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P. gingivalis Regulates the Expression of Cathepsin B and Cystatin C
1 PAROGÈNE, Bâtiment 3, Etage 7, 11, rue Humann, 67085, Strasbourg cedex, France; Correspondence: * corresponding author, htenen{at}gmail.com
Porphyromonas gingivalis is a major etiological agent of periodontitis that could affect the expression of Cathepsins B and C by disrupting the balance between these enzymes and their inhibitor, Cystatin C. We tested this hypothesis by infecting human oral epithelial cells with P. gingivalis or activating solely by its lipopolysaccharide. The mRNA level, the enzymatic activity, and the protein expression of Cathepsin B were increased (three-fold) in a dose-dependent manner, while those of Cystatin C decreased (five-fold). No changes were observed for Cathepsin C. Although activation by lipopolysaccharides led to a delayed imbalance (2 days) between Cathepsin B and Cystatin C, this imbalance took place very rapidly during the infection (< 6 hrs), indicating that the whole bacterium contains components that initiate rapid changes in the transcription rates of Cathepsin B and Cystatin C and selectively modify the molecular pathways that lead to this imbalance.
Key Words: cathepsins • cystatins • Porphyromonas gingivalis • periodontitis
Periodontitis is an inflammatory disease initiated by anaerobic bacteria such as Porphyromonas gingivalis. In vitro and clinical studies have provided evidence that the destructive process in periodontitis is a consequence of an imbalance of the homeostasis between degradative enzymes such as MMPs and their inhibitors, the Tissue Inhibitors of Metalloproteinases (TIMPs) (Chang et al., 2002; Soell et al., 2002; Kubota et al., 2008). This imbalance can also take place with other types of degradative enzymes, such as the lysosomal cysteine proteinases, Cathepsins, and their inhibitors, Cystatins (Abrahamson et al., 1997; Blankenvoorde et al., 1997; Mogi and Otogoto, 2007). Collectively, Cathepsins participate in multiple host systems that are active in healthy and disease situations, such as tissue remodeling, turnover of the extracellular matrix, immune system function, and apoptosis, or in antigen and pro-protein processing (Dickinson, 2002). We know relatively little about the role of the Cathepsins in periodontitis, although it has been shown that loss-of-function mutations in the gene encoding Cathepsin C result in Papillon-Lefèvre syndrome, a rare autosomal-recessive disease characterized by severe forms of early-onset periodontitis associated with loss of teeth (Toomes et al., 1999). In polymorphonuclear leukocytes (PMN) derived from individuals with this syndrome, another Cathepsin, Cathepsin G, was identified, along with Elastase and Proteinase 3, as being crucial for initiating a proper innate immune response to Aggregatibacter actinomycetemcomitans, another periodontal pathogen (de Haar et al., 2006). In addition, the concentration of Cathepsin C has been shown to decrease significantly in gingival crevicular fluid (GCF) from persons with periodontitis as compared with that in healthy individuals (Soell et al., 2002). Other studies have shown an increase in the Cathepsin B concentration in GCF from persons affected with periodontitis as compared with those treated for periodontitis (Ichimaru et al., 1996; Chen et al., 1998). In contrast, a simultaneous inhibition of Cystatin C was observed in inflamed gingiva and fluids (Lah et al., 1993; Blankenvoorde et al., 1997). Although these in vivo results tend to confirm an imbalance between Cathepsin B and Cystatin C in pathological situations, we do not know precisely how both types of proteins or other types of Cathepsins/Cystatins can be regulated upon infection of epithelial cells by P. gingivalis. To our knowledge, this problem has not been investigated in the context of periodontitis, but may be an interesting topic of study, because epithelial cells are a central component of the barrier between oral microflora and internal tissues. Consequently, the aim of our study was to investigate, in human oral epithelial cells, the effect of P. gingivalis and its lipopolysaccharide (LPS) on the expression of Cathepsins B and C and Cystatin C, all proteins known to be implicated in periodontitis.
All of the products and techniques are detailed in the APPENDIX.
