Tricellular Tight Junctions in the Inner Ear

Tight junctions (TJs) are structures that seal the space between the epithelial cell sheets. In the inner ear, the barrier function of TJs is indispensable for the separation of the endolymphatic and perilymphatic spaces, which is essential for the generation and maintenance of the endocochlear potential (EP). TJs are formed by the intercellular binding of membrane proteins, known as claudins, and mutations in these proteins cause deafness in humans and mice. Within the epithelial cell sheet, however, a bound structure is present at the site where the corners of three cells meet (tricellular tight junctions (tTJs)), and the maintenance of the barrier function at this location cannot be explained by the claudins alone. Tricellulin and the angulin family of proteins (angulin-1/LSR, angulin-2/ILDR1, and angulin-3/ILDR2) have been identified as tTJ-associated proteins. Tricellulin and ILDR1 are localized at the tTJ and alterations in these proteins have been reported to be involved in deafness. In this review, we will present the current state of knowledge for tTJs

Deafness in occludin-deficient mice with dislocation of tricellulin and progressive apoptosis of the hair cells

Occludin is the first identified protein in the tight junction (TJ), but its function has remained for the most part obscure. TJs have been demonstrated to play important roles in the inner ear function, and occludin is expressed in all the epithelial TJs in the inner ear. Thus, we examined the inner ears of occludin-deficient (Occ(-/-)) mice. Although inner ears initially developed normally in Occ(-/-) mice, apoptosis occurs in hair cells in the organ of Corti around day 12 after birth, and deafness develops. Since hair cell degeneration was not observed in cochlear explant cultures of Occ(-/-) mice, environmental changes were considered to be the trigger of cell death. As for the vestibular system, both the morphologies and functions are normal in Occ(-/-) mice. These phenotypes of Occ(-/-) mice are very similar with those of claudin-14 or claudin-9 deficient mice, leading us to speculate on the existence of imbalance induced by TJ abnormalities, such as localized ionic components. Moreover, the occludin deficiency led to dislocalization of tricellulin, a gene responsible for human deafness DFNB49. The deafness in Occ(-/-) mice may be due to this dislocalization of tricellulin.ArticleBIOLOGY OPEN.3(8):759-766(2014)journal articl

Methanol stimulates the colony-formation rate in a human hepatoma cell line (HLE).

&#60;P&#62;Effects of methanol on colony-formation of human hepatoma cells were investigated. Among five human hepatoma cell lines (Hep G2, HLE, HuH-6, HuH-7, and PLC/PRF/5), only HLE cells showed enhanced colony formation due to methanol. The effective concentrations of methanol were around 1%. The enhancement occurred in a greater degree when the cells were seeded in the culture medium containing methanol than when methanol was added 24h after the cells were seeded. Methanol itself, however, did not enhance the cell proliferation.</p

Digenic inheritance of mutations in EPHA2 and SLC26A4 in Pendred syndrome

Enlarged vestibular aqueduct (EVA) is one of the most commonly identified inner ear malformations in hearing loss patients including Pendred syndrome. While biallelic mutations of the SLC26A4 gene, encoding pendrin, causes non-syndromic hearing loss with EVA or Pendred syndrome, a considerable number of patients appear to carry mono-allelic mutation. This suggests faulty pendrin regulatory machinery results in hearing loss. Here we identify EPHA2 as another causative gene of Pendred syndrome with SLC26A4. EphA2 forms a protein complex with pendrin controlling pendrin localization, which is disrupted in some pathogenic forms of pendrin. Moreover, point mutations leading to amino acid substitution in the EPHA2 gene are identified from patients bearing mono-allelic mutation of SLC26A4. Ephrin-B2 binds to EphA2 triggering internalization with pendrin inducing EphA2 autophosphorylation weakly. The identified EphA2 mutants attenuate ephrin-B2- but not ephrin-A1-induced EphA2 internalization with pendrin. Our results uncover an unexpected role of the Eph/ephrin system in epithelial function

Tricellular Tight Junctions in the Inner Ear

Tight junctions (TJs) are structures that seal the space between the epithelial cell sheets. In the inner ear, the barrier function of TJs is indispensable for the separation of the endolymphatic and perilymphatic spaces, which is essential for the generation and maintenance of the endocochlear potential (EP). TJs are formed by the intercellular binding of membrane proteins, known as claudins, and mutations in these proteins cause deafness in humans and mice. Within the epithelial cell sheet, however, a bound structure is present at the site where the corners of three cells meet (tricellular tight junctions (tTJs)), and the maintenance of the barrier function at this location cannot be explained by the claudins alone. Tricellulin and the angulin family of proteins (angulin-1/LSR, angulin-2/ILDR1, and angulin-3/ILDR2) have been identified as tTJ-associated proteins. Tricellulin and ILDR1 are localized at the tTJ and alterations in these proteins have been reported to be involved in deafness. In this review, we will present the current state of knowledge for tTJs

