TGF-ß1 Triggers Salivary Hypofunction via Attenuating Protein Secretion and AQP5 Expression in Human Submandibular Gland Cells.

Aging-related salivary gland degeneration usually causes poor oral health. Periductal fibrosis frequently occurs in the submandibular gland of the elderly. Transforming growth factor ß1 (TGF-ß1) is the primary driving factor for fibrosis, which exhibits an increase in the fibrotic submandibular gland tissue. This study aimed to investigate the effects of TGF-ß1 on the human submandibular gland (HSG) cell secretory function and its influences on aquaporin 5 (AQP5) expressions and distribution. We found that TGF-ß1 reduces the protein secretion amount of HSG and leads to the abundance alteration of 151 secretory proteins. Data are available via ProteomeXchange with the identifier PXD043185. The majority of HSG secretory proteins (84.11%) could be matched to the human saliva proteome. Meanwhile, TGF-ß1 enhances the expression of COL4A2, COL5A1, COL7A1, COL1A1, COL2A1, and α-SMA, hinting that TGF-ß1 possesses the potential to drive HSG fibrosis-related events. Besides, TGF-ß1 also attenuates the AQP5 expression and its membrane distribution in HSGs. The percentage for TGF-ß1-induced AQP5 reduction (52.28%) is much greater than that of the TGF-ß1-induced secretory protein concentration reduction (16.53%). Taken together, we concluded that TGF-ß1 triggers salivary hypofunction via attenuating protein secretion and AQP5 expression in HSGs, which may be associated with TGF-ß1-driven fibrosis events in HSGs.

Induction of salivary gland-like cells from epithelial tissues transdifferentiated from mouse embryonic fibroblasts.

Salivary gland hypofunction due to radiation therapy for head and neck cancer or Sjögren syndrome may cause various oral diseases, which can lead to a decline in the quality of life. Cell therapy using salivary gland stem cells is a promising method for restoring hypofunction. Herein, we show that salivary gland-like cells can be induced from epithelial tissues that were transdifferentiated from mouse embryonic fibroblasts (MEFs). We introduced four genes, Dnp63a, Tfap2a, Grhl2, and Myc (PTMG) that are known to transdifferentiate fibroblasts into oral mucosa-like epithelium in vivo into MEFs. MEFs overexpressing these genes showed epithelial cell characteristics, such as cobblestone appearance and E-cadherin positivity, and formed oral epithelial-like tissue under air-liquid interface culture conditions. The epithelial sheet detached from the culture dish was infected with adenoviruses encoding Sox9 and Foxc1, which we previously identified as essential factors to induce salivary gland formation. The cells detached from the cell sheet formed spheres 10 days after infection and showed a branching morphology. The spheres expressed genes encoding basal/myoepithelial markers, cytokeratin 5, cytokeratin 14, acinar cell marker, aquaporin 5, and the myoepithelial marker α-smooth muscle actin. The dissociated cells of these primary spheres had the ability to form secondary spheres. Taken together, our results provide a new strategy for cell therapy of salivary glands and hold implications in treating patients with dry mouth.

FGF2-dependent mesenchyme and laminin-111 are niche factors in salivary gland organoids.

Epithelial progenitor cells are dependent upon a complex 3D niche to promote their proliferation and differentiation during development, which can be recapitulated in organoids. The specific requirements of the niche remain unclear for many cell types, including the proacinar cells that give rise to secretory acinar epithelial cells that produce saliva. Here, using ex vivo cultures of E16 primary mouse submandibular salivary gland epithelial cell clusters, we investigated the requirement for mesenchymal cells and other factors in producing salivary organoids in culture. Native E16 salivary mesenchyme, but not NIH3T3 cells or mesenchymal cell conditioned medium, supported robust protein expression of the progenitor marker Kit and the acinar/proacinar marker AQP5, with a requirement for FGF2 expression by the mesenchyme. Enriched salivary epithelial clusters that were grown in laminin-enriched basement membrane extract or laminin-111 together with exogenous FGF2, but not with EGF, underwent morphogenesis to form organoids that displayed robust expression of AQP5 in terminal buds. Knockdown of FGF2 in the mesenchyme or depletion of mesenchyme cells from the organoids significantly reduced AQP5 levels even in the presence of FGF2, suggesting a requirement for autocrine FGF2 signaling in the mesenchyme cells for AQP5 expression. We conclude that basement membrane proteins and mesenchyme cells function as niche factors in salivary organoids.

