Chloride channel accessory 2 (CLCA2) is a transmembrane protein, which promotes adhesion of keratinocytes and their survival in response to hyperosmotic stress. Here we show that CLCA2 is transported to the nucleus of keratinocytes via extracellular vesicles. The nuclear localization is functionally relevant, since wild-type CLCA2, but not a mutant lacking the nuclear localization signal, suppressed migration of keratinocytes and protected them from hyperosmotic stress-induced cell death. In the nucleus, CLCA2 bound to and activated β-catenin, resulting in enhanced expression of Wnt target genes. Mass-spectrometry-based interaction screening and functional rescue studies identified RNA binding protein 3 as a key effector of nuclear CLCA2. This is of likely relevance in vivo because both proteins co-localize in the human epidermis. Together, these results identify an unexpected nuclear function of CLCA2 in keratinocytes under homeostatic and stress conditions and suggest a role of extracellular vesicles and their nuclear transport in the control of key cellular activities.

Cellular senescence, a state of irreversible growth arrest, is implicated in various age-related pathologies, including skin aging. In this study, we investigated the role of CLCA2, a calcium-activated chloride channel accessory protein, in cellular senescence and its implications for skin aging. Utilizing UVB and Nutlin3a-induced senescence models, we observed the upregulation of CLCA2 at both transcriptomic and proteomic levels, suggesting its involvement in senescence pathways. Further analysis revealed that the depletion of CLCA2 led to accelerated senescence onset, characterized by classic senescence markers and a unique secretome profile. In 3D skin equivalent models, SEs constructed with CLCA2 knockdown fibroblasts exhibited features reminiscent of aged skin, underscoring the importance of CLCA2 in maintaining skin homeostasis. Our findings highlight CLCA2 as a novel regulator of cellular senescence and its potential implications for skin aging mechanisms.

BACKGROUND: Premature ovarian insufficiency (POI) is a severe disorder leading to female infertility. Genetic mutations are important factors causing POI. TP63-truncating mutation has been reported to cause POI by increasing germ cell apoptosis, however what factors mediate this apoptosis remains unclear.
METHODS: Ninety-three patients with POI were recruited from Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Whole-exome sequencing (WES) was performed for each patient. Sanger sequencing was used to confirm potential causative genetic variants. A minigene assay was performed to determine splicing effects of TP63 variants. A TP63-truncating plasmid was constructed. Real-time quantitative PCR, western blot analyses, dual luciferase reporter assays, immunofluorescence staining, and cell apoptosis assays were used to study the underlying mechanism of a TP63-truncating mutation causing POI.
RESULTS: By WES of 93 sporadic patients with POI, we found a 14-bp deletion covering the splice site in the TP63 gene. A minigene assay demonstrated that the 14-bp deletion variant led to exon 13 skipping during TP63 mRNA splicing, resulting in the generation of a truncated TP63 protein (TP63-mut). Overexpression of TP63-mut accelerated cell apoptosis. Mechanistically, the TP63-mut protein could bind to the promoter region of CLCA2 and activate the transcription of CLCA2 several times compared to that of the TP63 wild-type protein. Silencing CLCA2 using a specific small interfering RNA (siRNA) or inhibiting the Ataxia Telangiectasia Mutated (ATM) pathway using the KU55933 inhibitor attenuated cell apoptosis caused by TP63-mut protein expression.
CONCLUSIONS: Our findings revealed a crucial role for CLCA2 in mediating apoptosis in POI pathogenesis, and suggested that CLCA2 is a potential therapeutic target for POI.

Human CLCA2 regulates store-operated calcium entry (SOCE) by interacting with Orai1 and STIM1. It is expressed as a 943aa type I transmembrane protein that is cleaved at amino acid 708 to produce a diffusible 100 kDa product. The N-terminal ectodomain contains a hydrolase-like subdomain with a conserved HEXXH zinc-binding motif that is proposed to cleave the precursor autoproteolytically. Here, we tested this hypothesis and its link to SOCE. We first studied the conditions for autocleavage in isolated membranes and then in a purified protein system. Cleavage was zinc-dependent and abolished by mutation of the E in the HEXXH motif to Q, E165Q. Cleavage efficiency increased with CLCA2 concentration, implying that it occurs in trans. Accordingly, the E165Q mutant was cleaved by co-transfected wildtype CLCA2. Moreover, CLCA2 precursors with different epitope tags co-immunoprecipitated. In a membrane-free system utilizing immunopurified protease and target, no cleavage occurred unless the target was first denatured, implying that membranes provide essential structural or conformational cues. Unexpectedly, cleavage caused a conformational shift: an N-terminal antibody that immunoprecipitated the precursor failed to precipitate the N-terminal product unless the product was first denatured with an ionic detergent. The E165Q mutation abolished the stimulation of SOCE caused by wildtype CLCA2, establishing that the metalloprotease activity is required for this regulatory function.

Cervical cancer is an important malignant tumor threatening the physical and mental health of women in the world. As a new calcium activated chloride channel protein, calcium activated chloride channel (CLCA2) plays an important role in tumorigenesis and development. But its role and exact regulatory mechanism in cervical cancer are still unclear. In our study, we found CLCA2 was significantly decreased in cervical cancer cells, and overexpression of CLCA2 inhibited the proliferation, migration and invasion, and promotes apoptosis of cervical cancer cells, and CLCA2 inhibited EMT (Epithelial-mesenchymal transition) through an p38 / JNK / ERK pathway. The results in vivo were consistent with those in vitro. In conclusion, overexpression of CLCA2 inhibited the progression of cervical cancer in vivo and in vitro. This may provide a theoretical basis for CLCA2 as a new indicator of clinical diagnosis and prognosis of cervical cancer or as a potential target of drug therapy.

