Distribution of Tight Junction Proteins in Adult Human Salivary Glands

Tight junctions (TJs) are an essential structure of fluid-secreting cells, such as those in salivary glands. Three major families of integral membrane proteins have been identified as components of the TJ: claudins, occludin, and junctional adhesion molecules (JAMs), plus the cytosolic protein zonula occludens (ZO). We have been working to develop an orally implantable artificial salivary gland that would be suitable for treating patients lacking salivary parenchymal tissue. To date, little is known about the distribution of TJ proteins in adult human salivary cells and thus what key molecular components might be desirable for the cellular component of an artificial salivary gland device. Therefore, the aim of this study was to determine the distribution of TJ proteins in human salivary glands. Salivary gland samples were obtained from 10 patients. Frozen and formalin-fixed paraffin-embedded sections were stained using IHC methods. Claudin-1 was expressed in ductal, endothelial, and ∼25% of serous cells. Claudins-2, -3, and -4 and JAM-A were expressed in both ductal and acinar cells, whereas claudin-5 was expressed only in endothelial cells. Occludin and ZO-1 were expressed in acinar, ductal, and endothelial cells. These results provide new information on TJ proteins in two major human salivary glands and should serve as a reference for future studies to assess the presence of appropriate TJ proteins in a tissue-engineered human salivary gland. (J Histochem Cytochem 56:1093–1098, 2008)     

Epidermal growth factor receptor activation differentially regulates claudin expression and enhances transepithelial resistance in Madin-Darby canine kidney cells

Tight junctions (TJs) are the most apical cell-cell junctions, and claudins, the recently identified TJ proteins, are critical for maintaining cell-cell adhesion in epithelial cell sheets. Based on their in vivo distribution and the results of overexpression studies, certain claudins, including claudin-1 and -4, are postulated to increase, whereas other claudins, especially claudin-2, are postulated to decrease the overall transcellular resistance. The overall ratio among claudins expressed in a cell/tissue has been hypothesized to define the complexity of TJs. Disruption of the TJs contributes to various human diseases, and a correlation between reduction of TJ function and tumor dedifferentiation has been postulated. The epidermal growth factor (EGF) receptor (EGFR) is overexpressed in a wide spectrum of epithelial cancers, and its expression correlates with a more metastatic cancer phenotype. However, normal functioning of EGFR is essential for normal epithelial cell proliferation and differentiation. The role of EGFR-dependent signaling in the development and maintenance of epithelial TJ integrity has not been studied in detail. This study demonstrates that, in polarized Madin-Darby canine kidney II cells, EGF-induced EGFR activation significantly inhibited claudin-2 expression while simultaneously inducing cellular redistribution and increased expression of claudin-1, -3, and -4. Accompanying these EGF-induced changes in claudin expression was a 3-fold increase in transepithelial resistance, a functional measure of TJs. In contrast, there were no alterations in protein expression and/or intracellular localization of other TJ-related proteins (ZO-1 and occludin) or adherens junction-associated proteins (E-cadherin and beta-catenin), suggesting that EGF regulates TJ function through selective and differential regulation of claudins.     

Proinflammatory cytokine-induced tight junction remodeling through dynamic self-assembly of claudins

Tight junctions (TJs) are dynamic, multiprotein intercellular adhesive contacts that provide a vital barrier function in epithelial tissues. TJs are remodeled during physiological development and pathological mucosal inflammation, and differential expression of the claudin family of TJ proteins determines epithelial barrier properties. However, the molecular mechanisms involved in TJ remodeling are incompletely understood. Using acGFP-claudin 4 as a biosensor of TJ remodeling, we observed increased claudin 4 fluorescence recovery after photobleaching (FRAP) dynamics in response to inflammatory cytokines. Interferon γ and tumor necrosis factor α increased the proportion of mobile claudin 4 in the TJ. Up-regulation of claudin 4 protein rescued these mobility defects and cytokine-induced barrier compromise. Furthermore, claudins 2 and 4 have reciprocal effects on epithelial barrier function, exhibit differential FRAP dynamics, and compete for residency within the TJ. These findings establish a model of TJs as self-assembling systems that undergo remodeling in response to proinflammatory cytokines through a mechanism of heterotypic claudin-binding incompatibility.     

