Nkx2.3 transcription factor is a key regulator of mucous cell identity in salivary glands.

Saliva is vital to oral health, fulfilling multiple functions in the oral cavity. Three pairs of major salivary glands and hundreds of minor salivary glands contribute to saliva production. The secretory acinar cells within these glands include two distinct populations. Serous acinar cells secrete a watery saliva containing enzymes, while mucous acinar cells secrete a more viscous fluid containing highly glycosylated mucins. Despite their shared developmental origins, the parotid gland (PG) is comprised of only serous acinar cells, while the sublingual gland (SLG) contains predominantly mucous acinar cells. The instructive signals that govern the identity of serous versus mucous acinar cell phenotypes are not yet known. The homeobox transcription factor Nkx2.3 is uniquely expressed in the SLG. Disruption of the Nkx2.3 gene was reported to delay the maturation of SLG mucous acinar cells. To examine whether Nkx2.3 plays a role in directing the mucous cell phenotype, we analyzed SLG from Nkx2.3-/- mice using RNAseq, immunostaining and proteomic analysis of saliva. Our results indicate that Nkx2.3, most likely in concert with other transcription factors uniquely expressed in the SLG, is a key regulator of the molecular program that specifies the identity of mucous acinar cells.

Allosteric modulation of ß-cell M3 muscarinic acetylcholine receptors greatly improves glucose homeostasis in lean and obese mice.

Given the global epidemic in type 2 diabetes, novel antidiabetic drugs with increased efficacy and reduced side effects are urgently needed. Previous work has shown that M3 muscarinic acetylcholine (ACh) receptors (M3Rs) expressed by pancreatic ß cells play key roles in stimulating insulin secretion and maintaining physiological blood glucose levels. In the present study, we tested the hypothesis that a positive allosteric modulator (PAM) of M3R function can improve glucose homeostasis in mice by promoting insulin release. One major advantage of this approach is that allosteric agents respect the ACh-dependent spatiotemporal control of M3R activity. In this study, we first demonstrated that VU0119498, a drug known to act as a PAM at M3Rs, significantly augmented ACh-induced insulin release from cultured ß cells and mouse and human pancreatic islets. This stimulatory effect was absent in islets prepared from mice lacking M3Rs, indicative of the involvement of M3Rs. VU0119498 treatment of wild-type mice caused a significant increase in plasma insulin levels, accompanied by a striking improvement in glucose tolerance. These effects were mediated by ß-cell M3Rs, since they were absent in mutant mice selectively lacking M3Rs in ß cells. Moreover, acute VU0119498 treatment of obese, glucose-intolerant mice triggered enhanced insulin release and restored normal glucose tolerance. Interestingly, doses of VU0119498 that led to pronounced improvements in glucose homeostasis did not cause any significant side effects due to activation of M3Rs expressed by other peripheral cell types. Taken together, the data from this proof-of-concept study strongly suggest that M3R PAMs may become clinically useful as novel antidiabetic agents.

Loss of the disease-associated glycosyltransferase Galnt3 alters Muc10 glycosylation and the composition of the oral microbiome.

The importance of the microbiome in health and its disruption in disease is continuing to be elucidated. However, the multitude of host and environmental factors that influence the microbiome are still largely unknown. Here, we examined UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 (Galnt3)-deficient mice, which serve as a model for the disease hyperphosphatemic familial tumoral calcinosis (HFTC). In HFTC, loss of GALNT3 activity in the bone is thought to lead to altered glycosylation of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23), resulting in hyperphosphatemia and subdermal calcified tumors. However, GALNT3 is expressed in other tissues in addition to bone, suggesting that systemic loss could result in other pathologies. Using semiquantitative real-time PCR, we found that Galnt3 is the major O-glycosyltransferase expressed in the secretory cells of salivary glands. Additionally, 16S rRNA gene sequencing revealed that the loss of Galnt3 resulted in changes in the structure, composition, and stability of the oral microbiome. Moreover, we identified the major secreted salivary mucin, Muc10, as an in vivo substrate of Galnt3. Given that mucins and their O-glycans are known to interact with various microbes, our results suggest that loss of Galnt3 decreases glycosylation of Muc10, which alters the composition and stability of the oral microbiome. Considering that oral findings have been documented in HFTC patients, our study suggests that investigating GALNT3-mediated changes in the oral microbiome may be warranted.

