Clinical and histopathologic characteristics of submandibular gland in Stevens-Johnson syndrome: A comparative study.

OBJECTIVE: The objective of this study was to investigate the clinical manifestations and pathologic appearances of the submandibular gland (SMG) in Stevens-Johnson syndrome (SJS). STUDY DESIGN: Patients with autologous transplantation of SMG for treatment of severe dry eye between March 1998 and May 2018 were divided into the SJS group (70 cases) and non-SJS group (50 cases) according to the history of SJS. The SMG weight and computed tomography volume and salivary flow rate were measured. The concentration index and secretion index were estimated using scintigraphy with technetium-99m-pertechnetate. Histopathology studies of SMG tissues were conducted, and the acini parameters were measured using a digital image analyzer. RESULTS: A decreased computed tomography volume and weight was observed in 48.57% the SJS group and 2% in the non-SJS group (P < .01). The rest whole, acid-stimulated whole, and SMG rest salivary flow rates decreased in the SJS group (P < .05). The normal SMG concentration index (37.5% vs 96.67%, P < .001) and secretion index (35% vs 96.67%, P < .001) rates were lower in the SJS group than in the non-SJS group. The glandular parenchyma was reduced, the acinar space was widened, and the fat content was increased in the SJS group. CONCLUSION: SMG atrophic and degenerative changes occurred in the SJS group, with a decrease in salivary secretion function in more than half of the patients.

A mechanogenetic role for the actomyosin complex in branching morphogenesis of epithelial organs.

The actomyosin complex plays crucial roles in various life processes by balancing the forces generated by cellular components. In addition to its physical function, the actomyosin complex participates in mechanotransduction. However, the exact role of actomyosin contractility in force transmission and the related transcriptional changes during morphogenesis are not fully understood. Here, we report a mechanogenetic role of the actomyosin complex in branching morphogenesis using an organotypic culture system of mouse embryonic submandibular glands. We dissected the physical factors arranged by characteristic actin structures in developing epithelial buds and identified the spatial distribution of forces that is essential for buckling mechanism to promote the branching process. Moreover, the crucial genes required for the distribution of epithelial progenitor cells were regulated by YAP and TAZ through a mechanotransduction process in epithelial organs. These findings are important for our understanding of the physical processes involved in the development of epithelial organs and provide a theoretical background for developing new approaches for organ regeneration.

LPS-induced epithelial barrier disruption via hyperactivation of CACC and ENaC.

Gram-negative bacterial lipopolysaccharide (LPS) increases the susceptibility of cells to pathogenic diseases, including inflammatory diseases and septic syndrome. In our experiments, we examined whether LPS induces epithelial barrier disruption in secretory epithelia and further investigated its underlying mechanism. The activities of Ca2+-activated Cl- channels (CACC) and epithelial Na+ channels (ENaC) were monitored with a short-circuit current using an Ussing chamber. Epithelial membrane integrity was estimated via transepithelial electrical resistance and paracellular permeability assays. We found that the apical application of LPS evoked short-circuit current (Isc) through the activation of CACC and ENaC. Although LPS disrupted epithelial barrier integrity, this was restored with the inhibition of CACC and ENaC, indicating the role of CACC and ENaC in the regulation of paracellular pathways. We confirmed that LPS, CACC, or ENaC activation evoked apical membrane depolarization. The exposure to a high-K+ buffer increased paracellular permeability. LPS induced the rapid redistribution of zonula occludens-1 (ZO-1) and reduced the expression levels of ZO-1 in tight junctions through apical membrane depolarization and tyrosine phosphorylation. However, the LPS-induced epithelial barrier disruption and degradation of ZO-1 were largely recovered by blocking CACC and ENaC. Furthermore, although LPS-impaired epithelial barrier became vulnerable to secondary bacterial infections, this vulnerability was prevented by inhibiting CACC and ENaC. We concluded that LPS induces the disruption of epithelial barrier integrity through the activation of CACC and ENaC, resulting in apical membrane depolarization and the subsequent tyrosine phosphorylation of ZO-1.

CXCR4 Regulates Temporal Differentiation via PRC1 Complex in Organogenesis of Epithelial Glands.

