Abstract
Prevotella intermedia, a major periodontal pathogen, plays important roles in the initiation and development of periodontitis by stimulating the release of proinflammatory cytokines, proteinases and matrix metalloproteinases (MMPs). Our previous study demonstrated that P. intermedia induced MMP-9 expression in human periodontal ligament (hPDL) cells. In this study, we examined the effects of P. intermedia on other MMPs' expression. Semi-quantitative reverse transcriptase (RT)-PCR analysis revealed that P. intermedia ATCC 25611 supernatant increased MMP-1 and MMP-8 mRNA expression in a concentration- and time-dependent manner. Enzyme-linked immunosorbent assay and Western blot results confirmed the RT-PCR results at the protein level. Cyclooxygenase inhibitor indomethacin significantly attenuated the upregulatory effects of P. intermedia on MMP-1 and MMP-8 expression. Extracellular signal-related kinase inhibitor PD98059 and c-Jun N-terminal kinase inhibitor SP600125 considerably decreased the upregulated level of MMP-1, whereas p38 inhibitor SB203580 markedly inhibited MMP-8 expression, suggesting that prostaglandin E2 and mitogen-activated protein kinase signaling pathways are involved in P. intermedia-induced MMP-1 and MMP-8 upregulation. Our results indicate that P. intermedia might contribute to periodontal connective tissue and bone matrix destruction through upregulating MMP production.
Introduction
Periodontitis, the major reason of adult tooth loss, is a chronic inflammatory disease initiated by a group of periodontal pathogens and characterized by periodontal connective tissue destruction and alveolar bone resorption. After successful colonization, periodontal pathogens proliferate in the host and stimulate immune responses of circulating immune cells and local resident cells to produce proinflammatory cytokines, prostaglandins, proteinases as well as matrix metalloproteinases (MMPs) (Nishihara & Koseki, 2004; Feng & Weinberg, 2006).
MMPs are a group of structurally and functionally related, but genetically distinct zinc-dependent proteinases responsible for the proteolytic degradation of extracellular matrix (ECM) and basement membrane components. MMPs play important roles in fetal development, morphogenesis, tissue repair, wound healing and immunomodulation (Birkedal-Hansen et al., 1993; Nagase & Woessner, 1999; Visse & Nagase, 2003). Excessive production of MMPs leads to accelerated matrix degradation associated with pathological conditions such as rheumatoid arthritis, cancer and periodontitis (Birkedal-Hansen, 1993; Visse & Nagase, 2003; Sorsa et al., 2004, 2006). Based on their molecular structure and substrate specificity, MMPs can be divided into collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs (MT-MMPs) and other MMPs.
MMP-1 and MMP-8 are collagenases, both of which have a broad substrate spectrum. Besides cleaving type I, II, III, VII, VIII and X collagen, they can also digest other ECM and non-ECM molecules. MMP-1 degrades collagen III more effectively, whereas MMP-8 is most effective in collagen I cleavage (Birkedal-Hansen et al., 1993). Significantly elevated levels of MMP-1 and MMP-8 were detected in gingival crevicular fluids (GCF), gingival tissues and periodontal ligament cells from patients with periodontitis. MMP-8 was positively correlated with the severity of chronic periodontitis (Golub et al., 1995, 1997; Ingman et al., 1996; Kubota et al., 1996; Romanelli et al., 1999; Kiili et al., 2002; Tuter et al., 2002; Sorsa et al., 2004; Soder et al., 2006).
Prevotella intermedia is one of the major periodontal pathogens (Socransky et al., 1998). This black-pigmented anaerobic rod possesses various virulence factors, such as adhesin, hemolysin, hemagglutinin, proteolytic and hydrolytic enzymes, which allow them to colonize in the oral cavity, evade host defense, modulate immune response and cause tissue destruction (Eley & Cox, 2003). Prevotella intermedia can induce pro-MMP-2 and pro-MMP-9 expression in fetal mouse osteoblasts (Pelt et al., 2002). Moreover, our recent work demonstrated that it can induce MMP-9 production in human periodontal ligament (hPDL) cells (Guan et al., 2008). However, the effects of P. intermedia on other MMPs' expression in hPDL cells have not been examined. Therefore, the aim of this study was to investigate the effects of P. intermedia on other MMPs, especially MMP-1 and MMP-8 expression, in hPDL cells and to examine the possible signaling pathways and molecular mechanisms involved in P. intermedia-regulated MMP-1 and MMP-8 expression.
