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Epigenetic regulation of human β-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria.

Yin L, Chung WO - Mucosal Immunol (2011)

Bottom Line: Pretreatment with trichostatin A and sodium butyrate, which increase acetylation of chromatin histones, significantly enhanced the gene expression of antimicrobial proteins human β-defensin 2 (hBD2) and CC chemokine ligand 20 (CCL20) in response to both bacterial challenges.Furthermore, we observed a differential pattern of protein levels of H3K4me3, which has been associated with chromatin remodeling and activation of gene transcription, in response to P. gingivalis vs.F. nucleatum.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Biology, University of Washington, Seattle, Washington, USA. leiyin@u.washington.edu

ABSTRACT
Gingival epithelia utilize multiple signaling pathways to regulate innate immune responses to various oral bacteria, but little is understood about how these bacteria alter epithelial epigenetic status. In this study we report that DNA methyltransferase (DNMT1) and histone deacetylase expression were decreased in gingival epithelial cells treated with oral pathogen Porphyromonas gingivalis and nonpathogen Fusobacterium nucleatum. Pretreatment with trichostatin A and sodium butyrate, which increase acetylation of chromatin histones, significantly enhanced the gene expression of antimicrobial proteins human β-defensin 2 (hBD2) and CC chemokine ligand 20 (CCL20) in response to both bacterial challenges. Pretreatment with DNMT inhibitor 5'-azacytidine increased hBD2 and CCL20 expression in response to F. nucleatum, but not to P. gingivalis. Furthermore, we observed a differential pattern of protein levels of H3K4me3, which has been associated with chromatin remodeling and activation of gene transcription, in response to P. gingivalis vs. F. nucleatum. This study provides a new insight into the bacteria-specific innate immune responses via epigenetic regulation.

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Differential mRNA expression of HDAC1, HDAC2 and DNMT1 in human TERT cells in response to oral bacteria. Differential mRNA expression of (a) histone deacetylases 1 and 2 (HDAC1 and HDAC2) and (b) DNA methyltransferase (DNMT1) in human TERT cells in response to Porphyromonas gingivalis vs. Fusobacterium nucleatum. Human TERT cells were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 10:1, 50:1, and 100:1 for 4 or 24 h. Unstimulated cells at 4 and 24 h served as controls. Changes in mRNA expression were evaluated by quantitative real-time PCR (QRT-PCR) and results are expressed as fold change in gene expression compared with the corresponding unstimulated controls (4 and 24 h) after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). The experiment was repeated twice using TERT cells. Error bars indicate s.e.m. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (*P<0.05, **P<0.01).
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fig1: Differential mRNA expression of HDAC1, HDAC2 and DNMT1 in human TERT cells in response to oral bacteria. Differential mRNA expression of (a) histone deacetylases 1 and 2 (HDAC1 and HDAC2) and (b) DNA methyltransferase (DNMT1) in human TERT cells in response to Porphyromonas gingivalis vs. Fusobacterium nucleatum. Human TERT cells were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 10:1, 50:1, and 100:1 for 4 or 24 h. Unstimulated cells at 4 and 24 h served as controls. Changes in mRNA expression were evaluated by quantitative real-time PCR (QRT-PCR) and results are expressed as fold change in gene expression compared with the corresponding unstimulated controls (4 and 24 h) after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). The experiment was repeated twice using TERT cells. Error bars indicate s.e.m. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (*P<0.05, **P<0.01).

