Yuki Hamajima

Id1 induces the proliferation of cochlear sensory epithelial cells via the nuclear factor‐κB/cyclin D1 pathway in vitro

Inhibitors of differentiation (Id) play an essential role in the neurogenesis of the central nervous system. However, the expression and function of Id in the development of cochlear sensory epithelial cells have yet to be elucidated. In this study, we demonstrate the Id1 gene was expressed in the rapidly growing otocyst on embryonic day 12 (E12) and in the organ of Corti, spiral ganglions, and stria vascularis on postnatal day 1 (P1) by cellular and molecular biologic techniques. Knockdown of the Id1 gene with short interfering RNA (siRNA) in a cochlear sensory epithelial cell line (OC1) significantly reduced its proliferation, whereas overexpression of Id1 in OC1 significantly increased the proliferation of OC1, suggesting a role of Id1 in the development of cochlear sensory epithelial cells. The proliferative action of Id1 on OC1 was mediated by nuclear factor‐κB (NF‐κB) and cyclin D1 (a downstream molecule of NF‐κB). Blockage of the NF‐κB activity with pyrrolidine dithiocarbamate (PDTC) or enhancement of the NF‐κB activity with p65 (a subunit of NF‐κB) in OC1 significantly inhibited or increased, respectively, the cell proliferation and transcription of cyclin D1 induced by Id1. Truncation of the NF‐κB binding site in the cyclin D1 promoter fully abrogated the transcription of cyclin D1, suggesting that the cyclin D1 transcription is dependent on NF‐κB. We concluded from this study that Id1 induces the proliferation of OC1 via the NF‐κB/cyclin D1 pathway.

Impaired olfactory function in mice with allergic rhinitis

OBJECTIVE It has been reported that olfactory function is impaired in patients with allergic rhinitis. However, the mechanism of olfactory dysfunction in allergic rhinitis remains poorly understood. Because of difficulties in obtaining and analyzing human olfactory mucosa due to both technical and ethical issues, an animal model needs to be established to clarify the mechanism of olfactory dysfunction in allergic rhinitis. The purpose of this study was to study olfactory function and changes in olfactory mucosa using allergic rhinitis mice. METHODS A model of allergic rhinitis mice with olfactory dysfunction was developed by sensitizing with ovalbumin (OVA), and intranasally challenging with the same allergen. Olfactory function of mice with or without allergic rhinitis was assessed by odor detection ability test with cycloheximide and local field potential (LFP) with 1-octanal. We also evaluated histological changes in the olfactory mucosa of allergic rhinitis mice by both light and electron microscopy. RESULTS Both of odor detection ability test and LFP showed that olfactory function was impaired in mice with allergic rhinitis, but not in mice without allergic rhinitis. Histopathological findings showed prominent infiltration of eosinophils, plasma cells, neutrophils, mast cells, and macrophages in lamina propria of olfactory mucosa of mice with allergic rhinitis, although infiltration of these cells was not seen in control mice. Allergic rhinitis also increased the number and size of glands in olfactory mucosa, suggesting an elevated amount of mucin in olfactory mucosa. CONCLUSION This study showed for the first time that mice with allergic rhinitis have impaired olfactory function, increased size and number of olfactory glands, and infiltration of eosinophils, neutrophils, mast cells, plasma cells, and macrophages in the olfactory mucosa. This suggests that allergic reactions are seen in olfactory mucosa of mice with allergic rhinitis, and that greater olfactory gland activity is associated with olfactory dysfunction. Also, this mouse model could provide an expedient system for analyzing mechanisms of olfactory dysfunction.

Sonic hedgehog (SHH) promotes the differentiation of mouse cochlear neural progenitors via the Math1-Brn3.1 signaling pathway in vitro

Sonic hedgehog (SHH) is essential for the development of the cochlear duct that harbors the organ of Corti. However, little is known about the molecular signaling pathway through which SHH promotes the development of the organ of Corti, especially cochlear sensory epithelial cells. In this study, we demonstrated that SHH contributes to the differentiation of cochlear neural progenitors (CNPs), which are derived from the postnatal day 1 organ of Corti in mice. Addition of SHH to CNPs increased the formation of epithelial cell islands, simultaneously activated the expression of Math1 that is a transcription factor for the initial differentiation of auditory hair cells. The increased expression of Math1 then regulated the promoter activity of Brn3.1, another transcription factor that controls the further differentiation and survival of auditory hair cells. Taken together, our data suggest that SHH plays an important role in the promotion of auditory hair cell differentiation via the Math1‐Brn3.1 signaling pathway.