Materials and methods
Bacterial strains and culture conditions
Prevotella intermedia ATCC 25611 was plated on trypticase agar supplemented with 5% defibrinated rabbit blood. When black-pigmented colonies were visible, a single colony was inoculated into 10 mL trypticase soy broth (BBL Microbiology Systems, Cockeysville, MD) supplemented with yeast extract (5 mg mL−1), menadione (5 μg mL−1) and hemin (5 μg mL−1) (TSB) at 37 °C in a Bugbox anaerobic workstation (Ruskinn Life Sciences Ltd, Wales, UK) with an atmosphere of 80% N2, 10% CO2 and 10% H2. After 24 h of incubation, the bacteria were further inoculated by 5% into 200 mL TSB. They were harvested after 20 h of growth (OD600 nm=1.5) by centrifugation at 10 000 g for 15 min (4 °C). The culture supernatant was filter sterilized with a 0.22-μM filter (Millipore, Bedford, MA) and stored at −80 °C until use.
Cell culture
Healthy hPDL tissue was obtained from the extracted premolars of three subjects (age ranges from 11 to 15 years) for orthodontic reasons. Experimental protocols were approved by the Ethics Committee of School of Stomatology, The Fourth Military Medical University, China. Informed consents were obtained before tooth extraction. Freshly extracted teeth were immediately placed in 20 mM phosphate-buffered saline (PBS, pH 7.2) supplemented with antibiotics (100 U mL−1 penicillin and 100 μg mL−1 streptomycin) and rinsed thoroughly with Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL, Carlsbad, CA). Periodontal ligament was carefully removed from the middle third of the root surface with a scalpel. The aseptically removed tissue was then placed in a 60-mm Petri dish (Nunc, Roskilde, Denmark). The tissues were minced with a blade into approximately 1-mm3 fragments and grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco BRL) and antibiotics. The cell cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. After reaching confluence, the cells were detached with 0.25% trypsin and 0.2% EDTA and subcultured in 1 : 3 ratios. Cells between the 7th and the 11th passages were used in this study.
Stimulation of hPDL cells with P. intermedia supernatant
The hPDL cells were plated in six-well plates at a density of 1 × 106 cells per well (approximately 1 × 105 cells cm−2) and were grown in DMEM containing 5% FBS until confluence. The cells were subsequently starved with serum-free DMEM overnight. After two brief washes with cold PBS, the hPDL cells were challenged with P. intermedia supernatant for 24 h. Cells without treatment served as controls.
The concentration effects were observed by treating the hPDL cells with 1–10%P. intermedia supernatant, concentrations previously shown to be noncytotoxic (Guan et al., 2008) for 24 h. To determine the time-course effects, the hPDL cells were treated with 5%P. intermedia supernatant for indicated times (3–48 h). All the above experiments were conducted in duplicate and in three different cell lines.
A pharmacological approach was adopted to examine the signaling pathways and molecular mechanisms involved in P. intermedia-regulated MMP expression. The hPDL cells were pretreated with specific extracellular signal-related kinase (ERK) inhibitor PD98059 (20 μM), c-Jun N-terminal kinase (JNK) inhibitor SP600125 (10 μM), p38 inhibitor SB203580 (20 μM) and nonselective cyclooxygenase (COX) inhibitor indomethacin (20 μM) (Sigma-Aldrich, St. Louis, MO) for 1 h before 5%P. intermedia supernatant stimulation. After 24 h of incubation, conditioned culture media were collected for enzyme-linked immunosorbant assay (ELISA) and total RNA was extracted for reverse transcription (RT)-PCR.
MMP mRNA detection by semi-quantitative RT-PCR
Total RNA from hPDL cells (cultured for 3–48 h with 1–10%P. intermedia supernatant treatment) was extracted with Omega Total RNA extraction kit I (Omega Bio-Tek, Norcross, GA) according to the manufacturer's protocols and was quantified spectrophotometrically at 260/280 nm. cDNA was synthesized using 500 ng total RNA and Oligodt(18) primers with the BioRT Two Step RT-PCR Kit (BioER, Hangzhou, China) in a 20-μL reaction system. The reverse transcription was conducted with incubation of the mixture at 50 °C for 45 min and termination at 95 °C for 5 min. PCR was carried out using 2 μL of the reverse transcript in a 25 μL reaction system with 1 U Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA). MMP primer sequences have been described previously (Song & Windsor, 2005) and PCR conditions are shown in Table 1. Amplification was performed in a thermal cycler (Eastwin, Beijing, China) with denaturation at 94 °C for 35 s, annealing at respective temperatures for 35 s, extension at 72 °C for 45 s for indicated cycles and a final extension at 72 °C for 10 min. After PCR amplification, 5 μL of the PCR products were subjected to electrophoresis on 1.5% agarose gels and stained with ethidium bromide. The stained gels were visualized under UV illumination with Bio-Rad Gel DocTM XR system (Bio-Rad, Hercules, CA) and band intensities were analyzed with alphaview v.1.3.0 software (Alpha Innotech Corp., San Leandro, CA).
