• International Journal of Oral Biology
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• v.46 no.4
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• pp.190-199
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• 2021

Despite evidence that bacteria-sensing Toll-like receptors (TLRs) are activated in salivary gland tissues of Sjogren syndrome (SS) patients, the role of oral bacteria in SS etiopathogenesis is unclear. We previously reported that two SS-associated oral bacteria, Prevotella melaninogenica (Pm) and Rothia mucilagenosa (Rm), oppositely regulate the expression of major histocompatibility complex class I (MHC I) in human salivary gland (HSG) cells. Here, we elucidated the mechanisms underlying the differential regulation of MHC I expression by these bacteria. The ability of Pm and Rm to activate TLR2, TLR4, and TLR9 was examined using TLR reporter cells. HSG cells were stimulated by the TLR ligands, Pm, and Rm. The levels of MHC I expression, bacterial invasion, and viability of HSG cells were examined by flow cytometry. The hypoxic status of HSG cells was examined using Hypoxia Green. HSG cells upregulated MHC I expression in response to TLR2, TLR4, and TLR9 activation. Both Pm and Rm activated TLR2 and TLR9 but not TLR4. Rm-induced downregulation of MHC I strongly correlated with bacterial invasion and cell death. Rm-induced cell death was not rescued by inhibitors of the diverse cell death pathways but was associated with hypoxia. In conclusion, Pm upregulated MHC I likely through TLR2 and TLR9 activation, while Rm-induced hypoxia-associated cell death and the downregulation of MHC I, despite its ability to activate TLR2 and TLR9. These findings may provide new insight into how oral dysbiosis can contribute to salivary gland tissue damage in SS.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.4
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• pp.235-240
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• 2010

Toll-like receptors (TLRs) functionally expressed in salivary epithelial cells, but their roles remain elusive. Among TLRs family, TLR3 is activated by dsRNA, a byproduct of viral infection. The aim of this study was to investigate the role of TLR3 in the inflammatory immune responses using HSG cells. Reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR and ELISA were performed to identify expression of TLRs and TLR3-mediated chemokine inductions. The chemotaxis assay of activated T lymphocytes was also performed. Treatment of HSG cells with polyinosinic: polycytidylic acid (poly(I:C)) significantly increased interferon-$\gamma$-inducible protein 10 (IP-10), interferoninducible T-cell $\alpha$ chemoattractant (I-TAC), and regulated on activation, normal T-cells expressed and secreted (RANTES) gene expressions in a concentration-dependent manner. Anti-TLR3 antibody blocked the increases of IP-10 and I-TAC genes. Poly(I:C)-induced increases of IP-10 and I-TAC were also confirmed at protein levels from cell lysates, but their release into extracellular medium was detected only in IP-10. We found that the culture media from HSG cells stimulated with poly(I:C) significantly increases T lymphocyte migration. Our results suggest that TLR3 plays an important role in chemokine induction, particularly IP-10, in salivary epithelial cells.

• International Journal of Oral Biology
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• v.44 no.2
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• pp.62-70
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• 2019

Xylitol is well-known to have an anti-caries effect by inhibiting the replication of cariogenic bacteria. In addition, xylitol enhances saliva secretion. However, the precise molecular mechanism of xylitol on saliva secretion is yet to be elucidated. Thus, in this study, we aimed to investigate the stimulatory effect of xylitol on saliva secretion and to further evaluate the involvement of xylitol in muscarinic type 3 receptor (M3R) signaling. For determining these effects, we measured the saliva flow rate following xylitol treatment in healthy individuals and patients with dry mouth. We further tested the effects of xylitol on M3R signaling in human salivary gland (HSG) cells using real-time quantitative reverse-transcriptase polymerase chain reaction, immunoblotting, and immunostaining. Xylitol candy significantly increased the salivary flow rate and intracellular calcium release in HSG cells via the M3R signaling pathway. In addition, the expressions of M3R and aquaporin 5 were induced by xylitol treatment. Lastly, we investigated the distribution of M3R and aquaporin 5 in HSG cells. Xylitol was found to activate M3R, thereby inducing increases in $Ca^{2+}$ concentration. Stimulation of the muscarinic receptor induced by xylitol activated the internalization of M3R and subsequent trafficking of aquaporin 5. Taken together, these findings suggest a molecular mechanism for secretory effects of xylitol on salivary epithelial cells.

• Journal of the korean academy of Pediatric Dentistry
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• v.35 no.1
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• pp.47-56
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• 2008

Risperidone is a widely prescribed atypical antipsychotic agent. Approved by the FDA as the first drug to treat irritability associated with autism in children, it is also used to treat tic disorder and Tourette's syndrome. Its adverse reactions related to dentistry include dry mouth, the mechanism of which is yet to be identified. The aim of this study is to identify, at the cellular level, how and to what extent risperidone affects intracellular free calcium concentration ($[Ca^{2+}]_i$), an primary intracellular factor in the regulation of fluid secretion in salivary gland cells. The human salivary gland cell line (HSG) was grown in MEM supplemented with 10% BCS. In order to measure $[Ca^{2+}]_i$, Fura-2/AM was loaded in the HSG, and fluorescence at 340 nm/380 nm excitation was measured in the 500 nm emission ratio. After every experiment, a calibration experiment was conducted in order to readjust the ratio to the actual $[Ca^{2+}]_i$. Changes in $[Ca^{2+}]_i$ were measured in the presence of carbachol, ATP and histamine. The researcher then explored how the pretreatment of risperidone affected such changes. Findings of this study include: 1. In HSG, $[Ca^{2+}]_i$ increased due to the addition of carbachol, ATP and histamine. The presence of risperidone inhibited the action of histamine on this process, while making little effect on that of carbachol and ATP. 2. A quantification of $[Ca^{2+}]_i$ in relation to histamine of different concentrations indicates that the effect of histamine was concentration dependent with an $EC_{50}$ of $3.3{\pm}0.5\;{\mu}M$. 3. The inhibitory effect of risperidone on histamine-induced $[Ca^{2+}]_i$ was concentration-dependent with an $IC_{50}$ of $104.4{\pm}14\;nM$. 4. Risperidone inhibits histamine-induced Ca2+ release from endoplasmic reticulum and influx of extracellular $Ca^{2+}$ in HSG cells(p<0.05).