CXC-chemokine receptor type 4 (CXCR4), a 7-transmembrane receptor family member, displays multifaceted roles, participating in immune cell migration, angiogenesis, and even adipocyte metabolism. However, the activity of such a ubiquitously expressed receptor in epithelial gland organogenesis has not yet been fully explored. To investigate the relationship between CXCL12/CXCR4 signaling and embryonic glandular organogenesis, we used an ex vivo culture system with live imaging and RNA sequencing to elucidate the transcriptome and protein-level signatures of AMD3100, a potent abrogating reagent of the CXCR4-CXCL12 axis, imprinted on the developing organs. Immunostaining results showed that CXCR4 was highly expressed in embryonic submandibular gland, lung, and pancreas, especially at the periphery of end buds containing numerous embryonic stem/progenitor cells. Despite no significant increase in apoptosis, AMD3100-treated epithelial organs showed a retarded growth with significantly slower branching and expansion. Further analyses with submandibular glands revealed that such responses resulted from the AMD3100-induced precocious differentiation of embryonic epithelial cells, losing mitotic activity. RNA sequencing analysis revealed that inhibition of CXCR4 significantly down-regulated polycomb repressive complex (PRC) components, known as regulators of DNA methylation. Treatment with PRC inhibitor recapitulated the AMD3100-induced precocious differentiation. Our results indicate that the epigenetic modulation by the PRC-CXCR12/CXCR4 signaling axis is crucial for the spatiotemporal regulation of proliferation and differentiation of embryonic epithelial cells during embryonic glandular organogenesis.

Targeting Actomyosin Contractility Suppresses Malignant Phenotypes of Acute Myeloid Leukemia Cells.

Actomyosin-mediated contractility is required for the majority of force-driven cellular events such as cell division, adhesion, and migration. Under pathological conditions, the role of actomyosin contractility in malignant phenotypes of various solid tumors has been extensively discussed, but the pathophysiological relevance in hematopoietic malignancies has yet to be elucidated. In this study, we found enhanced actomyosin contractility in diverse acute myeloid leukemia (AML) cell lines represented by highly expressed non-muscle myosin heavy chain A (NMIIA) and increased phosphorylation of the myosin regulatory light chain. Genetic and pharmacological inhibition of actomyosin contractility induced multivalent malignancy- suppressive effects in AML cells. In this context, perturbed actomyosin contractility enhances AML cell apoptosis through cytokinesis failure and aryl hydrocarbon receptor activation. Moreover, leukemic oncogenes were downregulated by the YAP/TAZ-mediated mechanotransduction pathway. Our results provide a theoretical background for targeting actomyosin contractility to suppress the malignancy of AML cells.

Epigenetic Modification of CFTR in Head and Neck Cancer.

Cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP (cAMP)-regulated chloride channel, is critical for secretion and absorption across diverse epithelia. Mutations or absence of CFTR result in pathogeneses, including cancer. While CFTR has been proposed as a tumor suppressing gene in tumors of the intestine, lung, and breast cancers, its effects in head and neck cancer (HNC) have yet to be investigated. This study aimed to define expression patterns and epigenetic modifications of CFTR in HNC. CFTR was expressed in normal but not in HNC cells and tissues. Treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR) was associated with rescued expression of CFTR, whose function was confirmed by patch clamp technique. Further experiments demonstrated that CFTR CpG islands were hypermethylated in cancer cells and tissues and hypomethylated in normal cells and tissue. Our results suggest that CFTR epigenetic modifications are critical in both down-regulation and up-regulation of CFTR expression in HNC and normal cells respectively. We then investigated the impact of CFTR on expressions and functions of cancer-related genes. CFTR silencing was closely associated with changes to other cancer-related genes, suppressing apoptosis while enhancing proliferation, cell motility, and invasion in HNC. Our findings demonstrate that hypermethylation of CFTR CpG islands and CFTR deficiency is closely related to HNC.

Epigenetic Modification as a Regulatory Mechanism for Spatiotemporal Dynamics of ANO1 Expression in Salivary Glands.

