Involvement of CCL2 in Salivary Gland Response to Hyperosmolar Stress Related to Sjögren's Syndrome.

In primary Sjögren's syndrome (pSS) patients, salivary gland (SG) epithelial cells (SGECs) could be exposed to chronic hyperosmotic stress (HOS), consecutive to their destruction and deregulation, that exacerbates an inflammatory response. The aims of this study were to assess the mechanism accounting for C-C motif chemokine ligand 2 (CCL2) expression in an immortalized human salivary gland epithelial acinar cell line (NS-SV-AC) subjected to HOS, as well as the involvement of CCL2 in pSS. CCL2 mRNA and protein levels were determined via RT-qPCR and ELISA. Reporter plasmids and a promoter pull-down assay were used to identify transcription factors associated with CCL2 mRNA increase. Our data showed that HOS-induced CCL2 mRNA increase was independent of the nuclear factor of activated T-cells 5 (NFAT5) and nuclear factor-kappa B (NFkB) but involved Kruppel-like factor 5 (KLF5). CCL2 protein levels, quantified by enzyme-linked immunosorbent assay (ELISA) in sera samples from pSS patients, correlated with the European Alliance of Associations for Rheumatology's Sjogren's syndrome disease activity index (ESSDAI) score for systemic activity. In addition, CCL2 protein levels were higher in patients with biological activity, cutaneous manifestations, and ESSDAI score superior or equal to five. Our data suggest that chronic HOS could exacerbate pSS disease by contributing to the inflammatory process induced by the expression and secretion of CCL2.

Hyperosmolar environment and salivary gland epithelial cells increase extra-cellular matrix remodeling and lymphocytic infiltration in Sjögren's syndrome.

Salivary gland epithelial cells (SGECs) play an active role in primary Sjogren's syndrome (pSS) pathogenesis. Quantitative and qualitative abnormalities of saliva might expose SGECs to chronic hyperosmolarity. We aimed to decipher the links between hyperosmolar stimulation of SGECs and lymphocytic infiltration of the salivary glands (SG) observed in pSS. RNAseq was performed on NS-SV-AC cells stimulated with hyperosmolar media containing NaCl (100 mM) or sucrose (200 mM), or with iso-osmolar (Iso) medium. RNAseq was performed on primary cultured SGECs from pSS and controls, in the presence or not of B cells. Hyperosmolar stimulation of NS-SV-AC-cells identified an upregulation of interferon-induced (MX1, IFIT2) and MMPs genes. Enrichment analysis revealed an over-representation of fibrosis pathway. In parallel, RNAseq of SGECs comparing pSS to controls identified an over-representation of a pathway involving MMPs. Given the unexpected upregulation of collagen (COL3A1, COL1A2) and ADAMTS genes in pSS SGECs, we hypothesized that SGECs might undergo epithelial-mesenchymal transition. ZEB2 was upregulated and SLUG was down regulated in SGECs from pSS versus controls. MMP24 and ZEB2 were higher in SGECs from pSS with a focus score ≥1 versus <1. Lastly, SGECs cocultured with B cells expressed higher levels of COL1A2. These results suggest the existence of a vicious circle. Alteration of SGECs in pSS participates in the establishment of a hyperosmolar microenvironment, which in turn promotes SGECs transcriptomic modifications. These modifications include extracellular matrix remodeling and promote SG lymphocytic infiltration.

Aquaporins in Glandular Secretion.

Exocrine and endocrine glands deliver their secretory product, respectively, at the surface of the target organs or within the bloodstream. The release of their products has been shown to rely on secretory mechanisms often involving aquaporins (AQPs). This chapter will provide insight into the role of AQPs in secretory glands located within the gastrointestinal tract, including salivary glands, gastric glands, duodenal Brunner's glands, liver, gallbladder, intestinal goblets cells, and pancreas, as well and in other parts of the body, including airway submucosal glands, lacrimal glands, mammary glands, and eccrine sweat glands. The involvement of AQPs in both physiological and pathophysiological conditions will also be highlighted.

