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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.

Saliva secretion requires effective translocation of aquaporin 5 (AQP5) water channel to the salivary glands (SGs) acinar apical membrane. Patients with Sjögren's syndrome (SS) display abnormal AQP5 localization within acinar cells from SGs that correlate with sicca manifestation and glands hypofunction. Several proteins such as Prolactin-inducible protein (PIP) may regulate AQP5 trafficking as observed in lacrimal glands from mice. However, the role of the AQP5-PIP complex remains poorly understood. In the present study, we show that PIP interacts with AQP5 in vitro and in mice as well as in human SGs and that PIP misexpression correlates with an altered AQP5 distribution at the acinar apical membrane in PIP knockout mice and SS hMSG. Furthermore, our data show that the protein-protein interaction involves the AQP5 C-terminus and the N-terminal of PIP (one molecule of PIP per AQP5 tetramer). In conclusion, our findings highlight for the first time the role of PIP as a protein controlling AQP5 localization in human salivary glands but extend beyond due to the PIP-AQP5 interaction described in lung and breast cancers.

Connexins (Cxs) are transmembrane proteins involved in the formation of hemichannels and gap junctions (GJs). GJs are involved in various physiological functions, including secretion in glandular tissue. It has been demonstrated that Cx26, Cx32, and Cx43 are mainly expressed in glands, but no data are available in human salivary glands to date. The aim of our study was to investigate the presence and the localization of Cxs in human minor labial salivary glands. Immunofluorescence and immunoelectron microscopy were employed to evaluate the Cx26, Cx32, and Cx43 protein in human labial salivary gland biopsies (hLSGBs). RT-PCR was also used to detect their mRNA expression. Cx expression was found at both the mRNA and protein levels in all hLSGBs analysed. Cxs were observed at the level of the duct and acinar cells, as well as in myoepithelial cells. The localization of the three Cx types was very similar, suggesting colocalization of these Cxs in the same connexons. These results demonstrated the presence of Cxs in human salivary glands for the first time. Moreover, the few samples with primary Sjögren's Syndrome analysed only by immunofluorescence showed an alteration of the Cx expression, indicating that these proteins could be involved in salivary gland dysfunctions. [ABSTRACT FROM AUTHOR]

Introduction: Connexins (Cxs) are transmembrane proteins involved in the formation of connexons/hemichannels. Two connexons of adjacent cells can dock one each other form- ing an intercellular channel at gap junction (GJ), by which ions and small metabolites can pass between cells. Single hemichannels participate in the exchange of small metabolites between cells and the extracellular space [1]. There are at least 21 isoforms of Cxs expressed in humans and they are localized in almost every tissue. More than one Cx can be expressed in each tissue and connexons can be made by a single Cx or by different types of Cxs. GJ are involved in various physiological functions including secretion in glandular tissue [2-5]. Cx26, Cx32 and Cx43 are the mainly expressed Cxs in glands, but no data are available about their expression in human salivary glands. Aim: The aim of our study is to investigate the presence and the localization of these Cxs in human minor salivary glands. Methods: human minor salivary gland biopsies (hMSGBs) were obtained as part of the routine diagnostic procedures when primary Sjögren’s Syndrome (pSS) was suspected. Samples without pSS were selected for the study. Immunofluorescence and immunoelectron microscopy were employed to evaluated Cx26, Cx32 and Cx43 protein expression while RT-PCR was used to detect their mRNA expression. Results and Conclusions: Cx expression was found at both protein and mRNA level in all hMSGBs analysed. Cxs were observed at level of the duct and acinar cell membranes, as well as in myoepithelial cells. The localization of the three Cx types was very similar, suggesting a colocalization of these Cxs in the same connexons. Further studies are necessary to confirm this hypothesis. However, these preliminary results are important because demonstrate for the first time the presence of Cxs in human salivary glands. Moreover, in the few analysed pSS samples we observed an alteration of Cx expression indicating that these proteins may be involved in salivary gland dysfunctions. [ABSTRACT FROM AUTHOR]