Anoctamin1 (ANO1), a calcium activated chloride channel, is known to play a critical role in salivary secretion. In the salivary gland, ANO1 is expressed exclusively in the acinar cells, with no expression in the ductal cells. However, the mechanisms that determine this distinctive cell type-dependent expression pattern of ANO1 remain unknown. In this study, we discovered that the cell-dependent expression of ANO1 during salivary gland organogenesis is regulated by DNA methylation of ANO1 CpG islands. ANO1 CpG islands in e12 embryonic submandibular glands (eSMG) are highly methylated, but those in e14 eSMG or adult SMG are significantly unmethylated. The differential expression pattern of ANO1 in duct and acini is defined at e14. Artificial demethylation by treatment with the demethylating agent 5-aza-2'-deoxycytidine (5-Aza-CdR), induced the expression of ANO1 in both the ductal cell line Human Submandibular Gland (HSG) and in the duct cells of adult mouse SMG. During the trans-differentiation in Matrigel of duct-origin HSG cells into acinar-like phenotype, significant demethylation of ANO1 CpG islands is observed. This may be due to the reduced expression of DNA methyltransferase (DNMT) 3a and 3b. These results suggest that the differential expression of ANO1 in salivary glands during organogenesis and differentiation is mainly regulated by epigenetic demethylation of the ANO1 gene.

DNMT and HDAC inhibitors modulate MMP-9-dependent H3 N-terminal tail proteolysis and osteoclastogenesis.

BACKGROUND: MMP-9-dependent proteolysis of histone H3 N-terminal tail (H3NT) is an important mechanism for activation of gene expression during osteoclast differentiation. Like other enzymes targeting their substrates within chromatin structure, MMP-9 enzymatic activity toward H3NT is tightly controlled by histone modifications such as H3K18 acetylation (H3K18ac) and H3K27 monomethylation (H3K27me1). Growing evidence indicates that DNA methylation is another epigenetic mechanism controlling osteoclastogenesis, but whether DNA methylation is also critical for regulating MMP-9-dependent H3NT proteolysis and gene expression remains unknown. RESULTS: We show here that treating RANKL-induced osteoclast progenitor (OCP) cells with the DNMT inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) induces CpG island hypomethylation and facilitates MMP-9 transcription. This increase in MMP-9 expression results in a significant enhancement of H3NT proteolysis and OCP cell differentiation. On the other hand, despite an increase in levels of H3K18ac, treatment with the HDAC inhibitor trichostatin A (TSA) leads to impairment of osteoclastogenic gene expression. Mechanistically, TSA treatment of OCP-induced cells stimulates H3K27ac with accompanying reduction in H3K27me1, which is a key modification to facilitate stable interaction of MMP-9 with nucleosomes for H3NT proteolysis. Moreover, hypomethylated osteoclastogenic genes in 5-Aza-CdR-treated cells remain transcriptionally inactive after TSA treatment, because H3K27 is highly acetylated and cannot be modified by G9a. CONCLUSIONS: These findings clearly indicate that DNA methylation and histone modification are important mechanisms in regulating osteoclastogenic gene expression and that their inhibitors can be used as potential therapeutic tools for treating bone disorders.

Characterization of Primary Epithelial Cells Derived from Human Salivary Gland Contributing to in vivo Formation of Acini-like Structures.

Patients with head and neck cancer are treated with therapeutic irradiation, which can result in irreversible salivary gland dysfunction. Because there is no complete cure for such patients, stem cell therapy is an emerging alternative for functional restoration of salivary glands. In this study, we investigated in vitro characteristics of primarily isolated epithelial cells from human salivary gland (Epi-SGs) and in vivo formation of acini-like structures by Epi-SGs. Primarily isolated Epi-SGs showed typical epithelial cell-like morphology and expressed E-cadherin but not N-cadherin. Epi-SGs expressed epithelial stem cell (EpiSC) and embryonic stem cell (ESC) markers. During long-term culture, the expression of EpiSC and ESC markers was highly detected and maintained within the core population with small size and low cytoplasmic complexity. The core population expressed cytokeratin 7 and cytokeratin 14, known as duct markers indicating that Epi-SGs might be originated from the duct. When Epi-SGs were transplanted in vivo with Matrigel, acini-like structures were readily formed at 4 days after transplantation and they were maintained at 7 days after transplantation. Taken together, our data suggested that Epi-SGs might contain stem cells which were positive for EpiSC and ESC markers, and Epi-SGs might contribute to the regeneration of acini-like structures in vivo. We expect that Epi-SGs will be useful source for the functional restoration of damaged salivary gland.

Effect of anti-muscarinic autoantibodies on leukocyte function in Sjögren's syndrome.

