and 0.05% Nonidet P-40). The specificity of the complex was analyzed by incubation with an excess of unlabeled competitor oligonucleotides (100-fold molar excess of labeled probe). Samples were run on 7.5% polyacrylamide gels. Next, gels were dried at 80°C for 2 hours then exposed to Kodak X-Omat AR-5 film (Eastman Kodak, Rochester, NY) at −70°C.

Western blot analysis of MMP-9 and IκBα.

Secretion of MMP-9 from treated and untreated cells and the contents of IκBα protein in cytosolic extracts obtained from treated and untreated cells were examined by Western blot analysis. Conditioned medium and cytosolic extract containing 20 μg of protein each were subjected to electrophoresis in 15% SDS–polyacrylamide gel electrophoresis, then transferred to a nitrocellulose membrane. The membranes were blocked with 3% bovine serum albumin and incubated with an anti-human MMP-9 antibody (Oncogene Research Products, Cambridge, MA) or with an antibody specific for IκBα (Rockland, Gilbertsville, PA). After intervening rinses with phosphate buffered saline, the antibody was detected using a chemiluminescence Western blotting kit (Amersham, Tokyo, Japan) according to the manufacturer's instructions.

RNA isolation and reverse transcriptase–polymerase chain reaction (RT-PCR).

Cells were pretreated for 24 hours with or without cepharanthine (10 μg/ml) and then were treated with either TNFα (10 ng/ml) alone or a combination of TNFα and cepharanthine. Total cellular RNA was isolated at 0 hours and 6 hours after treatment, using the method described by Sambrook et al (28). Complementary DNA was synthesized from 10 μg of total RNA using the Universal RiboClone cDNA Synthesis system (Promega, Madison, WI). The following sense and antisense primers, respectively, were used: for MMP-9, 5′-GGTCCCCCACTGCTGGCCCTTCTACGGCC-3′ and 5′-GCCCACCTCCACTCCTCCCTTTCCTCCAGA-3′ (29); for IκBα, 5′-CGGAATTCCAGGCGGCCGAGCGCCCC-3′ and 5′-GGGGTACCTCATAACGTCAGACGCTG-3′ (27); and for GAPDH, 5′-ACGCATTTGGCTGTATTGGG-3′ and 5′-TGATTTTGGAGGGATCTCGC-3′ (30).

The PCR reactions were conducted in a DNA Thermal Cycler TP-3000 (Takara, Otsu, Japan). After 5 minutes of denaturation at 94°C, 35 cycles of PCR were performed (94°C for 1 minute, 68°C for 1 minute, and 72°C for 1 minute), followed by a final 4-minute extension at 70°C. To visualize the PCR products, the samples were subjected to electrophoresis on a 1% agarose gel, followed by staining with ethidium bromide.

Chloramphenicol acetyltransferase (CAT) assay.

The CAT assay was performed using a transient transfection system. A total of 5 × 104 cells were plated in 60-mm plastic petri dishes (Falcon) and pretreated with or without cepharanthine (10 μg/ml) for 24 hours before transfection. Five micrograms of the 5′-flanking region of the MMP-9 gene (54 to −670) linked to the CAT gene as a reporter (−670-CAT) (kindly provided by Dr. Motoharu Seiki, Tokyo University, Tokyo, Japan) was transfected by using a Superfect reagent (Qiagen, Hilden, Germany). At 8 hours after transfection, the culture medium was changed to medium containing either TNFα (10 ng/ml) alone or a combination of TNFα and cepharanthine. Cell lysate was prepared at 12 hours after the change of medium, and CAT activity was determined with a CAT ELISA kit (Boehringer Mannheim, Mannheim, Germany). The assay was performed at least 3 times.

Morphologic assessment of cepharanthine-pretreated or untreated cells grown on type IV collagen in the presence and absence of TNFα and plasmin.

Cells were seeded on 35-mm tissue culture dishes that had been precoated with mouse type IV collagen (Becton Dickinson Labware, Bedford, MA) at a density of 5 × 105/dish. Twenty-four hours after seeding, cells were pretreated with or without cepharanthine (10 μg/ml) for 24 hours. Cells were then treated either with TNFα (50 ng/ml) and plasmin (8 μg/ml; Sigma, St. Louis, MO) (31) or with TNFα, plasmin, and cepharanthine for 96 hours as previously described (16). To monitor morphologic changes, cultures were observed every 24 hours under a phase-contrast microscope (Nikon ELWD 0.3; Nikon, Tokyo, Japan) and photographed with Nikon optics.

