OBJECTIVE: Sox2 plays crucial roles in tissues homeostasis and regeneration. However, there are lack of a comprehensive examination of Sox2 expression and its functional role in submandibular gland regeneration. Therefore, we aimed to elucidate the impact of Sox2 on submandibular gland regeneration.
METHODS: A Sprague-Dawley rat submandibular gland duct ligation/de-ligation regeneration model was conducted in this study. Sox2-shRNA vectors were retro-ductally administered into the submandibular gland to establish a stable Sox2 knockdown model. Conventional histopathological and molecular biological methods were used to investigate phenotypic changes.
RESULTS: The submandibular gland normalized completely 28 days after ligature removal (following 7 days of duct ligation). AQP5 expression gradually increased after ligation removal until returning to normal levels. In submandibular gland regeneration, Sox2 re-expressed and co-expressed with AQP5+ acinar cells, and Sox2 expression peaked on day 14, recovered to normal on day 28, reproducing the developmental pattern. Sox2 knockdown hindered gland regeneration and induced irreversible fibrosis. The AQP5 expression was significantly lower than the contemporaneous solely ligated group, while the blue collagen deposition and the Vimentin expression increased prominently. The expression of CD68, IL-1β, TNF-α and IL-17A increased significantly, and epithelial cells in the Sox2 knockdown group expressed higher levels of IL-17A.
CONCLUSIONS: These findings highlight Sox2 as a crucial regulator of the acinar cell lineage. Sox2+ progenitor cells are pivotal for acinar cell maintenance, which is indispensable for submandibular gland regeneration. Collectively, our findings may help develop targeted interventions for enhancing tissue repair and preventing irreversible fibrosis in salivary gland disorders.

The Ezrin/Radixin/Moesin (ERM) family of proteins act as cross-linkers between the plasma membrane and the actin cytoskeleton. This mechanism plays an essential role in processes related to membrane remodeling and organization, such as cell polarization, morphogenesis and adhesion, as well as in membrane protein trafficking and signaling pathways. For several human aquaporin (AQP) isoforms, an interaction between the ezrin band Four-point-one, Ezrin, Radixin, Moesin (FERM)-domain and the AQP C-terminus has been demonstrated, and this is believed to be important for AQP localization in the plasma membrane. Here, we investigate the structural basis for the interaction between ezrin and two human AQPs: AQP2 and AQP5. Using microscale thermophoresis, we show that full-length AQP2 and AQP5 as well as peptides corresponding to their C-termini interact with the ezrin FERM-domain with affinities in the low micromolar range. Modelling of the AQP2 and AQP5 FERM complexes using ColabFold reveals a common mode of binding in which the proximal and distal parts of the AQP C-termini bind simultaneously to distinct binding sites of FERM. While the interaction at each site closely resembles other FERM-complexes, the concurrent interaction with both sites has only been observed in the complex between moesin and its C-terminus which causes auto-inhibition. The proposed interaction between AQP2/AQP5 and FERM thus represents a novel binding mode for extrinsic ERM-interacting partners.

