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TNF-α inhibits aquaporin 5 expression in human salivary gland acinar cells via suppression of histone H4 acetylation.

Yamamura Y, Motegi K, Kani K, Takano H, Momota Y, Aota K, Yamanoi T, Azuma M - J. Cell. Mol. Med. (2012)

Bottom Line: The mechanisms underlying these reductions remain unclear.TNF-α-treatment of NS-SV-AC cells significantly suppressed the expression levels of AQP5 mRNA and protein, and reduced the net fluid secretion rate.However, interestingly, chromatin immunoprecipitation analysis demonstrated a remarkable decrease in levels of acetylated histone H4 associated with the AQP5 gene promoter after treatment with TNF-α in NS-SV-AC cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Medicine, Institute of Health Biosciences, The University of Tokushima Graduate Faculty of Dentistry, Tokushima, Japan.

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Expression and activation levels of DNA methyltransferases in TNF-α-treated NS-SV-AC acinar cells. (A) RT-PCR analysis of the expression of Dnmt1, Dnmt3a and Dnmt3b mRNAs in TNF-α-treated NS-SV-AC acinar cells. NS-SV-AC acinar cells expressed Dnmt1, Dnmt3a and Dnmt3b mRNAs under the basal condition. After exposure to 10 ng/ml TNF-α, the expression levels of the three Dnmt mRNAs showed no significant changes. (B) Western blot analysis of Dnmt1, Dnmt3a and Dnmt3b proteins in TNF-α-treated NS-SV-AC acinar cells. There were no marked increases in the expression levels of these three Dnmts with TNF-α treatment. These results were similar to those observed by RT-PCR analysis. (C) Methylation activity assay of TNF-α-treated NS-SV-AC acinar cells. The methylation activity of TNF-α-treated NS-SV-AC cells was measured by using the EpiQuik™ DNA Methyltransferase Activity Assay Kit. The results are expressed as optical density at a wavelength of 450 nm, and are the means ± S.D. of three separate experiments. The results were analysed using the Mann–Whitney U-test. *P < 0.05 compared with control cells.
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fig03: Expression and activation levels of DNA methyltransferases in TNF-α-treated NS-SV-AC acinar cells. (A) RT-PCR analysis of the expression of Dnmt1, Dnmt3a and Dnmt3b mRNAs in TNF-α-treated NS-SV-AC acinar cells. NS-SV-AC acinar cells expressed Dnmt1, Dnmt3a and Dnmt3b mRNAs under the basal condition. After exposure to 10 ng/ml TNF-α, the expression levels of the three Dnmt mRNAs showed no significant changes. (B) Western blot analysis of Dnmt1, Dnmt3a and Dnmt3b proteins in TNF-α-treated NS-SV-AC acinar cells. There were no marked increases in the expression levels of these three Dnmts with TNF-α treatment. These results were similar to those observed by RT-PCR analysis. (C) Methylation activity assay of TNF-α-treated NS-SV-AC acinar cells. The methylation activity of TNF-α-treated NS-SV-AC cells was measured by using the EpiQuik™ DNA Methyltransferase Activity Assay Kit. The results are expressed as optical density at a wavelength of 450 nm, and are the means ± S.D. of three separate experiments. The results were analysed using the Mann–Whitney U-test. *P < 0.05 compared with control cells.

Mentions: We have previously demonstrated that an immortalized NS-SV-DC clone that lacks AQP5 expression acquires AQP5 gene expression in response to treatment with 5-aza-2′ -deoxycytidine (5-Aza-CdR) via the hypomethylation of the AQP5 promoter region [16]. Because hypermethylation of the promoter region of the target genes is known to inhibit gene expression, we hypothesized that the suppression of AQP5 in the TNF-α-treated cells may have been caused by the induction of DNA methyltransferases. Therefore, we examined the expression levels of three Dnmt members: Dnmt1, Dnmt3a and Dnmt3b, in NS-SV-AC cells by using both RT-PCR and Western blot analyses. As shown in Figure 3A and B, no significant changes were observed in the mRNA and protein expression levels of the three Dnmts in the TNF-α-treated NS-SV-AC cells. Although TNF-α did not induce an up-regulation of Dnmt mRNA or protein expression, it is possible that TNF-α activates Dnmt enzymatic activity. Thus, we investigated Dnmt activity by using a DNA Methyltransferase Activity Kit. As shown in Figure 3C, no changes in enzymatic activity were detected in the TNF-α-treated NS-SV-AC cells. This suggests that DNA methylation is not involved in the TNF-α-induced suppression of AQP5 expression in NS-SV-AC cells.