Human Oral Epithelial Cells
Infection of Human Oral Epithelial Cells by P. gingivalis and Activation by Its Lipopolysaccharide In preparation for infection, 24 hrs before the beginning of the experiment, the cells were washed with PBS, and a 5-mL quantity of the K-SFM medium without antibiotics was added. On the day of infection, cells were washed twice with PBS and infected with P. gingivalis at a multiplicity of infection (MOI) ranging from 2 to 200 bacteria per cell in 5 mL of the K-SFM medium without antibiotics. Uninfected cells served as controls. The percentage of dead cells did not exceed 5% at any point of the time-course infection (data not shown). Viability of P. gingivalis was confirmed by the determination of colony-forming units (CFUs) from extracts of infected epithelial cells obtained at each point of the time-course infection (Yamatake et al., 2007) (data not shown). For activation with P. gingivalis-LPS, epithelial cells were washed twice with PBS, and 5 mL of the K-SFM medium containing increasing doses (1 to 10 µg/mL) of P. gingivalis-LPS were added. Cells were cultured for 3 days in humidified air with 5% CO2. Cells without P. gingivalis-LPS served as controls.
Reverse-transcriptase/Polymerase Chain-reaction (RT-PCR)
Immunodetection of Cathepsins B and C and Quantification of Cystatin C The Cystatin C content was estimated on 20 µL of the cellular extracts by enzyme-linked immunosorbent assay (ELISA). Measurements were performed in triplicate.
Enzymatic Activities Assays
Statistical Analysis
Expression of the mRNAs Encoding for Cathepsins B and C and Cystatin C in Human Oral Epithelial Cells Activated by P. gingivalis-LPS or Infected by P. gingivalis When epithelial cells were activated with P. gingivalis-LPS ranging from 1 to 10 µg/mL, we observed a dose-dependent increase over time in the level of Cathepsin B mRNA (Figs. 1A, 1B  ). The increase reached significant levels after 1 day at 10 µg/mL of P. gingivalis-LPS and reached a maximum (threefold) after 3 days. Although the level of Cystatin C mRNA was not as strongly expressed as the mRNA encoding for Cathepsin B, it decreased in a dose-dependent manner (Figs. 1A, 1C). The decrease reached significant levels after 1 day at 2 µg/mL of P. gingivalis-LPS and was almost completed (five-fold) after 3 days with the higher P. gingivalis-LPS dose (10 µg/mL). We did not observe any significant modifications in the expression of Cathepsin C mRNA during the time-course activation of epithelial cells (Fig. 1A).
To verify if these effects were amplified or appeared more rapidly when the whole bacterium was used, we proceeded to a time-course infection of epithelial cells with P. gingivalis at an MOI ranging from 2 to 200 bacteria per cell. The level of Cathepsin B mRNA increased in an MOI-dependent manner (Fig. 2A ), reached significance after 4 hrs at an MOI of 20, and reached an optimum (three-fold) level similar to the one measured during activation. We also observed a dose-dependent decrease in the Cystatin C mRNA level, which reached significant levels after 4 hrs at an MOI of 20 and optimal levels (five-fold) at an MOI of 200 at the same time (Fig. 2B). Here again, we did not observe any significant modifications in the level of Cathepsin C mRNA at any point during the time-course infection (data not shown).
Enzymatic Activities of Cathepsins B and C and Concentration of Cystatin C We determined whether the appearance of an imbalance between Cathepsin B and Cystatin C resulted in an increase of the enzymatic activities of this enzyme and of Cathepsin C. The enzymatic activity of Cathepsin B increased in a dose-dependent manner with either the P. gingivalis-LPS doses or the MOI concentrations (Table ). This increase was in the same range (three-fold) as quantified by RT-PCR on the corresponding mRNA; however, we cannot rule out the possibility that another inhibitor, different from Cystatin C, was produced to minimize this measured increase. The enzymatic activity of Cathepsin C did not vary as a consequence of the decrease in expression of Cystatin C.
The concentration of Cystatin C decreased significantly after 4 hrs of infection and was almost completely undetectable after 24 hrs of activation.
Western Blotting of Cathepsins B and C
With activated epithelial cells, we observed a dose-dependent increase in the intensity of the 24-kDa band corresponding to Cathepsin B (Fig. 3B  ). This increase was significant after 2 days at 2 µg/mL and maximal (three-fold) after 3 days at 10 µg/mL of P. gingivalis-LPS.