• The Korean Journal of Physiology and Pharmacology
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• v.19 no.3
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• pp.249-255
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• 2015

Wnk kinase maintains cell volume, regulating various transporters such as sodium-chloride cotransporter, potassium-chloride cotransporter, and sodium-potassium-chloride cotransporter 1 (NKCC1) through the phosphorylation of oxidative stress responsive kinase 1 (OSR1) and STE20/SPS1-related proline/alanine-rich kinase (SPAK). However, the activating mechanism of Wnk kinase in specific tissues and specific conditions is broadly unclear. In the present study, we used a human salivary gland (HSG) cell line as a model and showed that $Ca^{2+}$ may have a role in regulating Wnk kinase in the HSG cell line. Through this study, we found that the HSG cell line expressed molecules participating in the WNK-OSR1-NKCC pathway, such as Wnk1, Wnk4, OSR1, SPAK, and NKCC1. The HSG cell line showed an intracellular $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) increase in response to hypotonic stimulation, and the response was synchronized with the phosphorylation of OSR1. Interestingly, when we inhibited the hypotonically induced $[Ca^{2+}]_i$ increase with nonspecific $Ca^{2+}$ channel blockers such as 2-aminoethoxydiphenyl borate, gadolinium, and lanthanum, the phosphorylated OSR1 level was also diminished. Moreover, a cyclopiazonic acid-induced passive $[Ca^{2+}]_i$ elevation was evoked by the phosphorylation of OSR1, and the amount of phosphorylated OSR1 decreased when the cells were treated with BAPTA, a $Ca^{2+}$ chelator. Finally, through that process, NKCC1 activity also decreased to maintain the cell volume in the HSG cell line. These results indicate that $Ca^{2+}$ may regulate the WNK-OSR1 pathway and NKCC1 activity in the HSG cell line. This is the first demonstration that indicates upstream $Ca^{2+}$ regulation of the WNK-OSR1 pathway in intact cells.

• Journal of Life Science
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• v.21 no.4
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• pp.529-533
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• 2011

Metallothioneins (MT) are a group of low-molecular weight, cysteine-rich, intracellular proteins that are encoded by a family of genes containing at least 10 functional isoforms in human. The expression and induction of these proteins is associated with protection against DNA damage, oxidative stress, and apoptosis. Many studies have shown increased expression of MT in various human tumors, whereas MT is down-regulated in certain tumors such as hepatocellular carcinoma and liver adenocarcinoma. Hence, the expression of MT is not universal to all human tumors but may depend on the differentiation status and proliferative index of tumors, along with other tissue factors and gene mutations. Using Northern blot analysis, we found that laminin induced expression of MT-1 in HSG and PC12 cells, which can be differentiated by laminin, but had no effect on MB-231, MDA-435, and PC-3 cells, which cannot be differentiated by laminin. In addition, we analyzed the expression level of the MT-1 gene in five prostate cancer cell lines possessing different metastatic potential. The expression of MT-1 in normal and less malignant cells (RWPE-1 and WPE1-NA22) was high and up-regulated by laminin, whereas the expression of MT-1 in WPE1-NB14, WPE1-NB11, and WPE1-NB26 cells (malignant) was extremely low and not elevated by laminin. These results suggest that the MT-1 gene is involved in laminin-mediated differentiation and affects the metastatic potential of tumor cells.

• The Korean Journal of Physiology and Pharmacology
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• v.18 no.6
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• pp.525-530
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• 2014

Transient receptor potential vanilloid subtype 1 (TRPV1) was originally found in sensory neurons. Recently, it has been reported that TRPV1 is expressed in salivary gland epithelial cells (SGEC). However, the physiological role of TRPV1 in salivary secretion remains to be elucidated. We found that TRPV1 is expressed in mouse and human submandibular glands (SMG) and HSG cells, originated from human submandibular gland ducts at both mRNA and protein levels. However, capsaicin (CAP), TRPV1 agonist, had little effect on intracellular free calcium concentration ($[Ca^{2+}]_i$) in these cells, although carbachol consistently increased $[Ca^{2+}]_i$. Exposure of cells to high temperature (> $43^{\circ}C$) or acidic bath solution (pH5.4) did not increase $[Ca^{2+}]_i$, either. We further examined the role of TRPV1 in salivary secretion using TRPV1 knock-out mice. There was no significant difference in the pilocarpine (PILO)-induced salivary flow rate between wild-type and TRPV1 knock-out mice. Saliva flow rate also showed insignificant change in the mice treated with PILO plus CAP compared with that in mice treated with PILO alone. Taken together, our results suggest that although TRPV1 is expressed in SGEC, it appears not to play any direct roles in saliva secretion via transcellular pathway.