Aquaporins in salivary glands and pancreas

Background

Salivary glands and pancreas are involved in saliva secretion, pancreatic fluid secretion and insulin secretion. These functions are essential for proper oral, pancreatic and glucose homeostasis. Aquaporins are water-permeable transmembrane protein involved in the physiology of these secretory gland functions.

Scope of review

This review gives an overview of the morphology of salivary glands and pancreas, the expression and localization of aquaporins, the secretion roles and mechanisms, the physiological roles of aquaporins, and the role of aquaporins in pathophysiological conditions.

Major conclusions

Several aquaporins are expressed in salivary glands and pancreas, and some play important physiological roles. Modulation of aquaporin expression and/or trafficking may contribute to the pathogenesis of diseases affecting salivary glands and pancreas glands such as xerostomic conditions, pancreatic insufficiencies and diabetes.

General significance

Aquaporins are involved in physiological and pathophysiological processes in salivary glands and pancreas. They could represent therapeutic targets for the treatment of diseases affecting the salivary glands and pancreas. This article is part of a Special Issue entitled Aquaporins.     

Integrin expression in developing human salivary glands

The development and complete differentiation of salivary glands is a complex process that involves a large number of co-ordinated events. Little is known about the molecular basis for salivary gland development. However, we have reported previously that integrins appear to play a role. Integrins are heterodimeric transmembrane receptors consisting of one α and one β subunit that play a pivotal role in the interaction of cells with the extracellular matrix. Such interactions regulate the organisation of cells of tissues and organs during development as well as cell proliferation and differentiation. Using immunohistochemistry and Western and Northern blot analysis, we mapped the localisation and expression of integrins β1, β3 and β4 in human salivary glands obtained from foetuses ranging from weeks 4–24 of gestation and compared it with adult salivary glands. Integrin β1 first appeared during the canalisation stage and during the differentiation stage. A message first appeared at week 6 of development. The expression of β4 integrin protein and message was observed only in the late stage of differentiation. Integrin β3 was not detected in the developing glands; however, integrins β1, β3 and β4 were all expressed in adult salivary gland tissues. The data suggest that integrins, particularly β1, have a role to play in salivary gland development and differentiation.     

Enhancement of barrier function by overexpression of claudin-4 in tight junctions of submandibular gland cells

In salivary glands, primary saliva is produced by acini and is modified by the reabsorption and secretion of ions in the ducts. Thus, the permeability of intercellular junctions in the ducts is considered to be lower than in the acini. We have examined the relationship between the expressed claudin isotypes and the barrier functions of tight junctions in a submandibular gland epithelial cell line, SMIE. SMIE cells were originally derived from rat submandibular duct cells, but their barrier functions are not as efficient as those of Madin-Darby canine kidney cells. Large molecules, such as 70-kDa dextran, diffuse across the monolayers, although E-cadherin and occludin, adherens junction and tight junction proteins, respectively, are expressed in SMIE cells. Claudin-3 protein has also been detected, but the expression level of claudin-3 mRNA is much lower than in the original submandibular glands. Other claudins including claudin-4 (originally expressed in the duct cells) have not been detected. Because of the limited expression of claudins, SMIE cells are suitable for studying the role(s) of claudins. To examine the function of claudin-4 in submandibular glands, we have overexpressed green fluorescence protein (GFP)-fused claudin-4 in SMIE cells. Cells that express GFP-fused claudin-4 have a higher transepithelial electrical resistance and a lower permeability of 70-kDa dextran, although the expression levels of occludin and claudin-3 are hardly affected. Therefore, claudin-4 plays a role in the regulation of the barrier function of tight junctions in submandibular glands. This work was supported by Grants-in-Aid for scientific research from the Ministry of Education, Science, Culture, Sports, and Technology of Japan (16591868), by a Nihon University Multidisciplinary Research Grant for 2006 and 2007, and by a Grant-in-Aid for a 2003 Multidisciplinary Research Project from MEXT.     

