Variants of human CLDN9 cause mild to profound hearing loss.

Hereditary deafness is clinically and genetically heterogeneous. We investigated deafness segregating as a recessive trait in two families. Audiological examinations revealed an asymmetric mild to profound hearing loss with childhood or adolescent onset. Exome sequencing of probands identified a homozygous c.475G>A;p.(Glu159Lys) variant of CLDN9 (NM_020982.4) in one family and a homozygous c.370_372dupATC;p.(Ile124dup) CLDN9 variant in an affected individual of a second family. Claudin 9 (CLDN9) is an integral membrane protein and constituent of epithelial bicellular tight junctions (TJs) that form semipermeable, paracellular barriers between inner ear perilymphatic and endolymphatic compartments. Computational structural modeling predicts that substitution of a lysine for glutamic acid p.(Glu159Lys) alters one of two cis-interactions between CLDN9 protomers. The p.(Ile124dup) variant is predicted to locally misfold CLDN9 and mCherry tagged p.(Ile124dup) CLDN9 is not targeted to the HeLa cell membrane. In situ hybridization shows that mouse Cldn9 expression increases from embryonic to postnatal development and persists in adult inner ears coinciding with prominent CLDN9 immunoreactivity in TJs of epithelia outlining the scala media. Together with the Cldn9 deaf mouse and a homozygous frameshift of CLDN9 previously associated with deafness, the two bi-allelic variants of CLDN9 described here point to CLDN9 as a bona fide human deafness gene.

NDRG1 is important to maintain the integrity of airway epithelial barrier through claudin-9 expression.

Impairment of epithelial barrier integrity caused by environmental triggers is associated with the pathogenesis of airway inflammation. Using human airway epithelial cells, we attempted to identify molecule(s) that promote airway epithelial barrier integrity. Microarray analyses were conducted using the Affimetrix human whole genome gene chip, and we identified the N-myc downstream-regulated gene 1 (NDRG1) gene, which was induced during the development of the epithelial cell barrier. Immunohistochemical analysis revealed strong NDRG1 expression in ciliated epithelial cells in nasal tissues sampled from patients with chronic rhinosinusitis (CRS), and the low expression of NDRG1 was observed in goblet cells or damaged epithelial cells. NDRG1 gene knockdown with its specific siRNA decreased the transepithelial electrical resistance and increased the dextran permeability. Immunocytochemistry revealed that NDRG1 knockdown disrupted tight junctions of airway epithelial cells. Next, we analyzed the effects of NDRG1 knockdown on the expression of tight and adhesion junction molecules. NDRG1 knockdown significantly decreased only claudin-9 expression, but did not decrease other claudin family molecules, such as E-cadherin, and ZO-1, -2, or -3. Knockdown of claudin-9 markedly impaired the barrier function in airway epithelial cells. These results suggest that NDRG1 is important for the barrier integrity in airway epithelial cells.

Differences in the expression profiles of claudin proteins in human nasopharyngeal carcinoma compared with non-neoplastic mucosa.

BACKGROUND: Several studies have suggested that claudin proteins, which are the main components of tight junction structures, are related to the regulation of cell polarity and cell differentiation. METHOD: To explore the expression profiles of the tight junction proteins claudin-2, - 5, - 8 and - 9 in nasopharyngeal carcinoma, IHC (immunohistochemical analysis), Western blot and real-time PCR were used to detect the expression profiles of these claudin proteins in nasopharyngeal carcinoma tissues and in non-neoplastic mucosal tissues. RESULTS: According to our study, the expression levels of claudin-2 and claudin-5 were reduced, while the expression of claudin-8 was increased in nasopharyngeal carcinoma tissues in comparison with non-neoplastic mucosal tissues. Correlations between claudin-2 and -5 expression and metastatic progression in nasopharyngeal carcinoma patients were also found. CONCLUSION: In summary, our research reveals distinct expression profiles of claudin-2, - 5 and - 8 in non-neoplastic mucosal tissues and nasopharyngeal carcinoma tissues. In addition, the expression of these claudin proteins was highly correlated with metastatic progression and prognosis in patients with nasopharyngeal carcinoma and had predictive value for the metastasis and survival of nasopharyngeal carcinoma patients.

A spontaneous metastasis model reveals the significance of claudin-9 overexpression in lung cancer metastasis.

Metastasis causes most cancer related mortality but the mechanisms governing metastatic dissemination are poorly defined. Metastasis involves egression of cancer cells from the primary tumors, their survival in circulation and colonization at the secondary sites. Cancer cell egression from the primary tumor is the least defined process of metastasis as experimental metastasis models directly seed cancer cells in circulation, thus bypassing this crucial step. Here, we developed a spontaneous metastasis model that retains the egression step of metastasis. By repeated in vivo passaging of the poorly metastatic Lewis lung carcinoma (3LL) cells, we generated a cell line (p-3LL) that readily metastasizes to lungs and liver from subcutaneous (s.c.) tumors. Interestingly, when injected intravenously, 3LL and p-3LL cells showed a similar frequency of metastasis. This suggests enhanced egression of p-3LL cells may underlie the enhanced metastatic spread from primary tumors. Microarray analysis of 3LL and p-3LL cells as well as the primary tumors derived from these cells revealed altered expression of several genes including significant upregulation of a tight junction protein, claudin-9. Increased expression of claudin-9 was confirmed in both p-3LL cells and tumors derived from these cells. Knockdown of claudin-9 expression in p-3LL cells by si-RNA significantly reduced their motility, invasiveness in vitro and metastasis in vivo. Conversely, transient overexpression of claudin-9 in 3LL cells enhanced their motility. These results suggest an essential role for claudin-9 in promoting lung cancer metastasis.

Expression of claudin-5, -7, -8 and -9 in cervical carcinoma tissues and adjacent non-neoplastic tissues.

Recent data indicate that the tight junction proteins are abnormally regulated in several human cancers and the expression of these proteins is involved in the etiology and progression of cancer. To explore the expression distinction of the tight junction proteins claudin-5, -7, -8 and -9 in the adjacent non-neoplastic tissues and cervical carcinoma tissues, 72 cervical carcinoma tissues and the samples of non-neoplastic tissues adjacent to the tumors were examined for expression of claudin-5, -7, -8 and -9 by streptavidin-perosidase immunohistochemical staining method. The positive expression rates of claudin-5 in cervical carcinoma tissues and adjacent non-neoplastic tissues were 31.9% (23/72) and 51.4% (37/72) respectively (P < 0.05). The positive expression rates of claudin-7 in cervical carcinoma tissues and adjacent non-neoplastic tissues were 47.2% and 50.0% respectively (P = 1.000). The positive expression rates of claudin-8 in cervical carcinoma tissues and adjacent non-neoplastic tissues were 54.2 % and 27.8% respectively (P < 0.01). The positive expression rates of claudin-9 in cervical carcinoma tissues and adjacent non-neoplastic tissues were 38.9% and 56.9% respectively (P < 0.05). Thus in our study, the expression of claudin-5 and claudin-9 was down-regulated while the expression of claudin-8 was up-regulated in cervical carcinoma tissues compared with adjacent non-neoplastic tissues. The expression of claudin-7 has no obviously difference between cervical carcinoma tissues and adjacent non-neoplastic tissues. In addition, correlations between claudin-5, -8 and -9 expression with lymphatic metastasis were observed. Our study reveals that the expression of claudin-5, -8 and -9 altered between in cervical carcinoma tissues and adjacent non-neoplastic tissues.