Cordycepin attenuates Salivary Hypofunction through the Prevention of Oxidative Stress in Human Submandibular Gland Cells.

Xerostomia (dry mouth) is a significant age-related condition. Meanwhile, cordycepin, the natural therapeutic agent, has demonstrated an anti-aging effect. Therefore, the present study aimed to investigate the preventive effects of cordycepin on secretory function in an in vitro model of hydrogen peroxide (H2O2)-induced salivary hypofunction. After being exposed to H2O2, human submandibular gland (HSG) cells were treated with various concentrations of cordycepin (6.25-50 µM) for 24, 48, and 72h. To evaluate cell proliferation and reactive oxygen species (ROS) generation, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and 2, 7'-dichlorodihydrofluorescein diacetate assays were performed. The amylase activity was kinetically measured by 2-chloro-p-nitrophenol linked with maltotrioside. The expression of salivary, antioxidant and apoptotic markers at mRNA and protein levels were performed by reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence analysis, respectively. We demonstrated that cordycepin (6.25-25 µM) contributed to significant increases in expression of the salivary marker genes, alpha-amylase 1 (AMY1A) and aquaporin-5 (AQP5), and in amylase secretion without changes in cell viability. Under oxidative stress, HSG cells showed remarkable dysfunction. Cordycepin rescued the protective effects partially by decreasing ROS generation and restoring the expression of the salivary proteins, AMY and AQP5 via anti-oxidant and anti-apoptotic activity. In addition, the amount of amylase that was secreted from HSG cells cultured in cordycepin was increased. In conclusion, cordycepin demonstrated a protective effect on H2O2 -induced HSG cells by decreasing ROS generation and upregulating the salivary function markers, AMY1A and AQP5, at both the transcriptional and translational levels.

Application of anti-vascular endothelial growth factor antibody restores the function of saliva secretion in a type 2 diabetes mouse model.

OBJECTIVES: Xerostomia, a common complication of type 2 diabetes, leads to an increased risk of caries, dysphagia, and dysgeusia. Although anti-vascular endothelial growth factor (VEGF) antibodies, such as ranibizumab (RBZ), have been used to treat diabetic retinopathy, their effects on the salivary glands are unknown. This study evaluated the effects of RBZ on salivary glands to reduce inflammation and restore salivary function in a mouse model of type 2 diabetes. METHODS: Male KK-Ay mice with type 2 diabetes (10-12 weeks old) were used. The diabetes mellitus (DM) group received phosphate-buffered saline, while the DM + RBZ group received an intraperitoneal administration of RBZ (100 µg/kg) 24 h before the experiment. RESULTS: Ex vivo perfusion experiments showed a substantial increase in salivary secretion from the submandibular gland (SMG) in the DM + RBZ group. In addition, the mRNA expression levels of TNF-α and IL-1ß were considerably lower in this group. In contrast, those of aquaporin 5 were substantially higher in the DM + RBZ group, as revealed by quantitative reverse transcription PCR. Furthermore, the number of lymphocyte infiltration spots in the SMG was notably lower in the DM + RBZ group. Finally, intracellular Ca2+ signaling in acinar cells was considerably higher in the DM + RBZ group than that in the DM group. CONCLUSION: Treating a type 2 diabetic mouse model with RBZ restored salivary secretion through its anti-inflammatory effects.

Downregulated GPX4 in salivary gland epithelial cells contributes to salivary secretion dysfunction in Sjogren's syndrome via lipid ROS/pSTAT4/AQP5 axis.

Sjogren's syndrome (SS) is an autoimmune disease characterized by dysfunction of exocrine glands, such as salivary glands. However, the molecular mechanism of salivary secretion dysfunction in SS is still unclear. Given the significance of glutathione peroxidase 4 (GPX4) in cellular redox homeostasis, we hypothesized that dysregulation of GPX4 may play a pivotal role in the pathogenesis of salivary secretion dysfunction observed in SS. The salivary gland of SS patients and the SS mouse model exhibited reduced expression of the ferroptosis inhibitor GPX4 and the important protein aquaporin 5 (AQP5), which is involved in salivary secretion. GPX4 overexpression upregulated and GPX4 knockdown downregulated AQP5 expression in salivary gland epithelial cells (SGECs) and salivary secretion. Bioinformatics analysis of GSE databases from SS patients' salivary glands revealed STAT4 as a key intermediary regulator between GPX4 and AQP5. A higher level of nuclear pSTAT4 was observed in the salivary gland of the SS mouse model. GPX4 overexpression inhibited and GPX4 knockdown promoted STAT4 phosphorylation and nuclear translocation in SGECs. CHIP assay confirmed the binding of pSTAT4 within the promoter of AQP5 inhibiting AQP5 transcription. GPX4 downregulation accumulates intracellular lipid ROS in SGECs. Lipid ROS inhibitor ferrostatin-1 treatment during in vitro and in vivo studies confirmed that lipid ROS activates STAT4 phosphorylation and nuclear translocation in SGECs. In summary, the downregulated GPX4 in SGECs contributes to salivary secretion dysfunction in SS via the lipid ROS/pSTAT4/AQP5 axis. This study unraveled novel targets to revitalize the salivary secretion function in SS patients.

