Modulatory mechanisms on the inflammation and muscarinic receptor function in salivary gland epithelial cells

Abstract

Salivary gland epithelial cells (SGEC) release several cytokines that play important roles in the inflammatory process. Muscarinic receptors, particularly the type 3 subtype (M3R), play an important role in exocrine secretion. However, the regulatory mechanism of the inflammation and the functional expression of M3R in SGEC remains to be elucidated. In chapterⅠ, I examined whether capsaicin can modulate the cytokine release in SGEC. These findings demonstrated that the increases in TNFα and IL-6 mRNA transcripts were highest at 3h and 1h after incubation with poly(I:C) and LPS, respectively. Pretreatment of the cells with 10 μΜ capsaicin, however, significantly inhibited mRNA transcripts and its protein levels. The simultaneous application of 10 μΜ capsazepine with capsaicin did not block the inhibitory effect of capsaicin. Furthermore, the inhibitory effect of capsaicin was also shown in primary cultured cells from TRPV1−/− mice. I found that both poly(I:C) and LPS induce IκB-α degradation and phosphorylation, which results in NF-κB activation and capsaicin inhibits this NF-κB pathway. These results demonstrate that SGEC release pro-inflammatory cytokines by TLR stimulation, and capsaicin inhibits this process by inhibiting the NF-κB pathway. In chapter Ⅱ, I examined A253 cells derived from human salivary gland tumor tissue, in which muscarinic receptor function is suppressed. In this study, I examined whether M3R function is suppressed by epigenetic modulation of the receptor. I found that A253 cells expressed all subtypes of muscarinic receptors, except subtype 3, at the mRNA and protein level. However, treatment of cells with 5-aza-2'-deoxycytidine (5-Aza-CdR) restored the functional expression of the M3R. Treatment of cells with 5-Aza-CdR completely restored the carbachol-induced calcium response, which was not observed in untreated A253 cells. Global methylation levels in A253 cells were also reduced by 5-Aza-CdR-treatment. I also examined whether 5-Aza-CdR treatment induced demethylation of the M3R CpG island, and found that one of the methylated CGs was demethylated by bisulfite sequencing. Thus, I conclude that suppression of M3R function in A253 cells results from hypermethylation of the CpG island

moreover, M3R function can be restored by DNA demethylation. This study suggests that capsaicin could potentially alleviate the inflammation and 5-Aza-CdR could potentially be used to restore function to the M3R, which is suppressed in salivary gland epithelial cells.