Search

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the endothelial barrier dysfunction is related to many salivary gland secretory disorders. The present protocol describes an in vivo paracellular permeability detection method to evaluate the function of endothelial tight junctions (TJs) in mouse submandibular glands (SMG). First, fluorescence-labeled dextrans with different molecular weights (4 kDa, 40 kDa, or 70 kDa) were injected into the angular veins of mice. Afterward, the unilateral SMG was dissected and fixed in the customized holder under a two-photon laser-scanning microscope, and then images were captured for blood vessels, acini, and ducts. Utilizing this method, the real-time dynamic leakage of the different-sized tracers from blood vessels into the basal sides of the acini and even across the acinar epithelia into the ducts was monitored to evaluate the alteration of the endothelial barrier function under physiological or pathophysiological conditions.

Tight junctions (TJs) play an important role in water, ion, and solute transport through the paracellular pathway of epithelial cells; however, their role in diabetes-induced salivary gland dysfunction remains unknown. Here, we found that the TJ proteins claudin-1 and claudin-3 were significantly increased in the submandibular glands (SMGs) of db/db mice and high glucose (HG)-treated human SMGs. HG decreased paracellular permeability and increased claudin-1 and claudin-3 expression in SMG-C6 cells. Knockdown of claudin-1 or claudin-3 reversed the HG-induced decrease in paracellular permeability. MiR-22-3p was significantly downregulated in diabetic SMGs and HG-treated SMG-C6 cells. A miR-22-3p mimic suppressed claudin-1 and claudin-3 expression and abolished the HG-induced increases in claudin-1 and claudin-3 levels in SMG-C6 cells, whereas a miR-22-3p inhibitor produced the opposite effects. Specificity protein-1 (Sp1) was enhanced in diabetic SMGs and HG-treated SMG-C6 cells, which promoted claudin-1 and claudin-3 transcription through binding to the corresponding promoters. A luciferase reporter assay confirmed that miR-22-3p repressed Sp1 by directly targeting the Sp1 mRNA 3'-untranslated region (3'-UTR). Consistently, the miR-22-3p mimic suppressed, whereas the miR-22-3p inhibitor enhanced, the effects of HG on Sp1 expression. Taken together, our results demonstrate a new regulatory pathway through which HG decreases the paracellular permeability of SMG cells by inhibiting miR-22-3p/Sp1-mediated claudin-1 and claudin-3 expression.

Objective: Hyposalivation is a common symptom of diabetes. Although microRNAs (miRNAs) play a role in the pathogenesis of diabetes, the specific effects of miRNAs on diabetic salivary glands are not clear., Design: We used high-throughput technologies to screen differentially expressed (DE) miRNAs and mRNAs in submandibular gland (SMG) tissues from db/db mice and db/m mice. DE miRNAs and mRNAs were confirmed using quantitative real-time polymerase chain reaction (qRT-PCR)., Results: Twenty-eight DE miRNAs and 1146 DE mRNAs were identified between the SMG tissues of db/db mice and db/m mice. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that the DE miRNAs were highly associated with terms related to diverse biological processes and signalling pathways. Of the related pathways, the tight junction pathway, autophagy pathway and transforming growth factor-β (TGF-β) signalling pathway were notable. AKT serine/threonine kinase 3 (AKT3) and phosphoinositide-3 kinase catalytic subunit delta (PIK3CD) may also play important roles in the development of diabetes-mediated hyposalivation., Conclusions: Our research described the miRNA-mRNA expression profiles and miRNA-mRNA network in the SMG tissues of db/db mice. These results provide possible molecular mechanisms of diabetes-induced hyposalivation and information for further studies., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Muscarinic acetylcholine receptors (mAChRs), including M1-M5 subtypes, are classic receptors in regulating water, ion, and solute transport in salivary gland. Our work focuses on the studies on the expression pattern and function of mAChR in the submandibular gland (SMG), and the underlying mechanism involved in the mAChR-regulated secretion, together with the effect of parasympathectomy on the salivary secretion. Microvascular autotransplantation of SMG into the temporal fossa provides a continuous and endogenous source of fluids, and is currently an effective method for treating severe keratoconjunctivitis sicca. By using RT-PCR, Western blotting, and immunofluorescence, our data demonstrated that the expression of M1 and M3 subtypes were decreased in latent period in rabbit SMG autotransplantation model, whereas carbachol stimulation promoted the salivary secretion, as well as M1 and M3 expressions. By contrast, mAChRs were hypersensitive in epiphora SMGs, whereas atropine gel and botulinum toxin A application significantly inhibited the hypersecretion in both animal models and patients. Furthermore, the possible intracellular signal molecules involved in the mAChR-modulated salivary secretion were explored. Activation of mAChR upregulated the expression of aquaporin 5 (AQP5), the main transporter that mediated water secretion through transcellular pathway, and led to AQP5 trafficking from lipid rafts to non-lipid microdomain. Extracellular signal-regulated kinase 1/2 (ERK1/2) was involved in the mAChR-regulated AQP5 content. mAChR activation also modulated the expression, distribution, and function of tight junction proteins, and increased paracellular permeability. ERK1/2/β-arrestin2/clathrin/ubiquitin signaling pathway was responsible for the mAChR-regulated downregulation of tight junction molecule claudin-4. Cytoskeleton filamentous actin (F-actin) was also involved in the distribution and barrier function of epithelial tight junctions. Besides, endothelial tight junctions were opened by mAChR agonist-evoked salivation in the mice. Furthermore, parasympathetic denervation increased resting salivary secretion in the long terminrats and minipigs. Taken together, our work demonstrated that mAChR regulated saliva secretion via transcellular and paracellular pathways in SMG epithelium as well as tight junction opening in SMG endothelium. Modulation of mAChR might be a promising strategy to ameliorate SMG dysfunction.