E2f1-deficient NOD/SCID mice have dry mouth due to a change of acinar/duct structure and the down-regulation of AQP5 in the salivary gland.

Non-obese diabetic (NOD) mice have been used as a model for dry mouth. NOD mice lacking the gene encoding E2f1, a transcription factor, develop hyposalivation more rapidly progressively than control NOD mice. However, the model mice are associated with an underlying disease such as diabetes. We have now established E2f1-deficient NOD/severe combined immunodeficiency disease (NOD/SCID.E2f1(-/-)) mice to avoid the development of diabetes (Matsui-Inohara et al., Exp Biol Med (Maywood) 234(12):1525-1536, 2009). In this study, we investigated the pathophysiological features of dry mouth using NOD/SCID.E2f1(-/-) mice. In NOD/SCID.E2f1(-/-) mice, the volume of secreted saliva stimulated with pilocarpine is about one third that of control NOD/SCID mice. In behavioral analysis, NOD/SCID.E2f1(-/-) mice drank plenty of water when they ate dry food, and the frequency and time of water intake were almost double compared with control NOD/SCID mice. Histological analysis of submandibular glands with hematoxylin-eosin stain revealed that NOD/SCID.E2f1(-/-) mice have more ducts than NOD/SCID mice. In western blot analysis, the expression of aquaporin 5 (AQP5), a marker of acinar cells, in parotid and in submandibular glands of NOD/SCID.E2f1(-/-) mice was lower than in NOD/SCID mice. Immunohistochemical analysis of parotid and submandibular acini revealed that the localization of AQP5 in NOD/SCID.E2f1(-/-) mice differs from that in NOD/SCID mice; AQP5 was leaky and diffusively localized from the apical membrane to the cytosol in NOD/SCID.E2f1(-/-) mice. The ubiquitination of AQP5 was detected in submandibular glands of NOD/SCID.E2f1(-/-) mice. These findings suggest that the change of acinar/duct structure and the down-regulation of AQP5 in the salivary gland cause the pathogenesis of hyposalivation in NOD/SCID.E2f1(-/-) mice.

Role of aquaporin 5 and glandular blood flow in the acetylcholine-induced secretion of saliva in rats.

To clarify the role of the aquaporin 5 (AQP5) in salivary secretion, we evaluated acetylcholine (ACh)-induced secretion in Sprague-Dawley (SD) rats, rats expressing a low level of AQP5 protein (AQP5/low SD) which developed from SD rats, and Wistar/ST rats. The salivary secretion in AQP5/low SD rats in response to infusions of low-dose ACh (60-120 nmol/min) was 27-42% of that in SD rats. By contrast, Wistar/ST rats exhibited comparable secretion to that of SD rats in response to low-doses ACh, despite their low-level expression of AQP5. Experiments using spectrofluorometry and RT-PCR demonstrated no differences in the ACh-induced Ca2+ responses or the mRNA expression of muscarinic receptor, Cl- channel, or cotransporter between these strains. These findings imply that factors other than the function of salivary acinar cells regulates the secretion in response to weak stimuli. Monitoring of the hemodynamics in the submandibular gland revealed that low-doses ACh induced different patterns of the fluctuations in the blood flow in these strains. The blood flow decreased below the resting level in AQP5/low SD rats, but remained mostly above the resting level in Wistar/ST rats. The present study reveals that the contribution of AQP5-dependent transport of water is altered by stimulus intensity and blood flow.

Involvement of aquaporin 5 in Sjögren's syndrome.