Primer sequences and PCR conditions
| Gene | Forward primer | Reverse primer | bp | Accession no. | Annealing temperature (Tm) (°C) | Cycles |
| MMP-1 | 5′-GAAGGTGATGAAGCAGCCCAGATGT-3′ | 5′-CAGTTGTGGCCAGAAAACAGAAATGAAA-3′ | 716 | NM 002421 | 56 | 28 |
| MMP-8 | 5′-GCTGCTTATGAAGATTTTGACAGAG-3′ | 5′-ACAGCCACATTTGATTTTGCTTCAG-3′ | 435 | NM 002424 | 60 | 35 |
| MMP-13 | 5′-GCTTAGAGGTGACTGGCAAAC-3′ | 5′-CCGGTGTAGGTGTAGATAGGAAA-3′ | 517 | NM 002427 | 56 | 35 |
| MMP-3 | 5′-GACACCAGCATGAACCTTGTT-3′ | 5′-GGAACCGAGTCAGGTCTGTG-3′ | 667 | NM 004530 | 56 | 28 |
| MMP-2 | 5′-ATCCGTGGTGAGATCTTCTTCTT-3′ | 5′-AGCCAGGATCCATTTTCTTCTT-3′ | 374 | NM 004530 | 56 | 28 |
| MMP-9 | 5′-TGCCAGTTTCCATTCATCTTCCAA-3′ | 5′-CTGCGGTGTGGTGGTGGTT-3′ | 519 | NM 004994 | 56 | 32 |
| MMP-14 | 5′-GATAAACCCAAAAACCCCACCTA-3′ | 5′-CCCTCCTCGTCCACCTCAATG-3′ | 680 | NM 004995 | 56 | 28 |
| MMP-15 | 5′-ACAACCACCATCTGACCTTTAGCA-3′ | 5′-AGCTTGAAGTTGTCAACGTCCTTC-3′ | 454 | NM 002428 | 57 | 35 |
| TIMP-1 | 5′-CCTTCTGCAATTCCGACCTCGTC-3′ | 5′-CGGGCAGGATTCAGGCTATCTGG-3′ | 534 | NM 003254 | 56 | 28 |
| TIMP-2 | 5′-TGGAAACGACATTTATGGCAACC-3′ | 5′-ACAGGAGCCGTCACTTCTCTTGAT-3′ | 433 | NM 003255 | 56 | 28 |
| GAPDH | 5′-CAACTACATGGTTTACATGTTC-3′ | 5′-GCCAGTGGACTCCACGAC-3′ | 181 | NM002046 | 57 | 27 |
| Gene | Forward primer | Reverse primer | bp | Accession no. | Annealing temperature (Tm) (°C) | Cycles |
| MMP-1 | 5′-GAAGGTGATGAAGCAGCCCAGATGT-3′ | 5′-CAGTTGTGGCCAGAAAACAGAAATGAAA-3′ | 716 | NM 002421 | 56 | 28 |
| MMP-8 | 5′-GCTGCTTATGAAGATTTTGACAGAG-3′ | 5′-ACAGCCACATTTGATTTTGCTTCAG-3′ | 435 | NM 002424 | 60 | 35 |
| MMP-13 | 5′-GCTTAGAGGTGACTGGCAAAC-3′ | 5′-CCGGTGTAGGTGTAGATAGGAAA-3′ | 517 | NM 002427 | 56 | 35 |
| MMP-3 | 5′-GACACCAGCATGAACCTTGTT-3′ | 5′-GGAACCGAGTCAGGTCTGTG-3′ | 667 | NM 004530 | 56 | 28 |
| MMP-2 | 5′-ATCCGTGGTGAGATCTTCTTCTT-3′ | 5′-AGCCAGGATCCATTTTCTTCTT-3′ | 374 | NM 004530 | 56 | 28 |
| MMP-9 | 5′-TGCCAGTTTCCATTCATCTTCCAA-3′ | 5′-CTGCGGTGTGGTGGTGGTT-3′ | 519 | NM 004994 | 56 | 32 |
| MMP-14 | 5′-GATAAACCCAAAAACCCCACCTA-3′ | 5′-CCCTCCTCGTCCACCTCAATG-3′ | 680 | NM 004995 | 56 | 28 |
| MMP-15 | 5′-ACAACCACCATCTGACCTTTAGCA-3′ | 5′-AGCTTGAAGTTGTCAACGTCCTTC-3′ | 454 | NM 002428 | 57 | 35 |
| TIMP-1 | 5′-CCTTCTGCAATTCCGACCTCGTC-3′ | 5′-CGGGCAGGATTCAGGCTATCTGG-3′ | 534 | NM 003254 | 56 | 28 |
| TIMP-2 | 5′-TGGAAACGACATTTATGGCAACC-3′ | 5′-ACAGGAGCCGTCACTTCTCTTGAT-3′ | 433 | NM 003255 | 56 | 28 |
| GAPDH | 5′-CAACTACATGGTTTACATGTTC-3′ | 5′-GCCAGTGGACTCCACGAC-3′ | 181 | NM002046 | 57 | 27 |
Primer sequences and PCR conditions
| Gene | Forward primer | Reverse primer | bp | Accession no. | Annealing temperature (Tm) (°C) | Cycles |