BACKGROUND: Progressive cardiac conduction defect (PCCD), also known as Lenegre-Lev disease, is one of the most common heart conduction abnormalities. Previous studies have screened for known mutation sites that cause heart block in a 68-person family with a history of PCCD, revealed no mutations.
OBJECTIVE: To screen pathogenic genes of the PCCD family and to study the function of the gene mutations related to heart block diseases.
METHODS: Whole exome sequencing (WES) was performed on two PCCD patients and one non-PCCD family member to find the related pathogenic gene. After family co-segregation and preliminary functional analysis, we identified the mutant gene CLCA2. To study the function of this gene, we constructed mutant-gene mice using CRISPR-Cas9 technology, and electrocardiogram monitoring was performed after genotype verification.
RESULTS: The CLCA2 c.G1725T mutation was identified and co-segregated with the phenotype. The analysis showed that the CLCA2 c.G1725T mutation is harmful and mainly affects protein glycosylation. Immunofluorescence staining revealed that CLCA2 was highly expressed in the sinoatrial node (SAN) tissues. Electrocardiogram monitoring of the mice revealed that CLCA2 point mutations induced mild conduction block and ectopic pacemakers.
CONCLUSIONS: Our findings indicate that a novel heterozygous missense mutation c.G1725T of the CLCA2 gene may be associated with heart block disease and the mutation in this gene may lead to sinus node lesions and conduction blocking.

Prostate cancer (PCa) is the most common cancer among men. Metabolic syndrome (MeS) is associated with increased PCa aggressiveness and recurrence. Previously, we proposed C-terminal binding protein 1 (CTBP1), a transcriptional co-repressor, as a molecular link between these two conditions. Notably, CTBP1 depletion decreased PCa growth in MeS mice. The aim of this study was to investigate the molecular mechanisms that explain the link between MeS and PCa mediated by CTBP1. We found that CTBP1 repressed chloride channel accessory 2 (CLCA2) expression in prostate xenografts developed in MeS animals. CTBP1 bound to CLCA2 promoter and repressed its transcription and promoter activity in PCa cell lines. Furthermore, we found that CTBP1 formed a repressor complex with ZEB1, EP300 and HDACs that modulates the CLCA2 promoter activity. CLCA2 promoted PCa cell adhesion inhibiting epithelial-mesenchymal transition (EMT) and activating CTNNB1 together with epithelial marker (CDH1) induction, and mesenchymal markers (SNAI2 and TWIST1) repression. Moreover, CLCA2 depletion in PCa cells injected subcutaneously in MeS mice increased the circulating tumor cells foci compared to control. A microRNA (miRNA) expression microarray from PCa xenografts developed in MeS mice, showed 21 miRNAs modulated by CTBP1 involved in angiogenesis, extracellular matrix organization, focal adhesion and adherents junctions, among others. We found that miR-196b-5p directly targets CLCA2 by cloning CLCA2 3\'UTR and performing reporter assays. Altogether, we identified a new molecular mechanism to explain PCa and MeS link based on CLCA2 repression by CTBP1 and miR-196b-5p molecules that might act as key factors in the progression onset of this disease.

BACKGROUND: CLCA2 was reported as a tumor suppressor and disregulated in breast cancer. However, its function in tumor growth and metastasis in NPC has rarely been reported. In this study, we investigated the functional and molecular mechanisms by which CLCA2 influences NPC.
METHODS: CLCA2 expression in human NPC cell lines and tissues was examined via real-time PCR (RT-PCR), Western blot and IHC. The biological roles of CLCA2 in proliferative, migration and invasion of NPC cell lines was evaluated in 5-8F, S18, S26 and SUNE-1 cells. Cell viability, migration and invasion were assessed in vitro by MTS, colony formation and transwell assay, respectively. CLCA2 in growth and metastasis of NPC were evaluated in vivo through NPC xenograft tumor growth, lung metastatic mice model and popliteal lymph node (LN) metastasis model.
RESULTS: Overexpression of CLCA2 significantly decreased proliferation, migration and invasion of NPC cells. In contrast, knockdown of CLCA2 elicited the opposite effects. CLCA2 overexpression suppressed xenograft tumor growth and lung, popliteal lymph node (LN) metastasis in vivo. CLCA2 inhibited tumor metastasis through suppressing epithelial-Mesenchymal transition (EMT) and in-activating FAK/ERK1/2 signaling pathway in NPC cells. Immunohistochemical staining of 143 NPC samples revealed that CLCA2 expression was an independent, favorable prognostic factor for overall survival and distant metastasis-free survival of patients. In addition, inhibition of FAK and ERK1/2 reversed CLCA2 silencing-induced tumor cell migration. Furthermore, inhibitors against chloride channels suppressed NPC cellular migration which could have been enhanced by the presence of CLCA2.
CONCLUSIONS: CLCA2 suppress NPC proliferation, migration, invasion and epithelial-mesenchymal transition through inhibiting FAK/ERK signaling.