Sjögren's syndrome (SS) is a chronic autoimmune disease with the pathological hallmark of lymphoplasmacytic infiltration of exocrine glands - more specifically salivary and lacrimal glands - resulting in a diminished production of tears and saliva (sicca syndrome). The pathophysiology underscoring the mechanisms of the sicca symptoms in SS has still yet to be unraveled but recent advances have identified a cardinal role of aquaporin-5 (AQP5) as a key player in saliva secretion as well as salivary gland epithelial cell dysregulation. AQP5 expression and localization are significantly altered in salivary glands from patients and mice models of the disease, shedding light on a putative mechanism accounting for diminished salivary flow. Furthermore, aberrant expression and localization of AQP5 protein partners, such as prolactin-inducible protein and ezrin, may account for altered AQP5 localization in salivary glands from patients suffering from SS and are considered as new players in SS development. This review provides an overview of the role of AQP5 in SS salivary gland epithelial cell dysregulation, focusing on its trafficking and protein-protein interactions.

Relationship between aquaporin-5 expression and saliva flow in streptozotocin-induced diabetic mice?

OBJECTIVE: To investigate the expression and distribution of AQP5 in submandibular acinar cells from sham- and streptozotocin (STZ)-treated mice in relation to the salivary flow. METHODS: Mice were sham or STZ injected. Distribution of AQP5 subcellular expression in submandibular glands was determined by immunohistochemistry. AQP5 labelling indices (LI), reflecting AQP5 subcellular distribution, were determined in acinar cells. Western blotting was performed to determine the expression of AQP5 in submandibular glands. Blood glycaemia and osmolality and saliva flow rates were also determined. RESULTS: AQP5 immunoreactivity was primarily located at the apical and apical-basolateral membranes of submandibular gland acinar cells from sham- and STZ-treated mice. No significant differences in AQP5 protein levels were observed between sham- and STZ-treated mice. Compared to sham-treated mice, STZ-treated mice had significant increased glycaemia, while no significant differences in blood osmolality were observed. Saliva flow rate was significantly decreased in STZ-treated mice as compared to sham-treated mice. CONCLUSIONS: In STZ-treated mice, significant reduction in salivary flow rate was observed without any concomitant modification in AQP5 expression and localization.

Genetic and environmental factors associated with dental caries in children: the Iowa Fluoride Study.

Dental caries remains the most common chronic childhood disease. Despite strong evidence of genetic components, there have been few studies of candidate genes and caries. In this analysis we tried to assess genetic and environmental factors contributing to childhood caries in the Iowa Fluoride Study. Environmental factors (age, sex, race, tooth-brushing frequencies and water fluoride level) and three dental caries scores (d(2)fs-total, d(2)fs-pit/fissure, and d(2)fs-smooth surface) were assessed in 575 unrelated children (mean age 5.2 years). Regression analyses were applied to assess environmental correlates. The Family-Based Association Test was used to test genetic associations for 23 single nucleotide polymorphism (SNP) markers in 7 caries candidate genes on 333 Caucasian parent-child trios. We evaluated the associations between caries status and the level of both single and multiple SNPs (haplotype) respectively. Permutation procedure was performed for correction of inflated type I errors due to multiple testing. Age, tooth-brushing frequency and water fluoride level were significantly correlated to at least one carious score. Caries on pit and fissure surfaces was substantially higher than on smooth surfaces (61 vs. 39%). SNPs in three genes (DSPP, KLK4 and AQP5) showed consistent associations with protection against caries. Of note, KLK4 and AQP5 were also highlighted by subsequent haplotype analysis. Our results support the concept that genes can modify the susceptibility of caries in children. Replication analysis in independent cohorts is highly needed in order to verify the validity of our findings.

Expression of ICOS in the salivary glands of patients with primary Sjogren's syndrome and its molecular mechanism.