Aquaporin-5 Dynamic Regulation.

Aquaporin-5 (AQP5), belonging to the aquaporins (AQPs) family of transmembrane water channels, facilitates osmotically driven water flux across biological membranes and the movement of hydrogen peroxide and CO2. Various mechanisms have been shown to dynamically regulate AQP5 expression, trafficking, and function. Besides fulfilling its primary water permeability function, AQP5 has been shown to regulate downstream effectors playing roles in various cellular processes. This review provides a comprehensive overview of the current knowledge of the upstream and downstream effectors of AQP5 to gain an in-depth understanding of the physiological and pathophysiological processes involving AQP5.

Experimental Models to Study Epithelial-Mesenchymal Transition in Proliferative Vitreoretinopathy.

Proliferative vitreoretinal diseases (PVDs) encompass proliferative vitreoretinopathy (PVR), epiretinal membranes, and proliferative diabetic retinopathy. These vision-threatening diseases are characterized by the development of proliferative membranes above, within and/or below the retina following epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) and/or endothelial-mesenchymal transition of endothelial cells. As surgical peeling of PVD membranes remains the sole therapeutic option for patients, development of in vitro and in vivo models has become essential to better understand PVD pathogenesis and identify potential therapeutic targets. The in vitro models range from immortalized cell lines to human pluripotent stem-cell-derived RPE and primary cells subjected to various treatments to induce EMT and mimic PVD. In vivo PVR animal models using rabbit, mouse, rat, and swine have mainly been obtained through surgical means to mimic ocular trauma and retinal detachment, and through intravitreal injection of cells or enzymes to induce EMT and investigate cell proliferation and invasion. This review offers a comprehensive overview of the usefulness, advantages, and limitations of the current models available to investigate EMT in PVD.

Bioengineering in salivary gland regeneration.

Salivary gland (SG) dysfunction impairs the life quality of many patients, such as patients with radiation therapy for head and neck cancer and patients with Sjögren's syndrome. Multiple SG engineering strategies have been considered for SG regeneration, repair, or whole organ replacement. An in-depth understanding of the development and differentiation of epithelial stem and progenitor cells niche during SG branching morphogenesis and signaling pathways involved in cell-cell communication constitute a prerequisite to the development of suitable bioengineering solutions. This review summarizes the essential bioengineering features to be considered to fabricate an engineered functional SG model using various cell types, biomaterials, active agents, and matrix fabrication methods. Furthermore, recent innovative and promising approaches to engineering SG models are described. Finally, this review discusses the different challenges and future perspectives in SG bioengineering.

Insight into the Mammalian Aquaporin Interactome.

Aquaporins (AQPs) are a family of transmembrane water channels expressed in all living organisms. AQPs facilitate osmotically driven water flux across biological membranes and, in some cases, the movement of small molecules (such as glycerol, urea, CO2, NH3, H2O2). Protein-protein interactions play essential roles in protein regulation and function. This review provides a comprehensive overview of the current knowledge of the AQP interactomes and addresses the molecular basis and functional significance of these protein-protein interactions in health and diseases. Targeting AQP interactomes may offer new therapeutic avenues as targeting individual AQPs remains challenging despite intense efforts.

The Involvement of Innate and Adaptive Immunity in the Initiation and Perpetuation of Sjögren's Syndrome.

Sjogren's syndrome (SS) is a chronic autoimmune disease characterized by the infiltration of exocrine glands including salivary and lachrymal glands responsible for the classical dry eyes and mouth symptoms (sicca syndrome). The spectrum of disease manifestations stretches beyond the classical sicca syndrome with systemic manifestations including arthritis, interstitial lung involvement, and neurological involvement. The pathophysiology underlying SS is not well deciphered, but several converging lines of evidence have supported the conjuncture of different factors interplaying together to foster the initiation and perpetuation of the disease. The innate and adaptive immune system play a cardinal role in this process. In this review, we discuss the inherent parts played by both the innate and adaptive immune system in the pathogenesis of SS.