Inhibition of Src and p38 MAP kinases suppresses the change of claudin expression induced on dedifferentiation of primary cultured parotid acinar cells

Sj?gren's syndrome and therapeutic radiation for head and neck cancers result in irreversible changes in the parenchyma of salivary glands, loss of acinar cells, prominence of duct cells, and fibrosis. To clarify mechanisms of salivary gland dysfunction, we identified a signaling pathway involved in the dedifferentiation of primary cultures of parotid acinar cells. We reported previously that the expression pattern of claudins changes during culture, is related to the three-dimensional organization of the cells, and reflects their ability to function as acinar cells. In this study, we found that this change of claudin expression is a process of dedifferentiation, because expression of other differentiation markers also changes during culture. The expression levels of claudins-4 and -6, cytokeratin 14, and vimentin are increased, and those of claudin-10, aquaporin 5, and amylase are decreased. Inhibitors of Src and p38 MAP kinases suppress these changes and increase the expression of acinar marker proteins. Differences in extracellular matrix components have no effect. Activation of p38 MAP kinase occurs during cell isolation from the parotid glands and is retained up to 6 h after the isolation. In contrast, activation of Src kinases does not increase during the cell isolation. The Src inhibitor PP1 suppresses the activation of p38 MAP kinase. Therefore, cellular stresses induced during cell isolation cause dedifferentiation and transition to duct-like cells through activation of p38 MAP kinase and constitutively active Src kinases.     

Proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma alter tight junction structure and function in the rat parotid gland Par-C10 cell line

Sj?gren's syndrome (SS) is an autoimmune disorder characterized by inflammation and dysfunction of salivary glands, resulting in impaired secretory function. The production of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) is elevated in exocrine glands of patients with SS, although little is known about the effects of these cytokines on salivary epithelial cell functions necessary for saliva secretion, including tight junction (TJ) integrity and the establishment of transepithelial ion gradients. The present study demonstrates that chronic exposure of polarized rat parotid gland (Par-C10) epithelial cell monolayers to TNF-alpha and IFN-gamma decreases transepithelial resistance (TER) and anion secretion, as measured by changes in short-circuit current (I(sc)) induced by carbachol, a muscarinic cholinergic receptor agonist, or UTP, a P2Y(2) nucleotide receptor agonist. In contrast, TNF-alpha and IFN-gamma had no effect on agonist-induced increases in the intracellular calcium concentration [Ca(2+)](i) in Par-C10 cells. Furthermore, treatment of Par-C10 cell monolayers with TNF-alpha and IFN-gamma increased paracellular permeability to normally impermeant proteins, altered cell and TJ morphology, and downregulated the expression of the TJ protein, claudin-1, but not other TJ proteins expressed in Par-C10 cells. The decreases in TER, agonist-induced transepithelial anion secretion, and claudin-1 expression caused by TNF-alpha, but not IFN-gamma, were reversible by incubation of Par-C10 cell monolayers with cytokine-free medium for 24 h, indicating that IFN-gamma causes irreversible inhibition of cellular activities associated with fluid secretion in salivary glands. Our results suggest that cytokine production is an important contributor to secretory dysfunction in SS by disrupting TJ integrity of salivary epithelium.     