Ano6 disruption impairs acinar cell regulatory volume decrease and protein secretion in murine submandibular salivary glands.

The widely expressed Anoctamin 6 (Ano6) supports different Ca2+ -dependent functions, but little is known about its role in salivary glands. Mouse submandibular gland (SMG) acinar cells exhibited a robust regulatory volume decrease (RVD) following cell swelling that was reduced approximately 70% in Ano6-/- mice. Ca2+ -free conditions nearly eliminated the RVD response suggesting that Ano6 is an obligatory component of the cell volume-activated, Ca2+ -dependent RVD pathway in salivary gland acinar cells. Ex vivo agonist-stimulated secretion of water and ions was unaffected by Ano6 disruption under both isotonic and hypotonic conditions suggesting that Ano6 does not play a major role in fluid and electrolyte secretion. In contrast, the total amount of ß-adrenergic-dependent protein secretion by the SMG was significantly reduced in Ano6-/- mice. Closer inspection of these latter results revealed that protein secretion was affected only in the female SMG by Ano6 disruption. These results indicate that Ano6 modulates the RVD response and protein secretion by salivary gland acinar cells.

Knockout of the LRRC26 subunit reveals a primary role of LRRC26-containing BK channels in secretory epithelial cells.

Leucine-rich-repeat-containing protein 26 (LRRC26) is the regulatory γ1 subunit of Ca2+- and voltage-dependent BK-type K+ channels. BK channels that contain LRRC26 subunits are active near normal resting potentials even without Ca2+, suggesting they play unique physiological roles, likely limited to very specific cell types and cellular functions. By using Lrrc26 KO mice with a ß-gal reporter, Lrrc26 promoter activity is found in secretory epithelial cells, especially acinar epithelial cells in lacrimal and salivary glands, and also goblet and Paneth cells in intestine and colon, although absent from neurons. We establish the presence of LRRC26 protein in eight secretory tissues or tissues with significant secretory epithelium and show that LRRC26 protein coassembles with the pore-forming BK α-subunit in at least three tissues: lacrimal gland, parotid gland, and colon. In lacrimal, parotid, and submandibular gland acinar cells, LRRC26 KO shifts BK gating to be like α-subunit-only BK channels. Finally, LRRC26 KO mimics the effect of SLO1/BK KO in reducing [K+] in saliva. LRRC26-containing BK channels are competent to contribute to resting K+ efflux at normal cell membrane potentials with resting cytosolic Ca2+ concentrations and likely play a critical physiological role in supporting normal secretory function in all secretory epithelial cells.

Slc26a6 is an apical membrane anion exchanger that drives HCO3--dependent fluid secretion in murine pancreatic acinar cells.

The nonselective anion exchanger Slc26a6, also known as putative anion transporter 1 and chloride/formate exchanger, is thought to play a major role in HCO3- transport in exocrine glands. In this study, Slc26a6 null mice were used to explore the function of Slc26a6 in the exocrine pancreas. Slc26a6 primarily localized to the apical membrane of pancreatic exocrine acinar cells. The volume of stimulated juice secretion by the ex vivo pancreas was significantly reduced ~35% in Slc26a6-/- mice, but no changes occurred in the gross structure or gland weights of Slc26a6 null mice. The secretion of pancreatic juice by Slc26a6+/+ mice was dependent on HCO3- while, in contrast, fluid secretion by Slc26a6-/- mice was independent of HCO3-, suggesting that Slc26a6 mediates the HCO3--dependent component of fluid secretion. Consistent with these observations, disruption of Slc26a6 also significantly reduced HCO3- secretion by the pancreas ~35%. Taken together, these results demonstrate that the apical Slc26a6 anion exchanger in acinar cells is involved in HCO3--dependent fluid secretion but that another major HCO3--independent pathway is the primary driver of the fluid secretion process in the mouse pancreas.

The apical anion exchanger Slc26a6 promotes oxalate secretion by murine submandibular gland acinar cells.