UNASSIGNED: Salivary gland dysfunction, often resulting from salivary gland obstruction-induced inflammation, is a prevalent condition. Corticosteroid, known for its anti-inflammatory and immunomodulatory properties, is commonly prescribed in clinics. This study investigates the therapeutic implications and potential side effects of dexamethasone on obstructive sialadenitis recovery using duct ligation mice and salivary gland organoid models.
UNASSIGNED: Functional and pathological changes were assessed after administering dexamethasone to the duct following deligation 2 weeks after maintaining ligation of the mouse submandibular duct. Additionally, lipopolysaccharide- and tumor necrosis factor-induced salivary gland organoid inflammation models were established to investigate the effects and underlying mechanisms of action of dexamethasone.
UNASSIGNED: Dexamethasone administration facilitated SG function restoration, by increasing salivary gland weight and saliva volume while reducing saliva lag time. Histological evaluation revealed, reduced acinar cell atrophy and fibrosis with dexamethasone treatment. Additionally, dexamethasone suppressed pro-inflammatory cytokines IL-1β and TNF expression. In a model of inflammation in salivary gland organoids induced by inflammatory substances, dexamethasone restored acinar markers such as AQP5 gene expression levels, while inhibiting pro-inflammatory cytokines TNF and IL6, as well as chemokines CCL2, CXCL5, and CXCL12 induction. Macrophages cultured in inflammatory substance-treated media from salivary gland organoid cultures exhibited pro-inflammatory polarization. However, treatment with dexamethasone shifted them towards an anti-inflammatory phenotype by reducing M1 markers (Tnf, Il6, Il1b, and Cd86) and elevating M2 markers (Ym1, Il10, Cd163, and Klf4). However, high-dose or prolonged dexamethasone treatment induced acino-ductal metaplasia and had side effects in both in vivo and in vitro models.
UNASSIGNED: Our findings suggest the effectiveness of corticosteroids in treating obstructive sialadenitis-induced salivary gland dysfunction by regulating pro-inflammatory cytokines.

UNASSIGNED: Abnormalities in aquaporins are implicated in the pathological progression of dry eye syndrome. Retinoic acid (RA) regulates cellular proliferation, differentiation, and apoptosis in the cornea, thereby being associated with dry eye disease (DED). The objective of this study is to explore the underlying mechanisms responsible for RA metabolic abnormalities in corneas lacking aquaporin 5 (AQP5).
UNASSIGNED: Dry eye (DE) models were induced via subcutaneous scopolamine hydrobromide. Aqp5 knockout (Aqp5-/-) mice and DE mice were utilized to assess corneal epithelial alterations. Tear secretion, goblet cell counts, and corneal punctate defects were evaluated. The impact of Aqp5 on RA-related enzymes and receptors was investigated using pharmacological RA or SR (A JunB inhibitor), a transcription factor JunB inhibitor, treatment in mouse corneal epithelial cells (CECs), or human corneal epithelial cells (HCECs). The HCECs and NaCl-treated HCECs underwent quantitative real-time PCR (qRT-PCR), immunofluorescent, Western blot, and TUNEL assays. The regulation of transcription factor JunB on Aldh1a1 was explored via ChIP-PCR.
UNASSIGNED: Aqp5 and Aldh1a1 were reduced in both CECs of DE mice and NaCl-induced HCECs. Aqp5-/- mice exhibited DE phenotype and reduced Aldh1a1. RA treatment reduced apoptosis, promoted proliferation, and improved the DE phenotype in Aqp5-/- mice. JunB enrichment in the Aldh1a1 promoter was identified by ChIP-PCR. SR significantly increased Aldh1a1 expression, Ki67, and ΔNp63-positive cells, and decreased TUNEL-positive cells in CECs and HCECs.
UNASSIGNED: Our findings demonstrated the downregulation of Aqp5 expression and aberrant RA metabolism in DE conditions. Knockout of Aqp5 resulted in reduced production of RA through activation of JunB, subsequently leading to the manifestation of DE symptoms.

Aquaporins (AQPs) are a family of water permeable channels expressed on the plasma membrane with AQP5 being the major channel expressed in several human tissues including salivary and lacrimal glands. Anti-AQP5 autoantibodies have been observed in patients with Sjögren\'s syndrome who are characterised by dryness of both salivary and lacrimal glands, and they have been implicated in the underlying mechanisms of glandular dysfunction. AQP5 is formed by six transmembrane helices linked with three extracellular and two intracellular loops. Develop antibodies against membrane protein extracellular loops can be a challenge due to the difficulty in maintaining these proteins as recombinant in their native form. Therefore, in this work we aimed to generate an efficient stable-transfected cell line overexpressing human AQP5 (CHO-K1/AQP5) to perform primarily cell-based phage display biopanning experiments to develop new potential recombinant antibodies targeting AQP5. We also showed that the new CHO-K1/AQP5 cell line can be used to study molecular mechanisms of AQP5 sub-cellular trafficking making these cells a useful tool for functional studies.

