Mapping of DNA methylationsensitive cellular processes in gingival and periodontal ligament fibroblasts in the context of periodontal tissue homeostasis

Interactions between gingival fibroblasts (GFs) and oral pathogens contribute to the chronicity of inflammation in periodontitis. Epigenetic changes in DNA methylation are involved in periodontitis pathogenesis, and recent studies indicate that DNA methyltransferase (DNMT) inhibitors may protect against epithelial barrier disruption and bone resorption. To assess the impact of DNMT inhibition on GFs, cells were cultured with decitabine (5-aza-2’-deoxycytidine, DAC) for 12 days to induce DNA hypomethylation. We observed several potentially detrimental effects of DAC on GF biological functions. First, extended treatment with DAC reduced GF proliferation and induced necrotic cell death. Second, DAC amplified Porphyromonas gingivalis- and cytokine-induced expression and secretion of the chemokine CCL20 and several matrix metalloproteinases (MMPs), including MMP1, MMP9, and MMP13. Similar pro-inflammatory effects of DAC were observed in periodontal ligament fibroblasts. Third, DAC upregulated intercellular adhesion molecule-1 (ICAM-1), which was associated with increased P. gingivalis adherence to GFs and may contribute to bacterial dissemination. Finally, analysis of DAC-induced genes identified by RNA sequencing revealed increased expression of CCL20, CCL5, CCL8, CCL13, TNF, IL1A, IL18, IL33, and CSF3, and showed that the most affected processes were related to immune and inflammatory responses. In contrast, the genes downregulated by DAC were associated with extracellular matrix and collagen fibril organization. Our observations demonstrate that studies of DNMT inhibitors provide important insights into the role of DNA methylation in cells involved in periodontitis pathogenesis. However, the therapeutic potential of hypomethylating agents in periodontal disease may be limited due to their cytotoxic effects on fibroblast populations and stimulation of pro-inflammatory pathways.</p

Utilization of transposon-based system "Sleeping Beauty" in order to express MCPIP2-D196A protein

System SB "Sleeping Beauty" jest mało poznanym systemem służącym do ekspresji białek. Bazuje on na wykorzystaniu właściwości transpozazy oraz transpozonu. Dzięki dostępności różnego rodzaju wektorów (od wektorów konstytutywnych do indukowanych np. tetracykliną) umożliwia on uzyskanie nadekspresji białek o różnych właściwościach (również tych potencjalnie toksycznych dla komórek). W niniejszej pracy wprowadzono do komórek ludzkiej linii astrocytomy U251 system SB w celu uzyskania zmodyfikowanej formy białka MCPIP2. MCPIP2 jest słabo poznanym przedstawicielem rodziny białek MCPIP. Posiada ono między innymi domenę palca cynkowego typu CCCH oraz domenę PIN, która odpowiada za aktywność RNA-zową białka. W niniejszej pracy wykorzystano białko MCPIP2D196A - jest to mutant pozbawiony aktywności RNA-zowej. Skonstruowano wektor konstytucyjny (SBbi) jak i indukowany (SBtet) zawierający fragment kodujący MPCIP2D196A. Uzyskane konstrukty zostały namnożone w komórkach prokariotycznych a następnie wektor SBbi został wykorzystany do transfekcji komórek U251. Stała synteza białka pozbawionego aktywności RNA-zowej nie jest toksyczna dla komórek.The SB "Sleeping Beauty" system is a protein expression system catalyzed by the transposase. The system includes a variety of vectors with constitutive and inducible promoters. Thanks to that diversity, it is possible to express different kinds of proteins, even proteins poisonous for cells. The aim of the performed experiments was to enter the SB system to the U251 astrocytoma cells in order to receive the modified version of the MCPIP2 protein. MCPIP2 is a weakly known protein which belongs to the MCPIP family but it is supposed that it has a role in inflammatory reactions. It has a zinc-finger motif (CCCH type) and PIN domain (responsible for RNase activity). In this research, MCPIP2D196A protein was used – a mutant form that doesn't has RNase activity. Firstly, vectors (both with constitutive and inducible promoter) with MCPIP2D196A-coding fragment were constructed. Next, in prokaryotic cells, those plasmids were amplified.. On the last stage, pSBbi-MCPIP2D196A was used to transect U251 cells. Constant expression of the protein without RNase activity is safe for cells

