hearing ͉ ion transport ͉ potassium

No abstract

Summary Nonmuscle myosin II (NMII) is thought to be the master integrator of force within epithelial apical junctions, mediating epithelial tissue morphogenesis and tensional homeostasis [1-3]. Mutations in NMII are associated with a number of diseases due to failures in cell-cell adhesion [4-8]. However, the organization and the precise mechanism by which NMII generates and responds to tension along the intercellular junctional line are still not known. We discovered that periodic assemblies of bipolar NMII filaments interlace with perijunctional actin and α-actinin to form a continuous belt of muscle-like sarcomeric units (~400 – 600 nm) around each epithelial cell. Remarkably, the sarcomeres of adjacent cells are precisely paired across the junctional line forming an integrated, transcellular contractile network. The contraction/relaxation of paired sarcomeres concomitantly impacts changes in apical cell shape and tissue geometry. We show differential distribution of NMII isoforms across heterotypic junctions and evidence for compensation between isoforms. Our results provide a model for how NMII force generation is effected along the junctional perimeter of each cell, and communicated across neighboring cells in the epithelial organization. The sarcomeric network also provides a well-defined target to investigate the multiple roles of NMII in junctional homeostasis as well as in development and disease.

Cytoplasmic aggregates of ubiquitinated TAR DNA-binding protein 43 (TDP-43) are a pathological hallmark of amyotrophic lateral sclerosis (ALS). However, the mechanism of TDP-43 polyubiquitination remains elusive. We investigated the effect of nuclear exclusion of TDP-43 on aggregate formation and fragmentation, using TDP-43 expression constructs for WT or mutant TDP-43 with a modified nuclear localizing signal (LQ-NLS). Overexpression of the LQ-NLS mutant alone induced no detectable cytoplasmic aggregates during a 72-hr period. Polyubiquitination of both WT TDP-43 and the LQ-NLS mutant was similar in total cell lysates exposed to the proteasome inhibitor lactacystin. However, analysis of subcellular fractions demonstrated a higher concentration of polyubiquitinated TDP-43 in the nuclear fraction than in the cytosol for WT, and vice versa for the LQ-NLS mutant. Polyubiquitin-charged WT and mutant TDP-43 were highly concentrated in the membrane/microsome fraction, which was also positive for the autophagosome marker LC3. In addition, the autophagy inhibitor 3-methyladenine (3MA) blocked degradation of both TDP-43 types, whereas lactacystin was minimally restorative. Furthermore, lactacystin plus 3MA induced prominent cytoplasmic aggregates. We also demonstrated mediation of TDP-43 polyubiquitination by lysine 48 of ubiquitin, indicating a degradation signal in both TDP-43 types. This is the first report delineating the distribution of polyubiquitinated TDP-43 and the degradation pathway of TDP-43 and clarifying the crucial role of autophagosomes in TDP-43 clearance. We also demonstrate that nuclear exclusion itself is not an immediate trigger for ALS pathology. Further clarification of the mechanism of polyubiquitination of TDP-43 and the role of autophagosomes may help in understanding and treating ALS.

Key points• The endocochlear potential (EP) of +80 mV in cochlear endolymph is essential for audition and controlled by K + transport across the lateral cochlear wall composed of two epithelial barrier layers, the syncytium containing the fibrocytes and the marginal cells.• The EP depends upon the diffusion potential elicited by a large K + gradient across the apical surface of the syncytium.• We examined by electrophysiological approaches an involvement of Na + ,K + -ATPase, which occurs at the syncytium's basolateral surface comprising the fibrocytes' membranes and would mediate K + transport across the lateral wall, in maintenance of the EP.• We show that the Na + ,K + -ATPase sustains the syncytium's high [K + ] that is crucial for the K + gradient across the apical surface of the syncytium.• The results help us better understand the mechanism underlying the establishment of the EP as well as the pathophysiological process for deafness induced by dysfunction of the ion transport apparatus.Abstract The endocochlear potential (EP) of +80 mV in the scala media, which is indispensable for audition, is controlled by K + transport across the lateral cochlear wall. This wall includes two epithelial barriers, the syncytium and the marginal cells. The former contains multiple cell types, such as fibrocytes, which are exposed to perilymph on their basolateral surfaces. The apical surfaces of the marginal cells face endolymph. Between the two barriers lies the intrastrial space (IS), an extracellular space with a low K + concentration ([K + ]) and a potential similar to the EP. This intrastrial potential (ISP) dominates the EP and represents the sum of the diffusion potential elicited by a large K + gradient across the apical surface of the syncytium and the syncytium's potential, which is slightly positive relative to perilymph. Although a K + transport system in fibrocytes seems to contribute to the EP, the mechanism remains uncertain. We examined the electrochemical properties of the lateral wall of guinea pigs with electrodes sensitive to potential and K + while perfusing into the perilymph of the scala tympani blockers of Na + ,K + -ATPase, the K + pump thought to be essential to the system. Inhibiting Na