Mentions: We first investigated any changes in the expression levels of chromatin-remodeling enzymes after GECs were exposed to oral bacteria. HDACs remove acetyl groups from histone, leading to suppression of genes. DNMTs catalyze transfer of methyl groups onto DNA, which also leads to gene suppression. First, the expression levels of HDAC1, HDAC2, and DNMT1 were investigated in human immortalized keratinocyte cell line (TERT) at different time points (4 and 24 h) and multiplicities of infection (MOIs; 10:1 and 100:1). Quantitative real-time PCR (QRT-PCR) analyses showed changes in the expression levels of DNMT1, HDAC1, and HDAC2 when TERT cells were treated with P. gingivalis (a pathogen) or F. nucleatum (a bridging organism between pathogens and nonpathogens) at MOIs of 10:1 and 100:1 (Figure 1) for 4 and 24 h. The gene expression of HDAC1 was decreased significantly at MOI 100:1 for 24 h in cells treated with P. gingivalis, but not with F. nucleatum. The gene expression of HDAC2 slightly increased with F. nucleatum at 4 h for MOI 10:1. Significant decreases of DNMT1 gene expression levels were observed in TERT cells treated with both bacteria at MOI of 100:1 for 24 h. As more significant changes were observed at 24 h, we further compared these results from human immortalized cell line with human primary GECs at 24 h and various MOIs (Figure 2). The expression level of DNMT1 decreased in response to both P. gingivalis and F. nucleatum (P=0.003 and 0.006, respectively; Figure 2a). This decrease was observed at all MOIs tested in response to both P. gingivalis and F. nucleatum. This decrease in DNMT1 gene expression was dose dependent in GECs in response to F. nucleatum. The gene expression level of HDAC1 significantly decreased in GECs when the cells were treated with P. gingivalis for 24 h at all MOIs compared with unstimulated control (P=0.001; Figure 2b). On the other hand, the expression of HDAC1 significantly decreased in GECs to the lowest level when treated with F. nucleatum for 24 h at MOI 10:1 (P=0.03; Figure 2b). The gene expression of HDCA2 significantly decreased in GECs in response to F. nucleatum at MOIs 50:1 and 100:1, whereas only at MOI 100:1 in response to P. gingivalis (Figure 2c). The decrease of DNMT1 and HDAC2 gene expression showed similar trends in GECs compared with what we observed in TERT cells, whereas the expression of HDAC1 in response to P. gingivalis and F. nucleatum differed between the two cell types. These data indicate that the gene expression of these chromatin-remodeling enzymes may have cell type-specific responses.


Epigenetic regulation of human β-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria.

Yin L, Chung WO - Mucosal Immunol (2011)