RESULTS

Effects of cepharanthine on cell growth. The growth kinetics of NS-SV-AC cells treated with various concentrations of cepharanthine was investigated by MTT assay for up to 4 days. No remarkable cytotoxicity was observed when NS-SV-AC cells were treated with 5 μg/ml or 10 μg/ml of cepharanthine. However, a relatively high dose of cepharanthine (20 μg/ml) significantly inhibited the growth of cells (data not shown). Thus, we selected a concentration of 10 μg/ml for the following experiments.

Effects of cepharanthine on the production of TNFα. Since TNFα stimulates the expression of MMP-9, we examined by ELISA the effect of cepharanthine on the production of TNFα by NS-SV-AC cells. We found that cepharanthine had no effect on the production of TNFα by NS-SV-AC cells (data not shown).

Suppression of MMP-9 activity by cepharanthine. As shown in Figure 1A, clearance of the gelatin substrate at a molecular weight of 92 kd was greatly enhanced in TNFα-treated NS-SV-AC cells (lane 2). However, pretreatment of NS-SV-AC cells with cepharanthine for 24 hours suppressed the activity of TNFα-induced MMP-9 (lane 3).

Figure 1.

Enzymatic activity of matrix metalloproteinase 9 (MMP-9) and amount of MMP-9 protein in conditioned medium from NS-SV-AC acinar cells treated for 24 hours without (basal level; lane 1) or with tumor necrosis factor α (TNFα; 10 ng/ml) alone (lane 2) or with TNFα plus cepharanthine (10 μg/ml) following a 24-hour pretreatment with cepharanthine (lane 3). A, Zymographic analysis revealed that the enzymatic activity of 92-kd MMP-9 was greatly enhanced in TNFα-treated cells compared with untreated cells, whereas pretreatment with cepharanthine followed by TNFα plus cepharanthine led to a drastic reduction of MMP-9 activity. B, Similarly, Western blot analysis revealed that although MMP-9 expression was greatly enhanced by TNFα treatment, pretreatment with cepharanthine significantly suppressed the expression of TNFα-induced MMP-9.

To further confirm the cepharanthine suppression of TNFα-induced MMP-9 production, conditioned medium was subjected to Western blot analysis. As can be seen in Figure 1B, MMP-9 expression in NS-SV-AC cells was largely augmented by TNFα treatment (lane 2). Consistent with the results of gelatin zymography, cepharanthine pretreatment of the cells prevented the ability of TNFα to stimulate the production of MMP-9 (lane 3). The enzymatic activity and the production of MMP-9 were not detectable at basal levels (lane 1, Figures 1A and B).

Inhibition of MMP-9 mRNA expression and MMP-9 gene promoter activity by cepharanthine. Figure 2 shows the expression of MMP-9 mRNA in NS-SV-AC cells pretreated with or without cepharanthine, followed by treatment for 6 hours with TNFα alone or a combination of cepharanthine and TNFα. The NS-SV-AC cells demonstrated a significant increase in the expression of MMP-9 mRNA (756 bp) in response to TNFα (lane 2). Although MMP-9 mRNA expression was not inhibited by simultaneous treatment with cepharanthine and TNFα (lane 3), cepharanthine pretreatment of the NS-SV-AC cells for 24 hours suppressed the expression of MMP-9 mRNA even after simultaneous treatment with cepharanthine and TNFα (lane 4). Consistent with the results obtained by the analysis at the protein level, MMP-9 mRNA expression was also not detected at the basal level (lane 1).

Figure 2.

Reverse transcriptase–polymerase chain reaction (RT-PCR) for the expression of matrix metalloproteinase 9 (MMP-9) mRNA in NS-SV-AC acinar cells treated for 6 hours without (basal level; lane 1) or with tumor necrosis factor α (TNFα; 10 ng/ml) alone (lane 2), with a combination of TNFα plus cepharanthine (10 μg/ml) (lane 3), or with a combination of TNFα plus cepharanthine following a 24-hour pretreatment with cepharanthine (lane 4). Treatment with TNFα alone and treatment with TNFα plus cepharanthine significantly induced MMP-9 mRNA expression. However, pretreatment with cepharanthine followed by treatment with TNFα plus cepharanthine almost completely suppressed the expression of MMP-9 mRNA. The mode of MMP-9 mRNA expression (756 bp) was similar to that detected by gelatin zymography and Western blot analysis.

Thus, RT-PCR analysis confirmed that at the mRNA level, cepharanthine can inhibit TNFα-induced production of MMP-9 protein in NS-SV-AC cells. Equal loading of RNA samples was demonstrated for the housekeeping gene GAPDH in these RT-PCR experiments.