| MMP-1 | 5′-GAAGGTGATGAAGCAGCCCAGATGT-3′ | 5′-CAGTTGTGGCCAGAAAACAGAAATGAAA-3′ | 716 | NM 002421 | 56 | 28 |
| MMP-8 | 5′-GCTGCTTATGAAGATTTTGACAGAG-3′ | 5′-ACAGCCACATTTGATTTTGCTTCAG-3′ | 435 | NM 002424 | 60 | 35 |
| MMP-13 | 5′-GCTTAGAGGTGACTGGCAAAC-3′ | 5′-CCGGTGTAGGTGTAGATAGGAAA-3′ | 517 | NM 002427 | 56 | 35 |
| MMP-3 | 5′-GACACCAGCATGAACCTTGTT-3′ | 5′-GGAACCGAGTCAGGTCTGTG-3′ | 667 | NM 004530 | 56 | 28 |
| MMP-2 | 5′-ATCCGTGGTGAGATCTTCTTCTT-3′ | 5′-AGCCAGGATCCATTTTCTTCTT-3′ | 374 | NM 004530 | 56 | 28 |
| MMP-9 | 5′-TGCCAGTTTCCATTCATCTTCCAA-3′ | 5′-CTGCGGTGTGGTGGTGGTT-3′ | 519 | NM 004994 | 56 | 32 |
| MMP-14 | 5′-GATAAACCCAAAAACCCCACCTA-3′ | 5′-CCCTCCTCGTCCACCTCAATG-3′ | 680 | NM 004995 | 56 | 28 |
| MMP-15 | 5′-ACAACCACCATCTGACCTTTAGCA-3′ | 5′-AGCTTGAAGTTGTCAACGTCCTTC-3′ | 454 | NM 002428 | 57 | 35 |
| TIMP-1 | 5′-CCTTCTGCAATTCCGACCTCGTC-3′ | 5′-CGGGCAGGATTCAGGCTATCTGG-3′ | 534 | NM 003254 | 56 | 28 |
| TIMP-2 | 5′-TGGAAACGACATTTATGGCAACC-3′ | 5′-ACAGGAGCCGTCACTTCTCTTGAT-3′ | 433 | NM 003255 | 56 | 28 |
| GAPDH | 5′-CAACTACATGGTTTACATGTTC-3′ | 5′-GCCAGTGGACTCCACGAC-3′ | 181 | NM002046 | 57 | 27 |
| Gene | Forward primer | Reverse primer | bp | Accession no. | Annealing temperature (Tm) (°C) | Cycles |
| MMP-1 | 5′-GAAGGTGATGAAGCAGCCCAGATGT-3′ | 5′-CAGTTGTGGCCAGAAAACAGAAATGAAA-3′ | 716 | NM 002421 | 56 | 28 |
| MMP-8 | 5′-GCTGCTTATGAAGATTTTGACAGAG-3′ | 5′-ACAGCCACATTTGATTTTGCTTCAG-3′ | 435 | NM 002424 | 60 | 35 |
| MMP-13 | 5′-GCTTAGAGGTGACTGGCAAAC-3′ | 5′-CCGGTGTAGGTGTAGATAGGAAA-3′ | 517 | NM 002427 | 56 | 35 |
| MMP-3 | 5′-GACACCAGCATGAACCTTGTT-3′ | 5′-GGAACCGAGTCAGGTCTGTG-3′ | 667 | NM 004530 | 56 | 28 |
| MMP-2 | 5′-ATCCGTGGTGAGATCTTCTTCTT-3′ | 5′-AGCCAGGATCCATTTTCTTCTT-3′ | 374 | NM 004530 | 56 | 28 |
| MMP-9 | 5′-TGCCAGTTTCCATTCATCTTCCAA-3′ | 5′-CTGCGGTGTGGTGGTGGTT-3′ | 519 | NM 004994 | 56 | 32 |
| MMP-14 | 5′-GATAAACCCAAAAACCCCACCTA-3′ | 5′-CCCTCCTCGTCCACCTCAATG-3′ | 680 | NM 004995 | 56 | 28 |
| MMP-15 | 5′-ACAACCACCATCTGACCTTTAGCA-3′ | 5′-AGCTTGAAGTTGTCAACGTCCTTC-3′ | 454 | NM 002428 | 57 | 35 |
| TIMP-1 | 5′-CCTTCTGCAATTCCGACCTCGTC-3′ | 5′-CGGGCAGGATTCAGGCTATCTGG-3′ | 534 | NM 003254 | 56 | 28 |
| TIMP-2 | 5′-TGGAAACGACATTTATGGCAACC-3′ | 5′-ACAGGAGCCGTCACTTCTCTTGAT-3′ | 433 | NM 003255 | 56 | 28 |
| GAPDH | 5′-CAACTACATGGTTTACATGTTC-3′ | 5′-GCCAGTGGACTCCACGAC-3′ | 181 | NM002046 | 57 | 27 |
MMP protein measurement by ELISA
Secreted MMP-1 and MMP-8 proteins in conditioned culture media were measured by commercially available ELISA kit according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). The detection limit is 15 pg mL−1. All determinations were performed in quadruplicate.
