Ezrin Is a Novel Protein Partner of Aquaporin-5 in Human Salivary Glands and Shows Altered Expression and Cellular Localization in Sjogren's Syndrome.
ABSTRACT: 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.
Project description: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.
Project description:<h4>Background</h4>While all salivary glands (SGs) can be involved in primary Sjögren's syndrome (pSS), their respective role in pathogenesis remains unclear. Our objective was to assess immunopathway activation in paired parotid and labial gland tissue from biopsy-positive and biopsy-negative pSS and non-SS sicca patients.<h4>Methods</h4>Paraffin-embedded, paired parotid and labial salivary gland tissue and peripheral blood mononuclear cells were obtained from 39 pSS and 20 non-SS sicca patients. RNA was extracted, complementary DNA libraries were prepared and sequenced. For analysis of differentially expressed genes (DEGs), patients were subdivided based on fulfillment of ACR-EULAR criteria and histopathology.<h4>Results</h4>With principal component analysis, only biopsy-positive pSS could be separated from non-SS sicca patients based on SG gene expression. When comparing the transcriptome of biopsy-positive pSS and biopsy-negative non-SS sicca patients, 1235 and 624 DEGs (FDR<0.05, log2FC<-1 or >1) were identified for parotid and labial glands, respectively. The number of DEGs between biopsy-negative pSS and non-SS sicca patients was scarce. Overall, transcript expression levels correlated strongly between parotid and labial glands (R<sup>2</sup> = 0.86, p-value<0.0001). Gene signatures present in both glands of biopsy-positive pSS patients included IFN-α signaling, IL-12/IL-18 signaling, CD3/CD28 T-cell activation, CD40 signaling in B-cells, DN2 B-cells, and FcRL4+ B-cells. Signature scores varied considerably amongst pSS patients.<h4>Conclusion</h4>Transcriptomes of paired major and minor SGs in pSS were overall comparable, although significant inter-individual heterogeneity in immunopathway activation existed. The SG transcriptome of biopsy-negative pSS was indistinguishable from non-SS sicca patients. Different patterns of SG immunopathway activation in pSS argue for personalized treatment approaches.
Project description:The autoimmune exocrinopathy, Sjögren's syndrome (SS), is associated with secretory defects in patients, including individuals with mild lymphocytic infiltration and minimal glandular damage. The mechanism(s) underlying the secretory dysfunction is not known. We have used minor salivary gland biopsies from SS patients and healthy individuals to assess acinar cell function in morphologically intact glandular areas. We report that agonist-regulated intracellular Ca(2+) release, critically required for Ca(2+) entry and fluid secretion, is defective in acini from SS patients. Importantly, these acini displayed reduction in IP3R2 and IP3R3, but not AQP5 or STIM1. Similar decreases in IP3R and carbachol (CCh)-stimulated [Ca(2+)]i elevation were detected in acinar cells from lymphotoxin-alpha (LT?) transgenic (TG) mice, a model for (SS). Treatment of salivary glands from healthy individuals with LT ?, a cytokine linked to disease progression in SS and IL14? mice, reduced Ca(2+) signaling. Together, our findings reveal novel IP3R deficits in acinar cells that underlie secretory dysfunction in SS patients.
