Olga J. Baker

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

Sjögrens 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.

Resolvin D1 prevents TNF-α-mediated disruption of salivary epithelial formation

Sjögrens syndrome is a chronic autoimmune disorder characterized by inflammation of salivary glands resulting in impaired secretory function. Our present studies indicate that chronic exposure of salivary epithelium to TNF-α and/or IFN-γ alters tight junction integrity, leading to secretory dysfunction. Resolvins of the D-series (RvDs) are endogenous lipid mediators derived from DHA that regulate excessive inflammatory responses leading to resolution and tissue homeostasis. In this study, we addressed the hypothesis that activation of the RvD1 receptor ALX/FPR2 in salivary epithelium prevents and/or resolves the TNF-α-mediated disruption of acinar organization and enhances monolayer formation. Our results indicate that 1) the RvD1 receptor ALX/FPR2 is present in fresh, isolated cells from mouse salivary glands and in cell lines of salivary origin; and 2) the agonist RvD1 (100 ng/ml) abolished tight junction and cytoskeletal disruption caused by TNF-α and enhanced cell migration and polarity in salivary epithelium. These effects were blocked by the ALX/FPR2 antagonist butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe. The ALX/FPR2 receptor signals via modulation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways since, in our study, blocking PI3K activation with LY294002, a potent and selective PI3K inhibitor, prevented RvD1-induced cell migration. Furthermore, Akt gene silencing with the corresponding siRNA almost completely blocked the ability of Par-C10 cells to migrate. Our findings suggest that RvD1 receptor activation promotes resolution of inflammation and tissue repair in salivary epithelium, which may have relevance in the restoration of salivary gland dysfunction associated with Sjögrens syndrome.

Interleukin‐1β enhances nucleotide‐induced and α‐secretase‐dependent amyloid precursor protein processing in rat primary cortical neurons via up‐regulation of the P2Y2 receptor

The heterologous expression and activation of the human P2Y2 nucleotide receptor (P2Y2R) in human 1321N1 astrocytoma cells stimulates α‐secretase‐dependent cleavage of the amyloid precursor protein (APP), causing extracellular release of the non‐amyloidogenic protein secreted amyloid precursor protein (sAPPα). To determine whether a similar response occurs in a neuronal cell, we analyzed whether P2Y2R‐mediated production of sAPPα occurs in rat primary cortical neurons (rPCNs). In rPCNs, P2Y2R mRNA and receptor activity were virtually absent in quiescent cells, whereas overnight treatment with the pro‐inflammatory cytokine interleukin‐1β (IL‐1β) up‐regulated both P2Y2R mRNA expression and receptor activity by four‐fold. The up‐regulation of the P2Y2R was abrogated by pre‐incubation with Bay 11‐7085, an IκB‐α phosphorylation inhibitor, which suggests that P2Y2R mRNA transcript levels are regulated through nuclear factor‐κ‐B (NFκB) signaling. Furthermore, the P2Y2R agonist Uridine‐5′‐triphosphate (UTP) enhanced the release of sAPPα in rPCNs treated with IL‐1β or transfected with P2Y2R cDNA. UTP‐induced release of sAPPα from rPCNs was completely inhibited by pre‐treatment of the cells with the metalloproteinase inhibitor TACE inhibitor (TAPI‐2) or the phosphatidylinositol 3‐kinase (PI3K) inhibitor LY294002, and was partially inhibited by the MAPK/extracellular signal‐regulated kinase inhibitor U0126 and the protein kinase C inhibitor GF109203. These data suggest that P2Y2R‐mediated release of sAPPα from cortical neurons is directly dependent on a disintegrin and metalloproteinase (ADAM) 10/17 and PI3K activity, whereas extracellular signal‐regulated kinase 1/2 and PI3K activity may indirectly regulate APP processing. These results demonstrate that elevated levels of pro‐inflammatory cytokines associated with neurodegenerative diseases, such as IL‐1β, can enhance non‐amyloidogenic APP processing through up‐regulation of the P2Y2R in neurons.

Tight Junctions in Salivary Epithelium

Epithelial cell tight junctions (TJs) consist of a narrow belt-like structure in the apical region of the lateral plasma membrane that circumferentially binds each cell to its neighbor. TJs are found in tissues that are involved in polarized secretions, absorption functions, and maintaining barriers between blood and interstitial fluids. The morphology, permeability, and ion selectivity of TJ vary among different types of tissues and species. TJs are very dynamic structures that assemble, grow, reorganize, and disassemble during physiological or pathological events. Several studies have indicated the active role of TJ in intestinal, renal, and airway epithelial function; however, the functional significance of TJ in salivary gland epithelium is poorly understood. Interactions between different combinations of the TJ family (each with their own unique regulatory proteins) define tissue specificity and functions during physiopathological processes; however, these interaction patterns have not been studied in salivary glands. The purpose of this review is to analyze some of the current data regarding the regulatory components of the TJ that could potentially affect cellular functions of the salivary epithelium.