The 5′-flanking region of the MMP-9 gene (54 to −670) was linked to the CAT gene as a reporter, and cepharanthine suppression of TNFα-induced MMP-9 gene promoter activity was monitored by determining the enzymatic activity expressed in the transiently transfected NS-SV-AC cells. As shown in Figure 3, basal promoter activity (control) was not detected in NS-SV-AC cells. CAT activity was clearly induced in these cells by treatment with TNFα alone as well as with TNFα and cepharanthine, although pretreatment with cepharanthine for 24 hours, followed by simultaneous treatment with TNFα and cepharanthine, resulted in a significant reduction of CAT activity. Thus, it became evident that cepharanthine suppresses the production of MMP-9 protein by inhibiting the TNFα-induced MMP-9 gene promoter activity.

Figure 3.

Transcriptional activity of the matrix metalloproteinase 9 (MMP-9) gene promoter in NS-SV-AC acinar cells, as determined by measurement of chloramphenicol acetyltransferase (CAT) activity. The reporter plasmid −670-CAT (5 μg) was transfected into the cells, and the cells were treated for 12 hours without (control) or with tumor necrosis factor α (TNFα; 10 ng/ml) alone, with a combination of TNFα plus cepharanthine (Cepha.; 10 μg/ml), or with a combination of TNFα plus cepharanthine following a 24-hour pretreatment with cepharanthine. No basal promoter activity (control) was detected. Consistent with the results obtained by gelatin zymography, Western blot, and RT-PCR analyses, treatment with TNFα alone or with a combination of TNFα plus cepharanthine significantly augmented CAT activity. However, pretreatment with cepharanthine, followed by treatment with TNFα plus cepharanthine caused a drastic reduction of CAT activity. Results are representative of 3 experiments. OD = optical density.

Inhibition of TNFα-induced NF-κB activation by cepharanthine. We previously showed that NS-SV-AC cells constitutively express active NF-κB at the basal level and that p65/p50 heterodimers and p50/p50 homodimers are components of NF-κB (32). In the present study, we examined whether cepharanthine treatment can suppress TNFα-induced NF-κB activity in these cells.

As shown in Figure 4A, when NS-SV-AC cells were treated with TNFα alone, NF-κB DNA binding activity was significantly enhanced, whereas cepharanthine pretreatment followed by treatment with TNFα plus cepharanthine efficiently suppressed the TNFα-induced NF-κB activity (Figure 4B). Accordingly, it became evident that cepharanthine can suppress NF-κB activity induced by TNFα in NS-SV-AC cells. The specific binding of NF-κB to DNA could be abrogated with an excess of unlabeled probe, indicating that this result actually demonstrated the NF-κB activity contained in cells.

Figure 4.

Electrophoretic mobility shift assay analyzing the suppressive effects of cepharanthine (10 μg/ml) on nuclear factor κB (NF-κB) activity in tumor necrosis factor α (TNFα; 10 ng/ml)–treated NS-SV-AC acinar cells. A, Cells were treated with TNFα for the indicated times. TNFα treatment caused an enhancement of NF-κB DNA binding activity. B, Following a 24-hour pretreatment with cepharanthine, cells were treated with TNFα plus cepharanthine for the indicated times. TNFα plus cepharanthine treatment caused a significant suppression of NF-κB activity, indicating that cepharanthine inhibits TNFα-induced NF-κB activity. The specificity of the complex was analyzed by incubation with a 100-fold excess of unlabeled κB oligonucleotide (Com).

Partial prevention of TNFα-induced degradation of IκBα protein by cepharanthine. The suppressed NF-κB DNA binding could be due to blocking of IκBα protein or to increased steady-state mRNA levels in cepharanthine-treated NS-SV-AC cells. To determine which of these two conditions occurred, we examined the expression of IκBα protein and mRNA in cepharanthine-treated cells.

Complete degradation of IκBα protein was observed at 30 minutes after treatment of NS-SV-AC cells with TNFα alone (Figure 5A). After 60 minutes of TNFα treatment, newly synthesized IκBα was detected in cytosols, indicating early NF-κB activation. Thus, IκBα protein underwent a cycle of proteolysis/resynthesis during 60 minutes of TNFα treatment of NS-SV-AC cells. However, cepharanthine pretreatment of NS-SV-AC cells followed by treatment with TNFα plus cepharanthine partially prevented the degradation of IκBα protein (Figure 5B).

Figure 5.