Western blot
hPDL cells were treated with or without 5%P. intermedia supernatant for 24 h. Conditioned culture media were collected and concentrated by vacuum freeze-drying. Protein concentrations in the concentrated media were determined using a BCATM protein assay kit (Thermo Scientific, Rockford, IL). Samples were heated for 10 min at 95 °C with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (62.5 mM Tris-HCl, pH 6.8, containing 2% SDS, 10% glycerol, 5%β-mercaptoethanol and 0.001% bromophenol blue) and same amounts of proteins (20 μg) were resolved with 10% SDS-PAGE separation gels at 30 mA. Precision Plus Protein Standards (Bio-Rad) were incorporated to determine molecular weights. Proteins separated by SDS-PAGE were electrotransferred to nitrocellulose membranes (0.45 μm, Millipore) by a semi-dry transfer system (Transblot, Bio-Rad). Following a brief wash with 20 mM PBS containing 0.05% Tween-20 (PBST), the membranes were blocked with 5% nonfat milk for 2 h at room temperature with gentle shaking. Subsequently, the membranes were, respectively, incubated with 1 : 200 rabbit anti-human MMP-1 polyclonal antibody (Biosynthesis Biotechnology Co. Ltd, Beijing, China) and 1 : 200 rabbit anti-human MMP-8 polyclonal antibody (Boster Biological Technology, Wuhan, China) overnight at 4 °C. After washing three times with PBST, the membranes were incubated with 1 : 5000 HRP-conjugated goat anti-rabbit secondary antibody (Zhongshan Goldenbridge Biotechnology, Beijing, China) for 1 h at room temperature. Following three washes with PBST, the membranes were developed with ChemiGlow West chemiluminescent substrate kit (Alpha Innotech Corp.) and the signals were captured with FluorChem FC2 (Alpha Innotech Corp.).
Statistical analysis
Data were presented as means ± SE. One-way or two-way anova, followed by post hoc Bonferroni's multiple comparison test, was used to compare the results by graphpad prism 5 (GraphPad Software Inc., San Diego, CA). P-values <0.05 were considered to be statistically significant.
Results
Effects of P. intermedia on MMP expression in hPDL cells
In order to observe the overall effects of P. intermedia on MMP expression in hPDL cells at the mRNA level, we first used a single dose of P. intermedia supernatant (5%) to stimulate the cells for 24 h. RT-PCR analysis revealed that MMP-1, -2, -3, -8, -14, -15, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-2 mRNA were constitutively expressed in hPDL cells. Prevotella intermedia treatment upregulated MMP-1, -8 and -15 expression by 1.73 (P<0.05), 2.42 (P<0.01) and 2.35 (P<0.01)-fold, respectively. MMP-9 expression was also induced by P. intermedia treatment. However, P. intermedia showed no significant influence on MMP-2, -3, -13, -14, TIMP-1 and TIMP-2 mRNA expression (Fig. 1 and Table 2).
Regulation of MMPs mRNA expression by Prevotella intermedia in hPDL cells. After hPDL cells were cultured in the absence (a) or presence (b) of 5%P. intermedia supernatant for 24 h, total RNA was extracted and subjected to RT-PCR analysis.
Comparison of RT-PCR results of Prevotella intermedia-treated vs. control hPDL cells
| Treated/control | |
| MMP-1 | 1.73 ± 0.08 |
| MMP-8 | 2.57 ± 0.15 |
| MMP-13 | Not detected |
| MMP-3 | 1.08 ± 0.03 |
| MMP-2 | 0.99 ± 0.02 |
| MMP-9 | Detected/not detected |
| MMP-14 | 1.04 ± 0.03 |
| MMP-15 | 2.13 ± 0.12 |
| TIMP-1 | 1.09 ± 0.04 |
| TIMP-2 | 0.98 ± 0.03 |
| Treated/control | |
| MMP-1 | 1.73 ± 0.08 |
| MMP-8 | 2.57 ± 0.15 |
| MMP-13 | Not detected |
| MMP-3 | 1.08 ± 0.03 |
| MMP-2 | 0.99 ± 0.02 |
| MMP-9 | Detected/not detected |
| MMP-14 | 1.04 ± 0.03 |
| MMP-15 | 2.13 ± 0.12 |
| TIMP-1 | 1.09 ± 0.04 |
| TIMP-2 | 0.98 ± 0.03 |
Average ± SD (n=3).