Deficiency of angulin-2/ILDR1, a tricellular tight junction-associated membrane protein, causes deafness with cochlear hair cell degeneration in mice

Tricellular tight junctions seal the extracellular spaces of tricellular contacts, where the vertices of three epithelial cells meet, and are required for the establishment of a strong barrier function of the epithelial cellular sheet. Angulins and tricellulin are known as specific protein components of tricellular tight junctions, where angulins recruit tricellulin. Mutations in the genes encoding angulin-2/ILDR1 and tricellulin have been reported to cause human hereditary deafness DFNB42 and DFNB49, respectively. To investigate the pathogenesis of DFNB42, we analyzed mice with a targeted disruption of Ildr1, which encodes angulin-2/ILDR1. Ildr1 null mice exhibited profound deafness. Hair cells in the cochlea of Ildr1 null mice develop normally, but begin to degenerate by two weeks after birth. Tricellulin localization at tricellular contacts of the organ of Corti in the cochlea was retained in Ildr1 null mice, but its distribution along the depth of tricellular contacts was affected. Interestingly, compensatory tricellular contact localization of angulin-1/LSR was observed in the organ of Corti in Ildr1 null mice although it was hardly detected in the organ of Corti in wild-type mice. The onset of hair cell degeneration in Ildr1 null mice was earlier than that in the reported Tric mutant mice, which mimic one of the tricellulin mutations in DFNB49 deafness. These results indicate that the angulin-2/ILDR1 deficiency causes the postnatal degenerative loss of hair cells in the cochlea, leading to human deafness DFNB42. Our data also suggest that angulin family proteins have distinct functions in addition to their common roles of tricellulin recruitment and that the function of angulin-2/ILDR1 for hearing cannot be substituted by angulin-1/LSR

Compensatory accumulation of angulin-1/LSR in the organ of Corti in <i>Ildr1</i>^<i>k-/-</i> mice.

<p>A. Immunofluorescence microscopic images of the organ of Corti in the middle turn in wild-type (+/+) and <i>Ildr1</i>^<i>k-/-</i> (-/-) mice at various time points using anti-angulin-1/LSR mAb (green) and anti-occludin pAb (red). Note that the angulin-1/LSR signals are evident at TCs in the organ of Corti in <i>Ildr1</i>^<i>k-/-</i> mice, but not in wild-type mice. B. The fluorescence intensities of angulin-1/LSR and occludin at TCs in the organ of Corti in wild-type mice and <i>Ildr1</i>^<i>k/-</i> mice at P10 were quantified. Error bars indicate s.d. *p<0.005 (Student’s t-test). Note that the intensity of angulin-1/LSR was significantly up-regulated at TCs in <i>Ildr1</i>^<i>k/-</i> mice, whereas that of occludin was unchanged. At least two wild-type mice and <i>Ildr1</i>^<i>k-/-</i> mice were analyzed for each time point and consistent results were obtained. Bars, 10 μm.</p

Hearing loss in <i>Ildr1</i>^<i>k-/-</i> mice.

<p>A. Hearing thresholds at sound frequencies of 10, 20 and 40 kHz of wild-type (+/+) (n = 3) and homozygous (-/-) mice (n = 4) at P35. <i>Ildr1</i>^<i>k-/-</i> mice showed increased thresholds (85- dB sound pressure level (SPL)) compared with wild-type mice (5–40 dB SPL). All average data on the graphs are shown as mean ± SEM. In all instances, P values were less than 0.01 and considered to be significant. B. ABRs to stimuli of 5–90 dB SPL at 20 kHz in P35 wild-type and <i>Ildr1</i>^<i>k-/-</i> mice. Typical data for a wild-type mouse and an <i>Ildr1</i>^<i>k-/-</i> mouse are shown. In wild-type mice, typical ABR waveform can be observed by small sound stimuli until 30dB. In contrast, <i>Ildr1</i>^<i>k-/-</i> mice did not respond to large sound stimuli over 90dB.</p