Effects of cepharanthine on the expression of inhibitor of nuclear factor κBα (IκBα) protein and mRNA in NS-SV-AC acinar cells. A, Treatment with tumor necrosis factor α (TNFα; 10 ng/ml) resulted in the complete disappearance of IκBα protein at 30 minutes after treatment, as shown by Western blotting. B, A 24-hour pretreatment with cepharanthine (10 μg/ml) prior to treatment with TNFα plus cepharanthine resulted in partial prevention of the IκBα protein degradation, as shown by Western blotting. C, Pretreatment with cepharanthine for 24 hours prior to treatment with TNFα alone resulted in stable expression of IκBα mRNA (951 bp), as shown by reverse transcriptase–polymerase chain reaction (RT-PCR) analysis of cDNA from the TNFα-treated cells. RT-PCR was performed using sense and antisense primers specific for IκBα and GAPDH.

RT-PCR analysis, on the other hand, revealed that NS-SV-AC cells did not show any change in the expression of IκBα mRNA in response to cepharanthine pretreatment (Figure 5C). These results therefore suggest that cepharanthine inhibited NF-κB activity in NS-SV-AC cells through partial blocking of the TNFα-induced degradation of IκBα protein.

Morphogenetic profiles of cells grown on type IV collagen. Twenty-four hours after seeding on type IV collagen, NS-SV-AC cells showed the rounded or polygonal-shaped morphology characteristic of the acinar cell phenotype (data not shown) (23). NS-SV-AC cells were cultured for 96 hours with type IV collagen as described in Materials and Methods and examined with phase-contrast microscopy. Following the addition of TNFα (50 ng/ml) and plasmin to the medium, NS-SV-AC cells revealed a loss of tight cell–substrate interactions (Figure 6B) compared with controls (Figure 6A). As shown in our previous study (16), floating cells obtained by treatment with both TNFα and plasmin for 96 hours were confirmed to be apoptotic on examination of Hoechst 33258–stained NS-SV-AC nuclei. In contrast, the in vitro morphogenesis of cepharanthine-pretreated NS-SV-AC cells was not affected by treatment with the combination of TNFα, plasmin, and cepharanthine, as observed with phase-contrast microscopy (Figure 6C).

Figure 6.

In vitro morphogenetic behavior of NS-SV-AC acinar cells cultured on type IV collagen–coated plates. Cells were cultured for 96 hours in serum-free keratinocyte medium without (control) or with tumor necrosis factor α (TNFα; 50 ng/ml) plus plasmin (8 μg/ml) or with TNFα, plasmin, and cepharanthine (10 μg/ml) following a 24-hour pretreatment with cepharanthine. Plates were then examined by phase-contrast microscopy. A, The morphologic appearance of control cells was a round or polygonal shape, with tight attachment to the substrate. B, Treatment with TNFα plus plasmin resulted in a loss of tight cell–substrate interactions. C, Cepharanthine pretreatment prior to TNFα, plasmin, and cepharanthine treatment resulted in a morphologic appearance similar to that of the control cells. (Original magnification × 100.)

DISCUSSION

We have previously shown that the expression of both MMP-9 and p65, one of the components of NF-κB, is up-regulated in SS acinar cells located near infiltrated lymphocytes, where destruction of the acinar structure seems to occur, compared with both the expression in cells distant from the infiltrated lymphocytes and the expression in cells from normal salivary glands (16). We also found evidence that although acinar (NS-SV-AC) cells entered apoptosis when cultured on type IV collagen–coated dishes in the presence of TNFα and plasmin, suppression of TNFα-induced MMP-9 production by the introduction of srIκBα cDNA corrected the aberrant in vitro morphogenesis of these cells (16). The importance of interactions between the cell and the basement membrane to the survival of cells has also been reported in normal endothelial and prostate cancer cells (33, 34). Our previous results therefore indicate that MMP-9 would be one of the causal molecules in the destruction of the acinar structure in the salivary glands of SS patients and that suppression of MMP-9 activity in acinar cells may provide a therapeutic strategy for clinical improvement in SS salivary glands.

In the present study, we found that pretreatment of acinar cells with cepharanthine enables them to survive on the type IV collagen substrate, even after treatment with TNFα and cepharanthine. We selected cepharanthine for our studies of the suppression of TNFα-induced NF-κB activity because cepharanthine has been shown to function as an anti-allergic agent and to reverse the anticancer drug resistance phenotype of cancer cells (19, 35, 36). The expression of proinflammatory cytokines, such as TNFα, IL-1, IL-6, and IL-8, is positively regulated by NF-κB activity (37, 38), and suppression of NF-κB in cancer cells augments sensitivity to anticancer drug–induced apoptosis (39–41). Based on the finding that the MMP-9 gene has a possible binding site for NF-κB in its promoter region (12), we speculated that cepharanthine could inhibit TNFα-induced MMP-9 production by suppressing NF-κB activity in human salivary acinar cells. The findings of our study showed that cepharanthine has the ability to inhibit TNFα-induced MMP-9 production via suppression of TNFα-stimulated NF-κB activity.