Comparison of RT-PCR results of Prevotella intermedia-treated vs. control hPDL cells
| Treated/control | |
| MMP-1 | 1.73 ± 0.08 |
| MMP-8 | 2.57 ± 0.15 |
| MMP-13 | Not detected |
| MMP-3 | 1.08 ± 0.03 |
| MMP-2 | 0.99 ± 0.02 |
| MMP-9 | Detected/not detected |
| MMP-14 | 1.04 ± 0.03 |
| MMP-15 | 2.13 ± 0.12 |
| TIMP-1 | 1.09 ± 0.04 |
| TIMP-2 | 0.98 ± 0.03 |
| Treated/control | |
| MMP-1 | 1.73 ± 0.08 |
| MMP-8 | 2.57 ± 0.15 |
| MMP-13 | Not detected |
| MMP-3 | 1.08 ± 0.03 |
| MMP-2 | 0.99 ± 0.02 |
| MMP-9 | Detected/not detected |
| MMP-14 | 1.04 ± 0.03 |
| MMP-15 | 2.13 ± 0.12 |
| TIMP-1 | 1.09 ± 0.04 |
| TIMP-2 | 0.98 ± 0.03 |
Average ± SD (n=3).
To evaluate the concentration effects of P. intermedia on the mRNA levels of MMP-1 and MMP-8, hPDL cells were stimulated with 1–10%P. intermedia supernatants for 24 h. A concentration-dependent increase in MMP-1 and MMP-8 mRNA expression was observed (Fig. 2a, c and e). Prevotella intermedia also increased MMP-1 and MMP-8 mRNA expression in a time-dependent manner. Marked increases in MMP-1 and MMP-8 mRNA expression were detected 3 h post-P. intermedia stimulation, and the stimulatory effects of P. intermedia were maintained for the whole stimulating period of 48 h (Fig. 2b, d and f).
Concentration- and time-dependent upregulation of MMP-1 and MMP-8 mRNA expression in response to Prevotella intermedia challenge. Representative RT-PCR results of three independent experiments are shown (a, b). hPDL cells were stimulated with 1–10%P. intermedia supernatant for 24 h (a) and with 5%P. intermedia supernatant for 3–48 h (b). Relative MMP-1 (c, d) and MMP-8 (e, f) mRNA expression as normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). n=3 (mean ± SE). *P<0.05, **P<0.01 vs. controls.
To investigate whether the P. intermedia-enhanced MMP-1 and MMP-8 mRNA expression resulted in increased protein production, the secreted protein levels of MMP-1 and MMP-8 in the culture media were measured by ELISA. In agreement with mRNA results, both MMP-1 and MMP-8 protein levels were increased in a concentration- and time-dependent manner (Fig. 3). Although 5%P. intermedia challenge for 24 h produced a 2.42-fold increase in MMP-8 mRNA expression (P<0.01), the same treatment led to only a 22% increase in protein secretion (P<0.05) (Fig. 3c and d), indicating that the stimulatory effect of P. intermedia on MMP-8 protein secretion was not as great as its effect on mRNA expression.
Increase of MMP-1 and MMP-8 protein secretion by Prevotella intermedia (Pi) as measured by ELISA. Concentration-dependent MMP-1 (a) and MMP-8 (c) protein secretion. Time course of MMP-1 (b) and MMP-8 (d) protein secretion. n=4 (mean ± SE). *P<0.05, **P<0.01, ***P<0.001 vs. 0% or controls.
Western blot analysis demonstrated that a protein band of approximately 53 kDa, which corresponded to the proform of MMP-1, was detected with rabbit anti-human MMP-1 antibody. Western blot confirmed the ELISA result that P. intermedia stimulation significantly upregulated MMP-1 expression at the protein level. Prevotella intermedia challenge did not significantly increase the 55-kDa proform of MMP-8. However, the 45-kDa active form and the 35-kDa degraded fragment of MMP-8 were increased by P. intermedia treatment (Fig. 4).
Prevotella intermedia upregulated MMP-1 expression and activated MMP-8 in hPDL cells. hPDL cells were cultured in the absence (−) or presence (+) of 5%P. intermedia supernatant for 24 h. Culture media were collected and concentrated. Equal amounts of protein (20 μg) were resolved by 10% SDS-PAGE and subjected to Western blotting. The blots were probed with anti-MMP-1 or anti-MMP-8 antibodies. glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.