The solute carrier family 26 (SLC26) gene family encodes at least 10 different anion exchangers. SLC26 member 6 (SLC26A6 or CFEX/PAT-1) and the cystic fibrosis transmembrane conductance regulator (CFTR) co-localize to the apical membrane of pancreatic duct cells, where they act in concert to drive HCO3- and fluid secretion. In contrast, in the small intestine, SLC26A6 serves as the major pathway for oxalate secretion. However, little is known about the function of Slc26a6 in murine salivary glands. Here, RNA sequencing-based transcriptional profiling and Western blots revealed that Slc26a6 is highly expressed in mouse submandibular and sublingual salivary glands. Slc26a6 localized to the apical membrane of salivary gland acinar cells with no detectable immunostaining in the ducts. CHO-K1 cells transfected with mouse Slc26a6 exchanged Cl- for oxalate and HCO3-, whereas two other anion exchangers known to be expressed in salivary gland acinar cells, Slc4a4 and Slc4a9, mediated little, if any, Cl-/oxalate exchange. Of note, both Cl-/oxalate exchange and Cl-/HCO3- exchange were significantly reduced in acinar cells isolated from the submandibular glands of Slc26a6-/- mice. Oxalate secretion in submandibular saliva also decreased significantly in Slc26a6-/- mice, but HCO3- secretion was unaffected. Taken together, our findings indicate that Slc26a6 is located at the apical membrane of salivary gland acinar cells, where it mediates Cl-/oxalate exchange and plays a critical role in the secretion of oxalate into saliva.

The apical Na+ -HCO3 - cotransporter Slc4a7 (NBCn1) does not contribute to bicarbonate transport by mouse salivary gland ducts.

The HCO3 - secretion mechanism in salivary glands is unclear but is thought to rely on the co-ordinated activity of multiple ion transport proteins including members of the Slc4 family of bicarbonate transporters. Slc4a7 was immunolocalized to the apical membrane of mouse submandibular duct cells. In contrast, Slc4a7 was not detected in acinar cells, and correspondingly, Slc4a7 disruption did not affect fluid secretion in response to cholinergic or ß-adrenergic stimulation in the submandibular gland (SMG). Much of the Na + -dependent intracellular pH (pH i ) regulation in SMG duct cells was insensitive to 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, S0859, and to the removal of extracellular HCO 3 - . Consistent with these latter observations, the Slc4a7 null mutation had no impact on HCO 3 - secretion nor on pH i regulation in duct cells. Taken together, our results revealed that Slc4a7 targets to the apical membrane of mouse SMG duct cells where it contributes little if any to pH i regulation or stimulated HCO 3 - secretion.

Submandibular gland-specific inflammaging-induced hyposalivation in the male senescence-accelerated mouse prone -1 line (SAM-P1).

Aging has pronounced effects on mammalian tissues and cells, but the impacts of aging on salivary gland function are relatively unknown. This study aims to evaluate the effects of aging on submandibular gland (SMG) and parotid gland (PG) functions in the male senescence-accelerated mouse. In vivo analysis at the systemic level revealed that salivary secretion induced by pilocarpine, a muscarinic agonist, from the SMG was significantly decreased in aged mice, whereas salivary secretion from the PG was not affected. To evaluate organ-level function, the SMG was perfused with the muscarinic agonists carbachol and calcium ionophore A23187 ex vivo to induce salivary secretion, and decreased saliva production was also observed in the aged SMG. Histological analysis revealed the presence of CD4-positive lymphocytes infiltrating the aged SMG. Furthermore, real-time PCR revealed that the aged SMG exhibited accelerated cell aging, increased levels of the inflammatory cytokine interleukin-6, and decreased mRNA levels of the water channel protein aquaporin-5 (AQP5). In summary, these results demonstrate that SMG function in aged mice was diminished, and that cell senescence, chronic inflammation, and the decreased gene expression of AQP5 are the likely causes of hyposalivation in the SMG of aged mice.

Is Anterior Guidance a Key Factor on Planning Implant Treatment for Free-End Missing in the Posterior Mandible?