Patients with primary Sjögren's syndrome, a systemic autoimmune disease, have been shown to have serum autoantibodies that react with the muscarinic acetylcholine type 3 receptor (M3R).Primary Sjögren's syndrome is a systemic autoimmune disease. Patients with primary Sjögren's syndrome have been shown to have serum autoantibodies that react with the muscarinic acetylcholine type 3 receptor (M3R). Leukopenia has been reported to be significantly more common in primary Sjögren's syndrome patients who have anti-M3R-autoantibodies in their sera. In this study, we investigated whether these anti-M3R autoantibodies have effects on M3R and MHCI expression in Jurkat T cells. Purified IgG antibodies were isolated from the serum of healthy individuals and primary Sjögren's syndrome patients. Jurkat cell line was used to represent T lymphocytes. In situ immunofluorescence confocal microscopy was used to confirm the binding reactivity of primary Sjögren's syndrome IgG antibodies to M3R. Co-immunoprecipitation and immunofluorescence results suggested a direct interaction between M3R and MHC I. Co-internalization of M3R and MHC I was observed when Jurkat cells were exposed to the primary Sjögren's syndrome IgG, but this primary Sjögren's syndrome IgG-induced co-internalization of M3R and MHC I was prevented by the presence of exogenous IFN-γ. Primary Sjögren's syndrome IgG itself did not affect the viability of Jurkat cells, but Jurkat cells exposed to primary Sjögren's syndrome IgG were observed to undergo significant cell death when co-cultured with primary Natural Killer cells. Our results suggest that anti-M3R autoantibodies in primary Sjögren's syndrome induce downregulation of plasma membrane-resident M3R and MHC class I molecules in leukocytes followed by NK cell-mediated cell death. This mechanism may explain the frequency of leukopenia occurrence in patients with primary Sjögren's syndrome.

P2X7 receptor and NLRP3 inflammasome activation in head and neck cancer.

In this study, we investigated purinergic receptor P2X7 and NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome expressions, and their role in head and neck cancer. We found upregulation of purinergic receptor P2X7 and all NLRP3 inflammasome components in biopsied head and neck squamous cell carcinoma tissues. Similarly, the expression of purinergic receptor P2X7, apoptosis-associated speck-like protein containing CARD, and pro-form caspase 1 in A253 cells derived from epidermoid carcinoma were highly upregulated in comparison to normal Human Salivary Gland cell line. Active caspase-1 and its final product, active interleukin-1ß, both increased in primed A253 cells stimulated with purinergic receptor P2X7 agonists, while this elevated NLRP3 inflammasome activity was suppressed by purinergic receptor P2X7 antagonists. However, we observed none of these effects in Human Salivary Gland cells. Inhibition of both NLRP3 inflammasome and purinergic receptor P2X7 led to the significant cell death of primed A253 cells, but had no effect on the viability of primed HSG cells or the primary cultured human fibroblast cells. Furthermore, inhibition of either purinergic receptor P2X7 or NLRP3 inflammasome decreased invasiveness of A253, and this effect became more evident when both purinergic receptor P2X7 and NLRP3 inflammasome were simultaneously blocked. Therefore, it is concluded that the purinergic receptor P2X7 and the activation of NLRP3 inflammasome play important roles in the survival and invasiveness of head and neck squamous cell carcinoma in humans.

Human salivary gland cells express bradykinin receptors that modulate the expression of proinflammatory cytokines.

Bradykinin is an important peptide modulator that affects the function of neurons and immune cells. However, there is no evidence of the bradykinin receptors and their functions in human salivary glands. Here we have identified and characterized bradykinin receptors on human submandibular gland cells. Both bradykinin B1 and B2 receptors are expressed on human submandibular gland cells, A253 cells, and HSG cells. Bradykinin increased the intracellular Ca2+ concentration ([Ca2+ ]i ) in a concentration-dependent manner. Interestingly, a specific agonist of the B1 receptor did not have any effect on [Ca2+ ]i in HSG cells, whereas specific agonists of the B2 receptor had a Ca2+ mobilizing effect. Furthermore, application of the B1 receptor antagonist, R715, did not alter the bradykinin-mediated increase in cytosolic Ca2+ , whereas the B2 receptor antagonist, HOE140, showed a strong inhibitory effect, which implies that bradykinin B2 receptors are functional in modulating the concentration of cytosolic Ca2+ . Bradykinin did not affect a carbachol-induced rise of [Ca2+ ]i and did not modulate translocation of aquaporin-5. However, bradykinin did promote the expression of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), implying the role of bradykinin in salivary gland inflammation. These data suggest that bradykinin receptors are involved in Ca2+ signaling in human submandibular gland cells and serve a unique role, which is separate from that of other salivary gland G protein-coupled receptors.