Parasympathectomy leads to retrogressive alteration and dysfunction of the submandibular gland (SMG) within 1 month, but its long-term effect is unclear. Excessive secretion is observed in half of the patients 4-6 months after SMG transplantation, which completely denervates the gland. Here, we investigated the long-term effect of parasympathectomy on the secretion of SMGs in minipigs. The results showed that the resting salivary secretion of SMGs decreased by 82.9% of that in control at 2 months after denervation, but increased by 156% at 6 months. Although experiencing an atrophic period, the denervated glands regained their normal morphology by 6 months. The expression of the function-related proteins, including muscarinic acetylcholine receptor (mAChR) 3, aquaporin 5 (AQP5), tight junction protein claudin-3, and claudin-4 was decreased at 2 months after denervation. Meanwhile, the protein expression of stem cell markers, including sex-determining region Y-box 2 and octamer-binding transcription factor 4, and the number of Ki67 + cells were significantly increased. However, at 6 months after denervation, the expression of mAChR3, AQP5, claudin-1, claudin-3, and claudin-4 was significantly raised, and the membrane distribution of these proteins was increased accordingly. The autonomic axonal area of the glands was reduced at 2 months after denervation but returned to the control level at 6 months, suggesting that reinnervation took place in the long term. In summary, parasympathectomy increases resting secretion of the SMGs in the long term with a possible mechanism involving improved transepithelial fluid transport. This finding may provide a new strategy for xerostomia treatment., (© 2018 Wiley Periodicals, Inc.)

Autophagy is a catabolic process which is involved in the development of many diseases including diabetes mellitus and its complications. Hyposalivation is a common complication of diabetes mellitus, whereas its mechanism remains unclear. Here, we observed that the stimulated salivary flow rate of SMG was significantly decreased in db/db mice, a diabetic mice model. The expressions of aquaporin 5 (AQP5), a water channel protein, were decreased, whereas the mRNA level of AQP5 was increased in SMGs of both diabetic patients and mice. Under transmission electron microcope, more autophagosomes were detected in diabetic SMGs. Expressions of autophagy related proteins LC3II, Beclin-1 and ATG5 were increased, meanwhile autophagy substrate p62 was decreased in SMGs of diabetic patients and mice, indicating that autophagy was activated in diabetic SMG. Double immunofluorescence staining showed that the colocalization of AQP5 and LC3 was increased in SMGs of diabetic mice. In cultured SMG-C6 cells, high glucose (HG), but not high osmotic pressure, reduced AQP5 protein expression and induced autophagy. Moreover, inhibition of autophagy by 3-methyladenin, an autophagy inhibitor, or by autophagy-related gene 5 siRNA, decreased HG-induced AQP5 reduction in SMG-C6 cells. Additionally, the expression of p-p85, p-Akt and p-mTOR were decreased in HG-treated SMG-C6 cells. Pretreatment with 740Y-P, a PI3K agonist, significantly suppressed HG-induced autophagy and AQP5 degradation. Taken together, these results indicate that autophagy plays a crucial role in AQP5 degradation in diabetic SMG via PI3K/Akt/mTOR signaling pathway, which contributes to the dysfunction of diabetic SMG. Our study provides a novel mechanism of diabetic hyposalivation.

Tight junction (TJ) proteins play a dynamic role in paracellular fluid transport in salivary gland epithelia. Most TJ studies are carried out in mice and rats. However, the morphology of rodent salivary glands differs from that of human glands. This study aimed to compare the histological features and the expression pattern of TJ proteins in porcine salivary glands with those of human and mouse. The results showed that porcine parotid glands were pure serous glands. Submandibular glands (SMGs) were serous acinar cell-predominated mixed glands, whereas sublingual glands were mucous acinar cell-predominated. Human SMGs were mixed glands containing fewer mucous cells than porcine SMGs, whereas the acinar cells of murine SMGs are seromucous. The histological features of the duct system in the porcine and human SMGs were similar and included intercalated, striated and excretory ducts, but the murine SMG contained a specific structure, the granular convoluted tubule. TJ proteins, including claudin-1 to claudin-12, occludin and zonula occludin-1 (ZO-1), were detected in the porcine major salivary glands and human SMGs by RT-PCR; however, claudin-6, claudin-9 and claudin-11 were not detected in the murine SMG. As shown by immunofluorescence, claudin-1, claudin-3, claudin-4, occludin and ZO-1 were distributed in both acinar and ductal cells in the porcine and human SMGs, whereas claudin-1 and claudin-3 were mainly present in acinar cells, and claudin-4 was mainly distributed in ductal cells in the murine SMG. In addition, 3D images showed that the TJ proteins arranged in a honeycomb-like structure on the luminal surface of the ducts, whereas their arrangements in acini were irregular in porcine SMGs. In summary, the expression pattern of TJ proteins in salivary glands is similar between human and miniature pig, which may be a candidate animal for studies on salivary gland TJ function., (© 2018 Anatomical Society.)

Autologous submandibular gland (SMG) transplantation has been proved to ameliorate the discomforts in patients with severe keratoconjunctivitis sicca. The transplanted glands underwent a hypofunctional period and then restored secretion spontaneously. This study aims to investigate whether autonomic nerves reinnervate the grafts and contribute to the functional recovery, and further determine the origin of these nerves. Parts of the transplanted SMGs were collected from the epiphora patients, and a rabbit SMG transplantation model was established to fulfill the serial observation on the transplanted glands with time. The results showed that autonomic nerves distributed in the transplanted SMGs and parasympathetic ganglionic cells were observed in the stroma of the glands. Low-dense and unevenly distributed cholinergic axons, severe acinar atrophy and fibrosis were visible in the patients' glands 4-6 months post-transplantation, whereas the cholinergic axon density and acinar area were increased with time. The acinar area or the secretory flow rate of the transplanted glands was statistically correlated with the cholinergic axon density in the rabbit model, respectively. Meanwhile, large cholinergic nerve trunks were found to locate in the temporal fascia lower to the gland, and sympathetic plexus concomitant with the arteries was observed both in the adjacent fascia and in the stroma of the glands. In summary, the transplanted SMGs are reinnervated by autonomic nerves and the cholinergic nerves play a role in the morphological and functional restoration of the glands. Moreover, these autonomic nerves might originate from the auriculotemporal nerve and the sympathetic plexus around the supplying arteries.