Project description:While all salivary glands (SGs) can be involved in primary Sjögren’s syndrome (pSS), their respective role in pathogenesis remains unclear. To assess immunopathway activation in paired parotid and labial gland tissue from biopsy-positive and biopsy-negative pSS and non-SS sicca patients, paraffin-embedded, paired parotid and labial salivary gland tissue and peripheral blood mononuclear cells were obtained from 39 pSS and 20 non-SS sicca patients. The patients were subdivided based on fulfillment of ACR-EULAR criteria and histopathology and the samples were analyzed for differentially expressed genes (DEGs). The principal component analysis of SG gene expression could only separate biopsy-positive pSS from non-SS sicca patients. However, when comparing the transcriptome of biopsy-positive pSS and biopsy-negative non-SS sicca patients, 1235 and 624 DEGs (FDR<-1 or >1) were identified for parotid and labial glands, respectively. The number of DEGs between biopsy negative pSS and non-SS sicca patients was scarce. The overall, transcript expression levels correlated strongly between parotid and labial glands (R2=0.86, p‐value<0.0001). Gene signatures present in both glands of biopsy‐positive pSS patients included IFN‐α signaling, IL-12/IL-18 signaling, CD3/CD28 T-cell activation, CD40 signaling in B-cells, DN2 B-cells, and FcRL4+ B-cells. Signature scores varied considerably amongst pSS patients. In conclusion, the transcriptomes of paired major and minor SGs in pSS were overall comparable, although significant inter-individual heterogeneity in immunopathway activation existed. The SG transcriptome of biopsy-negative pSS was indistinguishable from non-SS sicca patients. The different patterns of SG immunopathway activation in pSS argue for personalized treatment approaches. Overall design: Differential expression analysis of a cohort of 39 patients with primary Sjogren's syndrome and 20 non-SS sicca patients. characteristics: ACR EULAR = positive -> patients with primary Sjogren's syndrome characteristics: ACR EULAR = negative -> non-SS sicca patients
Project description:BACKGROUND:Gross cystic disease fluid protein-15(GCDFP-15)/prolactin-inducible protein (PIP) is a secretory acinar glycoprotein of 14 KDa which we have recently described as significantly lower in salivary samples of patients with primary Sjögren's syndrome (pSS) in comparison to healthy volunteers by proteomic analysis. AIMS OF THE STUDY:(1) to validate our previous data on the decrease of GCDFP-15/PIP protein in a larger number of subjects with pSS (2) to integrate the proteomic results with complementary immunoassays in order better clarify the pathophysiological relevance of GCDFP-15/PIP in pSS exocrinopathy (3) to assess both the glandular expression of the GCDFP-15/PIP and the levels of glandular GCDFP-15/PIP mRNA in the patients' minor salivary gland (MSG) biopsies in order to verify whether the observed reduction of GCDFP-15/PIP in saliva may be related to a decrease in the protein production. PATIENTS AND METHODS:A total of 123 salivary samples from patients affected by pSS, no-SS sicca syndrome and sex- age-matched healthy volunteers were analyzed by different proteomic techniques (SELDI-TOF-MS, 2DE, MALDI-TOF-MS). The expression of GCDFP-15/PIP was then validated by western blot analysis. Real Time PCR and immunohistochemistry for GCDFP-15/PIP in the minor salivary glands (MSG) biopsies were then carried out. RESULTS:By using complementary proteomic analysis we found that a putative peak of 16547 m/z was among the best independent biomarkers for pSS able to discriminate between patients and healthy controls with a sensitivity of 96 % and a specificity of 70%, with a global cross validated error of 29%. We identified the peak as the GCDFP-15/PIP protein and verified that the intensity of GCDFP-15/PIP was significantly lower in pSS patients when compared to both no-SS sicca subjects and healthy controls (p<0.0001). GCDFP-15/PIP expression also correlated with both the salivary flow rate (r=0.312, p=0.023) and MSG biopsies focus score (r=-0.377, p=0.04). Finally, immunohistochemistry confirmed that GCDFP-15/PIP staining was faint in mucus acini and Real Time PCR showed that GCDFP-15/PIP mRNA was significantly lower in pSS patients when compared to both no-SS sicca subjects and healthy controls (p=0.023) thus supporting the hypothesis that the observed reduction of GCDFP-15/PIP in pSS saliva may be related to a decrease in the protein production. CONCLUSION:In this study by different complementary-omic techniques we confirmed the potential role of GCDFP-15/PIP as a novel biomarker for pSS. This finding might also be functionally important as GCDFP-15/PIP has previously been shown to bind to Aquaporin 5 (AQP5), a salivary gland water channel, critical to saliva formation that is known to be downregulated in pSS. It is likely that exploring the GCDFP-15/PIP/AQP5 axis will help better understand the mechanism of salivary gland dysfunction in pSS.