Epigenetic regulation of CFTR in salivary gland.

Cystic fibrosis transmembrane conductance regulator (CFTR) plays a key role in exocrine secretion, including salivary glands. However, its functional expression in salivary glands has not been rigorously studied. In this study, we investigated the expression pattern and regulatory mechanism of CFTR in salivary glands using immunohistochemistry, western blot analysis, Ussing chamber study, methylation-specific PCR, and bisulfite sequencing. Using an organ culture technique, we found that CFTR expression was first detected on the 15th day at the embryonic stage (E15) and was observed in ducts but not in acini. CFTR expression was confirmed in HSG and SIMS cell lines, which both originated from ducts, but not in the SMG C-6 cell line, which originated from acinar cells. Treatment of SMG C-6 cells with 5-aza-2'-deoxycytidine (5-Aza-CdR) restored the expression level of CFTR mRNA in a time-dependent manner. Restoration of CFTR was further confirmed by a functional study. In the Ussing chamber study, 10 µM Cact-A1, a CFTR activator, did not evoke any currents in SMG C-6 cells. In contrast, in SMG C-6 cells pretreated with 5-Aza-CdR, Cact-A1 evoked a robust increase of currents, which were inhibited by the CFTR inhibitor CFTRinh-172. Furthermore, forskolin mimicked the currents activated by Cact-A1. In our epigenetic study, SMG C-6 cells showed highly methylated CG pairs in the CFTR CpG island and most of the methylated CG pairs were demethylated by 5-Aza-CdR. Our results suggest that epigenetic regulation is involved in the development of salivary glands by silencing the CFTR gene in a tissue-specific manner.

Role of flotillins in the endocytosis of GPCR in salivary gland epithelial cells.

Endocytosis has numerous functions in cellular homeostasis. Defects in the endocytic pathway of receptors may lead to dysfunction of salivary gland secretion. Therefore, elucidating the complex mechanisms of endocytosis may facilitate solutions for disease treatment and prevention. The muscarinic type 3 receptor (M3R), a G-protein-coupled receptor (GPCR) located in the plasma membrane, is involved in numerous physiological activities such as smooth muscle contraction and saliva secretion. M3R enters cells through clathrin-mediated endocytosis (CME), while flotillins (flot-1 and -2), highly conserved proteins residing in lipid-raft microdomains, make use of clathrin-independent endocytosis (CIE) for their internalization. Since these two proteins use two discrete pathways for endocytic entry, the association of flotillins with CME is poorly understood. We examined whether flotillins play a role in CME of M3R using immunoblotting, immunocytochemistry, confocal immunofluorescence microscopy, co-immunoprecipitation, and RNA interference techniques in secretory epithelial cells. Upon stimulation with a cholinergic agonist, M3R, flot-1, and flot-2 each internalized from the plasma membrane into intracellular sites. The addition of chlorpromazine and cytochalasin D, well-known inhibitors of CME, inhibited internalization of M3R via CME. Filipin III and methyl-ß-cyclodextrin (mßCD) acting as lipid raft inhibitors disrupted internalization of flot-1/2 via CIE. Interestingly, filipin III and mßCD slightly reduced expression level of M3R whereas chlorpromazine and cytochalasin D did not affect internalization of the flotillin isoforms. M3R and flot-1/2 colocalized and interacted with each other as they entered the cytosol during limited periods of incubation. Moreover, knockdown of flot-1 or -2 by flotillin-specific siRNA prevented internalization and reduced the endocytic efficiency of M3R. Our results suggest that flot-1 and -2 are partially involved in CME of M3R by facilitating its internalization.

Role of Sodium Bicarbonate Cotransporters in Intracellular pH Regulation and Their Regulatory Mechanisms in Human Submandibular Glands.