Ezrin Is a Novel Protein Partner of Aquaporin-5 in Human Salivary Glands and Shows Altered Expression and Cellular Localization in Sjögren's Syndrome.

Sjögren's syndrome (SS) is an exocrinopathy characterized by the hypofunction of salivary glands (SGs). Aquaporin-5 (AQP5); a water channel involved in saliva formation; is aberrantly distributed in SS SG acini and contributes to glandular dysfunction. We aimed to investigate the role of ezrin in AQP5 mislocalization in SS SGs. The AQP5-ezrin interaction was assessed by immunoprecipitation and proteome analysis and by proximity ligation assay in immortalized human SG cells. We demonstrated, for the first time, an interaction between ezrin and AQP5. A model of the complex was derived by computer modeling and in silico docking; suggesting that AQP5 interacts with the ezrin FERM-domain via its C-terminus. The interaction was also investigated in human minor salivary gland (hMSG) acini from SS patients (SICCA-SS); showing that AQP5-ezrin complexes were absent or mislocalized to the basolateral side of SG acini rather than the apical region compared to controls (SICCA-NS). Furthermore, in SICCA-SS hMSG acinar cells, ezrin immunoreactivity was decreased at the acinar apical region and higher at basal or lateral regions, accounting for altered AQP5-ezrin co-localization. Our data reveal that AQP5-ezrin interactions in human SGs could be involved in the regulation of AQP5 trafficking and may contribute to AQP5-altered localization in SS patients.

A Training Tool to support the management and diagnosis of Sjögren's syndrome.

OBJECTIVES: The objective of this work is to present a Training Tool designed to support healthcare professionals involved in the diagnosis and management of Sjögren's syndrome. METHODS: The Training Tool aims to fulfil the gap of targeted education by providing a structured protocol of training including state of the art guidelines and practices. For the development of the Training Tool, latest relevant technologies have been used to assure efficiency and usability. Core functionalities include training by a series of multimedia courses, testing during the learning process, and profiling for monitoring the progress. An iterative requirement analysis process was established involving a large number of clinical experts, with the objective to identify user's training needs. RESULTS: Comprehensive usability evaluation was performed by applying, an Unmoderated Remote Usability Test resulting to 97.2% Success Rate; and the well-established System Usability Scale, reaching a score of 90.4 which classifies the Training Tool as "A" graded-excellent. CONCLUSIONS: The Training Tool offers open-online training of healthcare professionals involved in the diagnosis and management of Sjögren's syndrome, using a well-designed training protocol in highly usable manner. To our knowledge, this is the first such tool for Sjögren's syndrome.

Aquaporins Involvement in Pancreas Physiology and in Pancreatic Diseases.

Aquaporins are a family of transmembrane proteins permeable to water. In mammals, they are subdivided into classical aquaporins that are permeable to water; aquaglyceroporins that are permeable to water, glycerol and urea; peroxiporins that facilitate the diffusion of H2O2 through cell membranes; and so called unorthodox aquaporins. Aquaporins ensure important physiological functions in both exocrine and endocrine pancreas. Indeed, they are involved in pancreatic fluid secretion and insulin secretion. Modification of aquaporin expression and/or subcellular localization may be involved in the pathogenesis of pancreatic insufficiencies, diabetes and pancreatic cancer. Aquaporins may represent useful drug targets for the treatment of pathophysiological conditions affecting pancreatic function, and/or diagnostic/predictive biomarker for pancreatic cancer. This review summarizes the current knowledge related to the involvement of aquaporins in the pancreas physiology and physiopathology.

Cross-contamination of the human salivary gland HSG cell line with HeLa cells: A STR analysis study.

