Genomic analysis of the function of the transcription factor gata3 during development of the Mammalian inner ear

We have studied the function of the zinc finger transcription factor gata3 in auditory system development by analysing temporal profiles of gene expression during differentiation of conditionally immortal cell lines derived to model specific auditory cell types and developmental stages. We tested and applied a novel probabilistic method called the gamma Model for Oligonucleotide Signals to analyse hybridization signals from Affymetrix oligonucleotide arrays. Expression levels estimated by this method correlated closely (p<0.0001) across a 10-fold range with those measured by quantitative RT-PCR for a sample of 61 different genes. In an unbiased list of 26 genes whose temporal profiles clustered most closely with that of gata3 in all cell lines, 10 were linked to Insulin-like Growth Factor signalling, including the serine/threonine kinase Akt/PKB. Knock-down of gata3 in vitro was associated with a decrease in expression of genes linked to IGF-signalling, including IGF1, IGF2 and several IGF-binding proteins. It also led to a small decrease in protein levels of the serine-threonine kinase Akt2/PKB beta, a dramatic increase in Akt1/PKB alpha protein and relocation of Akt1/PKB alpha from the nucleus to the cytoplasm. The cyclin-dependent kinase inhibitor p27(kip1), a known target of PKB/Akt, simultaneously decreased. In heterozygous gata3 null mice the expression of gata3 correlated with high levels of activated Akt/PKB. This functional relationship could explain the diverse function of gata3 during development, the hearing loss associated with gata3 heterozygous null mice and the broader symptoms of human patients with Hearing-Deafness-Renal anomaly syndrome

CLIMP-63 is a gentamicin-binding protein that is involved in drug-induced cytotoxicity

Aminoglycoside-induced nephrotoxicity and ototoxicity is a major clinical problem. To understand how aminoglycosides, including gentamicin, induce cytotoxicity in the kidney proximal tubule and the inner ear, we identified gentamicin-binding proteins (GBPs) from mouse kidney cells by pulling down GBPs with gentamicin–agarose conjugates and mass spectrometric analysis. Among several GBPs specific to kidney proximal tubule cells, cytoskeleton-linking membrane protein of 63 kDa (CLIMP-63) was the only protein localized in the endoplasmic reticulum, and was co-localized with gentamicin-Texas Red (GTTR) conjugate after cells were treated with GTTR for 1 h. In western blots, kidney proximal tubule cells and cochlear cells, but not kidney distal tubule cells, exhibited a dithiothreitol (DTT)-resistant dimer band of CLIMP-63. Gentamicin treatment increased the presence of DTT-resistant CLIMP-63 dimers in both kidney proximal (KPT11) and distal (KDT3) tubule cells. Transfection of wild-type and mutant CLIMP-63 into 293T cells showed that the gentamicin-dependent dimerization requires CLIMP-63 palmitoylation. CLIMP-63 siRNA transfection enhanced cellular resistance to gentamicin-induced toxicity, which involves apoptosis, in KPT11 cells. Thus, the dimerization of CLIMP-63 is likely an early step in aminoglycoside-induced cytotoxicity in the kidney and cochlea. Gentamicin also enhanced the binding between CLIMP-63 and 14-3-3 proteins, and we also identified that 14-3-3 proteins are involved in gentamicin-induced cytotoxicity, likely by binding to CLIMP-63

CLIMP-63 is a gentamicin-binding protein that is involved in drug-induced cytotoxicity

Aminoglycoside-induced nephrotoxicity and ototoxicity is a major clinical problem. To understand how aminoglycosides, including gentamicin, induce cytotoxicity in the kidney proximal tubule and the inner ear, we identified gentamicin-binding proteins (GBPs) from mouse kidney cells by pulling down GBPs with gentamicin–agarose conjugates and mass spectrometric analysis. Among several GBPs specific to kidney proximal tubule cells, cytoskeleton-linking membrane protein of 63 kDa (CLIMP-63) was the only protein localized in the endoplasmic reticulum, and was co-localized with gentamicin-Texas Red (GTTR) conjugate after cells were treated with GTTR for 1 h. In western blots, kidney proximal tubule cells and cochlear cells, but not kidney distal tubule cells, exhibited a dithiothreitol (DTT)-resistant dimer band of CLIMP-63. Gentamicin treatment increased the presence of DTT-resistant CLIMP-63 dimers in both kidney proximal (KPT11) and distal (KDT3) tubule cells. Transfection of wild-type and mutant CLIMP-63 into 293T cells showed that the gentamicin-dependent dimerization requires CLIMP-63 palmitoylation. CLIMP-63 siRNA transfection enhanced cellular resistance to gentamicin-induced toxicity, which involves apoptosis, in KPT11 cells. Thus, the dimerization of CLIMP-63 is likely an early step in aminoglycoside-induced cytotoxicity in the kidney and cochlea. Gentamicin also enhanced the binding between CLIMP-63 and 14-3-3 proteins, and we also identified that 14-3-3 proteins are involved in gentamicin-induced cytotoxicity, likely by binding to CLIMP-63

Gene Expression Analysis of Forskolin Treated Basilar Papillae Identifies MicroRNA181a as a Mediator of Proliferation

Auditory hair cells spontaneously regenerate following injury in birds but not mammals. A better understanding of the molecular events underlying hair cell regeneration in birds may allow for identification and eventually manipulation of relevant pathways in mammals to stimulate regeneration and restore hearing in deaf patients.Gene expression was profiled in forskolin treated (i.e., proliferating) and quiescent control auditory epithelia of post-hatch chicks using an Affymetrix whole-genome chicken array after 24 (n = 6), 48 (n = 6), and 72 (n = 12) hours in culture. In the forskolin-treated epithelia there was significant (p<0.05; >two-fold change) upregulation of many genes thought to be relevant to cell cycle control and inner ear development. Gene set enrichment analysis was performed on the data and identified myriad microRNAs that are likely to be upregulated in the regenerating tissue, including microRNA181a (miR181a), which is known to mediate proliferation in other systems. Functional experiments showed that miR181a overexpression is sufficient to stimulate proliferation within the basilar papilla, as assayed by BrdU incorporation. Further, some of the newly produced cells express the early hair cell marker myosin VI, suggesting that miR181a transfection can result in the production of new hair cells.These studies have identified a single microRNA, miR181a, that can cause proliferation in the chicken auditory epithelium with production of new hair cells

Genome-wide association meta-analysis identifies 48 risk variants and highlights the role of the stria vascularis in hearing loss

Hearing loss is one of the top contributors to years lived with disability and is a risk factor for dementia. Molecular evidence on the cellular origins of hearing loss in humans is growing. Here, we performed a genome-wide association meta-analysis of clinically diagnosed and self-reported hearing impairment on 723,266 individuals and identified 48 significant loci, 10 of which are novel. A large proportion of associations comprised missense variants, half of which lie within known familial hearing loss loci. We used single-cell RNA-sequencing data from mouse cochlea and brain and mapped common-variant genomic results to spindle, root, and basal cells from the stria vascularis, a structure in the cochlea necessary for normal hearing. Our findings indicate the importance of the stria vascularis in the mechanism of hearing impairment, providing future paths for developing targets for therapeutic intervention in hearing loss