Cochlear Stem Cells/Progenitors and Degenerative Hearing Disorders

Hearing loss (deafness) affects approximately 250 million people globally. The major cause of deafness is loss of hair cells and spiral ganglion neurons due to aging, antibiotic use, noise exposure, and genetic defects. At the present time, there is no effective method for restoration of hearing biologically. Cochlear stem cells/progenitors (CSCs), quiescent in the organ of Corti, are excellent candidates for restoration of cell types in the organ of Corti biologically. However, little is known about the biology of CSCs and developmental cues for CSCs to differentiate into hair cells and neurons at the present time. In this article, we briefly reviewed the isolation of CSCs from the postnatal organ of Corti in mice and their capability to differentiate into hair cells and neurons in vitro under the guidance of a group of growth factors: sonic hedgehog (SHH), epidermal growth factor (EGF), retinoic acid (RA), and brain-derived neurotrophic factor (BDNF), herein termed SERB. The identification of CSCs and their differentiation signals is potentially of clinical importance.

The role of inhibitor of DNA-binding (Id1) in hyperproliferation of keratinocytes: the pathological basis for middle ear cholesteatoma from chronic otitis media.

Objectives:  A hallmark of cholesteatoma is hyperproliferation of keratinocytes with abundant production of keratins in the middle ear under chronic inflammatory conditions. However, little is known about the driving force of cellular proliferation and keratin production of cholesteatomal matrix. The purpose of this study was to investigate the cellular proliferation and keratin production of keratinocytes under the influence of Id1, a candidate transcription factor to cell proliferation.

Expression of the integrin genes in the developing cochlea of rats.

Integrins play an important role in the development of the cochlea. However, little is known about the expression pattern of integrins in the developing cochlear tissue. In this study, we investigated the dynamic expression profile of the integrin genes in the developing cochlear tissue of rats by Affymetrix microarrays and explored the role of the integrin genes in vitro by using antisense oligonucleotides. It was demonstrated that the alpha1, alpha7, alphav, beta3, and beta4 genes were expressed in the developing cochlear tissue of rats. Inhibition of the integrin expression with antisense oligonucleotides against alphav, alpha7, beta3, and beta4, respectively, in cochlear sensorineural epithelial cells significantly decreased the [3H]thymidine incorporation, suggesting that these integrins are involved in cell growth and proliferation. Inhibition of the alphav and beta4 integrins significantly decreased the transcription of nuclear factor-kappa B (NF-kappaB, a signal molecule involved in cell growth and proliferation) induced by epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), respectively. It suggests that EGF-induced cell growth is dependent upon the alphav integrin whereas bFGF-induced cell growth is dependent upon the beta4 integrin in the cochlear tissue during the development of the inner ear.

Establishment and characterization of rat progenitor hair cell lines.

Cochlear progenitor hair cell lines are useful for studies of cellular specification, gene expression features, and signal transduction involved in the development of hair cells. To obtain embryonic and postnatal cochlear progenitor hair cell lines, we immortalized primary cultures of sensorineural epithelial cells from otocysts on embryonic day 12 (E12) and explants of the organ of Corti tissues on postnatal day 5 (P5). Primary cultures and explants were then transduced by the E6/E7 genes of human papilloma virus type 16. Transduced cells were passed for >50 passages and partial clonal cells were isolated from the above P5 organ of Corti explants by limiting dilution. The expression of neuronal, neural, epithelial, hair cell markers, and important transcription factors were then examined in these cell clones. Clones that express the above markers were considered as being progenitor hair cells. At least two representative cell lines, one from a mixed culture of otocyst epithelial cells and the other from the organ of Corti cells, ultimately expressed hair cell markers and neuronal/neural cell markers. The former only expressed the early hair cell marker oncomodulin and myosin VIIa, whereas the latter expressed oncomodulin, calretinin, myosin VIIa and Brn 3.1. These cell lines may represent progenitor hair cells at the different stages of cochlear development.

