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RESEARCH REPORT |
1 Departments of Periodontology and Oral Biology,
2 Medicine, and
3 Biochemistry, GI Section, X-510, 650 Albany Street, Boston University Medical Center, Boston, MA 02118, USA;
* corresponding author, goffner{at}bu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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), or interferon-gamma (IFN-
), or combinations of these, real-time PCR demonstrated that MUC1 mRNA increased 1.4- to 3.2-fold. Interestingly, a significant increase in levels of MUC1 protein was also observed. While no effect was observed when KB cells were incubated with LPS from Porphyromonas gingivalis, infection of KB monolayers with this oral pathogen caused a 2.85-fold increase in MUC1 transcript levels. These results suggest that increased MUC1 synthesis may be a key element in the host response to infection with oral pathogens.
KEY WORDS: mucin • gene expression • real-time PCR
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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There is increasing evidence that exposure of epithelial cells in the respiratory epithelium to bacteria or their secreted products leads to increased transcription of genes for the secreted mucins MUC2 and MUC5AC, thus enhancing the epithelial-protective barrier (Dohrman et al., 1998; Li et al., 1998a,b). These mucin genes, which are not expressed in oral tissues, are also up-regulated by pro-inflammatory mediators such as interleukin-1 beta and TNF alpha (Smirnova et al, 2000; Song et al., 2003). In the oral cavity, chronic infection by oral micro-organisms such as Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Candida albicans leads to the production of these mediators as well as IL-6 and IFN- (Kesavalu et al., 2002; Rouabhia et al., 2002). Pro-inflammatory cytokines have been shown to increase MUC1 expression in several epithelial and hematopoietic cell lines (Clark et al., 1994; Reddy et al., 2003), as well as in normal and malignant breast epithelial cells (Lagow and Carson, 2002). Taken together, these results suggested the possibility that up-regulation of MUC1 could be a component of the host response to bacterial infection in the oral cavity.
Nothing is known about the regulation of MUC1 expression in oral epithelial cells, and the present investigation used real-time PCR to quantify MUC1 transcript levels in KB cells in response to pro-inflammatory mediators, P. gingivalis and P. gingivalis-derived LPS.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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(20 ng/mL), or INF- (10 ng/mL), alone and in combination, for 4–8 hrs. All cytokines were purchased from Peprotech (Rocky Hill, NJ, USA). P. gingivalis LPS was kindly provided by Dr. Salomon Amar (Boston University Goldman School of Dental Medicine) and was used at a concentration of 10 µg/mL. P. gingivalis strain 381 cells were obtained from Dr. Caroline Genco (Department of Medicine, Boston University Medical Center) and cultured as described previously (Nassar et al., 2002). KB monolayers were infected with P. gingivalis cells at a Multiplicity of Infection (MOI) = 100 for 4 hrs in an atmosphere of 5% CO2 at 37°C.
RNA Isolation and cDNA Synthesis
RNA was isolated from control and cytokine-treated KB cells with the use of Tripure reagent (Roche, Indianapolis, IN, USA). First-strand cDNA was synthesized in 20-µL reactions containing 5 µg RNA, 1 µL 10 mM dNTP (ProMega, Madison, WI, USA), 100 pmol random hexamer primers (Amersham, Chicago, IL, USA), 4 µL 5x first-strand buffer, 1 µL 0.1 M DTT, 0.5 µL RNAse inhibitor (ProMega), and 2 µL M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). Reaction mixtures were incubated for 10 min at 25°C followed by incubation for 50 min at 37°C.
Northern Blot Analysis of MUC1 mRNA Expression
RNA from control and treated cells was electrophoresed on 1% agarose denaturing gels and transferred to nylon membranes (Hybond N+, Amersham) via capillary blotting for 16 hrs in 20 x SSC. Blots were hybridized overnight with a 32P-labeled 232-bp MUC1 tandem-repeat probe (Liu et al., 2002) at 42°C in 50% formamide, 5 x SSC, 10 x Denhardt’s solution, 2% SDS, 10% dextran sulfate, and 100 µg/mL of sheared salmon sperm DNA. Blots were washed twice in 2 x SSC, 0.1% SDS for 15 min at room temperature, followed by a final wash with 0.1 x SSC, 0.1% SDS at 65°C and were exposed to Kodak X-Omat film (Eastman Kodak, Rochester, NY, USA) at -80°C.