Severe dry eye is a refractory ophthalmologic disease. Our multidisciplinary research group treated severe dry eye by microvascular autologous transplantation of submandibular gland (SMG) during the past 20 years. The SMG, with its blood vessels and Wharton's duct, was harvested from the submandibular triangle and transferred to the temporal area. The blood vessels in the SMG were anastomosed with the temporal blood vessels using a microsurgical technique. Then, the distal end of Wharton's duct was sutured to form an opening in the upper lateral conjunctival fold. The tear was replaced by the secretion of the transplanted SMG to lubricate the ocular surface. In our study, the surgical techniques of blood vessel management were continuously modified to increase the survival rate of the transplanted SMG. A novel surgical modality of partial transplantation of SMG was established to prevent postoperative epiphora. A clinical study with the largest case number in the world was conducted and the effectiveness of transplantation of SMG for severe dry eye was fully confirmed. In order to resolve two main clinical problems including ductal obstruction resulted from low secretion rate during the latent period, and epiphora due to over secretion of the transplanted SMG in the later term of transplantation, the regulation of the secretion mechanism of the normal and transplanted SMG were investigated. New opinions on mechanisms of saliva secretion were provided. Based on the priniciple of translational medicine, the results of related basic research were applied in the clinic. The clinical guidelines for secretion regulation of transplanted SMG were established. A concept of chronic obstructive sialadenitis of transplanted SMG was provided and its diagnostic criteria, diagnostic technique of sialography, and therapeutic regimen were established. As a result, the surgical success rate was obviously elevated, the surgical complications were decreased, and life quality of the patients was greatly improved.

Autologous submandibular gland transplantation is an effective treatment for severe dry eye syndrome. However, the protein secretion in transplanted gland is altered by a mechanism that remains to be elucidated. In the present study, we found that β1-adrenoceptor (β1-AR) and β2-AR expression and the phosphorylation of the downstream molecule protein kinase A (PKA) were elevated in transplanted submandibular glands obtained from epiphora patients. Synaptobrevin/vesicle-associated membrane protein 2 (VAMP-2) interacted with syntaxin-4 and actin in human submandibular gland. The contents of syntaxin-4 and actin interacting with VAMP-2 were increased in transplanted gland. Moreover, VAMP-2 and syntaxin-4 expression in the secretory granule fraction, and VAMP-2 expression in the membrane protein fraction were increased in isoproterenol-treated and transplanted glands. Isoproterenol increased F-actin polymerization in the apical and lateral regions of the cytoplasm in both control and transplanted glands. Inhibiting PKA activity and/or F-actin formation abolished the isoproterenol-enhanced expression of VAMP-2 and syntaxin-4 in the secretory granule fraction and the isoproterenol-enhanced expression of VAMP-2 in the membrane protein fraction. Taken together, these results indicate that the activation of β-ARs induces secretory granules and cell membrane fusion via the interaction of VAMP-2 and syntaxin-4 in a PKA- and F-actin-dependent manner in human submandibular gland. Up-regulated β-ARs might participate in altering protein secretion in transplanted submandibular gland by promoting the interaction of VAMP-2 with syntaxin-4., (© 2018 The Author(s).)

Hypertension is a systemic disorder that affects numerous physiological processes throughout the body. Improper sodium transport is a common comorbidity of hypertension, and sodium transport is also critical for maintaining the secretion of submandibular glands, whether the function of submandibular glands is affected by hypertension remains unclear. To determine whether hypertension induces changes in the protein expression of submandibular glands, we compared the proteome of submandibular glands from 14-week-old spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats using LC-MS/MS. The results revealed that 95 proteins displayed different levels of expression between the submandibular glands from the SHRs and WKYs. Among these, 35 proteins were more abundant, and 60 proteins were less abundant in the SHR compared with the WKY rats. Specifically, aquaporin 5 and parvalbumin, which are correlated with water transport and intracellular Ca 2+ signal transduction, were verified to exhibit differences in protein abundance. Impaired Ca 2+ response to carbachol was confirmed in the acinar cells from SHRs, and hyposecretion by the submandibular glands was further confirmed by in vivo saliva collection. In conclusion, the proteomic analysis of the submandibular glands of SHRs revealed novel changes in protein abundance that provides possible mechanisms connecting hypertension and hyposecretion in submandibular glands.

Tight junctions (TJs) in salivary epithelium play an important role in regulating saliva secretion. Autologous transplantation of submandibular glands (SMGs) is an effective method to treat severe dry eye syndrome. However, epiphora occurs in some patients 6 months after transplantation. We previously found that the acinar TJs are enlarged in rabbit SMGs after long-term transplantation, but the exact TJ components involved in the epiphora are still unknown. Here, we found that the mRNA and protein expression of ZO-1 and occludin were increased in the transplanted SMGs obtained from epiphora patients, while other TJs were unchanged. The intensity of ZO-1 and occludin at the apicolateral membranes as well as occludin in the cytoplasm were increased in epiphora SMGs, but the interaction between ZO-1 and occludin was decreased as evidenced by both co-immunoprecipitation assay and co-immunofluorescence staining. Mechanically, the expression of casein kinase 2α (CK2α) and CK2β, which was reported to affect occludin modification and the interaction of occludin with ZO-1 in previous literatures, were increased in epiphora glands. Moreover, activation of muscarinic acetylcholine receptor (mAChR) by carbachol directly decreased the interaction between ZO-1 and occludin and increased the acinar TJ width in the freshly isolated human SMGs, whereas these effects were abolished by pretreatment with CK2 inhibitor. Taken together, our findings suggest that decreased interaction between ZO-1 and occludin might contribute to the epiphora occurred in the transplanted SMGs, and mAChR together with the intracellular molecule CK2 might be responsible for the alteration of TJs in epiphora glands.

Blood vessels provide the original supplies for the formation of primary saliva, which is regulated by the tight junctions (TJs) between endothelial cells. Previous studies have shown that blood flow increases with vasodilatation during cholinergic-evoked salivation. However, changes in vascular paracellular permeability and the role of endothelial TJs in salivation are unknown. Here, we established an in vivo paracellular permeability detection system and observed that the endothelial TJs were permeable to 4-kDa fluorescein isothiocyanate (FITC)-dextran while impermeable to 40- and 70-kDa FITC-dextran under an unstimulated condition in mouse submandibular glands (SMGs). Pilocarpine increased the flux of 4- and 40-kDa FITC-dextran out of blood vessels but did not affect 70-kDa FITC-dextran. Claudin 5, a TJ protein specifically localized in salivary endothelial cells, was redistributed from the apicolateral membranes to the lateral and basolateral membranes and cytoplasm in cholinergic-stimulated mouse SMGs and freshly cultured human SMG tissues. In the transplanted SMGs from epiphora patients, we found that claudin 5 was present in the basolateral membranes and cytoplasm, instead of the apical region in control SMGs. Moreover, the level of phospho-myosin light chain 2 increased within the blood vessels of the pilocarpine-stimulated mouse SMGs and transplanted human SMGs, while the downstream molecule F-actin was reorganized in the endothelial cells of the transplanted human SMGs. Taken together, our findings provide direct visual evidence that the opening of endothelial TJs and the redistribution of claudin 5 are essential events contributing to cholinergic-evoked salivation, thus enriching our understanding of the secretory mechanisms that link blood flow to primary saliva formation by regulating the endothelial paracellular permeability.