OBJECTIVES: The human salivary gland (HSG) cell line, labeled as a submandibular ductal cell line, is commonly used as in vitro models to study radiation therapy, Sjögren's syndrome, pleomorphic adenoma, mucocele, epithelial-to-mesenchymal transition, and epigenetics. However, the American Type Culture Collection (ATCC) has recently released a list of cross-contaminated cell lines that included HSG. Despite this notice, some research laboratories still use HSG as a salivary cell model. Therefore, this study examined the authenticity of HSG sampled from three different laboratories. METHODS: DNA was extracted from HSG and additional salivary cell lines (NS-SV-AC, NS-SV-DC, A253, HSY) and submitted for cell line authentication with short tandem repeat (STR) analysis. RESULTS: All HSG samples had STR profiles indicating >80% match with HeLa in both the ATCC and Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) databases. This confirmed that HSG sampled from three different laboratories and HSY shared a common ancestry (host) with HeLa, whereas NS-SV-AC, NS-SV-DC, and A253 had unique STR profiles. CONCLUSION: Short tandem repeat analysis revealed that HSG was contaminated by the HeLa cell line. Furthermore, because genotyping of the original HSG cell line was not performed during its establishment, it will be difficult to authenticate an uncontaminated sample of HSG.

Involvement of Aquaporins in the Pathogenesis, Diagnosis and Treatment of Sjögren's Syndrome.

Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by lymphocytic infiltration of salivary and lacrimal glands resulting in diminished production of saliva and tears. The pathophysiology of SS has not yet been fully deciphered. Classically it has been postulated that sicca symptoms in SS patients are a double step process whereby lymphocytic infiltration of lacrimal and salivary glands (SG) is followed by epithelial cell destruction resulting in keratoconjunctivitis sicca and xerostomia. Recent advances in the field of the pathophysiology of SS have brought in new players, such as aquaporins (AQPs) and anti AQPs autoantibodies that could explain underlying mechanistic processes and unveil new pathophysiological pathways offering a deeper understanding of the disease. In this review, we delineate the link between the AQP and SS, focusing on salivary glands, and discuss the role of AQPs in the treatment of SS-induced xerostomia.

Potential Interplay between Hyperosmolarity and Inflammation on Retinal Pigmented Epithelium in Pathogenesis of Diabetic Retinopathy.

Diabetic retinopathy is a frequent eyesight threatening complication of type 1 and type 2 diabetes. Under physiological conditions, the inner and the outer blood-retinal barriers protect the retina by regulating ion, protein, and water flux into and out of the retina. During diabetic retinopathy, many factors, including inflammation, contribute to the rupture of the inner and/or the outer blood-retinal barrier. This rupture leads the development of macular edema, a foremost cause of sight loss among diabetic patients. Under these conditions, it has been speculated that retinal pigmented epithelial cells, that constitute the outer blood-retinal barrier, may be subjected to hyperosmolar stress resulting from different mechanisms. Herein, we review the possible origins and consequences of hyperosmolar stress on retinal pigmented epithelial cells during diabetic retinopathy, with a special focus on the intimate interplay between inflammation and hyperosmolar stress, as well as the current and forthcoming new pharmacotherapies for the treatment of such condition.

Aquaporins and Gland Secretion.

Aquaporins (AQPs ) are expressed in most exocrine and endocrine secretory glands. Consequently, summarizing the expression and functions of AQPs in secretory glands represents a daunting task considering the important number of glands present in the body, as well as the number of mammalian AQPs - thirteen. The roles played by AQPs in secretory processes have been investigated in many secretory glands. However, despite considerable research, additional studies are clearly needed to pursue our understanding of the role played by AQPs in secretory processes. This book chapter will focus on summarizing the current knowledge on AQPs expression and function in the gastrointestinal tract , including salivary glands, gastric glands, Duodenal Brunner's gland, liver and gallbladder, intestinal goblets cells, exocrine and endocrine pancreas, as well as few other secretory glands including airway submucosal glands, lacrimal glands, mammary glands and eccrine sweat glands.

Lipopolysaccharide Modifies Glycerol Permeability and Metabolism in 3T3-L1 Adipocytes.

