Yu C

Effect of glucocorticoids on aquaporin-1 in guinea pigs with otitis media with effusion

The aim of this study was to explore the pathological changes in water homeostasis and the effects of glucocorticoids on aquaporin-1 (AQP1) in guinea pigs with otitis media with effusion (OME). Immunohistochemistry and western blotting were used to detect AQP1 in the bullae of OME models, which were induced by reversible Eustachian tube (ET) obstruction. Animals in the dexamethasone (dexa) group received dexa via intraperitoneal injection for 7 days and the pathological changes and expression patterns of AQP1 were compared with those in the OME group. In this study, 22 guinea pigs exhibited effusion 3–7 days after surgery, of which two were sacrificed. Six (60%) animals in the OME group and 9 (90%) in the dexa group presented no sign of effusion on postoperative day 14. AQP1 was detected as an 28-kDa protein in the two groups. Immunohistochemical analysis revealed that AQP1 was expressed in subepithelial fibroblasts and capillary endothelial cells. Western blot analysis revealed that the levels of AQP1 protein were markedly higher in the dexa group compared with the OME group. In conclusion, our study emphasized the significance of AQP1 in the pathophysiology of OME and suggests that glucocorticoids may regulate water homeostasis via an AQP1-regulated pathway.

Myosin light chain kinase regulates hearing in mice by influencing the F-actin cytoskeleton of outer hair cells and cochleae

Myosin light chain kinase (MLCK) phosphorylates myosin regulatory light chains to facilitate its interaction with actin filaments and produce contractile activity. The outer hair cells (OHCs) in the ear contain large amounts of actin and a variety myosins. The stereociliary and somatic motility of OHCs are closely related to hearing. It appears likely that MLCK may play an important role in acoustic trans-duction. In this study, we analyzed, both in vivo and in vitro, the OHCs of mice bearing a specific deletion of the MLCK gene and the OHCs of control mice. The phenotype was assessed by auditory function [acoustic brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs)], inner ear morphology and histology. MLCK-deficient mice aged 6-7 months showed impaired hearing, a 5- to 10-dB sound pressure level (SPL) increase in the ABR thresholds, when responding to clicks and tones of different frequencies (8 and 16 kHz) (P<0.05). The DPOAE amplitudes of 3-month-old MLCK-deficient mice decreased significantly (>10 dB SPL) at low frequencies (4, 5 and 6 kHz). The OHCs in the MLCK-deficient mice increased with abnormal stereocilia. The staining of F-actin and the phosphorylation of the regulatory light chain in MLCK-deficient OHCs was weak. Our results indicate that MLCK may regulate the structure and the motility of stereocilia through F-actin polymerization.

The role of FOXG1 in the postnatal development and survival of mouse cochlear hair cells

&NA; The development of therapeutic interventions for hearing loss requires a detailed understanding of the genes and proteins involved in hearing. The FOXG1 protein plays an important role in early neural development and in a variety of neurodevelopmental disorders. Previous studies have shown that there are severe deformities in the inner ear in Foxg1 knockout mice, but due to the postnatal lethality of Foxg1 knockout mice, the role of FOXG1 in hair cell (HC) development and survival during the postnatal period has not been investigated. In this study, we took advantage of transgenic mice that have a specific knockout of Foxg1 in HCs, thus allowing us to explore the role of FOXG1 in postnatal HC development and survival. In the Foxg1 conditional knockout (CKO) HCs, an extra row of HCs appeared in the apical turn of the cochlea and some parts of the middle turn at postnatal day (P)1 and P7; however, these HCs gradually underwent apoptosis, and the HC number was significantly decreased by P21. Auditory brainstem response tests showed that the Foxg1 CKO mice had lost their hearing by P30. The RNA‐Seq results and the qPCR verification both showed that the Wnt, Notch, IGF, EGF, and Hippo signaling pathways were down‐regulated in the HCs of Foxg1 CKO mice. The significant down‐regulation of the Notch signaling pathway might be the reason for the increased numbers of HCs in the cochleae of Foxg1 CKO mice at P1 and P7, while the down‐regulation of the Wnt, IGF, and EGF signaling pathways might lead to subsequent HC apoptosis. Together, these results indicate that knockout of Foxg1 induces an extra row of HCs via Notch signaling inhibition and induces subsequent apoptosis of these HCs by inhibiting the Wnt, IGF, and EGF signaling pathways. This study thus provides new evidence for the function and mechanism of FOXG1 in HC development and survival in mice. HIGHLIGHTSThe first study to conditionally knockout FoxG1 in hair cells that avoid the embryonic lethality of FoxG1 systemic knockout mice.The first study to assess the influence of FoxG1 knockout on hair cells survival in adult mice.The first study to analyse the molecular mechanism of FoxG1 regulation network in hair cells development and survival.