Fluid and ion secretion from the submandibular gland (SMG) is mainly regulated by parasympathetic nerves. This study evaluated the effect of parasympathectomy on salivary secretion from normal and irradiated rat SMGs from 1 to 24 wk after denervation. Although stimulated salivary secretion was significantly lower in denervated SMGs compared with contralateral self-controls, the resting salivary flow rates were markedly higher in the denervated SMGs at 1, 12, and 24 wk after denervation. The levels of muscarinic acetylcholine M1 and M3 receptors, as well as of aquaporin 5, were up-regulated. Notably, although irradiated SMGs showed significantly lower resting and stimulated salivary secretion rates than non-irradiated SMGs, the resting salivary secretion rates of the irradiated and denervated SMGs were markedly higher than seen in the irradiated self-control SMGs at 1, 12, and 24 wk after parasympathectomy, and were even higher than seen in the non-irradiated sham-operated rats. The expression of M1 and M3 receptors was similarly elevated. Taken together, our results suggest that parasympathetic denervation increases resting salivary secretion of both normal and irradiated SMGs. This approach might provide a potential modality for relieving radiation-induced xerostomia, which is a common complication following treatment of head and neck cancer., (© 2017 Eur J Oral Sci.)

Submandibular gland (SMG) autotransplantation is an effective therapy for treating severe dry eye syndrome. However, epiphora occurs in more than 40% of patients 6 months after operation. We previously found that muscarinic acetylcholine receptor (mAChR) plays a crucial role in regulating SMG secretion partially through the modulation on tight junction (TJ)-based paracellular pathway. Therefore, the present study aimed to investigate the possible involvement of mAChR and TJ in a rabbit long-term model of SMG transplantation. We found that SMG secretion was significantly increased on postoperative days 90 and 180, which imitated epiphora observed in the patients with SMG transplantation. Although the mRNA expression and fluorescence intensity of M1 and M3 mAChR subtypes were reversed to control levels on postoperative days 30, 90, and 180, the content of β-arrestin2, but not β-arrestin1, was gradually decreased after transplantation, which suggests that mAChR may be hypersensitive in late phase of SMG transplantation. The width of acinar TJs was enlarged and fluorescence intensity of F-actin in peri-apicolateral membranes were remarkably increased on postoperative days 90 and 180. Topical treatment with atropine gel significantly reduced SMG secretion, TJ width, as well as F-actin intensity in peri-apicolateral membranes on postoperative days 180. Moreover, in a perfused rabbit SMG, carbachol increased salivary secretion, enlarged TJ width, and induced F-actin rearrangement, whereas these responses were inhibited by atropine pretreatment. Taken together, our findings suggest that the hypersensitive mAChR may contribute to epiphora in late phase of SMG transplantation through modulating TJ-based paracellular permeability.

The tight junction-based paracellular pathway plays an important role in saliva secretion. Zonula occludens (ZO) proteins are submembranous proteins of tight junction complex; however, their function in salivary epithelium is poorly understood. Here, we found that activation of transient receptor potential vanilloid subtype 1 (TRPV1) by capsaicin increased rat saliva secretion both in vivo and ex vivo. Meanwhile, TRPV1 activation enlarged the width of tight junctions between neighboring acinar cells, increased the paracellular flux of 4-kDa fluorescein isothiocyanate (FITC)-dextran in submandibular gland (SMG) tissues, and decreased transepithelial electric resistance (TER) in SMG-C6 cells. ZO-1, -2, and -3 were distributed principally to the apical lateral region of acinar cells in SMG tissues and continuously encircled the peripheries of SMG-C6 cells in the untreated condition. TRPV1 activation obviously diminished ZO-1 and -2 staining, but not ZO-3 or β-catenin, at the cell-cell contacts ex vivo and in vitro. Moreover, in untreated SMG-C6 cells, ZO-1 and -2 single or double knockdown by small interfering RNA (siRNA) increased the paracellular flux of 4-kDa FITC-dextran. In capsaicin-treated cells, ZO-1 and -2 single or double knockdown abolished, whereas their re-expression restored, the capsaicin-induced increase in paracellular permeability. Furthermore, TRPV1 activation increased RhoA activity, and inhibition of either RhoA or Rho kinase (ROCK) abolished the capsaicin-induced TER decrease as well as ZO-1 and -2 redistribution. These results indicate that ZO-1 and -2 play crucial roles in both basal salivary epithelial barrier function and TRPV1-modulated paracellular transport. RhoA-ROCK signaling pathway is responsible for TRPV1-modulated paracellular permeability as well as ZO-1 and -2 redistribution., (© International & American Associations for Dental Research 2015.)

Intraglandular injection of botulinum toxin type A (BoNT/A) is an effective treatment for sialorrhea. Despite numerous experimental and clinical studies on inhibition of saliva section by BoNT/A, the proteolysis of synaptosomal-associated protein 25 (SNAP-25) following BoNT/A treatment has not yet been confirmed in the salivary gland after injection of BoNT/A. More important, it is not known whether BoNT/A exerts a direct effect in acinar cells. Here, we show that injection of BoNT/A into the rat submandibular gland (SMG) decreased salivary flow in a dose-dependent manner; the inhibitory effect lasted at least 4 wk, and salivary flow recovered to normal levels by 12 wk. During the inhibitory period, SMG neurons and synapses expressed lower levels of full-length SNAP-25, and cleavage of SNAP-25 was observed, as indicated by detection of reduced molecular weight SNAP-25 using Western blotting. In addition, the water channel aquaporin 5 (AQP5) was downregulated and abnormally distributed in rat SMG after injection of BoNT/A. The direct effects of BoNT/A on AQP5 expression and distribution were assessed in vitro to exclude the influence of BoNT/A-induced inhibitory neurotransmission. In stable GFP-AQP5-transfected SMG-C6 cells, treatment with BoNT/A reduced the cell surface protein level of AQP5 in a dose- and time-dependent manner without affecting total AQP5 protein expression. Cell surface biotinylation and immunofluorescence demonstrated translocation of AQP5 from the membrane to the cytoplasm, which was confirmed by decreased levels of AQP5 protein in the membrane fraction and increased levels in the cytoplasmic fraction, suggestive of AQP5 redistribution. Taken together, these results indicated that BoNT/A reversibly decreased saliva secretion in rat SMGs through not only the presynaptic SNAP-25 cleavage but also the postsynaptic AQP5 redistribution. These data provide the first evidence for a direct effect of BoNT/A on the salivary gland., (© International & American Associations for Dental Research 2015.)