To perform safe implant treatment, the anatomical structure and bone quality at implant placement sites are evaluated based on a patient's computerized tomography (CT) data, but there is no definite method to determine placement sites and the appropriate number of implants. The objective of this study was to investigate the influence of the number and arrangement of implants on the stress distribution in 3-unit posterior fixed partial dentures for the posterior mandible by mechanical analysis using the finite element method. Three-dimensional finite element analysis models were constructed from the CT data of a patient with missing mandibular teeth (Nos. 35, 36, 37). Implant placement was simulated under various conditions. Superstructures were connected and fixed with a titanium frame. As the loading conditions, 400 N vertical and lateral loads (45° on the lingual side and 45° on the buccal side) were applied to the upper areas of Nos. 35, 36, and 37, and the stress distribution and frame displacement were evaluated. When a vertical force was applied, no difference of the von Mises stress was noted among the 5 experimental conditions. When lateral force was applied from the lingual and buccal sides at 45°, the stress was higher than that induced by vertical force under all conditions, and it was especially high under mesial and distal cantilever conditions. When displacement of the titanium frame was measured, the displacement caused by lateral force was greater than that due to vertical force. In addition, comparison between long and short distal cantilever bridges revealed that displacement of the titanium frame tended to be smaller when the short cantilever was used. These findings suggested that the stress on peri-implant tissues and displacement of the titanium frame vary depending on the configuration and number of implants, with greater stress and more marked displacement of the titanium frame being induced by lateral force when the number of implants is reduced and a cantilever bridge is selected.

A prospective study of changes in oral health-related quality of life during immediate function implant procedures for edentulous individuals.

OBJECTIVE: The aim of this prospective clinical study was to evaluate longitudinal changes in oral health-related quality of life (OHRQoL) attributable to fixed dental prostheses during All-on-4(®) treatment in one or both jaws. MATERIALS AND METHODS: Ten patients underwent placement of four or six endosteal dental implants on the basis of the All-on-4(®) treatment concept in the edentulous maxilla or both jaws and immediate loading with acrylic interim prostheses. The prostheses were replaced after 3-6 months, and definitive prostheses with titanium framework and reinforced resin facing were fixed after another 5 months or more. The subjects completed the shortened Japanese version of the Oral Health Impact Profile (OHIP-J14) before the surgery (T0), 1 week after the initial (T1) and secondary (T2) interim prostheses were placed, and 3 months after definitive prosthesis placement (T3). Complete data of nine subjects were analyzed with the Wilcoxon signed-rank test. RESULTS: The total OHIP-J14 score significantly reduced only at T3 (P < 0.05). "Functional limitation," "physical pain," "physical disability," and "psychological disability" scores significantly decreased at T3, and "psychological discomfort" scores also significantly dropped at T2. "Social disability" and "handicap" scores remained unchanged throughout. CONCLUSION: Fixed definitive prostheses with metal framework are more effective than fixed all-acrylic prostheses in improving OHRQoL during All-on-4(®) treatment.

Soft tissue biological response to zirconia and metal implant abutments compared with natural tooth: microcirculation monitoring as a novel bioindicator.

INTRODUCTION: Zirconia is often used for implant abutments for esthetics. The aim of this clinical study was to compare the effects of zirconia and metal abutments on periimplant soft tissue. MATERIALS AND METHODS: Ten maxillary anterior implant patients, 5 with metal abutments and 5 with zirconia abutments, were enrolled in this trial. The soft tissue around the implant abutments was evaluated by 2-dimensional laser speckle imaging and thermography. The blood flow in soft tissue around natural teeth was also measured to correct for differences among the subjects. RESULTS: Significantly greater blood flow was detected in the zirconia abutment group (95.64 ± 5.17%) relative to the metal abutment group (82.25 ± 8.92%) in free gingiva (P = 0.0317). Reduced blood flow (by almost 18%) was detected in the tissue surrounding metal abutments compared with the tissue surrounding natural teeth. The surface temperature showed no significant difference for all measurements. CONCLUSIONS: These results suggest that blood flow in tissue surrounding zirconia abutments is similar to that in soft tissue around natural teeth. Moreover, zirconia abutments could be advantageous for the maintenance of immune function by improving blood circulation.

Na(+)-K(+)-2Cl(-) cotransporter-mediated fluid secretion increases under hypotonic osmolarity in the mouse submandibular salivary gland.

