Chong Ding

Occludin is required for TRPV1-modulated paracellular permeability in the submandibular gland.

Summary 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.

Claudin-4 is required for modulation of paracellular permeability by muscarinic acetylcholine receptor in epithelial cells.

ABSTRACT The epithelial cholinergic system plays an important role in water, ion and solute transport. Previous studies have shown that activation of muscarinic acetylcholine receptors (mAChRs) regulates paracellular transport of epithelial cells; however, the underlying mechanism is still largely unknown. Here, we found that mAChR activation by carbachol and cevimeline reduced the transepithelial electrical resistance (TER) and increased the permeability of paracellular tracers in rat salivary epithelial SMG-C6 cells. Carbachol induced downregulation and redistribution of claudin-4, but not occludin or ZO-1 (also known as TJP1). Small hairpin RNA (shRNA)-mediated claudin-4 knockdown suppressed, whereas claudin-4 overexpression retained, the TER response to carbachol. Mechanistically, the mAChR-modulated claudin-4 properties and paracellular permeability were triggered by claudin-4 phosphorylation through ERK1/2 (also known as MAPK3 and MAPK1, respectively). Mutagenesis assay demonstrated that S195, but not S199, S203 or S207, of claudin-4, was the target for carbachol. Subsequently, the phosphorylated claudin-4 interacted with β-arrestin2 and triggered claudin-4 internalization through the clathrin-dependent pathway. The internalized claudin-4 was further degraded by ubiquitylation. Taken together, these findings suggested that claudin-4 is required for mAChR-modulated paracellular permeability of epithelial cells through an ERK1/2, β-arrestin2, clathrin and ubiquitin-dependent signaling pathway. Highlighted Article: The epithelial cholinergic system activates a signaling pathway that regulates paracellular permeability by modulating the content and distribution of the tight junction protein claudin-4.

Activation of transient receptor potential vanilloid subtype 1 increases expression and permeability of tight junction in normal and hyposecretory submandibular gland

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.

Functional Vanilloid Receptor-1 in Human Submandibular Glands

Vanilloid receptor-1 (VR1) was originally found in the nervous system. Recent evidence indicates that VR1 is also expressed in various cell types. We hypothesized that VR1 exists in the human submandibular gland (SMG) and is involved in regulating salivary secretion. VR1 mRNA and protein were expressed in human SMGs and a human salivary intercalated duct cell line. VR1 was mainly located in serous acinar and ductal cells, but not in mucous acinar cells. Capsaicin, an agonist of VR1, increased intracellular free calcium, enhanced phosphorylation of extracellular signal-regulated kinase, and induced the trafficking of aquaporin 5 (AQP5) from the cytoplasm to the plasma membrane. These effects were abolished by pre-treatment with the VR1 antagonist capsazepine. Furthermore, capsaicin cream applied to the skin covering the submandibular area increased salivary secretion. These findings indicated that a functional VR1 is expressed in the human SMG and is involved in regulating salivary secretion by mediating AQP5 trafficking.

Adiponectin Increases Secretion of Rat Submandibular Gland via Adiponectin Receptors-Mediated AMPK Signaling

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.

Carbachol improves secretion in the early phase after rabbit submandibular gland transplantation.

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 Whartons duct in the early phase after SMG transplantation.

Hypersensitive mAChRs are Involved in the Epiphora of Transplanted Glands

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 [Ca2+]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.

Claudin-4 is required for AMPK-modulated paracellular permeability in submandibular gland cells

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.

Expression of ghrelin in human salivary glands and its levels in saliva and serum in Chinese obese children and adolescents.

OBJECTIVE The aim of the present study was to reveal the expression characteristics of ghrelin in human three major salivary glands and to investigate saliva and serum ghrelin level and the relation with weight and lipid indices in Chinese children. DESIGN Expression and distribution of ghrelin in parotid, submandibular, and sublingual glands were measured by reverse transcription-polymerase chain reaction and immunohistochemistry. Saliva and serum samples were collected from 194 Chinese children and adolescents (mean age 12.98 years). Ghrelin levels were tested by enzyme-linked immunosorbent assay. Significant differences were estimated by one-way ANOVA. RESULTS Ghrelin mRNA was expressed in parotid and submandibular glands, but was not detectable in sublingual glands. Ghrelin proteins were widespread in the cytoplasm of striated, intercalated and excretory ducts, as well as in serous acini of parotid and submandibular glands, but not in mucous acinar cells of sublingual glands. Saliva and serum ghrelin levels were increased along with BMI. There was positive correlation between saliva and serum ghrelin levels (r=0.534, P<0.01). Serum and saliva ghrelin levels were both significantly correlated with BMI (r=0.523, r=0.374, P<0.01, respectively), but not with blood lipid levels. CONCLUSIONS Parotid and submandibular glands were primary sources of ghrelin produced and released in saliva. Although whether salivary ghrelin could be useful in the diagnosis of obesity remains to be determined, salivary ghrelin might be a possible alternative to serum ghrelin for predicting obesity.

Proteomic analysis of secretion from human transplanted submandibular gland replacing lacrimal gland with severe keratoconjunctivitis sicca

PURPOSE Proteomic analysis of secretions from transplanted or non-transplanted submandibular glands in patients with severe keratoconjunctivitis sicca and tears from normal eyes. EXPERIMENTAL DESIGN Secretions from submandibular glands transplanted to replace lacrimal glands and non-transplanted submandibular glands were collected at 1year from 5 patients with severe keratoconjunctivitis sicca undergoing transplantation, and tears were collected from 3 normal subjects. 2-D electrophoresis (2-DE), then mass spectrometry was used to identify proteins. Western blot analysis was used to confirm protein expression. RESULTS We identified 34 and 11 distinct proteins in the saliva from transplanted submandibular glands and tears, respectively. The saliva from transplanted submandibular glands contained almost all the proteins abundant in tear fluid. The functions of identified proteins in the saliva from transplanted submandibular gland were mainly immune response and anti-bacterial. In total, 7 proteins showed differential expression between the saliva of transplanted and non-transplanted submandibular glands. The upregulation of short palate, lung and nasal epithelium carcinoma-associated protein 2 and carbonic anhydrase VI was confirmed by Western blot analysis. CONCLUSIONS Identified proteins in saliva from transplanted submandibular glands may protect ocular structures. These findings can help in understanding the functional status of transplanted submandibular glands.