Our work focused on the studies on the expression and function of transient receptor potential vanilloid subtype 1 (TRPV1) in the submandibular gland. By using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence, our data demonstrated the expression and distribution characteristics of TRPV1 in rabbit and human submandibular glands, as well as rat submandibular gland cell line SMG-C6. Furthermore, the possible intracellular signal molecules involved in the TRPV1-modulated saliva secretion were explored. Activation of TRPV1 increased the intracellular Ca(2+) concentration, upregulated the expression of aquaporin 5 (AQP5), the main transporter that mediate water secretion through transcellular pathway, and led to AQP5 redistribution. Extracellular signal-regulated kinase 1/2 (ERK1/2) was involved in the TRPV1-regulated AQP5 content. Besides, TRPV1 activation also modulated the expression, distribution, and function of tight junction protein, and increased paracellular permeability. ERK1/2 and myosin light chain 2 (MLC2) were responsible for the regulation of TRPV1on tight junction properties. Taken together, our work suggested that TRPV1 was a potential target to promote saliva secretion, and activation of TRPV1 might provide a new and safe therapeutic strategy to ameliorate submandibular gland hypofunction.

Tight junction plays an important role in mediating paracellular permeability in epithelia. We previously found that activation of AMP-activated protein kinase (AMPK) increased saliva secretion by modulating paracellular permeability in submandibular glands. However, the molecular mechanisms underlying AMPK-modulated paracellular permeability are unknown. In this study, we found that AICAR, an AMPK agonist, increased saliva secretion in the isolated rat submandibular glands, decreased transepithelial electrical resistance (TER), and increased 4 kDa FITC-dextran flux in cultured SMG-C6 cells. AICAR also induced redistribution of tight junction protein claudin-4, but not claudin-1, claudin-3, occludin, or ZO-1, from the cytoplasm to the membrane. Moreover, knockdown of claudin-4 by shRNA suppressed while claudin-4 re-expression restored the TER and 4 kDa FITC-dextran flux responses to AICAR. Additionally, AICAR increased ERK1/2 phosphorylation, and inhibition of ERK1/2 by U0126, an ERK1/2 kinase inhibitor, or by siRNA decreased AICAR-induced TER responses. AICAR induced the serine S199 phosphorylation of claudin-4 and enhanced the interaction of claudin-4 and occludin. Furthermore, pretreatment with U0126 significantly suppressed AMPK-modulated phosphorylation, redistribution, and interaction with occludin of claudin-4. Taken together, these results indicated that claudin-4 played a crucial role in AMPK-modulated paracellular permeability and ERK1/2 was required in AMPK-modulated tight junction barrier function in submandibular gland., (© The Author (2014). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.)

Objective: To investigate a possible role of adiponectin in the pathogenesis of autoimmune sialoadenitis in non-obese diabetic (NOD) mouse model of Sjögren's syndrome., Materials and Methods: Expression of adiponectin and its receptors (AdipoR1/2) was detected by PCR, immunoblotting, or immunofluorescence. The level of adiponectin was quantified by ELISA. Adiponectin-related signaling molecules and pro-inflammatory cytokines were examined by PCR or immunoblotting. Apoptosis was evaluated by TUNEL staining, flow cytometry, and caspase 3 activation., Results: Adiponectin and AdipoR1/2 mRNA and protein were expressed in submandibular glands. Adiponectin immunostaining was widely diffused in the cytoplasm of acinar and ductal cells. AdipoR1 was mainly distributed in acinar cytoplasm, while AdipoR2 was predominantly located at acinar cell membrane. Submandibular adiponectin levels were reduced during the progression of autoimmune sialoadenitis in 7-, 14-, and 21-week-old NOD mice, while AdipoR1/2 levels were unchanged. The levels of phosphorylated adenosine monophosphate-activated protein kinase, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase were decreased, while interferon (IFN)-γ and glandular apoptosis were temporally increased at all time points. Moreover, exogenous adiponectin supplement inhibited, whereas neutralizing endogenous adiponectin by its antibody promoted IFN-γ-induced apoptosis and caspase 3 activation in cultured submandibular acinar cells., Conclusions: Adiponectin plays a protective role on submandibular cells. Decreased adiponectin might promote glandular destruction in autoimmune sialoadenitis., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Autologous transplantation of the submandibular gland is an effective treatment for severe dry eye syndrome. However, more than 40% of patients experience epiphora 3 to 6 months after transplantation. The underlying mechanism of epiphora remains to be elucidated. To investigate the potential roles of muscarinic acetylcholine receptors (mAChRs) in the induction of epiphora in transplanted glands, we assessed and found elevated mRNA and protein expression of M1- and M3-mAChR in transplanted glands from epiphora patients. The content of inositol 1, 4, 5-trisphosphate was also elevated. Moreover, carbachol (5 and 10 µM) induced greater increase of [Ca(2+)]i in isolated epiphora submandibular cells than in controls. Although aquaporin-5 (AQP5) content and distribution in the apical and lateral plasma of epiphora glands did not change, AQP5 content was reduced in lipid microdomains (lipid rafts and caveolae) but increased in non-lipid microdomains compared with controls. Carbachol (10 µM) increased the ratio of non-lipid microdomain to total AQP5 in the cultured control submandibular gland tissue. Taken together, these results indicated that hypersensitive mAChRs might be involved in the epiphora of transplanted submandibular glands by modulating AQP5 trafficking.