Due to the low concentrations of Cystatin C detected in epithelial cells (Table In infected or activated epithelial cells, we did not observe any significant variations in the intensity of the 25-kDa band corresponding to mature Cathepsin C (data not shown).
In human oral epithelial cells, we have demonstrated that P. gingivalis creates an imbalance between Cathepsin B and the inhibitor Cystatin C. This imbalance occurred after 4 hrs of infection by the whole bacterium, while a delay (> 24 hrs) was required when solely its LPS was used, suggesting that other components of the bacterium were needed to initiate this effect. Candidates among these bacterial components are cysteine proteinases, termed gingipains, such as Arginine-specific gingipains (RgpA, RgpB) and Lysine-specific gingipain (Kgp) (Imamura et al., 2003). These enzymes are known to hydrolyze Cystatin C (Abrahamson et al., 1997), at least in vitro, with a probable effect on the physiological control of Cathepsin B, an enzyme whose level in gingival crevicular fluid correlates with pocket depth (Chen et al., 1998). Indeed, in infected human oral epithelial cells, we observed a rapid disappearance of Cystatin C, which probably resulted from an efficient proteolysis initiated by gingipains. However, other hypotheses can explain the activation of this cell type with P. gingivalis-LPS, which contributes to the disappearance (although delayed) or the inhibition of the transcription of Cystatin C mRNA. We have noticed that Cystatin C was expressed at low levels in our tested cell line, thus partly explaining its absence or its low concentration in gingival crevicular fluids (Abrahamson et al., 1997; Blankenvoorde et al., 1997). Our study also showed the enhanced expression of Cathepsin B, which is related to the mRNA level, the enzymatic activity, and the synthesis of new proteins. To be rapidly expressed with time, all these effects were dependent on components of the whole bacterium; activation of epithelial cells by P. gingivalis-LPS alone delayed them without modifying their final levels. The enhanced expression of the enzymatic activity of Cathepsin B resulted not only from the Cystatin C inhibition, but also from a de novo synthesis of the protein, as observed by Western blotting. Among other Cathepsins potentially affected by the decrease of Cystatin C, we also investigated Cathepsin C, due to its implications in severe forms of periodontitis, such as Papillon-Lefèvre syndrome. We did not observe any variation in the expression of this enzyme after infection or activation of oral epithelial cells. Additionally, although expressed at a low level, its enzymatic activity was not affected by the decrease of Cystatin C. This suggests that another type of Cystatin is certainly required for full inhibition of the enzymatic activity of Cathepsin C. To our knowledge, this is the first study showing that a pathogenic bacterium modifies, at least in vitro, the imbalance between Cystatin C and Cathepsin B in oral epithelial cells. A previous study showed an increased expression of this enzyme after infection by P. gingivalis, but this work was done with human aortic endothelial cells in an attempt to correlate periodontitis and atherosclerosis (Yamatake et al., 2007). Besides its involvement in periodontitis, Cathepsin B is implicated not only in other diseases, such as atherosclerosis (Lutgens et al., 2007) and cancer (Gocheva and Joyce, 2007), but also in various cellular events, such as antigen processing (Maekawa et al., 1998) and Cathepsin B-dependent cell death initiated by LPS (Tang et al., 2006).(AQ) Finally, this enzyme has been found to be associated with internalized P. gingivalis during its trafficking toward the endocytic pathway to lysosomes, where it likely contributes to bacterial destruction (Yamatake et al., 2007). It could be of clinical interest to know the precise role(s) of Cathepsin B, as well as other Cathepsins and Cystatins, in the molecular events observed during the progression of periodontitis.
This study was supported by INSERM, the Ministry of Education and Research, and the Université Louis Pasteur of Strasbourg (ERT 1061).
A supplemental appendix to this article is published electronically only at http://jdr.iadrjournals.org/cgi/content/full/87/10/932/DC1. Received for publication January 29, 2008. Revision received May 24, 2008. Accepted for publication June 27, 2008.
Journal of Dental Research, Vol. 87, No. 10,
932-936 (2008)
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