Treponema denticola upregulates MMP-2 activation in periodontal ligament cells: interplay between epigenetics and periodontal infection

Abstract

Objective: Periodontal pathogens initiate chronic dysregulation of inflammation and tissue homeostasis that characterize periodontal disease. To better understand oral microbe-host tissue interactions, we investigated expression and activation of MMP-2 in periodontal ligament cells following Treponema denticola challenge.

Design: Cultured PDL cells were challenged with T. denticola, and bacterial adherence, internalization and survival were assayed by immunofluorescence microscopy and antibiotic protection assays, respectively. MMP-2 activation was detected by zymography. MMP-2, MT1/MMP and TIMP-2 expression following T. denticola challenge was determined by qRT-PCR. Promoter methylation of MMP-2 and MT1/MMP was screened by methylation-sensitive restriction analysis and by bisulfite DNA sequencing.

Results: T. denticola adhered to and was internalized by PDL cells but did not survive intracellularly beyond 24h. Importantly, while dentilisin activity in PDL culture supernatants gradually decreased following T. denticola challenge, MMP-2 activation persisted for up to 5 days, suggesting involvement of other regulatory mechanisms. Transcription and expression of MT1/MMP and TIMP-2 increased in response to T. denticola challenge. However, consistent with previously reported constitutive pro-MMP-2 expression in PDL cells, the MMP-2 promoter was hypomethylated, independent of T. denticola challenge.

Conclusions: MMP-2 promoter hypomethylation is consistent with constitutive pro-MMP-2 expression in PDL cells. This, coupled with T. denticola-mediated upregulation of MMP-2-related genes and chronic activation of pro-MMP-2, mimics key in vivo mechanisms of periodontal disease chronicity, in particular MMP-2-dependent matrix degradation and bone resorption. Adherence and/or internalization of T. denticola may contribute to these processes by one or more regulatory mechanisms, including contact-dependent signal transduction or other epigenetic mechanisms.

Keywords: Epigenetics; MMP-2; MT1/MMP; Proteases; TIMP-2; Treponema.

Figure 1. Zymograms showing gelatinase activity of T. denticola dentilisin protease, pro-MMP-2 and activated MMP-2

PDL cells were challenged with T. denticola at MOI=100 for 2h, washed twice in PBS and incubated in serum- and antibiotic-free medium with daily changes. Panel A: Gelatinase activity in equal volumes of conditioned medium collected on the indicated days following T. denticola challenge. Panel B: Gelatinase activity in lysates of PDL cells collected on the indicated days following T. denticola challenge and medium replacement as in Panel A. Equal amounts of protein were loaded per lane. The locations of the active dentilisin complex (95-100 kDa), 72-kDa pro-MMP-2 and 64-kDa activated MMP-2 are indicated, as are the positions of relative molecular mass markers in kDa.

Figure 2. T. denticola adherence to and uptake by PDL cells

Panels A and B: T. denticola at MOI=100 was added to PDL cultures for 2h, after which PDL cells were treated (“uptake”) or not treated (“adherence + uptake”) with 200 μg ml^-1 gentamicin for 1h to kill extracellular bacteria. After washing and incubation in fresh αMEM for the indicated times, PDL cells were lysed with sterile water, and lysates were mixed with NOS semisolid medium and incubated anaerobically at 37°C. Panel A: T. denticola colony forming units recovered per well of PDL cells (approximately 10⁵ cells) after 0, 7 and 24 h post-challenge incubation. The data represent two independent experiments conducted in triplicate. Panel B: T. denticola colonies recovered from PDL cell lysates in a representative experiment. Panel C: Immunofluorescence microscopy of PDL cells with or without 2h T. denticola challenge (2h, MOI=100) followed by washes, with or without further incubation in culture medium and membrane permeabilization. Slides were probed with rabbit anti-T. denticola Msp IgG followed by Alexa 555-conjugated goat anti-rabbit IgG to detect T. denticola and phalloidin-647 to detect cytoskeletal actin in PDL cells. Panel D: Imunofluorescence microscopy of PDL cells challenged with T. denticola (2h, MOI=100) followed by washes and membrane permeabilization, probed with rabbit anti-T. denticola whole cell IgG and mouse anti-LAMP1 IgG followed by fluor-conjugated secondary antibodies.

Figure 3. Expression of MMP-2, MT1/MMP and TIMP-2 following T. denticola challenge

Panels A and B: Transcript levels in PDL cells after T. denticola challenge and incubation in fresh medium for indicated times, assayed by qRT-PCR. The Y-axis in each panel represents fold-expression level of each gene relative to unchallenged control at day 1 shown in Panel A. Panel A: gene expression after 2h T. denticola challenge and incubation in fresh medium for 1, 3 or 5 days. Panel B: gene expression after 4h, 8h or 24h T. denticola challenge and incubation in fresh medium for 24h. Data were analyzed using Student's t-test. Panel C: Western blots showing levels of MT1-MMP expression in PDL cells treated with purified dentilisin (50 ng/ml, 2h) or media control (Ctrl), then washed and maintained in fresh media for 5 days (MT1-MMP1 antibody; Abcam; actin antibody). Panel D: Western blot of TIMP-2 expression in PDL cells (conditioned media) treated for 2h with wildtype T. denticola, T. denticola dentilisin mutant (Td-ΔP) or media control (Ctrl), then washed and maintained in antibiotic supplemented media for 5 days (TIMP-2 antibody, Triple Point Biologics).

Figure 4. Analysis of MMP-2 promoter methylation in PDL cells following T. denticola challenge

PDL cells were challenged with T. denticola for 2h, washed, and incubated in fresh medium for 24 h prior to analysis. The vertical scale indicates percent of total DNA. Panel A: methylation analysis of the MMP-2 promoter region as determined by MethylDNA Restriction Screen (Qiagen). Results are expressed as the percentage of hyper-methylated and unmethylated DNA, which sum to 100%. Data were analyzed using Student's t-test. Panel B: DNA methylation analysis of the MMP-2 promoter region as determined by sequencing of independent PCR clones of bisulfite-converted DNA from T. denticola challenged and unchallenged PDL cell cultures. Each circle represents an individual CpG within residues 236-456 of MMP2-exon 1-transcript variant 1. Open and closed circles indicate unmethylated and methylated CpGs, respectively within residues 236-456 of MMP2-exon 1.