Adiponectin and adiponectin receptors (AdipoR1/2) are expressed in various tissues and are involved in the regulation of multiple functions such as energy metabolism and inflammatory responses. However, the effect of adiponectin and AdipoRs in submandibular glands has not been fully evaluated. In the present study, we found that mRNA and protein of both adiponectin and AdipoR1/2 were expressed in rat submandibular glands and in the SMG-C6 cell line, as evidenced by RT-PCR and Western blot analysis. Immunofluorescence staining showed that adiponectin was diffused in the cytoplasm, while AdipoR1/2 was concentrated in the membrane of acinar cells. Saliva flow was significantly increased by full length adiponectin (fAd) or globular adiponectin (gAd) perfusion in isolated rat submandibular glands. 5-Aminoimidazole-4-carboxamide-1-4-ribofuranoside (AICAR), an adenosine monophosphate activated protein kinase (AMPK) activator, also increased saliva secretion. fAd, gAd, and AICAR all increased the average width of apical tight junctions in perfused submandibular glands, and decreased transepithelial electrical resistance (TER) in SMG-C6 cells, suggesting that adiponectin promoted secretion by modulating paracellular permeability. fAd and gAd increased p-AMPK levels, while AraA, an AMPK antagonist, abolished fAd- and gAd-induced changes in secretion, tight junction ultrastructure, and TER. Moreover, both AdipoR1 and AdipoR2 were required for fAd- or gAd-induced p-AMPK and TER responses, suggesting from their inhibition following AdipoR1 or AdipoR2 knockdown, and co-knockdown of AdipoRs by RNA interference. Our results suggest that adiponectin functions as a promoter of salivary secretion in rat submandibular glands via activation of AdipoRs, AMPK, and paracellular permeability.

Occludin plays an important role in maintaining tight junction barrier function in many types of epithelia. We previously reported that activation of transient receptor potential vanilloid subtype 1 (TRPV1) in rabbit submandibular gland promoted salivary secretion, partly by an increase in paracellular permeability. We have now explored the role of occludin in TRPV1-modulated paracellular permeability in a rat submandibular gland cell line SMG-C6. Both TRPV1 and occludin were expressed in SMG-C6 cells, and capsaicin induced redistribution of occludin, but not claudin-3, claudin-4 or E-cadherin, from the cell membrane into the cytoplasm. Capsaicin also decreased transepithelial electrical resistance (TER) and increased the Trypan Blue and FITC-dextran flux. Capsazepine (CPZ), a TRPV1 antagonist, inhibited the capsaicin-induced occludin redistribution and TER decrease. Moreover, occludin knockdown by shRNA suppressed, whereas occludin re-expression restored, the TER response to capsaicin. Mechanistically, TRPV1 activation increased ERK1/2 and MLC2 phosphorylation. PD98059, an ERK1/2 kinase inhibitor, abolished the capsaicin-induced MLC2 phosphorylation, whereas ML-7, an MLC2 kinase inhibitor, did not affect ERK1/2 phosphorylation, suggesting that ERK1/2 is the upstream signaling molecule of MLC2. Capsaicin also induced F-actin reorganization, which was abolished by CPZ, PD98059 and ML-7, indicating that TRPV1 activation altered F-actin organization in an ERK1/2- and MLC2-dependent manner. Furthermore, either PD98059 or ML-7 could abolish the capsaicin-induced TER response and occludin redistribution, whereas knockdown of ERK1/2 further confirmed that the TRPV1-modulated paracellular permeability was ERK1/2 dependent. Taken together, these results identified a crucial role of occludin in submandibular epithelial cells, and more importantly, demonstrated that occludin was required to mediate TRPV1-modulated paracellular permeability.

Background: Ischemic preconditioning (IPC) can reduce ischemia/reperfusion (I/R) injury in multiple organs and species. However, the effect of IPC on transplanted submandibular glands remains unknown. We explored the protection of IPC in transplanted submandibular glands in the rabbit and the underlying mechanism., Methods: IPC was performed by clamping the lingual artery for 10 min, with 10 min of reperfusion before transplantation. Male rabbits were randomly divided into control, transplantation, and IPC groups (n = 6 each). Saliva secretion, oxidative stress, pro-inflammatory cytokine levels, and apoptosis-related protein levels were determined at 1, 12, and 24 h after reperfusion., Results: Salivary flow was significantly increased at 12 h and decreased at 24 h in the transplanted glands. IPC treatment prevented the reduced saliva secretion at 24 h after reperfusion (P < 0.01). The mRNA levels of tumor necrosis factor-α, interleukin-1β, and reactive oxygen species, as well as malondialodehyde (MDA) and myeloperoxidase activity, were significantly increased and superoxide dismutase activity was decreased in the transplanted glands. However, these changes were all attenuated with IPC treatment (all P < 0.05). Also, acinar cell apoptosis and Bax protein expression were decreased and Bcl-2 protein expression was increased in the IPC-treated glands at 1 and 12 h after reperfusion (all P < 0.05)., Conclusions: IPC protects the secretory function of transplanted submandibular gland in the rabbit by reducing the inflammatory response, attenuating oxidative stress, and an anti-apoptosis process., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Tight junction (TJ) is an important structure that regulates material transport through the paracellular pathway across the epithelium, but its significance in salivary physiology and pathogenesis of salivary dysfunctional diseases is not fully understood. We previously demonstrated that a functional transient receptor potential vanilloid subtype 1 (TRPV1) expresses in submandibular gland (SMG). However, association of TRPV1-induced saliva secretion with TJ remains unknown. Here we explored the effect of TRPV1 activation on expression and function of TJ of rabbit SMG in vitro and in vivo. RT-PCR and western blot analysis revealed that capsaicin upregulated expression of zonula occludin-1 (ZO-1), claudin (Cldn)-3, and -11, but not Cldn-1, -2, -4, -5, and -7 in cultured SMG cells. Capsaicin also increased the entering of 4 kDa FITC-dextran into the acinar lumen, induced redistribution of cytoskeleton F-actin under confocal microscope, and these effects were abolished by preincubation of capsazepine, a TRPV1 antagonist, indicating that activation of TRPV1 increases expression and permeability of TJ in SMG. Additionally, in a hyposecretory model induced by rabbit SMG transplantation, the expression of ZO-1, Cldn-3, and -11 was decreased, whereas other TJs remained unaltered. The structure of TJ was impaired and the width of apical TJs was reduced under transmission electron microscope, concomitant with diminished immunofluorescence of F-actin in peri-apicolateral region, indicating impaired TJ expression and decreased paracellular permeability in the transplanted SMG. Moreover, topical capsaicin cream increased secretion, decreased TJ structural injury, reversed TJ expression levels, and protected F-actin morphology from disarrangement in transplanted SMGs. These data provide the first evidence to demonstrate that TJ components, particularly ZO-1, Cldn-3, and -11 have important roles in secretion of SMG under both physiological and pathophysiological conditions. The injury in TJ integrity was involved in the hypofunctional SMGs, and TRPV1 might be a potential target to improve saliva secretion through modulating expression and function of TJs.