P2Y2 Nucleotide Receptors Mediate Metalloprotease-dependent Phosphorylation of Epidermal Growth Factor Receptor and ErbB3 in Human Salivary Gland Cells

The G protein-coupled receptor P2Y2 nucleotide receptor (P2Y2R) has been shown to be up-regulated in a variety of tissues in response to stress or injury. Recent studies have suggested that P2Y2Rs may play a role in immune responses, wound healing, and tissue regeneration via their ability to activate multiple signaling pathways, including activation of growth factor receptors. Here, we demonstrate that in human salivary gland (HSG) cells, activation of the P2Y2R by its agonist induces phosphorylation of ERK1/2 via two distinct mechanisms, a rapid, protein kinase C-dependent pathway and a slower and prolonged, epidermal growth factor receptor (EGFR)-dependent pathway. The EGFR-dependent stimulation of UTP-induced ERK1/2 phosphorylation in HSG cells is inhibited by the adamalysin inhibitor tumor necrosis factor-α protease inhibitor or by small interfering RNA that selectively silences ADAM10 and ADAM17 expression, suggesting that ADAM metalloproteases are required for P2Y2R-mediated activation of the EGFR. G protein-coupled receptors have been shown to promote proteolytic release of EGFR ligands; however, neutralizing antibodies to known ligands of the EGFR did not inhibit UTP-induced EGFR phosphorylation. Immunoprecipitation experiments indicated that UTP causes association of the EGFR with another member of the EGF receptor family, ErbB3. Furthermore, stimulation of HSG cells with UTP induced phosphorylation of ErbB3, and silencing of ErbB3 expression inhibited UTP-induced phosphorylation of both ErbB3 and EGFR. UTP-induced phosphorylation of ErbB3 and EGFR was also inhibited by silencing the expression of the ErbB3 ligand neuregulin 1 (NRG1). These results suggest that P2Y2R activation in salivary gland cells promotes the formation of EGFR/ErbB3 heterodimers and metalloprotease-dependent neuregulin 1 release, resulting in the activation of both EGFR and ErbB3.

ALX/FPR2 receptor for RvD1 is expressed and functional in salivary glands

Sjögrens syndrome (SS) is an autoimmune disorder characterized by chronic inflammation and destruction of salivary and lacrimal glands, leading to dry mouth, dry eyes, and the presence of anti-nuclear antibodies. Despite modern advances, the current therapies for SS have no permanent benefit. A potential treatment could involve the use of resolvins, which are highly potent endogenous lipid mediators that are synthesized during the resolution of inflammation to restore tissue homeostasis. Our previous studies indicate that ALX/FPR2, the receptor for RvD1, is expressed and active in the rat parotid cell line Par-C10. Specifically, activation of ALX/FPR2 with RvD1 blocked inflammatory signals caused by TNF-α and enhanced salivary epithelial integrity. The goal of this study was to investigate RvD1 receptor expression and signaling pathways in primary salivary cells. Additionally, we determined the role of the aspirin-triggered 17R analog (AT-RvD1, a more chemically stable RvD1 epimeric form) in prevention of TNF-α-mediated salivary inflammation in mouse submandibular glands (mSMG). Our results indicate that ALX/FPR2 is expressed in mSMG and is able to elicit intracellular Ca2+ responses and phosphorylation of Erk1/2, as well as Akt. Given that these signaling pathways are linked to cell survival, we investigated whether AT-RvD1 was able to prevent programmed cell death in mSMG. Specifically, we determined that AT-RvD1 prevented TNF-α-mediated caspase-3 activation. Finally, we show that ALX/FPR2 is expressed in human minor salivary glands with and without SS, indicating the potential therapeutic use of AT-RvD1 for this condition.

Current cell models for bioengineering a salivary gland: a mini-review of emerging technologies

Saliva plays a major role in maintaining oral health. Patients afflicted with a decrease in saliva secretion (symptomatically, xerostomia) exhibit difficulty in chewing and swallowing foods, tooth decay, periodontal disease, and microbial infections. Despite recent improvements in treating xerostomia (e.g., saliva stimulants, saliva substitutes, and gene therapy), there is a need of more scientific advancements that can be clinically applied toward restoration of compromised salivary gland function. Here we provide a summary of the current salivary cell models that have been used to advance restorative treatments via development of an artificial salivary gland. These models represent initial steps toward clinical and translational research, to facilitate creation of clinically safe salivary glands. Further studies in salivary cell lines and primary cells are necessary to improve survival rates, cell differentiation, and secretory function. Additionally, the characterization of salivary progenitor and stem cell markers are necessary. Although these models are not fully characterized, their improvement may lead to the construction of an artificial salivary gland that is in high demand for improving the quality of life of many patients suffering from salivary secretory dysfunction.

