ABSTRACT
OBJECTIVE: Age-related hearing loss (AHL), characterized by degeneration of cochlea structures, is the most common sensory disorder among the elderly worldwide. The calcium channel is considered to contribute to normal hearing. However, the role of the T-type voltage-activated calcium channel, Cav3.1, remains unclear in AHL. Here, we investigate the age-related change of Cav3.1 expression in the cochlea and D-gal-induced senescent HEI-OC1 cells. METHODS: Cochleae from C57BL/6 mice at 2 months and 12 months of age were assessed. Senescence in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells was induced by D-gal treatment. The immunofluorescence technique was employed to investigate the distribution of Cav3.1 in vivo and in vitro. Quantitative assessment was achieved by Western blotting and real-time PCR. RESULTS: In comparison with 2-month-old animals, 12-month old C57BL/6 mice exhibited great loss of hair cells and elevated auditory brainstem threshold. The Cav3.1 was located in hair cells, spiral ganglion cells, lateral walls, and the expression of Cav3.1 protein and mRNA decreased in the aged cochleae. D-gal-induced senescence assay confirmed the down-regulation of Cav3.1 expression in senescent HEI-OC1 cells. CONCLUSION: Our results show that age-related down-regulated expression of Cav3.1 in the cochleae is associated with AHL and may contribute to the pathogenesis of AHL.
Subject(s)
Calcium Channels, T-Type/genetics , Cochlea/metabolism , Presbycusis/genetics , Animals , Cochlea/diagnostic imaging , Cochlea/pathology , Disease Models, Animal , Gene Expression Regulation/genetics , Hair Cells, Auditory/metabolism , Hair Cells, Auditory/pathology , Humans , Mice , Organ of Corti/diagnostic imaging , Organ of Corti/metabolism , Organ of Corti/pathology , Presbycusis/pathology , Spiral Ganglion/diagnostic imaging , Spiral Ganglion/metabolism , Spiral Ganglion/pathologyABSTRACT
To interrogate endogenous p21(WAF1/CIP1) (p21) promoter activity under basal conditions and in response to various forms of stress, knock-in imaging reporter mice in which expression of firefly luciferase (FLuc) was placed under the control of the endogenous p21 promoter within the Cdkn1a gene locus were generated. Bioluminescence imaging (BLI) of p21 promoter activity was performed noninvasively and repetitively in mice and in cells derived from these mice. We demonstrated that expression of FLuc accurately reported endogenous p21 expression at baseline and under conditions of genotoxic stress and that photon flux correlated with mRNA abundance and, therefore, bioluminescence provided a direct readout of p21 promoter activity in vivo. BLI confirmed that p53 was required for activation of the p21 promoter in vivo in response to ionizing radiation. Interestingly, imaging of reporter cells demonstrated that p53 prevents the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway from activating p21 expression when quiescent cells are stimulated with serum to reenter the cell cycle. In addition, low-light BLI identified p21 expression in specific regions of individual organs that had not been observed previously. This inducible p21(FLuc) knock-in reporter strain will facilitate imaging studies of p53-dependent and -independent stress responses within the physiological context of the whole animal.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/genetics , Luciferases, Firefly/genetics , Promoter Regions, Genetic , Tumor Suppressor Protein p53/metabolism , Animals , Blotting, Western , Cell Cycle , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation , Gene Knock-In Techniques , Hepatocytes/metabolism , Luciferases, Firefly/biosynthesis , Mice , Mice, Inbred C57BL , Polymerase Chain Reaction , RNA Interference , RNA, Messenger , RNA, Small Interfering , Radiation, Ionizing , Transcriptional ActivationABSTRACT
The CDC25 protein phosphatases drive cell cycle advancement by activating cyclin-dependent protein kinases (CDKs). Humans and mice encode three family members denoted CDC25A, -B and -C and genes encoding these family members can be disrupted individually with minimal phenotypic consequences in adult mice. However, adult mice globally deleted for all three phosphatases die within one week after Cdc25 disruption. A severe loss of absorptive villi due to a failure of crypt epithelial cells to proliferate was observed in the small intestines of these mice. Because the Cdc25s were globally deleted, the small intestinal phenotype and loss of animal viability could not be solely attributed to an intrinsic defect in the inability of small intestinal stem and progenitor cells to divide. Here, we report the consequences of deleting different combinations of Cdc25s specifically in intestinal epithelial cells. The phenotypes arising in these mice were then compared with those arising in mice globally deleted for the Cdc25s and in mice treated with irinotecan, a chemotherapeutic agent commonly used to treat colorectal cancer. We report that the phenotypes arising in mice globally deleted for the Cdc25s are due to the failure of small intestinal stem and progenitor cells to proliferate and that blocking cell division by inhibiting the cell cycle engine (through Cdc25 loss) versus by inducing DNA damage (via irinotecan) provokes a markedly different response of small intestinal epithelial cells. Finally, we demonstrate that CDC25A and CDC25B but not CDC25C compensate for each other to maintain the proliferative capacity of intestinal epithelial stem and progenitor cells.
Subject(s)
Cell Proliferation , Epithelial Cells/cytology , Intestine, Small/cytology , Stem Cells/cytology , cdc25 Phosphatases/physiology , Animals , Gene Deletion , Mice , Mice, Knockout , cdc25 Phosphatases/geneticsABSTRACT
Bioluminescence, positron emission tomography (PET), and fluorescence modalities are currently available for noninvasive imaging in vivo, each with its own merits. To exploit the combined strengths of each and facilitate multimodality imaging, we engineered a dual-reporter construct in which firefly luciferase (FLuc) and a 12-amino acid nonstructural linker were fused in frame to the N-terminus of a mutant herpes simplex virus thymidine kinase (mNLS-SR39TK) kinetically enhanced for positron emission tomography (PET). Furthermore, a triple-reporter construct was developed in which monster green fluorescent protein (MGFP), a recently available enhanced fluorescent protein, was introduced into the fusion vector downstream of an internal ribosome entry site (IRES) to allow analysis by fluorescence microscopy or flow cytometry without compromising the specific activities of the upstream fusion components. FLuc bioluminescence was measured with a cooled charge-coupled device camera and mNLS-SR39TK activity by 9-[4-[(18)F]fluoro-3-(hydroxymethyl) butyl guanine ((18)F-FHBG) microPET or (3)H-penciclovir net accumulation. Importantly, HeLa cells transiently transfected with the FLuc-mNLS-SR39TK-IRES-MGFP triple reporter retained the same specific activities of the FLuc-mNLS-SR39TK heteroenzyme and the individual unfused enzymes with no change in protein half-lives. The presence of the IRES-MGFP modestly decreased upstream heteroprotein expression. In living mice, somatic gene transfer of a ubiquitin promoter-driven FLuc-mNLS-SR39TK-IRES-MGFP plasmid showed a > 1,000-fold increase in liver photon flux and a > 2-fold increase in liver retention of (18)F-FHBG by microPET compared with mice treated with control plasmid. Multifocal hepatocellular fluorescence was readily observed by standard confocal microscopy. This second-generation triple reporter incorporating enhanced components enables bioluminescence, PET, and fluorescence imaging of cells and living animals.