Project description:OBJECTIVE:Salivary gland (SG) progenitor cells (SGPCs) maintain SG homeostasis. We have previously shown that in primary Sjögren's syndrome (pSS), SGPCs are likely to be senescent, and may underpin SG dysfunction. This study assessed the extent of senescence of cells in a SGPC niche in pSS patients' SGs, and its correlation with functional and clinical parameters. METHODS:The expression of p16 and p21 as markers of senescence in both total SG epithelium and a SGPC niche (basal striated duct cells, BSD) was examined in SGs of pSS (n?=?35), incomplete pSS (n?=?13) (patients with some signs of pSS, but not fulfilling all classification criteria) and non-SS sicca control (n?=?21) patients. This was correlated with functional and clinical parameters. RESULTS:pSS patient SGs contained significantly more p16+ cells both in the epithelium in general (P <0.01) and in the BSD layer (P <0.001), than non-SS SGs. Significant correlations were found in pSS patients between p16+ BSD cells and secretion of unstimulated whole saliva, stimulated whole saliva, stimulated parotid saliva, CD45+ infiltrate, ultrasound total score and ACR-EULAR classification score, but not with EULAR Sjögren's syndrome disease activity index (ESSDAI) and EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI) scores. Correlations with total epithelium p16+ cells were weaker. Incomplete pSS patients also had increased numbers of p16+ epithelial and BSD cells. Based on protein and mRNA expression, p21+ appears not to play a significant role in the SG in pSS. CONCLUSION:These findings suggest SGPC senescence may be an early feature of primary Sjögren's syndrome and may contribute to defective SG function in pSS but not to systemic disease activity.
Project description:Neurotransmitter regulation of salivary fluid secretion is mediated by activation of Ca(2+) influx. The Ca(2+)-permeable transient receptor potential canonical 1 (TRPC1) channel is crucial for fluid secretion. However, the mechanism(s) involved in channel assembly and regulation are not completely understood. We report that Caveolin1 (Cav1) is essential for the assembly of functional TRPC1 channels in salivary glands (SG) in vivo and thus regulates fluid secretion. In Cav1(-/-) mouse SG, agonist-stimulated Ca(2+) entry and fluid secretion are significantly reduced. Microdomain localization of TRPC1 and interaction with its regulatory protein, STIM1, are disrupted in Cav1(-/-) SG acinar cells, whereas Orai1-STIM1 interaction is not affected. Furthermore, localization of aquaporin 5 (AQP5), but not that of inositol (1,4,5)-trisphosphate receptor 3 or Ca(2+)-activated K(+) channel (IK) in the apical region of acinar cell was altered in Cav1(-/-) SG. In addition, agonist-stimulated increase in surface expression of AQP5 required Ca(2+) influx via TRPC1 channels and was inhibited in Cav1(-/-) SG. Importantly, adenovirus-mediated expression of Cav1 in Cav1(-/-) SG restored interaction of STIM1 with TRPC1 and channel activation, apical targeting and regulated trafficking of AQP5, and neurotransmitter stimulated fluid-secretion. Together these findings demonstrate that, by directing cellular localization of TRPC1 and AQP5 channels and by selectively regulating the functional assembly TRPC1-STIM1 channels, Cav1 is a crucial determinant of SG fluid secretion.