PKCαβγ- and PKCδ-dependent endocytosis of NBCe1-A and NBCe1-B in salivary parotid acinar cells

We examined membrane trafficking of NBCe1-A and NBCe1-B variants of the electrogenic Na(+)-HCO(3)(-) cotransporter (NBCe1) encoded by the SLC4A4 gene, using confocal fluorescent microscopy in rat parotid acinar cells (ParC5 and ParC10). We showed that yellow fluorescent protein (YFP)-tagged NBCe1-A and green fluorescent protein (GFP)-tagged NBCe1-B are colocalized with E-cadherin in the basolateral membrane (BLM) but not with the apical membrane marker zona occludens 1 (ZO-1). We inhibited constitutive recycling with monensin and W13 and detected that NBCe1-A and NBCe1-B accumulated in vesicles marked with the early endosomal marker early endosome antigen-1 (EEA1), with a parallel loss from the BLM. We observed that NBCe1-A and NBCe1-B undergo massive carbachol (CCh)-stimulated redistribution from the BLM into early endosomes. We showed that internalization of NBCe1-A and NBCe1-B was prevented by the general PKC inhibitor GF-109203X, the PKCalphabetagamma-specific inhibitor Gö-6976, and the PKCdelta-specific inhibitor rottlerin. We verified the involvement of PKCdelta by blocking CCh-induced internalization of NBCe1-A-cyan fluorescent protein (CFP) in cells transfected with dominant-negative kinase-dead (Lys376Arg) PKCdelta-GFP. Our data suggest that NBCe1-A and NBCe1-B undergo constitutive and CCh-stimulated endocytosis regulated by conventional PKCs (PKCalphabetagamma) and by novel PKCdelta in rat epithelial cells. To help develop a more complete model of the role of NBCe1 in parotid acinar cells we also investigated the initial phase of the secretory response to cholinergic agonist. In an Ussing chamber study we showed that inhibition of basolateral NBCe1 with 5-chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-1H-indole-1-carboxamide (tenidap) significantly decreases an initial phase of luminal anion secretion measured as a transient short-circuit current (I(sc)) across ParC10 cell monolayers. Using trafficking and functional data we propose a model that describes a physiological role of NBC in salivary acinar cell secretion.

Stem Cell–Soluble Signals Enhance Multilumen Formation in SMG Cell Clusters

Saliva plays a major role in maintaining oral health. Patients with salivary hypofunction exhibit difficulty in chewing and swallowing foods, tooth decay, periodontal disease, and microbial infections. At this time, treatments for hyposalivation are limited to medications (e.g., muscarinic receptor agonists: pilocarpine and cevimeline) that induce saliva secretion from residual acinar cells as well as artificial salivary substitutes. Therefore, advancement of restorative treatments is necessary to improve the quality of life in these patients. Our previous studies indicated that salivary cells are able to form polarized 3-dimensional structures when grown on growth factor–reduced Matrigel. This basement membrane is rich in laminin-III (L1), which plays a critical role in salivary gland formation. Mitotically inactive feeder layers have been used previously to support the growth of many different cell types, as they provide factors necessary for cell growth and organization. The goal of this study was to improve salivary gland cell differentiation in primary cultures by using a combination of L1 and a feeder layer of human hair follicle–derived mesenchymal stem cells (hHF-MSCs). Our results indicated that the direct contact of mouse submandibular (mSMG) cell clusters and hHF-MSCs was not required for mSMG cells to form acinar and ductal structures. However, the hHF-MSC conditioned medium enhanced cell organization and multilumen formation, indicating that soluble signals secreted by hHF-MSCs play a role in promoting these features.

Expression of Resolvin D1 Biosynthetic Pathways in Salivary Epithelium

Resolvins are potent anti-inflammatory mediators derived from ω-3 fatty acids. Results from our previous studies indicated that resolvin D1 (RvD1) blocks pro-inflammatory responses in salivary glands. Furthermore, RvD1 enhances salivary epithelial integrity, demonstrating its potential use for the restoration of salivary gland function in Sjögren’s syndrome (SS). We investigated whether the RvD1 biosynthetic machinery (e.g., cytosolic phospholipase A2, calcium-independent phospholipase A2, 12/15 and 5-lipoxygenase) is expressed in mouse submandibular glands (mSMG), using qPCR and Western blot analyses. Additionally, we determined the localization of RvD1 biosynthetic machinery in mSMG and human minor salivary glands (hMSG), with and without SS, using confocal microscopy. Finally, we measured RvD1 levels in cell supernatants from mSMG cell cultures and freshly isolated mSMG cells, with and without SS, using ELISA. Our results indicate that: (1) RvD1 machinery is expressed in mouse and human salivary glands; (2) polar distribution of RvD1 biosynthetic machinery is lost in hMSG with SS; (3) RvD1 levels in mSMG cell culture supernatants increased with time; and (4) RvD1 levels in mSMG cell supernatants, with and without SS, were similar. These studies demonstrate that the RvD1 biosynthesis machinery is expressed and functional in salivary glands with and without SS.