Involvement of mitogen-activated protein kinase (MAPK) signaling pathways and prostaglandin E2 (PGE2) in P. intermedia-evoked upregulation of MMP-1 and MMP-8
To study whether the ERK, JNK and p38 MAPK signaling pathways are involved in P. intermedia-evoked MMP-1 and MMP-8 upregulation, we pretreated hPDL cells for 1 h with PD98059 (ERK inhibitor), SP600125 (JNK inhibitor) and SB203580 (p38 inhibitor) for 1 h, respectively. As shown in Fig. 5, pretreatment of hPDL cells with PD98059, SP600125 and SB203580 produced differential inhibitory effects on the increased protein levels of MMP-1 and MMP-8. All three inhibitors showed decreasing effects on P. intermedia-increased MMP-1 and MMP-8 protein secretion. However, PD98059 and SP600125 significantly inhibited MMP-1 secretion (P<0.001 and P<0.05, respectively), whereas SB203580 significantly reduced MMP-8 secretion (P<0.05).
Involvement of MAPK signaling pathways and PGE2 in Prevotella intermedia-induced MMP-1 and MMP-8 upregulation. hPDL cells were pretreated with PD (PD98059), SP (SP600125), SB (SB203580) and Indo (Indomethacin) for 1 h before P. intermedia challenge. After 24 h incubation, culture media were subjected to ELISA. n=4 (mean ± SE). *P<0.05, **P<0.01, ***P<0.001 vs. P. intermedia (Pi)-treated group.
PGE2 plays important roles in bone resorption, a characteristic of periodontal disease, and it is implicated in MMP regulation. Indomethacin, a nonselective COX inhibitor, was used to examine the roles of PGE2 in P. intermedia-induced MMP-1 and MMP-8 upregulation. Preincubation of hPDL cells with indomethacin resulted in a marked decrease in MMP-1 (P<0.001) and MMP-8 (P<0.01) protein secretion (Fig. 5).
Discussion
In this study, we examined the expression of multiple MMPs in hPDL cells and explored the effects of P. intermedia on these MMPs. We found that hPDL cells constitutively expressed MMP-1, -2, -3, -8, -14, -15, TIMP-1 and TIMP-2 mRNA. The novel finding is that P. intermedia stimulation increased the expression of MMP-1, -8 and -15. MAPK signaling pathways and PGE2 were involved in P. intermedia-elicited MMP-1 and MMP-8 upregulation. The present work extended our knowledge about how P. intermedia affect MMP expression, which plays multiple roles in the pathology of periodontal disease.
MMP-1 is produced by fibroblasts in a variety of connective tissues (Birkedal-Hansen, 1993). Previous studies indicated that MMP-1 levels were elevated in GCF and gingival tissues of periodontitis patients (Ingman et al., 1996; Kubota et al., 1996). Although previous studies showed that Porphyromonas gingivalis and proinflammatory cytokines induced the expression of MMP-1 in human gingival fibroblasts (Domeij et al., 2002; Zhou & Windsor, 2006), our study is the first one to show that P. intermedia increased MMP-1 expression in hPDL cells.
MMP-8 is the predominant collagenase present in the gingival tissue, GCF and saliva in periodontitis patients. Physiological levels of MMP-8 have been shown to exhibit protective and anti-inflammatory effects (Kuula et al., 2009). However, pathologically elevated MMP-8 has been found to be one of the key mediators of tissue destruction in periodontal inflammation (Golub et al., 1995; Ingman et al., 1996; Romanelli et al., 1999; Sorsa et al., 2004). MMP-8 was previously thought to be expressed solely by polymorphonuclear leukocytes (Visse & Nagase, 2003). However, it was recently found that MMP-8 can also be de novo expressed in non-polymorphonuclear lineage cells, such as gingival fibroblasts, odontoblasts, synovial fibroblasts, endothelial cells, plasma cells, periodontal ligament cells and gingival sulcular epithelium (Hanemaaijer et al., 1997; Palosaari et al., 2000; Tervahartiala et al., 2000; Wahlgren et al., 2001; Takahashi et al., 2003; Zhou & Windsor, 2006). Although periodontal pathogens Treponema denticola, Actinobacillus actinomycetemcomitans, P. gingivalis and Fusobacterium nucleatum were shown to induce the production, release and activation of MMP-8 in human neutrophils (Sorsa et al., 1992; Claesson et al., 2002; Shin et al., 2008), we demonstrated for the first time that P. intermedia could upregulate MMP-8 expression and activate the enzyme in hPDL cells. This may contribute to the destruction of periodontal tissues because MMP-8 effectively cleaves not only various forms of collagen but also a wide range of noncollagenous substrates, such as laminin, fibrinogen, fibronectin and proteoglycans.