Aquaglyceroporins-aquaporin membrane channels (AQP) that conduct glycerol and other small neutral solutes in addition to water-play major roles in obesity. In adipocytes, aquaglyceroporins mediate glycerol uptake and release across the plasma membrane, which are two key steps for triacylglycerols (TAGs) synthesis (lipogenesis) and hydrolysis (lipolysis). The aim of this study was to assess both glycerol permeability and metabolism in undifferentiated 3T3-L1 cells (UDCs) as well as in untreated (CTL-DCs) versus lipopolysaccharide (LPS-DCs)-treated differentiated 3T3-L1 adipocytes. Glycerol release, TAGs content and whole membrane glycerol permeability were significantly increased in DCs as compared to UDCs. Moreover, in DCs, LPS treatment significantly increased TAGs content and decreased glycerol permeability. In addition, a significant reduction in whole membrane glycerol permeability was observed in LPS-DCs as compared to CTL-DCs. The relative contributions of AQP3, AQP7 and AQP9 (facilitated diffusion), as well as that of the phospholipid bilayer (simple diffusion), to the whole membrane glycerol permeability, were estimated biophysically in UDCs, CTL-DCs and LPS-DCs, using selective AQP inhibitors. Further studies will be required to determine if modifications in either subcellular localization and/or activity of aquaglyceroporins could account for the data herein. Nevertheless, our findings provide novel insights in understanding the LPS-induced adipocyte hypertrophy that accompanies obesity.

Involvement of TonEBP/NFAT5 in osmoadaptative response of human retinal pigmented epithelial cells to hyperosmolar stress.

PURPOSE: Macular edema, a frequently encountered complication of diabetic retinopathy (DR), results from alterations of the blood retinal barrier (BRB) and leads to modifications of the retinal pigmented epithelium (RPE) functions. Osmolar changes of the surrounding medium could be responsible for modifications of the RPE functions leading to disturbance of retinal homeostasis. The expression, activation and function of the key hyperosmolar response factor Tonicity Enhancer Binding Protein (TonEBP also called nuclear factor of activated T-cell 5 - NFTA5) was investigated in ARPE-19 cells, derived from human RPE, in response to hyperosmolar stimulation. METHODS: ARPE-19 cells were exposed to hyperosmolar medium. TonEBP mRNA and protein levels were quantified by qRT-PCR and semi-quantitative Western blot. TonEBP nuclear translocation was investigated by immunofluorescence. TonEBP transactivation activity was measured using a reported plasmid containing TonEBP binding sites. RESULTS: In response to hyperosmolar stimulation of ARPE-19 cells, a dose-dependent increase in TonEBP mRNA and protein levels, as well as TonEBP nuclear translocation were observed. TonEBP transactivation activity was further demonstrated using a reporter plasmid containing TonEBP binding sites. A dominant negative form of TonEBP abolished NaCl-induced increase in TonEBP transactivation activity, and inhibited the increase of the target genes aldose reductase and sodium-dependent taurine transporter mRNA levels. SB203580, an inhibitor of two of the p38 protein kinase's isoforms (p38α and p38ß) inhibited the TonEBP nuclear translocation and transactivation activity in ARPE-19 cells exposed to hyperosmolar stimulation. CONCLUSIONS: Our data demonstrates the involvement of TonEBP in the mechanisms responsible for osmoadaptation to hyperosmolar stress in RPE cells. Given the emerging role of TonEBP in different pathological pathways, these data open new perspectives for the analysis of the mechanisms involved in the modification of functions of the RPE during macular edema.

Forskolin Inhibits Lipopolysaccharide-Induced Modulation of MCP-1 and GPR120 in 3T3-L1 Adipocytes through an Inhibition of NFκB.