Myoepithelial cells (MECs) mainly surround acini and intercalated ducts in the human salivary glands. The contraction of MECs provides the expulsive force to promote salivation. We previously found functional transient receptor potential vanilloid subtype 1 (TRPV1) was expressed in rabbit and human submandibular glands and increased saliva secretion. However, it was unknown whether TRPV1 was expressed in MECs of submandibular glands. In this study, we observed the immunoflourescence of TRPV1 was not only located in serous acini and ducts but also surround the basal layer of the acinus and intercalated ducts of human submandibular glands. Double immunofluorescence staining revealed colocalization of TRPV1 with calponin, vimentin, and α-smooth muscle actin, which indicated the myoepithelial expression of TRPV1. Treating submandibular gland tissues with capsaicin, an agonist of TRPV1, substantially increased the phosphorylation of the 20-kDa regulatory light-chain subunit of myosin (MLC(20) ), a crucial molecule for contraction of smooth muscle cells, in MECs. Pretreatment with capsazepine, a specific TRPV1 inhibitor, blocked capsaicin-induced MLC(20) phosphorylation. These results suggest that TRPV1 is expressed in MECs of the human submandibular gland and mediates myoepithelial contraction via a mechanism involving MLC(20) phosphorylation., (Copyright © 2011 Wiley Periodicals, Inc.)

Submandibular gland autotransplantation is effective for treating severe dry eye syndrome. However, more than 40% of patients show epiphora within 3-6 months after treatment. The mechanism underlying the hypersecretion in epiphora remains to be elucidated for developing novel interventions. Since salivary gland secretion is dependent on a variety of proteins, we analyzed the changes in protein expression in transplanted glands of epiphora patients with 2-D gel electrophoresis and electrospray ionization quadrupole/time-of-flight mass spectrometry and evaluated their possible roles in epiphora. There were 23 proteins that showed altered expression in the glands of epiphora patients, 15 being up-expressed and 8 being down-expressed. The expression of secretory proteins was decreased in these glands, including alpha-amylase, cystatin S, SA, and SN. In contrast, cytoskeletal proteins were all up-regulated, including actin and vimentin. Immunofluorescence revealed that the intensity ratio of F-actin in apical and lateral cytoplasm to total F-actin in acini was decreased in the glands of epiphora patients. Carbachol stimulation induced a similar redistribution of F-actin in the control glands. Phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) was increased in both carbachol-stimulated and epiphora glands. Preincubation of submandibular glands with ERK1/2 inhibitors PD98059 or U0126 inhibited carbachol-induced F-actin redistribution. These results indicated that differentially expressed proteins participated in the hypersecretion of transplanted submandibular glands and the redistribution of F-actin might be involved in this hypersecretion in an ERK1/2-dependent manner.

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.

Objectives: To investigate the changes in the muscarinic receptor signaling pathway with submandibular gland (SMG) transplantation and whether carbachol improves secretion in transplanted SMGs., Materials and Methods: SMG autotransplantation was performed in a rabbit model. Carbachol (1 microM) was infused into the transplanted glands from postoperative day 1-7. The expression of the M1 and M3 muscarinic receptors, aquaporin-5 (AQP5), and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was measured by RT-PCR, immunoblotting or immunofluorescence. The content of inositol 1, 4, 5-trisphosphate (IP(3)) was measured by radioimmunoassay., Results: Salivary flow of the transplanted SMGs was decreased after transplantation. As well, the expressions of M1 and M3 receptors and their downstream signaling molecules, IP(3), p-ERK1/2 and AQP5, were all reduced. Atrophy of acinar cells was shown in transplanted glands. However, all these alterations were reversed after carbachol treatment for 7 days. Furthermore, carbachol directly increased the mRNA expression of AQP5 and phosphorylation of ERK1/2 in cultured neonatal rabbit SMG cells., Conclusion: A lack of acetylcholine and downregulation of the muscarinic receptor signaling pathway is involved in the early hypofunction of transplanted SMGs. Carbachol treatment could be a new therapeutic strategy to improve secretion and prevent the obstruction of Wharton's duct in the early phase after SMG transplantation.

Autotransplantation of the submandibular gland is effective for severe keratoconjunctivitis sicca. However, most transplants show decreased secretion shortly after the operation, which leads to obstruction of Wharton's duct. The hypothesis that decreased catecholamine release due to denervation contributes to hypofunction in the early phase was tested in transplanted glands in rabbits. We found that salivary flow, expression of beta(1)- and beta(2)-adrenoceptor, and the maximum binding capacity were markedly decreased in the transplanted glands. Isoproterenol significantly reversed the decreased secretion, enhanced the expressions of beta(1)- and beta(2)-adrenoceptor, and ameliorated the atrophy of acinar cells. The contents of cAMP and phospho-ERK 1/2 were increased after isoproterenol treatment. These results indicate that lack of beta-adrenoceptor stimulation is involved in early dysfunction of the transplanted gland. Isoproterenol treatment moderates structural injury and improves secretory function in the transplanted submandibular gland through up-regulating beta(1)- and beta(2)-adrenoceptor expression and post-receptor signal transduction.

Tight junction proteins play a crucial role in paracellular transport in salivary gland epithelia. It is clear that severe xerostomia in patients with HELIX syndrome is caused by mutations in the claudin-10 gene. However, little is known about the expression pattern and role of claudin-10 in saliva secretion in physical and disease conditions. In the present study, we found that only claudin-10b transcript was expressed in human and mouse submandibular gland (SMG) tissues, and claudin-10 protein was dominantly distributed at the apicolateral membranes of acini in human, rat, and mouse SMGs. Overexpression of claudin-10 significantly reduced transepithelial electrical resistance and increased paracellular transport of dextran and Na+ in SMG-C6 cells. In C57BL/6 mice, pilocarpine stimulation promoted secretion and cation concentration in saliva in a dose-dependent increase. Assembly of claudin-10 to the most apicolateral portions in acini of SMGs was observed in the lower pilocarpine (1 mg/kg)–treated group, and this phenomenon was much obvious in the higher pilocarpine (10 mg/kg)–treated group. Furthermore, 7-, 14-, and 21-wk-old nonobese diabetic (NOD) and BALB/c mice were used to mimic the progression of hyposalivation in Sjögren syndrome. Intensity of claudin-10 protein was obviously lower in SMGs of 14- and 21-wk-old NOD mice compared with that of age-matched BALB/c mice. In the cultured mouse SMG tissues, interferon-γ (IFN-γ) downregulated claudin-10 expression. In claudin-10–overexpressed SMG-C6 cells, paracellular permeability was decreased. Furthermore, IFN-γ stimulation increased p-STAT1 level, whereas pretreatment with JAK/STAT1 antagonist significantly alleviated the IFN-γ–induced claudin-10 downregulation. These results indicate that claudin-10 functions as a pore-forming component in acinar epithelia of SMGs, assembly of claudin-10 is required for saliva secretion, and downregulation of claudin-10 induces hyposecretion. These findings may provide new clues to novel therapeutic targets on hyposalivation. [ABSTRACT FROM AUTHOR]