Although pretreatment of NS-SV-AC cells with cepharanthine followed by treatment with TNFα plus cepharanthine almost completely inhibited both the transactivation of the MMP-9 gene promoter activity and the production of MMP-9, it seemed likely that the degree of blocking of IκBα degradation by cepharanthine treatment did not necessarily parallel the effects observed in the MMP-9 experiments. We do not currently have a precise explanation for this discrepancy, although Han and Brasier (42) demonstrated that while IκBα appears to be the main regulator of TNFα-induced NF-κB activation in most of the cells, another member of the IαB protein family, IκBβ, also plays a key role in the NF-κB activation by TNFα. This indicates that several IκB proteins, such as IκBα, IκBβ, and IκBε (43), may cooperatively participate in the cepharanthine-mediated prevention of protein degradation.

The novel result among our findings is that cepharanthine suppresses TNFα-induced MMP-9 production through inhibition of NF-κB activity in NS-SV-AC cells. Although we have not yet identified in detail the mechanism involved in the cepharanthine-induced inhibition of NF-κB activity, except to discern that blocking of the degradation of IκBα protein is involved, several possibilities can be suggested. More specifically, a decrease in the activity of IκB kinase or in the ubiquitination or proteasome-mediated degradation of IκBα, IκBβ, or IκBε could explain the cepharanthine-mediated inhibition of TNFα-induced NF-κB activation (44). Further investigation is necessary to clarify the mechanism by which cepharanthine inhibits the TNFα-stimulated degradation of IκB.

An unexpected result from the EMSA study was that although NS-SV-AC cells expressed relatively high levels of NF-κB activity at the basal level, the transcriptional activity of the MMP-9 gene and the expression of MMP-9 protein were extremely low. Although in this study, we did not identify the mechanisms involved in the suppression of MMP-9 expression in NS-SV-AC cells at the basal level, it has recently been shown that transcription of IL-8 and IFNβ genes, both of which are reported to have a κB motif in their promoter regions (45, 46), is inhibited at the basal level via binding of NF-κB–repressing factor (NRF) to their negative regulatory element (NRE). That is, under unstimulated conditions, transcription of IL-8 and IFNβ genes is constitutively repressed by NRF in their promoter regions, which partly overlap with the NF-κB response element. Conversely, upon stimulation by external stimuli, by binding to the NRE of the IL-8 and IFNβ promoters, NRF plays an additional role and acts as a coactivator of cytokine-induced IL-8 and IFNβ gene expression (47, 48). Thus, since we also have shown that MMP-9 expression is significantly induced by TNFα in NS-SV-AC cells and that NRF has been detected in NS-SV-AC cells (Azuma M et al: unpublished data), it seems likely that NRF may actually function in our in vitro system.

Similar to cepharanthine, the antiinflammatory drugs sodium salicylate and aspirin have been shown to inhibit the activation of NF-κB by preventing the degradation of IκBα protein (49). The effects of salicylate and aspirin on the suppression of NF-κB activity, however, were observed at suprapharmacologic concentrations (>5 mM). In contrast, we have found that cepharanthine is effective at a 2,900-fold lower concentration (10 μg/ml, or ∼1.7 μM), which suggests that cepharanthine is a potent inhibitor of MMP-9 activity. Selective gelatinase inhibitors could also suppress TNFα-induced MMP-9 activity (50) and may prove useful for the prevention of the destruction of acinar structure. However, in addition to the suppressive effect on TNFα-induced MMP-9 production, cepharanthine has the ability to inhibit the secretion of TNFα from infiltrated lymphocytes (51). Cepharanthine would therefore be effective in terms of both inhibiting the release of TNFα from lymphocytes and suppressing the TNFα-induced MMP-9 production. Furthermore, it is recognized that cepharanthine is a safe agent even if administered for a long period of time, and it is used widely in Japan.

Our results suggest that cepharanthine would be a promising agent for use in the treatment of salivary gland involvement in patients with SS. It may also have applications in various other diseases in which NF-κB activation has been shown to mediate pathogenesis, including arthritis and oral lichen planus. These possibilities warrant further investigation.

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