Protein Kinase C Activation Has Distinct Effects on the Localization, Phosphorylation and Detergent Solubility of the Claudin Protein Family in Tight and Leaky Epithelial Cells

We have previously shown that protein kinase C (PKC) activation has distinct effects on the structure and barrier properties of cultured epithelial cells (HT29 and MDCK I). Since the claudin family of tight junction (TJ)-associated proteins is considered to be crucial for the function of mature TJ, we assessed their expression patterns and cellular destination, detergent solubility and phosphorylation upon PKC stimulation for 2 or 18 h with phorbol myristate acetate (PMA). In HT29 cells, claudins 1, 3, 4 and 5 and possibly claudin 2 were redistributed to apical cell–cell contacts after PKC activation and the amounts of claudins 1, 3 and 5, but not of claudin 2, were increased in cell lysates. By contrast, in MDCK I cells, PMA treatment resulted in redistribution of claudins 1, 3, 4 and 5 from the TJ and in reorganization of the proteins into more insoluble complexes. Claudins 1 and 4 were phosphorylated in both MDCK I and HT29 cells, but PKC-induced changes in claudin phosphorylation state were detected only in MDCK I cells. A major difference between HT29 and MDCK I cells, which have low and high basal transepithelial electrical resistance, respectively, was the absence of claudin 2 in the latter. Our findings show that PKC activation targets in characteristic ways the expression patterns, destination, detergent solubility and phosphorylation state of claudins in epithelial cells with different capacities to form an epithelial barrier.     

Serial cultivation of epithelial cells from human and macaque salivary glands

Summary To study the regulation of human salivary-type gene expression we developed cell culture systems to support the growth and serial cultivation of salivary gland epithelial and fibroblastic cell types. We have established 22 independent salivary gland epithelial cell strains from parotid or submandibular glands of human or macaque origin. Nineteen strains were derived from normal tissues and three from human parotid gland tumors. Both the normal and the tumor-derived salivary gland epithelial cells could be serially cultivated with the aid of a 3T3 fibroblast feeder layer in a mixture of Ham’s F12 and Dulbecco’s modified Eagle’s media supplemented with fetal bovine serum, calcium, cholera toxin, hydrocortisone, insulin, and epidermal growth factor. Salivary gland epithelial cells cultured under these conditions continued to express the genes for at least two acinar-cell-specific markers at early passages. Amylase enzyme activity was detected in conditioned media from cultured rhesus parotid epithelial cells as late as Passage 5. Proline-rich-protein-specific RNAs were detected in primary cultures of both rhesus and human parotid epithelial cells. Neither amylase enzyme activity nor PRP-specific RNAs were detected in fibroblasts isolated from the same tissues. In addition, salivary gland epithelial cells cultured under our conditions retain the capacity to undergo dramatic morphologic changes in response to different substrata. The cultured salivary gland epithelial cells we have established will be important tools for the study of salivary gland differentiation and the tissue-specific regulation of salivary-type gene expression.     

Crosstalk of tight junction components with signaling pathways

Tight junctions (TJs) regulate the passage of ions and molecules through the paracellular pathway in epithelial and endothelial cells. TJs are highly dynamic structures whose degree of sealing varies according to external stimuli, physiological and pathological conditions. In this review we analyze how the crosstalk of protein kinase C, protein kinase A, myosin light chain kinase, mitogen-activated protein kinases, phosphoinositide 3-kinase and Rho signaling pathways is involved in TJ regulation triggered by diverse stimuli. We also report how the phosphorylation of the main TJ components, claudins, occludin and ZO proteins, impacts epithelial and endothelial cell function.     

Epidermal growth factor (EGF) expression in human salivary glands. An immunohistochemical study.

Epidermal growth factor (EGF) is a biologically active peptide involved in differentiation, growth, regeneration and repair of human and animal tissues. Quantitative biochemical studies showed in man the highest concentration of EGF in the parotid gland. The aim of the present study was to define EGF immunolocalization in the individual segments of the human major salivary glands (salivon). The material consisted of sections obtained from the surgically removed salivary glands: parotid, submaxillary and sublingual. Immunohistochemical studies were performed by PAP method using monoclonal antibody against human epidermal growth factor. EGF expression was found almost exclusively in the efferent pathways of the salivary glands, mostly in the intercalated ducts and Pflüger salivary tubules. These segments of the salivon are most developed in the parotid gland in which the staining was stronger than in other salivary glands.     