BACKGROUND: Emerging data identifies aquaporin 5 (AQP5) as a vital player in many kinds of cancers. Over expression of AQP5 was associated with increased metastasis and poor prognosis, suggesting that AQP5 may facilitate cancer cell proliferation and migration. Our previous studies also showed that AQP3 and AQP5 were highly expressed in triple-negative breast cancer (TNBC) and the expression of AQP3 and AQP5 in TNBC tissue was positive correlated with advanced clinical stage.
OBJECTIVE: We aim to investigate the role of AQP5 in TNBC oncogenesis and development.
METHODS: MDA-MB-231 cells were transfected with siRNA-AQP5 and AQP5 overexpression vector to establish a differential expression system for AQP5. Cell proliferation and apoptosis of MDA-MB-231 cells were detected by CCK-8 (Cell Counting Kit-8) and FCM (flow cytometry), respectively. Cell migration and invasion abilities were evaluated by wound healing assay and transwell assay. The qRT-PCR and western blot assays were used to study the effect of AQP5 expression level on the expression of epithelial-to-mesenchymal transition (EMT) related molecules. The effects of ICG-001, a Wnt/β-catenin signaling pathway inhibitor, on the invasive and migratory capabilities of overexpressed AQP5 cells and downstream molecules were measured.
RESULTS: 1. The expression of AQP5 in the MDA-MB-231 cells was significantly higher than that in the MCF-10A cells. 2. Up-regulation of AQP5 significantly promoted the proliferation, migration and invasion of TNBC cells, while inhibited the cell apoptosis; in addition, up-regulation of AQP5 increased the expression of Bcl-2 and decreased the expression of Caspase-3. However, knockdown of AQP5 presented the adverse effects of AQP5 overexpression. 3. Overexpressed AQP5 induced the overexpression of EMT-related factors, which further promoted the migration and invasion of cells. 4. Overexpression of AQP5 could up-regulate the expression of β-catenin in the nucleus followed by increasing the expression levels of downstream genes in Wnt/β-catenin signaling pathway. Moreover, ICG-001, the inhibitor of Wnt/β-catenin signaling pathway, could significantly attenuate the effect of overexpression of AQP5 on cells, further confirming that AQP5 may promote the proliferation, migration and invasion of TNBC cells by activating Wnt/β-catenin signaling pathway.
CONCLUSIONS: In the TNBC cells, AQP5 modulates the expression levels of EMT-related proteins through activation of Wnt/β-catenin signaling pathway, thus enhancing the cell proliferation, migration and invasion while inhibiting the cell apoptosis.

OBJECTIVE: Xerostomia, a common complication of type 2 diabetes, leads to an increased risk of caries, dysphagia, and dysgeusia. Although anti-vascular endothelial growth factor (VEGF) antibodies, such as ranibizumab (RBZ), have been used to treat diabetic retinopathy, their effects on the salivary glands are unknown. This study evaluated the effects of RBZ on salivary glands to reduce inflammation and restore salivary function in a mouse model of type 2 diabetes.
METHODS: Male KK-Ay mice with type 2 diabetes (10-12 weeks old) were used. The diabetes mellitus (DM) group received phosphate-buffered saline, while the DM + RBZ group received an intraperitoneal administration of RBZ (100 μg/kg) 24 h before the experiment.
RESULTS: Ex vivo perfusion experiments showed a substantial increase in salivary secretion from the submandibular gland (SMG) in the DM + RBZ group. In addition, the mRNA expression levels of TNF-α and IL-1β were considerably lower in this group. In contrast, those of aquaporin 5 were substantially higher in the DM + RBZ group, as revealed by quantitative reverse transcription PCR. Furthermore, the number of lymphocyte infiltration spots in the SMG was notably lower in the DM + RBZ group. Finally, intracellular Ca2+ signaling in acinar cells was considerably higher in the DM + RBZ group than that in the DM group.
CONCLUSIONS: Treating a type 2 diabetic mouse model with RBZ restored salivary secretion through its anti-inflammatory effects.