Mapping of DNA methylation-sensitive cellular processes in gingival and periodontal ligament fibroblasts in the context of periodontal tissue homeostasis

Interactions between gingival fibroblasts (GFs) and oral pathogens contribute to the chronicity of inflammation in periodontitis. Epigenetic changes in DNA methylation are involved in periodontitis pathogenesis, and recent studies indicate that DNA methyltransferase (DNMT) inhibitors may protect against epithelial barrier disruption and bone resorption. To assess the impact of DNMT inhibition on GFs, cells were cultured with decitabine (5-aza-2’-deoxycytidine, DAC) for 12 days to induce DNA hypomethylation. We observed several potentially detrimental effects of DAC on GF biological functions. First, extended treatment with DAC reduced GF proliferation and induced necrotic cell death. Second, DAC amplified Porphyromonas gingivalis- and cytokine-induced expression and secretion of the chemokine CCL20 and several matrix metalloproteinases (MMPs), including MMP1, MMP9, and MMP13. Similar pro-inflammatory effects of DAC were observed in periodontal ligament fibroblasts. Third, DAC upregulated intercellular adhesion molecule-1 (ICAM-1), which was associated with increased P. gingivalis adherence to GFs and may contribute to bacterial dissemination. Finally, analysis of DAC-induced genes identified by RNA sequencing revealed increased expression of CCL20, CCL5, CCL8, CCL13, TNF, IL1A, IL18, IL33, and CSF3, and showed that the most affected processes were related to immune and inflammatory responses. In contrast, the genes downregulated by DAC were associated with extracellular matrix and collagen fibril organization. Our observations demonstrate that studies of DNMT inhibitors provide important insights into the role of DNA methylation in cells involved in periodontitis pathogenesis. However, the therapeutic potential of hypomethylating agents in periodontal disease may be limited due to their cytotoxic effects on fibroblast populations and stimulation of pro-inflammatory pathways

Mapping of DNA methylation-sensitive cellular processes in gingival and periodontal ligament fibroblasts in the context of periodontal tissue homeostasis

Interactions between gingival fibroblasts (GFs) and oral pathogens contribute to the chronicity of inflammation in periodontitis. Epigenetic changes in DNA methylation are involved in periodontitis pathogenesis, and recent studies indicate that DNA methyltransferase (DNMT) inhibitors may protect against epithelial barrier disruption and bone resorption. To assess the impact of DNMT inhibition on GFs, cells were cultured with decitabine (5-aza-2’-deoxycytidine, DAC) for 12 days to induce DNA hypomethylation. We observed several potentially detrimental effects of DAC on GF biological functions. First, extended treatment with DAC reduced GF proliferation and induced necrotic cell death. Second, DAC amplified Porphyromonas gingivalis- and cytokine-induced expression and secretion of the chemokine CCL20 and several matrix metalloproteinases (MMPs), including MMP1, MMP9, and MMP13. Similar pro-inflammatory effects of DAC were observed in periodontal ligament fibroblasts. Third, DAC upregulated intercellular adhesion molecule-1 (ICAM-1), which was associated with increased P. gingivalis adherence to GFs and may contribute to bacterial dissemination. Finally, analysis of DAC-induced genes identified by RNA sequencing revealed increased expression of CCL20, CCL5, CCL8, CCL13, TNF, IL1A, IL18, IL33, and CSF3, and showed that the most affected processes were related to immune and inflammatory responses. In contrast, the genes downregulated by DAC were associated with extracellular matrix and collagen fibril organization. Our observations demonstrate that studies of DNMT inhibitors provide important insights into the role of DNA methylation in cells involved in periodontitis pathogenesis. However, the therapeutic potential of hypomethylating agents in periodontal disease may be limited due to their cytotoxic effects on fibroblast populations and stimulation of pro-inflammatory pathways