Crosstalk of tight junction components with signaling pathways

Tight junctions (TJs) regulate the passage of ions and molecules through the paracellular pathway in epithelial and endothelial cells. TJs are highly dynamic structures whose degree of sealing varies according to external stimuli, physiological and pathological conditions. In this review we analyze how the crosstalk of protein kinase C, protein kinase A, myosin light chain kinase, mitogen-activated protein kinases, phosphoinositide 3-kinase and Rho signaling pathways is involved in TJ regulation triggered by diverse stimuli. We also report how the phosphorylation of the main TJ components, claudins, occludin and ZO proteins, impacts epithelial and endothelial cell function.     

Decreased interaction between ZO-1 and occludin is involved in alteration of tight junctions in transplanted epiphora 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.     

Zonula Occludens-1, Occludin and E-cadherin Expression and Organization in Salivary Glands with Sj?gren’s Syndrome

Sjögren’s syndrome (SS) is a chronic inflammatory autoimmune disorder that causes secretory dysfunction of the salivary glands leading to dry mouth. Previous studies reported that tight junction (TJ) proteins are down-regulated and lose polarity in human minor salivary glands with SS, suggesting that TJ structure is compromised in SS patients. In this paper, we utilized the NOD/ShiLtJ mouse with the main goal of evaluating this model for future TJ research. We found that the organization of apical proteins in areas proximal and distal to lymphocytic infiltration remained intact in mouse and human salivary glands with SS. These areas looked comparable to control glands (i.e., with no lymphocytic infiltration). TJ staining was absent in areas of lymphocytic infiltration coinciding with the loss of salivary epithelium. Gene expression studies show that most TJs are not significantly altered in 20-week-old NOD/ShiLtJ mice as compared with age-matched C57BL/6 controls. Protein expression studies revealed that the TJ proteins, zonula occludens-1 (ZO-1), occludin, claudin-12, as well as E-cadherin, do not significantly change in NOD/ShiLtJ mice. Our results suggest that ZO-1, occludin and E-cadherin are not altered in areas without lymphocytic infiltration. However, future studies will be necessary to test the functional aspect of these results.     

Molecular composition of tight and adherens junctions in the rat olfactory epithelium and fila

Tight and adherens junctions (TJs, AJs) between neurons, epithelial and glial cells provide barrier and adhesion properties in the olfactory epithelium (OE), and subserve functions such as compartmentalization and axon growth in the fila olfactoria (FO). Immunofluorescence and immunoelectronmicroscopy were combined in sections of rat OE and FO to document the cellular and subcellular localization of TJ proteins occludin(Occl), claudins(Cl) 1-5 and zonula occludens(ZO) proteins 1-3, and of AJ proteins N-cadherin(cad), E-cad, and alpha-, beta- and p120-catenin(cat). With the exception of Cl2, all TJ proteins were colocalized in OE junctions. Differences in relative immunolabeling intensities were noted between neuronal and epithelial TJs. In the FO, Cl5-reactivity was localized in olfactory ensheathing cell (OEC) junctions, Cl1-reactivity in the FO periphery, with differential colocalization with ZOs. Supporting cells formed N-cad-immunoreactive (ir) AJs with olfactory sensory neurons, E-cad-ir junctions with microvillar and gland duct cells, and both N-cad and E-cad-ir junctions in homotypic contacts. Alpha, beta- and p120-cat were localized in all AJs of the OE. AJs were scarce in the globose basal cell layer. Immature and mature neurons formed numerous contacts. In the FO, AJs were documented between OECs, between OECs and axons, and between axons. Most AJs colocalized N-cad with catenins, occasionally E-cad-ir AJs were found in the FO periphery. Characteristics of molecular composition suggest differential properties of TJs formed by neuronal, epithelial and glial cells in the OE and FO. The presence and molecular composition of AJs are consistent with a role of AJ proteins in neuroplastic processes in the peripheral olfactory pathway.