The present study aimed to explore latent markers for identifying primary Sjogren's syndrome (pSS), as well as their expression and molecular mechanism. Hub inducible T cell co­stimulator genes were retrieved from the Gene Expression Omnibus. A total of 95 patients with pSS and 68 healthy individuals from Affiliated Hospital of Nantong University (Nantong, China) were included in the study. The expression of inducible T cell co­stimulator (ICOS) in whole blood and saliva was evaluated using ELISA. Western blotting was performed to investigate aquaporin 5 (AQP5) protein expression, as well as the inflammatory effects of ICOS in patients with pSS compared with healthy individuals. Differentially expressed mRNAs were analyzed in whole blood (GSE84844) and salivary gland (GSE40611) of patients with pSS. In total, 15 hub genes were identified, among which ICOS was indicated to serve a role in the most pivotal immunity pathways. pSS was markedly associated with inflammatory pathways. Results from the present study found that ICOS was upregulated in the salivary gland, whole blood and saliva of patients with pSS. Salivary weight was negatively correlated with the levels of ICOS in the saliva of patients with pSS. The expression of AQP5 was markedly lower in patients with pSS. The expression of AQP5 was negatively associated with ICOS. Compared with that of healthy individuals, the expression of ICOS and inflammatory factors was higher and the expression of AQP5 was lower in pSS patients as assessed by western blotting. These data demonstrated that ICOS may affect AQP5 expression by promoting inflammation in the salivary glands of patients with pSS.

Activation of transient receptor potential vanilloid subtype 1 increases secretion of the hypofunctional, transplanted submandibular gland.

Hyposecretion occurs in most patients early after submandibular gland autotransplantation for severe keratoconjunctivitis sicca. Endogenous transient receptor potential vanilloid subtype 1 (TRPV1) has been recently demonstrated in rabbit submandibular glands, and activation of TRPV1 by capsaicin increases secretion in isolated glands, but the TRPV1-mediated secretory mechanism remains to be elucidated. The purpose of this study was to verify whether activation of TRPV1 by capsaicin could improve the secretion of transplanted gland and its underlying mechanism. The salivary flow of the transplanted glands was significantly decreased, and the mRNA and protein levels of TRPV1 and aquaporin 5 (AQP5) were downregulated in the transplanted glands. Topical capsaicin cream increased secretion and upregulated levels of TRPV1 and AQP5 in transplanted glands. Moreover, in cultured submandibular gland cells, capsaicin increased the mRNA expression of AQP5 and led to redistribution of AQP5 from the cytoplasm to the plasma membrane via TRPV1 activation. Capsaicin enhanced the phosphorylation of extracellular signal-regulated kinase (ERK). Preincubation of cells with PD98059, an inhibitor of ERK kinase, suppressed the capsaicin-induced mRNA expression of AQP5. In summary, the capsaicin-induced secretory mechanism involved activation of TRPV1 and upregulation of AQP5 in an ERK-dependent manner and promoted the redistribution of AQP5 in submandibular gland cells. Activation of TRPV1 may provide a new therapeutic strategy to improve submandibular gland hypofunction.

Interactions with the aquaporin 5 gene increase the susceptibility to molar-incisor hypomineralization.

OBJECTIVE: The aim of this study was to evaluate whether individual genetic factors involved in amelogenesis, the immune response and water channel proteins may increase the susceptibility to Molar-Incisor Hypomineralization (MIH) in Chinese children. DESIGN: DNA samples were collected from 86 cases with MIH cases and 344 controls. Sixteen single-nucleotide polymorphisms (SNPs) were investigated. Logistic regression analysis was performed to assess association between SNPs and the risk of MIH. RESULTS: Our results showed that the risk of MIH in the rs13115627-AA genotype carriers and the rs1784418-TT genotype carriers were significantly higher than that among those with the rs13115627-GG genotype (OR (95 % CI)) = 4.942 (0.658-37.131) and the rs1784418-CT genotype (OR (95 % CI)) = 2.023 (1.63-3.521). The population with the rs1800972-CC genotype and the rs1800972-C allele had a higher risk to develop MIH, OR (95 % CI) = 2.284 (1.267-4.115), OR (95 % CI) = 2.427 (1.493-3.953) respectively. In the Aquaporin 5(AQP5) gene, we individually analyzed two SNPs, rs1996315 and rs923911. We found no significant associations between them and MIH. However, in the analysis of the gene-gene interactions, we discovered a significant two-locus model (P = 0.023) involving rs1996315 and rs923911. Participants with the rs1996315-AG and rs923911-AC genotypes had the highest MIH risk, compared to participants with the rs1996315-GG and rs923911-CC genotypes, OR (95 % CI) = 3.603 (1.147-11.318). CONCLUSION: This study showed that genetic variants in the AMBN, MMP20 and DEFB1 genes may contribute to MIH in the permanent dentition of children. Moreover, interactions among AQP5 gene may also increase the MIH susceptibility.

Postnatal changes in the development of rat submandibular glands in offspring of diabetic mothers: Biochemical, histological and ultrastructural study.