TNF-α inhibits aquaporin 5 expression in human salivary gland acinar cells via suppression of histone H4 acetylation.

Yamamura Y, Motegi K, Kani K, Takano H, Momota Y, Aota K, Yamanoi T, Azuma M - J. Cell. Mol. Med. (2012)

Expression and activation levels of DNA methyltransferases in TNF-α-treated NS-SV-AC acinar cells. (A) RT-PCR analysis of the expression of Dnmt1, Dnmt3a and Dnmt3b mRNAs in TNF-α-treated NS-SV-AC acinar cells. NS-SV-AC acinar cells expressed Dnmt1, Dnmt3a and Dnmt3b mRNAs under the basal condition. After exposure to 10 ng/ml TNF-α, the expression levels of the three Dnmt mRNAs showed no significant changes. (B) Western blot analysis of Dnmt1, Dnmt3a and Dnmt3b proteins in TNF-α-treated NS-SV-AC acinar cells. There were no marked increases in the expression levels of these three Dnmts with TNF-α treatment. These results were similar to those observed by RT-PCR analysis. (C) Methylation activity assay of TNF-α-treated NS-SV-AC acinar cells. The methylation activity of TNF-α-treated NS-SV-AC cells was measured by using the EpiQuik™ DNA Methyltransferase Activity Assay Kit. The results are expressed as optical density at a wavelength of 450 nm, and are the means ± S.D. of three separate experiments. The results were analysed using the Mann–Whitney U-test. *P < 0.05 compared with control cells.
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fig03: Expression and activation levels of DNA methyltransferases in TNF-α-treated NS-SV-AC acinar cells. (A) RT-PCR analysis of the expression of Dnmt1, Dnmt3a and Dnmt3b mRNAs in TNF-α-treated NS-SV-AC acinar cells. NS-SV-AC acinar cells expressed Dnmt1, Dnmt3a and Dnmt3b mRNAs under the basal condition. After exposure to 10 ng/ml TNF-α, the expression levels of the three Dnmt mRNAs showed no significant changes. (B) Western blot analysis of Dnmt1, Dnmt3a and Dnmt3b proteins in TNF-α-treated NS-SV-AC acinar cells. There were no marked increases in the expression levels of these three Dnmts with TNF-α treatment. These results were similar to those observed by RT-PCR analysis. (C) Methylation activity assay of TNF-α-treated NS-SV-AC acinar cells. The methylation activity of TNF-α-treated NS-SV-AC cells was measured by using the EpiQuik™ DNA Methyltransferase Activity Assay Kit. The results are expressed as optical density at a wavelength of 450 nm, and are the means ± S.D. of three separate experiments. The results were analysed using the Mann–Whitney U-test. *P < 0.05 compared with control cells.
Mentions: We have previously demonstrated that an immortalized NS-SV-DC clone that lacks AQP5 expression acquires AQP5 gene expression in response to treatment with 5-aza-2′ -deoxycytidine (5-Aza-CdR) via the hypomethylation of the AQP5 promoter region [16]. Because hypermethylation of the promoter region of the target genes is known to inhibit gene expression, we hypothesized that the suppression of AQP5 in the TNF-α-treated cells may have been caused by the induction of DNA methyltransferases. Therefore, we examined the expression levels of three Dnmt members: Dnmt1, Dnmt3a and Dnmt3b, in NS-SV-AC cells by using both RT-PCR and Western blot analyses. As shown in Figure 3A and B, no significant changes were observed in the mRNA and protein expression levels of the three Dnmts in the TNF-α-treated NS-SV-AC cells. Although TNF-α did not induce an up-regulation of Dnmt mRNA or protein expression, it is possible that TNF-α activates Dnmt enzymatic activity. Thus, we investigated Dnmt activity by using a DNA Methyltransferase Activity Kit. As shown in Figure 3C, no changes in enzymatic activity were detected in the TNF-α-treated NS-SV-AC cells. This suggests that DNA methylation is not involved in the TNF-α-induced suppression of AQP5 expression in NS-SV-AC cells.

Bottom Line: The mechanisms underlying these reductions remain unclear.TNF-α-treatment of NS-SV-AC cells significantly suppressed the expression levels of AQP5 mRNA and protein, and reduced the net fluid secretion rate.However, interestingly, chromatin immunoprecipitation analysis demonstrated a remarkable decrease in levels of acetylated histone H4 associated with the AQP5 gene promoter after treatment with TNF-α in NS-SV-AC cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Medicine, Institute of Health Biosciences, The University of Tokushima Graduate Faculty of Dentistry, Tokushima, Japan.

Show MeSH
Related in: MedlinePlus