Cell Lysate Preparation and Western Blots
Cells underwent lysis on ice for 45 min on a rocker platform in RIPA lysis buffer [1 x PBS, 1% Nonidet P-40 (NP-40), 0.1% SDS, 0.5% sodium deoxycholate, and complete mini-protease inhibitor cocktail tablets (Roche)], and cell lysates were passed through a 21-gauge needle to shear DNA. After centrifugation at 10,000 x g for 10 min at 4°C, the soluble protein fraction was recovered and total protein concentration was determined by BCA protein assay (Pierce, Rockford, IL, USA). Cell lysates were examined by SDS-PAGE on 6% polyacrylamide gels or on Tricine gels (Schagger and von Jagow, 1987), and proteins were transferred to nitrocellulose membrane. Membranes were blocked for 1 hr at room temperature in 5% non-fat dry milk dissolved in phosphate-buffered saline (137 mM NaCl, 12 mM NaH2PO4, 2 mM KH2PO4, and 0.2 mM KCl) containing 0.05% Tween 20 (PBST) and incubated with MUC1-specific primary antibodies for 1 hr at room temperature. The MUC1-specific antibodies were mouse monoclonal antibody VU-4H5 (1:500 dilution; NeoMarkers, Fremont, CA, USA), which recognizes a peptide epitope in the MUC1 tandem repeat, and hamster monoclonal antibody CT2 (1:300 dilution; kindly provided by Dr. Sandra Gendler, Mayo Clinic, Scottsdale, AZ, USA), which recognizes the cytoplasmic tail. After being washed 4 times for 5 min with PBST, blots were incubated with HRP-conjugated species-specific secondary antibodies, and immunoreactive bands were visualized by enhanced chemiluminescence (Perkin Elmer, Boston, MA, USA).
Real-time PCR
We used real-time PCR to quantify levels of MUC1 mRNA in control and treated cells relative to levels of actin mRNA. The forward and reverse primers used to generate a 207-bp MUC1 amplicon were: 5' CGCCGAAAGAACTACGGGCAGCTG and 5' CAAGTTGGCAGAAGTGGCTGCCAC, respectively. The forward and reverse primers used to generate a 231-bp actin amplicon were: 5' GCGGGAAATCGTGCGTGACATT and 5' GATGGAGTTGAAGGTAGTTTCGTG, respectively. Primers were designed to span intron/exon boundaries to verify that PCR products are amplified from cDNA.
Real-time PCR was performed on a DNA Engine Opticon System (MJ Research, Waltham, MA, USA). All 25-µL reactions contained cDNA (1 µL), QuantiTect SYBR Green PCR Master Mix (Qiagen, Valencia, CA, USA) (12.5 µL), and specific primers (15 pmol). Reaction components were assembled in strip tubes with ultra-clear strip caps (MJ Research). The cycle profile was: 1 cycle at 95°C for 15 min followed by 40 cycles at 95°C for 30 sec; 55°C (for MUC1) or 50°C (for ß-actin) for 30 sec; and at 72°C for 30 sec (for MUC1) or for 20 sec (for ß-actin). Standard curves for MUC1 and ß-actin were generated with the use of a serially diluted template of known copy number. To verify the specificity of the amplification reaction, we performed melting curve analysis by increasing the temperature from 55°C to 95°C at a temperature transition rate of 0.5°C/sec. In addition, reaction products were examined on agarose gels. We normalized data from triplicate reactions using the ratio of target MUC1 cDNA to that of ß-actin.
Statistical Analysis
Data are expressed as mean ± SEM. Statistical significance was determined by the Wilcoxon signed-rank test. P values of less than 0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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, lane 1). This pattern of bands closely resembles that on blots of RNA from human parotid, submandibular, and sublingual glands (Liu et al., 2002). The 6.4-kb transcript likely represents the full-length MUC1 mRNA, while the 4.7-kb transcript may represent an allelic variant with fewer tandem repeats (Gendler et al., 1990).
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, lane 2), perhaps indicating co-allelic expression of MUC1 (Treon et al., 1999). Blots of proteins in lysates subjected to electrophoresis on Tricine gels were probed with MUC1 antibody CT2 directed against the cytoplasmic tail. This antibody detected a series of immunoreactive bands with molecular weights ranging from 15 to 25 kDa which could represent different glycoforms or variously processed fragments (Fig. 1, lane 3).