Water-handling epithelia are sensitive to the osmotic environment. In this study, the effects of a hypo-osmotic challenge on carbachol (CCh)-induced fluid secretion was investigated using an ex vivo submandibular gland perfusion technique and intracellular pH and Ca(2+) measurements. The osmolality of the perfusion solution was altered to examine the response of the gland to a hypotonic challenge. The flow rate was increased by 34% with a 30% hypotonic solution (225 mosmol/kgH2O), although the Ca(2+) response was unchanged. The lowering of the external Cl(-) by 50% abolished this increase in the 30% hypotonic solution. Furthermore, bumetanide, an inhibitor of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1), completely inhibited the fluid secretion increase caused by the 30% hypotonic solution, and both the total amount of fluid and the flow rate were identical to those of the isotonic solution. This finding was confirmed by measuring the NKCC1 bumetanide-dependent NH4 (+) transport; Na(+)-K(+)-2Cl(-) transport was upregulated >40% by a 30% hypotonic challenge. Therefore, the increase in CCh-induced fluid secretion in response to hypotonic conditions can be attributed, to a large extent, to the specific activation of the NKCC1.

Evaluation of Rheological Properties of Saliva by Determining the Spinnbarkeit.

Saliva is crucial to maintaining oral health and facilitating chewing, swallowing, and speech functions. Decreased saliva secretion, known as hyposalivation, impairs these functions and increases the risk of dental caries and other infectious diseases in the oral cavity.Saliva exhibits various rheological properties, with mucin being a factor in determining these properties. Alterations in these properties can also affect the sensation of dry mouth. In this article, we focus on the spinnbarkeit of saliva using the Neva Meter instrument and provide a methodology for fully understanding the appropriate conditions for its use.

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.

Diagnostic potential of endothelin-1 in peri-implant diseases: a cross-sectional study.

PURPOSE: This study aimed to evaluate the potential of Endothelin-1 (ET-1), a peptide derived from vascular endothelial cells, as a biomarker for diagnosing peri-implant diseases. METHODS: A cohort of 29 patients with a total of 76 implants was included in this study and subsequently divided into three groups based on peri-implant clinical parameters and radiographic examination: healthy (peri-implant health) (n = 29), mucositis (n = 22), and peri-implantitis (n = 25) groups. The levels of ET-1 (ρg/site) and interleukin (IL)-1ß (ρg/site) in peri-implant sulcus fluid (PISF) samples were determined using enzyme immunoassay. Statistical analyses were conducted using Kruskal-Wallis and Steel-Dwass tests. Logistic regression and receiver operating characteristic (ROC) curve analyses were performed to evaluate the diagnostic performance of the biomarkers. RESULTS: ET-1 levels were significantly elevated in the peri-implantitis group compared to those in the healthy group, and were highest in the peri-implant mucositis group. Additionally, IL-1ß levels were significantly higher in the peri-implantitis group than those in the healthy group. ROC curve analysis indicated that ET-1 exhibited superior area under the curve values, sensitivity, and specificity compared to those of IL-1ß. CONCLUSIONS: Our findings suggest that the presence of ET-1 in PISF plays a role in peri-implant diseases. Its significantly increased expression in peri-implant mucositis indicates its potential for enabling earlier and more accurate assessments of peri-implant inflammation when combined with conventional examination methods.

Chronic kidney disease compromises structural and mechanical properties of maxillary cortical bone in a rat model.

PURPOSE: This study aimed to investigate the effects of chronic kidney disease (CKD) on the structural and mechanical properties of the maxillary and mandibular cortical bone. METHODS: The maxillary and mandibular cortical bones from CKD model rats were used in this study. CKD-induced histological, structural, and micro-mechanical alterations were assessed using histological analyses, micro-computed tomography (CT), bone mineral density (BMD) measurements, and nanoindentation tests. RESULTS: Histological analyses indicated that CKD caused an increase in the number of osteoclasts and a decrease in the number of osteocytes in the maxilla. Micro-CT analysis revealed that CKD induced a void volume/cortical volume (%) increase, which was more remarkable in the maxilla than in the mandible. CKD also significantly decreased the BMD in the maxilla. In the nanoindentation stress-strain curve, the elastic-plastic transition point and loss modulus were lower in the CKD group than that in the control group in the maxilla, suggesting that CKD increased micro fragility of the maxillary bone. CONCLUSIONS: CKD affected bone turnover in the maxillary cortical bone. Furthermore, the maxillary histological and structural properties were compromised, and micro-mechanical properties, including the elastic-plastic transition point and loss modulus, were altered by CKD.