In an obese state, Toll-like receptor-4 (TLR-4) upregulates proinflammatory adipokines secretion including monocyte chemotactic protein-1 (MCP-1) in adipose tissue. In contrast, G-protein coupled receptor 120 (GPR120) mediates antiobesity effects. The aim of this study was to determine the signaling pathway by which Forskolin (FK), a cyclic adenosine monophosphate- (cAMP-) promoting agent causing positive changes in body composition in overweight and obese adult men, affects MCP-1 and GPR120 expression during an inflammatory response induced by lipopolysaccharide (LPS) in adipocytes, such as in an obese state. 3T3-L1 cells differentiated into adipocytes (DC) were stimulated with LPS in the absence or presence of FK and inhibitors of TLR-4 and inhibitor of kappa B (IκBα). In DC, LPS increased MCP-1, TLR-4, and nuclear factor-κB1 (NFκB1) mRNA levels, whereas it decreased GPR120 mRNA levels. In DC, FK inhibited the LPS-induced increase in MCP-1, TLR-4, and NFκB1 mRNA levels and the LPS-induced decrease in GPR120 mRNA. BAY11-7082 and CLI-095 abolished these LPS-induced effects. In conclusion, FK inhibits LPS-induced increase in MCP-1 mRNA levels and decrease in GPR120 mRNA levels in adipocytes and may be a potential treatment for inflammation in obesity. Furthermore, TLR-4-induced activation of NFκB may be involved in the LPS-induced regulation of these genes.

Aquaporins in Salivary Glands: From Basic Research to Clinical Applications.

Salivary glands are involved in saliva secretion that ensures proper oral health. Aquaporins are expressed in salivary glands and play a major role in saliva secretion. This review will provide an overview of the salivary gland morphology and physiology of saliva secretion, and focus on the expression, subcellular localization and role of aquaporins under physiological and pathophysiological conditions, as well as clinical applications involving aquaporins. This review is highlighting expression and localization of aquaporins in human, rat and mouse, the most studied species and is pointing out possible difference between major salivary glands, i.e., parotid, submandibular and sublingual glands.

Involvement of JNK/NFκB Signaling Pathways in the Lipopolysaccharide-Induced Modulation of Aquaglyceroporin Expression in 3T3-L1 Cells Differentiated into Adipocytes.

Aquaglyceroporins, belonging to the family of aquaporins (AQPs), are integral plasma membrane proteins permeable to water and glycerol that have emerged as key players in obesity. The aim of this study was to investigate the expression profile of AQPs in undifferentiated and differentiated 3T3-L1 cells and to investigate the changes in expression of aquaglyceroporins in 3T3-L1 cells differentiated into adipocytes and subjected to lipopolysaccharide (LPS) mimicking inflammation occurring during obesity. Furthermore, the study aimed at identifying the signaling cascade involved in the regulation of aquaglyceroporins expression upon LPS stimulation. 3T3-L1 cells were grown as undifferentiated cells (UDC; preadipocytes) or cells differentiated into adipocytes (DC, adipocytes). DC were incubated in the presence or absence of LPS with or without inhibitors of various protein kinases. AQPs mRNA expression levels were measured by real-time quantitative polymerase chain reaction (RT-qPCR). AQP1, AQP2, AQP3, AQP9 and AQP11 mRNA were expressed in both UDC and DC, whereas AQP4, AQP7 and AQP8 mRNA were expressed only in DC. In DC, LPS up-regulated AQP3 mRNA levels (p < 0.05) compared to control; these effects were inhibited by CLI095, SP600125 and BAY11-7082 (p < 0.05). LPS decreased both AQP7 and AQP11 mRNA levels (p < 0.01) in DC as compared to control; this decrease was inhibited by CLI095 and BAY11-7082 (p < 0.05) and additionally by SP00125 for AQP7 (p < 0.05). SB203580 had no effect on LPS-induced AQP3, AQP7 and AQP11 mRNA levels modulations. In conclusion, our results clearly show that many AQPs are expressed in murine 3T3-L1 adipocytes. Moreover, in DCs, LPS led to decreased AQP7 and AQP11 mRNA levels but to increased AQP3 mRNA levels, resulting from the Toll-like receptor 4 (TLR4)-induced activation of JNK and/or NFκB pathway.