In the salivary glands, fibrosis occurs in many pathological conditions. Endothelial tight junction (TJ)–based barrier function plays a vital role in maintaining the homeostasis of the salivary glands. However, whether endothelial barrier function is changed and involved in the pathogenesis of glandular fibrosis is unknown. Here, by using a mouse model in which the main excretory duct of the submandibular gland (SMG) was ligated to induce inflammation and fibrosis, endothelial barrier function and TJ protein expression and distribution were examined. Both 4-kDa and 70-kDa fluorescence-labeled dextrans permeated more in the 1-, 3-, and 7-d ligated SMGs. Meanwhile, the mRNA level of claudin-5 was increased with an obvious redistribution from apicolateral membranes to lateral membranes and cytoplasm in the fibrotic glands. Notably, the TJ sealer AT1001 significantly attenuated the disrupted endothelial barrier function and thereby ameliorated the glandular fibrosis. Cytokine array detection showed that monocyte chemoattractant protein–1 (MCP-1) was highly enriched in the 3-d ligated SMGs, and MCP-1 directly impaired barrier function, increased claudin-5 expression, induced the relocalization of claudin-5, and activated p-ERK1/2 in cultured human endothelial cells. Furthermore, the upregulation and disorganization of claudin-5 as well as the elevation of MCP-1 and p-ERK1/2 signaling were also confirmed in fibrotic SMGs from patients with chronic sialadenitis and immunoglobulin G4–related sialadenitis. Altogether, our findings revealed that disrupted endothelial barrier function contributed to the progression of glandular fibrosis, and targeting endothelial TJs might be a promising approach to alleviate salivary gland fibrosis-related diseases. [ABSTRACT FROM AUTHOR]

Volume and composition of saliva are crucial for oral and systemic health. How substances, particularly macromolecules, are transported across the salivary gland epithelium has not been established in detail. Tricellulin is a component of tricellular tight junctions that form a central tube to serve as an important route for macromolecule transport. Whether tricellulin is expressed in the submandibular gland (SMG) and involved in salivation has been unknown. Here, by using Western blotting and immunofluorescence, tricellulin was found to be characteristically localized at tricellular contacts of human, rat, and mouse SMGs. Knockdown of tricellulin significantly increased, whereas overexpression of tricellulin decreased, paracellular permeability for 40-kDa but not for 4-kDa fluorescein isothiocyanate-dextran, while transepithelial electrical resistance was unaffected. Conversely, claudin-4 knockdown and overexpression affected transepithelial electrical resistance but not 40-kDa fluorescein isothiocyanate-dextran transport, suggesting that tricellulin regulated transport of macromolecules but not ions, which were mainly regulated by bicellular tight junctions (bTJs). Moreover, tricellulin was dynamically redistributed from tri- to bicellular membranes in cholinergically stimulated SMG tissues and cells. Immunoglobulin-like domain-containing receptor 1 (ILDR1) recruits tricellulin to tricellular contacts. The proportion of macromolecules in the saliva was increased, whereas the amount of stimulated saliva was unchanged in Ildr1-/- mice, which displayed abnormal tricellulin distribution in SMGs. Furthermore, tricellulin interacted with bTJ proteins, such as occludin, claudin-1, claudin-3, claudin-4, and ZO-1, in rat SMG epithelial polarized cell line SMG-C6. Knockdown of tricellulin decreased occludin levels. Thus, we revealed a specific expression pattern of tricellulin in SMG epithelium. Tricellulin not only functioned as a barrier for macromolecules but also modulated the connection of bTJs to the tight junction complex. Alterations in tricellulin expression and distribution could thereby change salivary composition. Our study provided novel insights on salivary gland tight junction organization and function. [ABSTRACT FROM AUTHOR]

Sjögren's syndrome (SS) is an inflammatory autoimmune disease that causes secretory dysfunction of the salivary glands. It has been reported that proinflammatory cytokine interleukin-17 (IL-17) was elevated and tight junction (TJ) integrity disrupted in minor salivary glands from SS patients. However, whether the elevated IL-17 in SS affects TJ integrity and thereby alters the function of salivary gland is unknown. Here, by using nonobese diabetic (NOD) mice as SS model, we found that the stimulated salivary flow rate was significantly decreased in NOD mice. Lymphocyte infiltration was mainly observed in submandibular glands (SMGs), but not parotid glands (PGs), of NOD mice. IL-17 was significantly increased and mainly located in lymphocytic-infiltrating regions in SMGs but not detectable in PGs of NOD mice. Meanwhile, the epithelial barrier function was disrupted, as evidenced by an increased paracellular tracer clearance and an enlarged acinar TJ width in SMGs of NOD mice. Furthermore, claudin-1 and -3 were elevated especially at the basolateral membranes, whereas claudin-4, occludin, and zonula occludens-1 (ZO-1) were reduced in SMGs of NOD mice. Moreover, occludin and ZO-1 were dispersed into cytoplasm in SMGs of NOD mice. However, no change in the expression and distribution of TJ proteins was found in PGs. In vitro, IL-17 significantly decreased the levels and apical staining of claudin-4 and ZO-1 proteins in the cultured SMG tissues, as well as claudin-1, occludin, and ZO-1 in PG tissues. Moreover, IL-17 activated the phosphorylation of IκBα and p65 in SMG cells, whereas pretreatment with NF-κB inhibitor pyrrolidine dithiocarbamate suppressed the IL-17-induced downregulation of claudin-4 and ZO-1 in SMG tissues. Taken together, these findings indicate that IL-17 derived from infiltrating lymphocyte impairs the integrity of TJ barrier through NF-κB signaling pathway, and thus might contribute to salivary gland dysfunction in SS. [ABSTRACT FROM AUTHOR]