Interleukin (IL)-27 is an IL-12 family cytokine playing a pivotal role in the induction of Th1 immune responses, although its action on natural killer (NK) cells has not been fully elucidated. Here, we show that IL-27 is capable of inducing phosphorylation of signal transducers and activators of transcription 1 and 3, as well as expression of T-bet and granzyme B in murine DX-5+ NK cells. IL-27 also enhances cytotoxic activity of NK cells both in vitro and in vivo, while the in vitro viability of NK cells is also improved by this cytokine. Therapeutic administration of the IL-27 gene drastically suppressed the growth of NK-unsusceptible SCCVII tumors that had been preestablished in syngenic mice, resulting in significant prolongation of the survival of the animals. This can likely be ascribed to the antibody-dependent cellular cytotoxicity machinery because IL-27 successfully induced tumor-specific IgG in the sera of the tumor-bearing mice, and supplementation of the sera enabled IL-27-activated NK cells to kill SCCVII cells in an Fc; receptor IIIdependent manner. These findings strongly suggest that IL-27 may offer a powerful immunotherapeutic tool to eradicate head and neck squamous cell carcinoma and other poorly immunogenic neoplasms through activating NK cells and inducing tumor-specific immunoglobulin that may cooperatively elicit antibody-dependent cellular cytotoxicity activity.

Type I allergic diseases such as allergic rhinitis are caused by IgE-mediated humoral immune responses, while eosinophils also fulfill important roles in the etiology of IgE-mediated allergy. IL-21 regulates growth, differentiation, and function of T, B, and NK cells, while the production of IgE is also influenced by IL-21. In this study we examined whether IL-21 is capable of controlling IgE-mediated allergic reactions in vivo by using the allergic rhinitis mouse model that was established by repetitive sensitization and intranasal challenge with OVA. Intranasal administration with recombinant mouse IL-21 (rmIL-21) significantly reduced the number of sneezes, as well as the serum concentration of OVA-specific IgE, in comparison with that of untreated allergic mice. The rmIL-21 treatment also suppressed germline Cε transcription in the nasal-associated lymphoid tissues, which may have, at least partly, resulted from the up-regulation of Bcl-6 mRNA caused by IL-21. Local expression of IL-4, IL-5, and IL-13 was also inhibited by the intranasal cytokine therapy whereas, in contrast, the expression of endogenous IL-21 mRNA was induced by exogenous rmIL-21. Moreover, IL-21 acted on nasal fibroblasts to inhibit production of eotaxin. This novel function of IL-21 may be associated with the attenuation of eosinophil infiltration into nasal mucosa that was revealed by histopathological observation. These results indicated that IL-21 nasal administration effectively ameliorated allergic rhinitis through pleiotropic activities, i.e., the prevention of IgE production by B cells and eotaxin production by fibroblasts.

BSTRACTCdc42 is a key regulator of dynamic actin organization. However, little is known about how Cdc42-dependent actin regulation influences steady-state actin structures in differentiated epithelia. We employed inner ear hair-cell-specific conditional knockout to analyze the role of Cdc42 in hair cells possessing highly elaborate stable actin protrusions (stereocilia). Hair cells of Atoh1-Cre;Cdc42 flox/flox mice developed normally but progressively degenerated after maturation, resulting in progressive hearing loss particularly at high frequencies. Cochlear hair cell degeneration was more robust in inner hair cells than in outer hair cells, and began as stereocilia fusion and depletion, accompanied by a thinning and waving circumferential actin belt at apical junctional complexes (AJCs). Adenovirus-encoded GFP-Cdc42 expression in hair cells and fluorescence resonance energy transfer (FRET) imaging of hair cells from transgenic mice expressing a Cdc42-FRET biosensor indicated Cdc42 presence and activation at stereociliary membranes and AJCs in cochlear hair cells. Cdc42-knockdown in MDCK cells produced phenotypes similar to those of Cdc42-deleted hair cells, including abnormal microvilli and disrupted AJCs, and downregulated actin turnover represented by enhanced levels of phosphorylated cofilin. Thus, Cdc42 influenced the maintenance of stable actin structures through elaborate tuning of actin turnover, and maintained function and viability of cochlear hair cells.