Project description:Infected Ixodes scapularis (black-legged tick) transmit a host of serious pathogens via their bites, including Borrelia burgdorferi, Babesia microti, and tick-borne flaviviruses (TBFVs), such as Powassan virus (POWV). Although the role of female I. scapularis ticks in disease transmission is well characterized, the role of male ticks is poorly understood. Because the pathogens are delivered in tick saliva, we studied the capacity of male salivary glands (SGs) to support virus replication. Ex vivo cultures of SGs from unfed male I. scapularis were viable for more than a week and maintained the characteristic tissue architecture of lobular ducts and acini. When SG cultures were infected with the TBFVs Langat virus (LGTV) or POWV lineage II (deer tick virus), the production of infectious virus was demonstrated. Using a green fluorescent protein-tagged LGTV and confocal microscopy, we demonstrated LGTV infection within SG acinus types II and III. The presence of LGTV in the acini and lobular ducts of the cultures was also shown via immunohistochemistry. Furthermore, the identification by in situ hybridization of both positive and negative strand LGTV RNA confirmed that the virus was indeed replicating. Finally, transmission electron microscopy of infected SGs revealed virus particles packaged in vesicles or vacuoles adjacent to acinar lumina. These studies support the concept that SGs of male I. scapularis ticks support replication of TBFVs and may play a role in virus transmission, and further refine a useful model system for developing countermeasures against this important group of pathogens.
Project description:Objective: Sjögren's syndrome (SS) is a chronic autoimmune disorder that affects mainly the exocrine glands. Endoplasmic reticulum (ER) stress proteins have been suggested to participate in autoimmune and inflammatory responses, either acting as autoantigens, or by modulating factors of inflammation. The chaperone protein ERdj5 is an ER-resident disulfide reductase, required for the translocation of misfolded proteins during ER-associated protein degradation. In this study we investigated the role of ERdj5 in the salivary glands (SGs), in association with inflammation and autoimmunity. Methods: In situ expression of ERdj5 and XBP1 activation were studied immunohistochemically in minor SG tissues from primary SS patients and non-SS sicca-complaining controls. We used the mouse model of ERdj5 ablation and characterized its features: Histopathological, serological (antinuclear antibodies and cytokine levels), and functional (saliva flow rate). Results: ERdj5 was highly expressed in the minor SGs of SS patients, with stain intensity correlated to inflammatory lesion severity and anti-SSA/Ro positivity. Moreover, SS patients demonstrated higher XBP1 activation within the SGs. Remarkably, ablation of ERdj5 in mice conveyed many of the cardinal features of SS, like spontaneous inflammation in SGs with infiltrating T and B lymphocytes, distinct cytokine signature, excessive cell death, reduced saliva flow, and production of anti-SSA/Ro and anti-SSB/La autoantibodies. Notably, these features were more pronounced in female mice. Conclusions: Our findings suggest a critical connection between the function of the ER chaperone protein ERdj5 and autoimmune inflammatory responses in the SGs and provide evidence for a new, potent animal model of SS.
Project description:The pathophysiology of exocrine dysfunction observed in Sjögren's syndrome (SS) is not fully understood. The purpose of this study was to investigate whether autoantibodies against human AQP5 are present in the sera of SS patients. Frozen sections of mouse submandibular salivary glands, CHO cells over-expressing a human AQP5-GFP fusion protein or GFP, and MDCK cells over-expressing AQP5 were used in the indirect immunofluorescence assay to detect anti-AQP5 autoantibodies in the sera from patients with primary SS. The lysates of HEK-293 cells over-expressing the AQP5-GFP fusion protein or GFP were used for immunoprecipitation. Serum IgG from the SS patients but not from the control subjects stained acinar cells in the mouse salivary glands, the signals of which colocalized with those of AQP5-specific antibodies. Serum IgG from the SS patients also selectively stained AQP5-GFP expressed in CHO cells. However, both the control and SS sera immunoprecipitated the AQP5-GFP, suggesting that autoantibodies against AQP5 were also present in the control sera. The screening of 53 control and 112 SS samples by indirect immunofluorescence assay using the AQP5-expressing MDCK cells revealed the presence of significantly higher levels of anti-AQP5 IgG in the SS samples than in the control samples with sensitivity of 0.73 and a specificity of 0.68. Furthermore, the presence of anti-AQP5 autoantibodies was associated with low resting salivary flow in SS patients. In conclusion, anti-AQP5 autoantibodies were detected in the sera from SS patients, which could be a novel biomarker of SS and provide new insight into the pathogenesis of SS.