Characterization of Lgr6+ Cells as an Enriched Population of Hair Cell Progenitors Compared to Lgr5+ Cells for Hair Cell Generation in the Neonatal Mouse Cochlea

Hair cell (HC) loss is irreversible because only very limited HC regeneration has been observed in the adult mammalian cochlea. Wnt/β-catenin signaling regulates prosensory cell proliferation and differentiation during cochlear development, and Wnt activation promotes the proliferation of Lgr5+ cochlear HC progenitors in newborn mice. Similar to Lgr5, Lgr6 is also a Wnt downstream target gene. Lgr6 is reported to be present in adult stem cells in the skin, nail, tongue, lung, and mammary gland, and this protein is very important for adult stem cell maintenance in rapidly proliferating organs. Our previous studies showed that Lgr6+ cells are a subpopulation of Lgr5+ progenitor cells and that both Lgr6+ and Lgr5+ progenitors can generate Myosin7a+ HCs in vitro. Thus we hypothesized that Lgr6+ cells are an enriched population of cochlear progenitor cells. However, the detailed distinctions between the Lgr5+ and Lgr6+ progenitors are unclear. Here, we systematically compared the proliferation, HC differentiation, and detailed transcriptome expression profiles of these two progenitor populations. We found that the same number of isolated Lgr6+ progenitors generated significantly more Myosin7a+ HCs compared to Lgr5+ progenitors; however, Lgr5+ progenitors formed more epithelial colonies and more spheres than Lgr6+ progenitors in vitro. Using RNA-Seq, we compared the transcriptome differences between Lgr5+ and Lgr6+ progenitors and identified a list of significantly differential expressed genes that might regulate the proliferation and differentiation of these HC progenitors, including 4 cell cycle genes, 9 cell signaling pathway genes, and 54 transcription factors. In conclusion, we demonstrate that Lgr6+ progenitors are an enriched population of inner ear progenitors that generate more HCs compared to Lgr5+ progenitors in the newborn mouse cochlea, and the our research provides a series of genes that might regulate the proliferation of progenitors and HC generation.

Loss of ARHGEF6 Causes Hair Cell Stereocilia Deficits and Hearing Loss in Mice

ARHGEF6 belongs to the family of guanine nucleotide exchange factors (GEFs) for Rho GTPases, and it specifically activates Rho GTPases CDC42 and RAC1. Arhgef6 is the X-linked intellectual disability gene also known as XLID46, and clinical features of patients carrying Arhgef6 mutations include intellectual disability and, in some cases, sensorineural hearing loss. Rho GTPases act as molecular switches in many cellular processes. Their activities are regulated by binding or hydrolysis of GTP, which is facilitated by GEFs and GTPase-activating proteins, respectively. RAC1 and CDC42 have been shown to play important roles in hair cell (HC) stereocilia development. However, the role of ARHGEF6 in inner ear development and hearing function has not yet been investigated. Here, we found that ARHGEF6 is expressed in mouse cochlear HCs, including the HC stereocilia. We established Arhgef6 knockdown mice using the clustered regularly interspaced short palindromic repeat-associated Cas9 nuclease (CRISPR-Cas9) genome editing technique. We showed that ARHGEF6 was indispensable for the maintenance of outer hair cell (OHC) stereocilia, and loss of ARHGEF6 in mice caused HC stereocilia deficits that eventually led to progressive HC loss and hearing loss. However, the loss of ARHGEF6 did not affect the synapse density and did not affect the mechanoelectrical transduction currents in OHCs at postnatal day 3. At the molecular level, the levels of active CDC42 and RAC1 were dramatically decreased in the Arhgef6 knockdown mice, suggesting that ARHGEF6 regulates stereocilia maintenance through RAC1/CDC42.

Expression and prognostic value of E-cadherin in laryngeal cancer.

Abstract Conclusion: Down-regulation of ECD may associate with infiltrative tumor growth and result in poor prognosis. However, ECD expression is not significantly associated with tumor metastasis. Aim: To investigate the prognostic role of E-cadherin (ECD) expression in laryngeal cancer. Methods: In a clinical study, a total of 79 laryngeal cancer patients who received surgery were included in this study. The expression of ECD in laryngeal cancer tissue was detected by immumohistochemical staining. In an in vitro study, ECD up-regulated cells (Ad-ECD) and ECD down-regulated cells (sh-ECD) were cultured, along with interference by siRNA. Cell apoptosis was detected by flow cytometry. In an in vivo study, nude mice were injected with Ad-ECD, sh-ECD, and Hep-2 cells. The ECD expression and tumor metastasis in the brain, liver, and lung tissues were detected. Results: Clinical data showed that expression of ECD was statistically correlated with survival rate (p < 0.05), but not with other clinical characteristics, such as age, gender, lymphatic metastasis, and TNM staging. ECD was down-regulated in sh-ECD cells and the down-regulation significantly reduced apoptosis of Hep-2 cells. Tumor growth was significantly inhibited in the Ad-ECD group. Except the tumor tissue, no positive ECD expression or significant tumor cells were found in the brain, liver, and lung tissues.