Cytokines and P. gingivalis Infection Modulate MUC1 Transcript Levels
KB cells were treated with pro-inflammatory mediators, RNA was isolated, and cDNA was synthesized and subjected to real-time PCR. Reactions were performed in the presence of SYBR Green with the use of MUC1 and ß-actin gene-specific primers. To generate standard curves, we amplified serially diluted MUC1 and ß-actin templates and plotted the log of the number of target molecules against Ct (threshold cycle). Regression analysis demonstrated R2 values of 0.999 and 1.00 for the MUC1 and ß-actin standard curves, respectively (data not shown). Representative real-time PCR plots of transcript levels of MUC1 and ß-actin in control and cytokine-treated cells show an increase in the fluorescent signal as a function of cycle number. Transcript levels in each sample were then quantified by reference to the standard curves, and MUC1 expression levels were normalized to levels of ß-actin. Melting curves of PCR products obtained with both MUC1 and ß-actin primers confirmed the presence of a single amplification product. When KB cells were treated for 4 hrs with IL-1ß, IL-6, TNF-, or IFN- alone, MUC1 transcripts exhibited an increase of 1.4- to 2.1-fold. However, when KB cells were treated with combinations of mediators, particularly IL-1ß/IFN- and TNF-/IFN-, MUC1 transcripts increased approximately three-fold compared with MUC1 transcripts in control cells (Fig. 2). No increase in MUC1 transcript levels was observed after treatment with the anti-inflammatory cytokine IL-10 or LPS from P. gingivalis. However, infection of KB monolayer cells with P. gingivalis cells for 4 hrs led to a 2.85-fold increase in MUC1 transcripts (Fig. 2).
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, IL-6/TNF-, or TNF-/IFN- for 8 hrs were significantly higher than those in untreated control cells (Fig. 3). Treatment of cells with the same combinations of mediators for 4 hrs yielded nearly identical results (data not shown). Increased levels of MUC1 protein are consistent with increased MUC1 mRNA levels observed after treatment with the same combinations of mediators shown in Fig. 2. These results show that cytokines promote transcription of the MUC1 gene, leading to high levels of stable MUC1 protein expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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In the present investigation, we have shown that MUC1 expression is up-regulated by treatment of oral epithelial cells with the pro-inflammatory mediators IL-1ß, IL-6, TNF-, and IFN-. Real-time PCR revealed that treatment with each of these cytokines alone caused an ca. two-fold increase in MUC1 mRNA levels, whereas treatment with combinations of mediators nearly doubled this effect. The additive effect of mediators suggests that MUC1 gene expression is up-regulated by one or more different signal transduction pathways. Interestingly, cytokine treatment resulted in even greater (ca. five- to 10-fold) increases in MUC1 protein levels, suggesting control at both the transcriptional and translational levels.
The effect of pro-inflammatory mediators on MUC1 RNA and protein expression observed in this study is important, since the same cytokines are produced in oral tissues in response to infection with the periodontal pathogens P. gingivalis (Sandros et al., 2000; Steffen et al., 2000; Imatani et al., 2001; Lourbakos et al., 2001) and A. actinomycetemcomitans (Sfakianakis et al., 2001; Uchida et al., 2001), the cariogenic dental pathogen S. mutans (Hahn et al., 2000), and the pathogenic yeast C. albicans (Rouabhia et al., 2002; Steele and Fidel, 2002). Both epithelial and non-epithelial cells appear to be capable of synthesizing pro-inflammatory cytokines in response to a microbial challenge (Sandros et al., 2000; Uchida et al., 2001; Gemmell et al., 2002). This indicates that increased MUC1 expression could be mediated by either autocrine or paracrine pathways and suggests a mechanism by which this mucin participates in oral host defense.
No effect on MUC1 expression was observed after treatment with either the anti-inflammatory cytokine IL-10 or P. gingivalis LPS, whereas a significant increase in MUC1 transcript levels was found after infection of KB cells with P. gingivalis cells for 4 hrs. KB cells, like gingival epithelial cells, express the toll-like receptors TLR2 and TLR4, but not CD14, which is a co-receptor for LPS (Uehara et al., 2001). Thus, while LPS is recognized as an important factor in the chronic inflammation associated with periodontal disease (Nonnenmacher et al., 2001; Wang and Ohura, 2002), its effect is primarily on monocytes and gingival fibroblasts. The present study shows that MUC1 expression can be stimulated either indirectly through the effects of inflammatory mediators or directly by bacterial binding or bacterial products other than LPS.
The relationship between mucins and bacteria is somewhat paradoxical. On the one hand, mucins protect epithelial surfaces from bacterial colonization, yet they may also serve as bacterial attachment sites (Lillehoj et al., 2001, 2002). A mechanism could be envisioned in which binding of bacteria to MUC1 on oral surfaces initiates a signaling cascade which leads to increased production of pro-inflammatory cytokines by the epithelial cells. Increased cytokine production could lead to up-regulation of MUC1 gene expression, resulting in increased numbers of mucin molecules on the cell surface. This would lead to strengthening of the protective barrier and diminished bacterial invasion. It is also possible that increased MUC1 density on the cell membrane could lead to enhanced shedding of the extracellular mucin subunit, together with adherent bacteria. Regulation of MUC1 expression could thus represent a novel mechanism for clearance of pathogenic organisms from the oral cavity.
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ACKNOWLEDGMENTS |
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Received March 25, 2003; Last revision September 9, 2003; Accepted September 10, 2003
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