Differential mRNA expression of HDAC1, HDAC2 and DNMT1 in human TERT cells in response to oral bacteria. Differential mRNA expression of (a) histone deacetylases 1 and 2 (HDAC1 and HDAC2) and (b) DNA methyltransferase (DNMT1) in human TERT cells in response to Porphyromonas gingivalis vs. Fusobacterium nucleatum. Human TERT cells were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 10:1, 50:1, and 100:1 for 4 or 24 h. Unstimulated cells at 4 and 24 h served as controls. Changes in mRNA expression were evaluated by quantitative real-time PCR (QRT-PCR) and results are expressed as fold change in gene expression compared with the corresponding unstimulated controls (4 and 24 h) after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). The experiment was repeated twice using TERT cells. Error bars indicate s.e.m. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (*P<0.05, **P<0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig1: Differential mRNA expression of HDAC1, HDAC2 and DNMT1 in human TERT cells in response to oral bacteria. Differential mRNA expression of (a) histone deacetylases 1 and 2 (HDAC1 and HDAC2) and (b) DNA methyltransferase (DNMT1) in human TERT cells in response to Porphyromonas gingivalis vs. Fusobacterium nucleatum. Human TERT cells were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 10:1, 50:1, and 100:1 for 4 or 24 h. Unstimulated cells at 4 and 24 h served as controls. Changes in mRNA expression were evaluated by quantitative real-time PCR (QRT-PCR) and results are expressed as fold change in gene expression compared with the corresponding unstimulated controls (4 and 24 h) after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). The experiment was repeated twice using TERT cells. Error bars indicate s.e.m. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (*P<0.05, **P<0.01).
Mentions: We first investigated any changes in the expression levels of chromatin-remodeling enzymes after GECs were exposed to oral bacteria. HDACs remove acetyl groups from histone, leading to suppression of genes. DNMTs catalyze transfer of methyl groups onto DNA, which also leads to gene suppression. First, the expression levels of HDAC1, HDAC2, and DNMT1 were investigated in human immortalized keratinocyte cell line (TERT) at different time points (4 and 24 h) and multiplicities of infection (MOIs; 10:1 and 100:1). Quantitative real-time PCR (QRT-PCR) analyses showed changes in the expression levels of DNMT1, HDAC1, and HDAC2 when TERT cells were treated with P. gingivalis (a pathogen) or F. nucleatum (a bridging organism between pathogens and nonpathogens) at MOIs of 10:1 and 100:1 (Figure 1) for 4 and 24 h. The gene expression of HDAC1 was decreased significantly at MOI 100:1 for 24 h in cells treated with P. gingivalis, but not with F. nucleatum. The gene expression of HDAC2 slightly increased with F. nucleatum at 4 h for MOI 10:1. Significant decreases of DNMT1 gene expression levels were observed in TERT cells treated with both bacteria at MOI of 100:1 for 24 h. As more significant changes were observed at 24 h, we further compared these results from human immortalized cell line with human primary GECs at 24 h and various MOIs (Figure 2). The expression level of DNMT1 decreased in response to both P. gingivalis and F. nucleatum (P=0.003 and 0.006, respectively; Figure 2a). This decrease was observed at all MOIs tested in response to both P. gingivalis and F. nucleatum. This decrease in DNMT1 gene expression was dose dependent in GECs in response to F. nucleatum. The gene expression level of HDAC1 significantly decreased in GECs when the cells were treated with P. gingivalis for 24 h at all MOIs compared with unstimulated control (P=0.001; Figure 2b). On the other hand, the expression of HDAC1 significantly decreased in GECs to the lowest level when treated with F. nucleatum for 24 h at MOI 10:1 (P=0.03; Figure 2b). The gene expression of HDCA2 significantly decreased in GECs in response to F. nucleatum at MOIs 50:1 and 100:1, whereas only at MOI 100:1 in response to P. gingivalis (Figure 2c). The decrease of DNMT1 and HDAC2 gene expression showed similar trends in GECs compared with what we observed in TERT cells, whereas the expression of HDAC1 in response to P. gingivalis and F. nucleatum differed between the two cell types. These data indicate that the gene expression of these chromatin-remodeling enzymes may have cell type-specific responses.

Bottom Line: Pretreatment with trichostatin A and sodium butyrate, which increase acetylation of chromatin histones, significantly enhanced the gene expression of antimicrobial proteins human β-defensin 2 (hBD2) and CC chemokine ligand 20 (CCL20) in response to both bacterial challenges.Furthermore, we observed a differential pattern of protein levels of H3K4me3, which has been associated with chromatin remodeling and activation of gene transcription, in response to P. gingivalis vs.F. nucleatum.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Biology, University of Washington, Seattle, Washington, USA. leiyin@u.washington.edu

ABSTRACT
Gingival epithelia utilize multiple signaling pathways to regulate innate immune responses to various oral bacteria, but little is understood about how these bacteria alter epithelial epigenetic status. In this study we report that DNA methyltransferase (DNMT1) and histone deacetylase expression were decreased in gingival epithelial cells treated with oral pathogen Porphyromonas gingivalis and nonpathogen Fusobacterium nucleatum. Pretreatment with trichostatin A and sodium butyrate, which increase acetylation of chromatin histones, significantly enhanced the gene expression of antimicrobial proteins human β-defensin 2 (hBD2) and CC chemokine ligand 20 (CCL20) in response to both bacterial challenges. Pretreatment with DNMT inhibitor 5'-azacytidine increased hBD2 and CCL20 expression in response to F. nucleatum, but not to P. gingivalis. Furthermore, we observed a differential pattern of protein levels of H3K4me3, which has been associated with chromatin remodeling and activation of gene transcription, in response to P. gingivalis vs. F. nucleatum. This study provides a new insight into the bacteria-specific innate immune responses via epigenetic regulation.

Show MeSH
Related in: MedlinePlus