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.

Characterization of inhibitor of differentiation (Id3) gene expression in the developing cochlear tissue of rats.

CONCLUSION: The Id3 gene is expressed in the developing cochlear tissue and participates in the development of cochlear progenitor hair cells during the embryonic stage. OBJECTIVES: Inhibitor of differentiation (Id3) is an important transcription factor expressed in the cochlear tissue and progenitor hair cells. It is likely to be involved in cell-cycle progression and the proliferation of cochlear progenitor hair cells during the embryonic stage. The dynamic expression of Id3 in the developing cochlear tissue is, however, poorly understood. In this study we sought to characterize the expression of Id3 in the developing cochlear tissue and to explore its role in the growth and proliferation of progenitor hair cells. MATERIAL AND METHODS: Expression of the Id3 gene in the developing cochlear tissue was examined by means of Northern blotting, in situ hybridization and immunohistochemistry. The effects of Id3 on primary cultures of otocyst epithelial cells and a progenitor hair cell line (OT12) were studied using specific antisense oligonucleotides. RESULTS: The Id3 gene was expressed in the rapidly growing otocyst on embryonic Day 12 and specifically in the fundamental structures of the cochlea, e.g. the organ of Corti, spiral ganglions and stria vascularis, on postnatal Day 1. Inhibition of Id3 gene expression with antisense oligonucleotides in cultured otocyst epithelial cells and OT12 reduced DNA synthesis and cell-cycle progression, suggesting that Id3 participates in the proliferation of cochlear progenitor hair cells.

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.

Identification of gene expression profiles in rat ears with cDNA microarrays.

The physiological processes of hearing implicate thousands of molecules acting in harmony; however, their identities are only partially understood. We used cDNA microarrays containing 1,176 genes to identify >150 genes expressed in rat middle and inner ear tissue. Expressed genes covered several gene families and biological pathways, many of which have previously not been described. Transcription factor genes that were expressed included inhibitors of DNA binding protein (Id). These were localized to the spiral ganglion, organ of Corti and stria vascularis, and they are possibly involved in neurogenesis and angiogenesis. Transcriptional factors that were highly expressed included Gax (homeobox) and I-kappaB, which inhibit cellular proliferation. Their presence suggests that inhibitory programs for cell proliferation are enforced in the ear. Ion channel genes that were expressed included voltage-dependent L-type calcium channels (LTCC) and proton-gated cation channels (PGCC). Genes involved in neurotransmitter production and release included glutamic acid decarboxylase (GAD1). Genes involved in postsynaptic inhibition included neuropeptide Y5 receptors (NPY5) and GAD1. Due to the existence of receptors and/or enzymes involved in their biochemical synthesis, neurotransmitters associated with these might include serotonin, glutamide, acetylcholine, gamma-aminobutyric acid (GABA), neurotensin, and dopamine.

Expression of platelet-derived growth factor in the developing cochlea of rats.

OBJECTIVE: Platelet-derived growth factor (PDGF) is involved in the control of cell proliferation, differentiation and survival in various tissues of vertebrates. However, little is known about the expression of PDGF in the developing cochlea of rodents. MATERIAL AND METHODS: We examined the expression of PDGF family genes in the developing cochlear tissue of rats using microarrays and tested their role in the proliferation of progenitor hair cells using cellular and molecular biology techniques. RESULTS: It was found that the genes for PDGF-A, PDGF receptor (R)-alpha and PDGFR-beta were highly expressed in the rapidly growing otocyst on embryonic Days 12-14 and weakly expressed thereafter. Reverse transcriptase polymerase chain reaction demonstrated the expression of PDGF-C and confirmed the expression of PDGF-A and PDGFR-alpha and -beta in the developing cochlear tissue of rats and the cultured progenitor hair cells. Inhibition of the expression of PDGFs in the cultured progenitor hair cells with antisense oligonucleotides reduced the DNA synthesis. CONCLUSION: PDGFs and their receptors may play a role in the proliferation of developing cochlear hair cells.

Feline immunodeficiency virus-mediated gene therapy of middle ear mucosa cells.

HYPOTHESIS: To investigate the feasibility of gene therapy of the middle ear mucosa using a novel vector. BACKGROUND: Given present medications are unable to affect chronic otitis media, cholesteatoma, or tympanic membrane perforation, newer methods of treatment like gene therapy for these diseases must be explored. These genes can then be used to alter cytokines in the middle ear, slow or stop cholesteatoma growth, or improve tympanic membrane perforation healing. Feline immunodeficiency virus (FIV), a new lentiviral vector has been found to have greater than 90% efficiency in transfecting epithelial cells. Therefore, in vivo gene therapy of middle ear mucosa cells was attempted. METHODS: Twenty microliter of 5x10(5) vectors per ml FIV carrying the gene for green fluorescence protein (GFP) was introduced into the middle ears of Sprague-Dawley rats via a bulla approach. RESULTS: Expression of the GFP gene was observed in the middle ear mucosa cells at 1 week post-inoculation indicating transfection. CONCLUSION: Gene therapy of the middle ear is feasible with a FIV-based vector.

Id1 induces the proliferation of cochlear sensory epithelial cells via the nuclear factor-kappaB/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-kappaB (NF-kappaB) and cyclin D1 (a downstream molecule of NF-kappaB). Blockage of the NF-kappaB activity with pyrrolidine dithiocarbamate (PDTC) or enhancement of the NF-kappaB activity with p65 (a subunit of NF-kappaB) in OC1 significantly inhibited or increased, respectively, the cell proliferation and transcription of cyclin D1 induced by Id1. Truncation of the NF-kappaB binding site in the cyclin D1 promoter fully abrogated the transcription of cyclin D1, suggesting that the cyclin D1 transcription is dependent on NF-kappaB. We concluded from this study that Id1 induces the proliferation of OC1 via the NF-kappaB/cyclin D1 pathway.