Development and maturation of submandibular salivary glands are influenced by intrauterine diabetic environment. Several studies investigated the effects of diabetes on the salivary glands. However, the effects of maternal diabetes on the submandibular glands of the offspring was not properly examined. Therefore, the present study was designed to describe the changes in the development of the submandibular glands of the offspring of diabetic mothers. The submandibular glands of the offspring of Streptozotocin (STZ)-induced diabetic female rats were examined at two and four weeks after birth. Detection of mRNA demonstrated that maternal diabetes affects the level of different indicators. The reduction of expression of epidermal growth factor (EGF); a protein mitogen, cytokeratin 5 (CK5); an epithelial cell progenitor, CK7 and aquaporin 5 (AQP5); differentiation markers and B cell lymphoma 2 (Bcl2); an antiapoptotic marker were found. Increase in Bcl2-associated X protein (Bax); an apoptotic marker was detected. These changes indicate their effects on saliva secretion, glands tumorigenesis, growth of normal oral flora and oral microbes, with decreased protein synthesis and production of xerostomia and dental caries. Loss of normal glandular architecture, significant increase in fibrosis, by the detection of collagen fibers, and stagnation of secretory granules were found with atrophic changes in the acinar cells. Marked defect of polysaccharides in the acinar cells, denoting functional changes, was manifested by significant reduction of the intensity of periodic acid-Schiff (PAS) reaction. The positive immunoreactivity of caspase-3, denoting cellular apoptosis, and minimal reaction of alpha-smooth muscle actin (α SMA) and proliferating cell nuclear antigen (PCNA) were evident in the offspring of diabetic mothers. We conclude that maternal diabetes produces degenerative effects in the structure and function of the submandibular salivary glands of the offspring, reflecting possible influences on their secretory activity affecting oral and digestive health.

Expression of aquaporin 3 and 5 as a potential marker for distinguishing dry mouth from Sjögren's syndrome.

A study was performed to investigate whether expression of aquaporin (AQP) 3 and 5 has potential as a marker for distinguishing dry mouth from Sjögren's syndrome. Twenty-five patients underwent labial minor salivary gland biopsy (dry mouth, n = 9; Sjögren's syndrome, n = 16; control, n = 8). All patients were interviewed about their medical history and subjective oral symptoms, and intraoral examinations were conducted. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry were used to examine the expression and localization of AQP3 and 5. Significant differences in oral dryness, dry eye, medical history, and Saxon test results were revealed among the groups. However, there were no significant inter-group differences in expression of mRNA for AQP3 and 5. Immunohistochemical staining for AQP3 was localized mainly in the basolateral and part of the ductal cell membrane, and was barely evident in the apical membrane of acinar cells. AQP5 was localized to the basolateral and apical membrane and cytoplasm, but not the ductal cell membrane. Staining intensity for AQP3 in the apical membrane was significantly stronger in Sjögren's syndrome, and that for AQP5 was significantly weaker in dry mouth. Taken together, the present data suggest that expression of AQP3 and 5 may be a marker for distinguishing between patients with dry mouth and those with Sjögren's syndrome.

The difference of aquaporin 5 distribution in acinar and ductal cells in lacrimal and parotid glands.

PURPOSE: This study was designed to clarify the physiological function and tissue distribution of aquaporin 5 (AQP5) in the lacrimal and parotid glands. METHODS: Saliva and tear volumes were compared in AQP5 knockout (AQP5-/-) mice and wild-type mice. Immunohistochemistry and immunoblot analysis were performed in wild-type and AQP5-/- mice. RESULTS: Immunofluorescence of AQP5 staining showed that AQP5 was localized mainly in the ductal cells rather than in the acinar cells of the lacrimal gland. In contrast, in the parotid gland, AQP5 was observed abundantly in acinar cells with undetectable staining in ductal cells. Tear secretion was not changed in AQP5-/- mouse, although saliva secretion was significantly reduced. CONCLUSIONS: AQP5 distribution in acinar cells and ductal cells was completely opposite in the lacrimal and parotid glands. The physiological role of AQP5 might be dependent on the characteristic tissue distribution of the protein in the lacrimal and parotid glands.

Characteristics of Labial Gland Mesenchymal Stem Cells of Healthy Individuals and Patients with Sjögren's Syndrome: A Preliminary Study.