Optimal duration of macrolide treatment for chronic sinusitis after endoscopic sinus surgery

OBJECTIVE The objective is to determine the appropriate duration of postoperative macrolide therapy for chronic rhinosinusitis to obtain a favourable outcome with endoscopic sinus surgery (ESS). METHODS The effectiveness of postoperative macrolide treatment was examined in patients with chronic rhinosinusitis who underwent ESS, by comparing 3-month (44 patients) and 6-month administration (66 patients) of clarithromycin (CAM) (200mg/day). Evaluation was made based on subjective symptoms and endoscopic findings at 3, 6 and 12 months after surgery. RESULTS Seventeen (3-month CAM group) and 22 (6-month CAM group) subjects were able to be followed up to 12 months after surgery. No difference in effectiveness was observed between the groups until 6 months after surgery, but the 6-month treatment group showed significantly higher disappearance rates and significantly lower visual analogue scale (VAS) scores in the subjective symptoms of rhinorrhea and postnasal drip at 12 months after surgery. The positive finding rate of postnasal drip by endoscopic examination was also significantly lower in the 6-month treatment group at 12 months after surgery. These changes over time indicated gradual deterioration after discontinuation of CAM treatment in the 3-month treatment group, whereas a small improvement was observed after discontinuation in the 6-month treatment group. CONCLUSION The results indicate that chronic sinusitis patients with rhinorrhea or postnasal drip should be treated with macrolides for 6 months after surgery in order to improve the long-term outcome of endoscopic sinus surgery.

Regulation of the angiogenesis of acquired middle ear cholesteatomas by inhibitor of DNA binding transcription factor.

IMPORTANCE The aggressive growth of cholesteatoma in the middle ear involves the angiogenesis of the cholesteatomal perimatrix. However, which transcription factor is involved in this process remains unclear. OBJECTIVE To identify a transcription factor that supports the aggressive growth of cholesteatoma by controlling the angiogenesis of cholesteatoma in the middle ear milieu. DESIGN We used clinical specimens for the profiling of angiogenic factors and their regulatory transcription factors in cholesteatoma. Human skin keratinocytes and endothelial cells were used for evaluation of the effects of candidate transcription factor on the angiogenic factor regulation and endothelial cell proliferation. SETTING University departments of otolaryngology-head and neck surgery. PARTICIPANTS Eight clinical cholesteatomal and 8 control specimens were used for cellular and molecular biologic evaluation. An additional 8 cholesteatomal and 8 aural skin specimens were used for microarray studies. MAIN OUTCOME MEASURES The expression of vascular endothelial growth factor, interleukin 8, and cyclooxygenase 2 as measured by means of immunohistochemistry and molecular biologic methods. RESULTS Human aural cholesteatomal specimens were rich in the expression of angiogenic factors such as vascular endothelial growth factor in the cholesteatomal matrix and perimatrix, accompanied by the transcription factor inhibitor of DNA binding (Id1). We found Id1 to be an essential regulator of vascular endothelial growth factor. In addition, potent angiogenic factors, including interleukin 8 and cyclooxygenase 2, were regulated by Id1 via different molecular mechanisms. CONCLUSIONS AND RELEVANCE The transcription factor Id1 controls the angiogenesis of cholesteatoma through the regulation of vascular endothelial growth factor, interleukin 8, and cyclooxygenase 2, which are responsible for the angiogenesis of cholesteatoma. Id1 may serve as a good target for the treatment of cholesteatomal progression in the middle ear milieu.

Identification of Id1 in Acquired Middle Ear Cholesteatoma

OBJECTIVES To determine (1) the relationship between chronic inflammatory changes in the ossicular chain area (OCA) and the formation of cholesteatoma and (2) the correlates between aberrant gene expression and abnormal proliferation of cholesteatoma. METHODS Two hundred sixty-four ears with chronic otitis media that had undergone ear surgery were included in this study for statistical analysis of the relationship between abnormalities in the OCA and cholesteatoma. Fourteen middle ear cholesteatoma specimens were collected for immunohistochemical analysis of candidate molecules involved in the abnormal proliferation of keratinocytes. A cell model was used for verification of candidate molecule involvement. RESULTS The formation of cholesteatoma was accompanied by chronic inflammatory changes in the OCA, including granulated tissue, adhesion, and stagnating effusion. The inhibitor of the DNA-binding (Id1) gene, which is involved in controlling cell cycle progression, was abundantly expressed in cholesteatoma epithelium. In vitro studies indicate that Id1 regulated the expression of nuclear factor kappaB, cyclin D1, proliferating cell nuclear antigen, and cell cycle progression of keratinocytes, CONCLUSIONS Chronic inflammation in the OCA is closely related to the formation of cholesteatoma. The Id1/nuclear factor kappaB/cyclin D1/proliferating cell nuclear antigen signaling pathway is involved in the abnormal proliferation of keratinocytes in acquired cholesteatoma.