Treponema denticolaincreases MMP-2 expression and activation in the periodontium via reversible DNA and histone modifications

Abstract

Host-derived matrix metalloproteinases (MMPs) and bacterial proteases mediate destruction of extracellular matrices and supporting alveolar bone in periodontitis. The Treponema denticola dentilisin protease induces MMP-2 expression and activation in periodontal ligament (PDL) cells, and dentilisin-mediated activation of pro-MMP-2 is required for cellular fibronectin degradation. Here, we report that T. denticola regulates MMP-2 expression through epigenetic modifications in the periodontium. PDL cells were treated with epigenetic enzyme inhibitors before or after T. denticola challenge. Fibronectin fragmentation, MMP-2 expression, and activation were assessed by immunoblot, zymography, and qRT-PCR, respectively. Chromatin modification enzyme expression in T. denticola-challenged PDL cells and periodontal tissues were evaluated using gene arrays. Several classes of epigenetic enzymes showed significant alterations in transcription in diseased tissue and T. denticola-challenged PDL cells. T. denticola-mediated MMP-2 expression and activation were significantly reduced in PDL cells treated with inhibitors of aurora kinases and histone deacetylases. In contrast, DNA methyltransferase inhibitors had little effect, and inhibitors of histone acetyltransferases, methyltransferases, and demethylases exacerbated T. denticola-mediated MMP-2 expression and activation. Chronic epigenetic changes in periodontal tissues mediated by T. denticola or other oral microbes may contribute to the limited success of conventional treatment of chronic periodontitis and may be amenable to therapeutic reversal.

Treponema denticoladentilisin triggered TLR2/MyD88 activation upregulates a tissue destructive program involving MMPs via Sp1 in human oral cells

“…Our RNA-Seq analysis revealed specific gene expression patterns that are associated with distinct pathologic features presented clinically (16), reported in in vivo studies (88) and previous studies from our lab (41,44). We recently reported that short term T. denticola challenge induced chronic upregulation of MMP-2 and 14 expression in vitro as long as 12 days.…”

“…Equivalent protein concentrations from samples were mixed with nonreducing sample buffer (0.25 M tris-base, 40% glycerol, 0.8% sodium dodecyl sulfate (SDS), and 0.05% bromophenol blue stain in distilled deionized water/ddH2O at pH 6.8) and loaded into 8% polyacrylamide gels that were co-polymerized with 0.4% SDS and 0.2% gelatin. Samples were electrophoretically resolved on gelatin-containing gels at 125 V for 110 min at 4 °C using a Power Pack supply (Bio-Rad, USA) Next, gels were washed in a series of buffers to facilitate re-constitution of endogenous protein and stained with Coomassie Blue as previously described (44). Briefly, Gels were then washed twice for 15 min under continuous agitation using renaturation/ washing buffer (2.5% v/v Triton-X100 and 0.05 M Tris-base in ddH2O at pH 7.5) to eliminate SDS and promote the renaturation of MMP enzymes.…”

Epigenetic regulation of inflammation in periodontitis: cellular mechanisms and therapeutic potential

“…These findings indicate that, at least in this model, the amelioration of disease severity by HDACi was unrelated to inflammation. In light of the well-documented ability of [53] TSA, butyrate Upregulation of hBD2, IL-8 and CCL20 in GECs infected with P. gingivalis or F. nucleatum [35] butyrate Suppression of LPS-induced TNF and IL-1β expression and ROS production in PDL cells [56] TSA, apicidin Suppression of T. denticola-induced MMP2 activation in PDL cells [29] TSA, butyrate Upregulation of osteoblast markers and induction of osteogenic differentiation of PDL cells [56,57] 1179.4b Reduction of alveolar bone destruction in experimental periodontitis in mice [69] TSA Reduction of inflammation and increased alveolar bone volume in experimental periodontitis in rats [68] BET proteins I-BET151, JQ1 Suppression of inflammatory mediator production by GECs and gingival fibroblasts [65] JQ1 Amelioration of inflammation and alveolar bone resorption in experimental periodontitis in mice [70] DNMTs AZA Induction of differentiation of gingival fibroblasts into osteoblasts and induction of ectopic bone formation in mice [120] Suppression of T. denticola-induced MMP2 activation in PDL cells [29] AZA, decitabine Modulation of inflammatory cytokine production by GECs [121] RG108 Prevention of P. gingivalis-mediated impairment of GEC barrier function [114] HDACi to suppress osteoclastogenesis [71], and recent evidence that these compounds induce osteogenic differentiation of PDL cells [56,57], the protective effect of 1179.4b may be attributed to the influence of HDACi on alveolar bone regeneration. Indeed, the bone-protective effects of TSA in ligature-induced periodontitis in rats were associated with the ability of HDACi to induce osteogenic differentiation of mesenchymal stem cells [68], though this effect has not been formally demonstrated in vivo.…”

“…Global HDAC expression profiling in the gingival tissue from patients with periodontitis and healthy controls produced conflicting data. Ateia et al [ 29 ] reported reduced expression of several classes of chromatin-modifying enzymes, including class II HDACs in periodontitis-affected gingival tissue, though the differences were not verified at the protein level. Conversely, an independent study identified elevated transcript levels of several HDAC family members, including HDAC1, HDAC5, HDAC8, and HDAC9, in periodontitis patients compared to healthy individuals, though only the increased HDAC1 expression was confirmed at the protein level in immunohistochemical studies of the gingival tissue [ 30 ].…”

Epigenetic regulation of matrix metalloproteinases in inflammatory diseases: a narrative review

“…Histone phosphorylation refers to the process of transferring the phosphoryl group of ATP to histone-specific amino acids through protein phosphokinase. Histone phosphorylation occurs mostly on serine(S), threonine(T) and tyrosine(Y) residues and constitutes an critical part of the "histone code, " or combinatory function of PTMs on chromatin Pretreatment or post-treatment of PDL cells with PF-03814735 (histone phosphorylation inhibitor) significantly prevented or reversed the increase of MMP-2 and the MT1-MMP/TIMP-2 complex mediated by Treponema denticola (T. denticola) [45].…”