Diabetes mellitus is a prevalent disease that is often accompanied by ocular surface abnormalities including delayed epithelial wound healing and decreased corneal sensitivity. The impact of diabetes on the lacrimal functional unit (LFU) and the structures responsible for maintaining tear homeostasis, is not completely known. It has been shown that the Opioid Growth Factor Receptor (OGFr), and its ligand, Opioid Growth Factor (OGF), is dysregulated in the ocular surface of diabetic rats leading to overproduction of the inhibitory growth peptide OGF. The opioid antagonist naltrexone hydrochloride (NTX) blocks the OGF-OGFr pathway, and complete blockade following systemic or topical treatment with NTX restores the rate of re-epithelialization of corneal epithelial wounds, normalizes corneal sensitivity, and reverses dry eye in diabetic animal models. These effects occur rapidly and within days of initiating treatment. The present study was designed to understand mechanisms related to the fast reversal (<5 days) of dry eye by NTX in type 1 diabetes (T1D) by investigating dysregulation of the LFU. The approach involved examination of the morphology of the LFU before and after NTX treatment. Male and female adult Sprague-Dawley rats were rendered hyperglycemic with streptozotocin, and after 6 weeks rats were considered to be a T1D model. Rats received topical NTX twice daily to one eye for 10 days. During the period of treatment, tear production and corneal sensitivity were recorded. On day 11, animals were euthanized and orbital tissues including conjunctiva, eyelids, and lacrimal glands, were removed and processed for histologic examination including immunohistochemistry. Male and female T1D rats had significantly decreased tear production and corneal insensitivity, significantly decreased number and size of lacrimal gland acini, decreased expression of aquaporin-5 (AQP5) protein and decreased goblet cell size. Thus, 10 days of NTX treatment restored tear production and corneal sensitivity to normal values, increased AQP5 expression, and restored the surface area of goblet cells to normal. NTX had no effect on the number of lacrimal gland acini or the number of conjunctival goblet cells. In summary, blockade of the OGF-OGFr pathway with NTX reversed corneal and lacrimal gland complications and restored some components of tear homeostasis confirming the efficacy of topical NTX as a treatment for ocular defects in diabetes.

Sjogren\'s syndrome (SS) is an autoimmune disease characterized by dysfunction of exocrine glands, such as salivary glands. However, the molecular mechanism of salivary secretion dysfunction in SS is still unclear. Given the significance of glutathione peroxidase 4 (GPX4) in cellular redox homeostasis, we hypothesized that dysregulation of GPX4 may play a pivotal role in the pathogenesis of salivary secretion dysfunction observed in SS. The salivary gland of SS patients and the SS mouse model exhibited reduced expression of the ferroptosis inhibitor GPX4 and the important protein aquaporin 5 (AQP5), which is involved in salivary secretion. GPX4 overexpression upregulated and GPX4 knockdown downregulated AQP5 expression in salivary gland epithelial cells (SGECs) and salivary secretion. Bioinformatics analysis of GSE databases from SS patients\' salivary glands revealed STAT4 as a key intermediary regulator between GPX4 and AQP5. A higher level of nuclear pSTAT4 was observed in the salivary gland of the SS mouse model. GPX4 overexpression inhibited and GPX4 knockdown promoted STAT4 phosphorylation and nuclear translocation in SGECs. CHIP assay confirmed the binding of pSTAT4 within the promoter of AQP5 inhibiting AQP5 transcription. GPX4 downregulation accumulates intracellular lipid ROS in SGECs. Lipid ROS inhibitor ferrostatin-1 treatment during in vitro and in vivo studies confirmed that lipid ROS activates STAT4 phosphorylation and nuclear translocation in SGECs. In summary, the downregulated GPX4 in SGECs contributes to salivary secretion dysfunction in SS via the lipid ROS/pSTAT4/AQP5 axis. This study unraveled novel targets to revitalize the salivary secretion function in SS patients.

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