Sodium bicarbonate cotransporters (NBCs) are involved in the pH regulation of salivary glands. However, the roles and regulatory mechanisms among different NBC isotypes have not been rigorously evaluated. We investigated the roles of two different types of NBCs, electroneutral (NBCn1) and electrogenic NBC (NBCe1), with respect to pH regulation and regulatory mechanisms using human submandibular glands (hSMGs) and HSG cells. Intracellular pH (pHi) was measured and the pHi recovery rate from cell acidification induced by an NH4Cl pulse was recorded. Subcellular localization and protein phosphorylation were determined using immunohistochemistry and co-immunoprecipitation techniques. We determined that NBCn1 is expressed on the basolateral side of acinar cells and the apical side of duct cells, while NBCe1 is exclusively expressed on the apical membrane of duct cells. The pHi recovery rate in hSMG acinar cells, which only express NBCn1, was not affected by pre-incubation with 5 µM PP2, an Src tyrosine kinase inhibitor. However, in HSG cells, which express both NBCe1 and NBCn1, the pHi recovery rate was inhibited by PP2. The apparent difference in regulatory mechanisms for NBCn1 and NBCe1 was evaluated by artificial overexpression of NBCn1 or NBCe1 in HSG cells, which revealed that the pHi recovery rate was only inhibited by PP2 in cells overexpressing NBCe1. Furthermore, only NBCe1 was significantly phosphorylated and translocated by NH4Cl, which was inhibited by PP2. Our results suggest that both NBCn1 and NBCe1 play a role in pHi regulation in hSMG acinar cells, and also that Src kinase does not regulate the activity of NBCn1.

Epigenetic alteration of the purinergic type 7 receptor in salivary epithelial cells.

Purinergic receptors, particularly type 7 (P2RX7), are involved in apoptotic cell death. However, the expression and function of P2RX7 are suppressed in HSG cells. In the present study, we explored whether P2RX7 function is regulated by epigenetic alteration of the receptors in two different cell lines, HSG cells derived from human submandibular ducts, and A253 cells, originated from human submandibular carcinoma. We discovered that HSG cells expressed all subtypes of purinergic receptors, excluding P2RX7, at the mRNA level. However, treatment of the cells with 5-Aza-CdR, a DNA demethylating agent, increased the mRNA expression levels of P2RX7 in a time-dependent manner. Furthermore, 5-Aza-CdR completely rescued the calcium response induced by P2RX7 agonist BzATP, a response that was absent in untreated HSG cells. In contrast, A253 cells showed a moderate methylation pattern in the P2RX7 CpG island. Most CG pairs from the first to the 21st were methylated in untreated HSG cells, but 5-Aza-CdR-treatment partially demethylated the methylated CG pairs. We obtained similar results when investigated human tissues; the CG pairs in the P2RX7 CpG islands showed hypermethylation and hypomethylation patterns in human normal and cancer tissues, respectively. Our results suggest that the expression level and function of P2RX7 are regulated by DNA methylation in epithelial cells.

Claudin-3 is required for modulation of paracellular permeability by TNF-α through ERK1/2/slug signaling axis in submandibular gland.

TNF-α plays an important role in the pathogenesis of salivary inflammatory diseases. Salivary dysfunction, which leads to impaired saliva secretion, can be caused by TNF-α-induced disrupted epithelial barrier. However, the signaling mechanism involved in TNF-α-modulated tight junction barrier in salivary gland remains unclear. Here, we found that TNF-α reduced transepithelial resistance (TER) and increased FITC-dextran flux in a rat submandibular cell line SMG-C6. Claudin (Cln)-3 was selectively downregulated and disrupted by TNF-α, whereas Cln-1, Cln-4, and ß-catenin were not affected. Overexpression of Cln-3 retained and Cln-3 knockdown abolished the TNF-α-induced alterations. Moreover, TNF-α increased extracellular signal-regulated kinase (ERK1/2) phosphorylation and the expression of transcriptional factor slug. ERK1/2 kinase inhibitor PD98059 abrogated TNF-α-induced increase in paracellular permeability, alterations of Cln-3, and elevation of slug. Overexpression of slug decreased and slug knockdown increased Cln-3 expression. In addition, slug bind to the E-box elements of Cln-3 promoter in TNF-α-treated cells, and this response was blocked by PD98059. Furthermore, TNF-α decreased Cln-3 expression and increased slug content in cultured human submandibular gland. Taken together, our data suggest that Cln-3 plays a vital role in TNF-α-modulated paracellular permeability in submandibular epithelium and ERK1/2/slug signaling axis is involved in alteration of Cln-3 redistribution and downregulation.

Epigenetic modulation of the muscarinic type 3 receptor in salivary epithelial cells.