Sjögren's syndrome (SS) is a systemic autoimmune disease that is characterized by focal lymphocytic infiltration into exocrine organs such as salivary and lacrimal glands, resulting in dry mouth and eyes, and other systemic injuries. There is no curative clinical therapy for SS, and stem cell therapy has shown great potential in this area. The mesenchymal stem cells (MSCs) in the salivary glands of healthy individuals and in patients with SS have not been extensively studied. The aim of this study was to elucidate the characteristics of MSCs from the labial glands of healthy controls and of those from patients with SS to elucidate the related pathogenesis and to uncover potential avenues for novel clinical interventions. Labial glands from patients with SS and healthy subjects were obtained, and MSCs were isolated and cultured by using the tissue adherent method. The MSC characteristics of the cultured cells were confirmed by using morphology, proliferation, colony forming-unit (CFU) efficiency, and multipotentiality, including osteogenic, adipogenic, and salivary gland differentiation. The MSCs from the healthy controls and SS patients expressed characteristic MSC markers, including CD29, CD44, CD73, CD90, and CD105; they were negative for CD34, CD45, and CD106, and also negative for the salivary gland epithelium markers (CD49f and CD117). Labial gland MSCs from both groups were capable of osteogenic and adipogenic differentiation. The CFU efficiency and adipogenic differentiation potential of MSCs were significantly lower in the SS group compared with the healthy controls. Cells from both groups could also be induced into salivary gland-like cells. Real-time polymerase chain reaction and immunofluorescence staining showed that the gene and protein expression of AMY1, AQP5, and ZO-1 in cells from the SS group was lower than that in cells from the healthy group. Thus, MSCs from the labial glands in patients with SS could lack certain characteristics and functions, especially related to salivary secretion. These preliminary data provided insights that could lead to the development of novel therapeutic strategies for the treatment of SS.

Aquaporin 5 Interacts with Fluoride and Possibly Protects against Caries.

Aquaporins (AQP) are water channel proteins and the genes coding for AQP2, AQP5, and AQP6 are clustered in 12q13. Since AQP5 is expressed in serous acinar cells of salivary glands, we investigated its involvement in caries. DNA samples from 1,383 individuals from six groups were studied. Genotypes of eight single nucleotide polymorphisms covering the aquaporin locus were tested for association with caries experience. Interaction with genes involved in enamel formation was tested. The association between enamel microhardness at baseline, after creation of artificial caries lesion, and after exposure to fluoride and the genetic markers in AQP5 was tested. Finally, AQP5 expression in human whole saliva, after exposure to fluoride in a mammary gland cell line, which is known to express AQP5, and in Wistar rats was also verified. Nominal associations were found between caries experience and markers in the AQP5 locus. Since these associations suggested that AQP5 may be inhibited by levels of fluoride in the drinking water that cause fluorosis, we showed that fluoride levels above optimal levels change AQP5 expression in humans, cell lines, and rats. We have shown that AQP5 is involved in the pathogenesis of caries and likely interacts with fluoride.

Deletion of ATG5 shows a role of autophagy in salivary homeostatic control.

Autophagy is a catabolic pathway utilized to maintain a balance among the synthesis, degradation, and recycling of cellular components, thereby playing a role in cell growth, development, and homeostasis. Previous studies revealed that a conditional knockout of essential member(s) of autophagy in a variety of tissues causes changes in structure and function of these tissues. Acinar cell-specific expression of knocked-in Cre recombinase through control of aquaporin 5 (Aqp5) promoter/enhancer (Aqp5-Cre) allows us to specifically inactivate Atg5, a protein necessary for autophagy, in salivary acinar cells of Atg5(f/f);Aqp5-Cre mice. There was no difference in apoptotic or proliferation levels in salivary glands of Atg5/Cre mice from each genotype. However, H&E staining and electron microscopy studies revealed modestly enlarged acinar cells and accumulated secretory granules in salivary glands of Atg5(f/f);Aqp5-Cre mice. Salivary flow rates and amylase contents of Atg5/Cre mice indicated that acinar-specific inactivation of ATG5 did not alter carbachol-evoked saliva and amylase secretion. Conversely, autophagy intersected with salivary morphological and secretory manifestations induced by isoproterenol administration. These results identified a role for autophagy as a homeostasis control in salivary glands. Collectively, Atg5(f/f);Aqp5-Cre mice would be a useful tool to enhance our understanding of autophagy in adaptive responses following targeted head and neck radiation or Sjögren syndrome.