The molecular mechanisms and signaling pathways involved in MMP production in hPDL cells are poorly understood. Previous works demonstrated that coordinated activation of ERK, JNK and p38 MAPK pathways was involved in increased MMP-1 expression in normal human skin fibroblasts by various stimuli (Reunanen et al., 1998; Westermarck et al., 1998). In line with these works, we found that specific inhibitors of ERK and JNK significantly decreased the augmented MMP-1 protein level, whereas p38 inhibitor strongly attenuated the increased MMP-8 secretion, indicating that subtypes of MAPKs are differentially involved in P. intermedia-induced MMP-1 and MMP-8 upregulation. Extensive studies have been conducted to investigate the roles of p38 on induced MMP-1 expression. Some studies indicated that p38 inhibition potently inhibited the induced MMP-1 expression in human gingival fibroblasts and skin fibroblasts (Reunanen et al., 1998, 2002; Westermarck et al., 1998; Domeij et al., 2002, 2005), while other reports showed that p38 inhibition had no effect on induced MMP-1 expression in skin fibroblasts and monocytes (Park et al., 2004; Domeij et al., 2005). Although the mediating roles of different subtypes of MAPKs in the regulation of MMPs are not fully understood, the differential roles might be cell-type related or due to the different extracellular stimuli used for different experiments.
PGE2 have been reported to regulate the production of MMPs in human gingival fibroblasts, synovial fibroblasts and hPDL fibroblasts (DiBattista et al., 1994; Noguchi et al., 2001, 2005; Domeij et al., 2002; Yan et al., 2005). In order to examine the roles of PGE2 in P. intermedia-induced MMP-1 and MMP-8 upregulation, we used indomethacin, a nonselective COX inhibitor, to block the synthesis of PGE2. Preincubation of hPDL cells with indomethacin resulted in a marked reduction in MMP-1 and MMP-8 expression, indicating that PGE2 was partly involved in the P. intermedia-modulated MMP-1 and MMP-8 upregulation. The roles of PGE2 on MMP expression appear to be conflicting. DiBattista et al. (1994) reported that PGE2 significantly inhibited the interleukin-1β (IL-1β)-induced MMP-1 expression in human synoviocytes. In contrast, Domeij et al. (2002) found NS-398, a selective COX2 inhibitor, reduced the tumor necrosis factor (TNF)-α and IL-1β-induced MMP-1 and MMP-3 expression in human gingival fibroblasts. Another interesting work showed that in normal human gingival fibroblasts, PGE2 decreased the IL-1β-induced MMP-3 expression, while in diseased human gingival fibroblasts, it increased the IL-1β-induced MMP-3 expression (Ruwanpura et al., 2004). Because PGE2 exerts its biological actions via PGE2 receptors, these contradictory results might result from the different receptors it binds.
IL-1β and TNF-α were reported to upregulate MMP-1 expression in human gingival fibroblasts (Domeij et al., 2002; Beklen et al., 2007) and MMP-8 expression in rheumatoid synovial fibroblasts (Hanemaaijer et al., 1997). However, the P. intermedia upregulation of MMP-1 and MMP-8 in hPDL cells does not seem to be attributable to IL-1β and TNF-α because we did not observe any stimulatory effects of P. intermedia on IL-1β and TNF-α expression (Guan et al., 2009). A previous study suggested that heat-shock-induced MMP-1 was through the autocrine IL-6 loop (Park et al., 2004). Moreover, IL-8 expressed by periodontitis-affected gingival sulcular epithelial cells can provoke the release of MMP-8 by recruited neutrophils (Uitto et al., 2003). We found that the P. intermedia supernatant significantly induced IL-6 and IL-8 mRNA expression and protein secretion (Guan et al., 2009), and this might contribute to the observed MMP-1 and MMP-8 upregulation in our study. Further studies are necessary to investigate in detail about the effects of IL-6 and IL-8 on MMP-1 and MMP-8 expression.
MMP-15, also called MT2-MMP, is a type I transmembrane protein capable of activating proMMP-2. It can also digest a number of ECM molecules, including fibronectin, tenascin, aggrecan, perlecan and laminin (Visse & Nagase, 2003). We found that P. intermedia upregulated the expression of MMP-15. MMP-15 upregulation might be favorable for activating proMMP-2 into active MMP-2 and enhancing periodontal connective tissue degradation.
In summary, the present study showed that P. intermedia upregulated the mRNA expression and protein secretion of MMP-1 and MMP-8 via MAPK signaling pathways and via PGE2 synthesis in hPDL cells. Prevotella intermedia may contribute to periodontal connective tissue and bone matrix degradation occurred in the process of chronic periodontitis through increasing the expression of multiple MMPs.
Acknowledgements
This study was supported by the Creative Project of the School of Stomatology, The Fourth Military Medical University and by the Shaanxi Province Science and Technology Development Program (2008K14-03 to Dr S.-M. Guan). The authors are grateful to Prof. Yan Jin (Tissue Engineering Center, School of Stomatology, The Fourth Military Medical University) for his generous support of the study.
References
Editor: William Wade