Muscarinic receptors, particularly the type 3 subtype (M3R), have an important role in exocrine secretion. M3R normally function in HSG cells originated from human submandibular gland ducts, but not in A253 and SGT cells, derived from human submandibular carcinoma and salivary gland adenocarcinoma. However, the underlying mechanism of this suppression has remained elusive. In this study, we examined whether M3R function is suppressed by epigenetic modulation of the receptor. To this end, we investigated the mRNA transcript and protein levels of the M3R using reverse transcriptase-PCR, western blot, and confocal microscopy analyses. Global DNA methylation assays, methylation-specific PCR, and bisulfite sequencing were also performed to understand the epigenetic status of the M3R CpG island. We found that A253 cells expressed all subtypes of muscarinic receptors, except M3R, on the mRNA level. However, treatment of cells with 5-aza-2'-deoxycytidine (5-Aza-CdR), a DNA-demethylating agent, increased the expression levels of both M3R mRNA transcript and protein in proportion to the incubation period. 5-Aza-CdR completely restored the carbachol-induced calcium response, which was not observed in untreated A253 cells. In untreated A253 cells, all CG pairs from the 1st to 14th were methylated and 5-Aza-CdR treatment demethylated one of the methylated CG pairs. We also examined the methylation pattern of M3R CpG island in human cancer tissue. Interestingly, the result was very similar to those of A253 cells. All CG pairs in M3R CpG island were also methylated. Another salivary gland tumor cell line, SGT, also showed the similar methylation pattern, heavy methylation in M3R CpG island. It is concluded that CpG island in M3R is hypermethylated in cancer cell lines and human cancer. Our results further suggest that 5-Aza-CdR could potentially be used to restore the function of M3R, suppressed in some cancer cell types.

Role of LPA and the Hippo pathway on apoptosis in salivary gland epithelial cells.

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid involved in numerous physiological responses. However, the expression of LPA receptors and the role of the Hippo signaling pathway in epithelial cells have remained elusive. In this experiment, we studied the functional expression of LPA receptors and the associated signaling pathway using reverse transcriptase-PCR, microspectrofluorimetry, western blotting and immunocytochemistry in salivary gland epithelial cells. We found that LPA receptors are functionally expressed and involved in activating the Hippo pathway mediated by YAP/TAZ through Lats/Mob1 and RhoA/ROCK. Upregulation of YAP/TAZ-dependent target genes, including CTGF, ANKRD1 and CYR61, has also been observed in LPA-treated cells. In addition, based on data suggesting that tumor necrosis factor (TNF)-α induces cell apoptosis, LPA upregulates TNF-induced caspase-3 and cleaved Poly(ADP-ribose)polymerase (PARP). However, small interfering RNA treatment to Yes-associated protein (YAP) or transcriptional co-activator with a PDZ-binding motif (TAZ) significantly decreased TNF-α- and LPA-induced apoptosis, suggesting that YAP and TAZ modulate the apoptotic pathway in salivary epithelial cells.

Antibodies interfering with the type 3 muscarinic receptor pathway inhibit gastrointestinal motility and cholinergic neurotransmission in Sjögren's syndrome.

OBJECTIVE: In primary Sjögren's syndrome (SS), impairment of the gastrointestinal (GI) tract is common, and includes reduced esophageal motor function, delayed gastric emptying, and abnormalities in colonic motility; the pathogenesis is as yet unknown. We undertook this study to investigate the role of functional antibodies to the type 3 muscarinic receptor (M3R) in GI dysfunction associated with primary SS. METHODS: Muscle strip and whole-organ functional assays were used to determine whether IgG with anti-M3R activity from patients with primary SS disrupted neurotransmission in tissue from throughout the mouse GI tract. Specificity of the autoantibody for the M3R was determined using knockout mice that were deficient in the expression of muscarinic receptor subtypes. RESULTS: Functional antibodies to the M3R inhibited neuronally mediated contraction of smooth muscle from throughout the GI tract and disrupted complex contractile motility patterns in the colon. The autoantibodies were not active on tissue from mice that lacked the M3R, providing compelling evidence of the direct interaction of patient autoantibodies with the M3R. CONCLUSION: Our results indicate that anti-M3R autoantibodies have the potential to mediate multiple dysfunctions of the GI tract in primary SS, ranging from reduced esophageal motor activity to altered colonic motility. We hypothesize that altered GI motility forms part of a broader autonomic dysfunction mediated by pathogenic anti-M3R autoantibodies in primary SS.