Mary D'Souza

BMP-6 Is an Autocrine Stimulator of Chondrocyte Differentiation

While parathyroid hormone‐related protein (PTHrP) has been characterized as an important negative regulator of chondrocyte maturation in the growth plate, the autocrine or paracrine factors that stimulate chondrocyte maturation are not well characterized. Cephalic sternal chondrocytes were isolated from 13‐day embryos, and the role of bone morphogenetic protein‐6 (BMP‐6) as a positive regulator of chondrocyte maturation was examined in monolayer cultures. Progressive maturation, which was accelerated in the presence of ascorbate, occurred in the cultures. During maturation, the cultures expressed high levels of BMP‐6 mRNA which preceded the induction of type X collagen mRNA. Treatment of the cultures with PTHrP (10−7 M) at the time of plating completely abolished BMP‐6 and type X collagen mRNA expression. Removal of PTHrP after 6 days was followed by the rapid (within 24 h) expression of BMP‐6 and type X collagen mRNA, with BMP‐6 again preceding type X collagen expression. The addition of exogenous BMP‐6 (100 ng/ml) to the cultures accelerated the maturation process both in the presence and absence of ascorbate and resulted in the highest levels of type X collagen. When exogenous BMP‐6 was added to PTHrP containing cultures, maturation occurred with the expression of high levels of type X collagen, despite the presence of PTHrP in the cultures. Furthermore, BMP‐6 did not stimulate expression of its own mRNA in the PTHrP treated cultures, but it did stimulate the expression of Indian hedgehog (Ihh) mRNA. These latter findings suggest that while PTHrP directly inhibits BMP‐6, it indirectly regulates Ihh expression through BMP‐6. Other phenotypic changes associated with chondrocyte differentiation were also stimulated by BMP‐6, including increased alkaline phosphatase activity and decreased proliferation. The results suggest that BMP‐6 is an autocrine factor that initiates chondrocyte maturation and that PTHrP may prevent maturation by inhibiting the expression of BMP‐6.

BMP signaling stimulates chondrocyte maturation and the expression of Indian hedgehog

Mutant BMP receptors were transfected into cultured embryonic upper sternal chrondrocytes using retroviral vectors to determine if BMP signaling is required for chondrocyte maturation and the expression of a key regulatory molecule, Indian hedgehog (Ihh). Chondrocytes infected with replication competent avian retroviruses (RCAS) viruses carrying constitutive active (CA) BMPR‐IA and BMPR‐IB had enhanced expression of type X collagen and Ihh mRNA. Addition of PTHrP, a known inhibitor of chondrocyte maturation, abolished the expression of type X collagen, BMP‐6, and Ihh mRNAs in control cells. In contrast, PTHrP treated cultures infected with of CA BMPR‐IA or CA BMPR‐IB had low levels of BMP‐6 and type X collagen, but high levels of Ihh expression. Although dominant negative (DN) BMPR‐IA had no effect, DN BMPR‐IB inhibited the expression of type X collagen and BMP‐6, and decreased alkaline phosphatase activity, even in the presence of exogenously added BMP‐2 and BMP‐6. DN BMPR‐IB also completely blocked Ihh expression. Overall, the effect of DN BMPR‐IB mimicked the effects of PTHrP. To determine if there is an autocrine role for the BMPs in chondrocyte maturation, the cultures were treated with noggin and follistatin, molecules that bind BMP‐2/‐4 and BMP‐6/‐7, respectively. While noggin and follistatin inhibited the effects of recombinant BMP‐2 and BMP‐6, respectively, they had only minimal effects on the spontaneous maturation of chondrocytes in culture, suggesting that more than one subgroup of BMPs regulates chondrocyte maturation. The results demonstrate that: (i) BMP signaling stimulates chondrocyte maturation; (ii) BMP signaling increases Ihh expression independent of maturational effects; and (iii) BMP signaling can partially overcome the inhibitory effects of PTHrP on maturation.

Glutamate-related gene expression changes with age in the mouse auditory midbrain.

Glutamate is the main excitatory neurotransmitter in both the peripheral and central auditory systems. Changes of glutamate and glutamate-related genes with age may be an important factor in the pathogenesis of age-related hearing loss-presbycusis. In this study, changes in glutamate-related mRNA gene expression in the CBA mouse inferior colliculus with age and hearing loss were examined and correlations were sought between these changes and functional hearing measures, such as the auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAEs). Gene expression of 68 glutamate-related genes was investigated using both genechip microarray and real-time PCR (qPCR) molecular techniques for four different age/hearing loss CBA mouse subject groups. Two genes showed consistent differences between groups for both the genechip and qPCR. Pyrroline-5-carboxylate synthetase enzyme (Pycs) showed down-regulation with age and a high-affinity glutamate transporter (Slc1a3) showed up-regulation with age and hearing loss. Since Pycs plays a role in converting glutamate to proline, its deficiency in old age may lead to both glutamate increases and proline deficiencies in the auditory midbrain, playing a role in the subsequent inducement of glutamate toxicity and loss of proline neuroprotective effects. The up-regulation of Slc1a3 gene expression may reflect a cellular compensatory mechanism to protect against age-related glutamate or calcium excitoxicity.

Serotonin 2B receptor: Upregulated with age and hearing loss in mouse auditory system

Serotonin (5-HT) is a monoamine neurotransmitter. Serotonin may modulate afferent fiber discharges in the cochlea, inferior colliculus (IC) and auditory cortex. Specific functions of serotonin are exerted upon its interaction with specific receptors; one of those receptors is the serotonin 2B receptor. The aim of this study was to investigate the differences in gene expression of serotonin 2B receptors with age in cochlea and IC, and the possible correlation between gene expression and functional hearing measurements in CBA/CaJ mice. Immunohistochemical examinations of protein expression of IC in mice of different age groups were also performed. Gene expression results showed that serotonin 2B receptor gene was upregulated with age in both cochlea and IC. A significant correlation between gene expression and functional hearing results was established. Immunohistochemical protein expression studies of IC showed more serotonin 2B receptor cells in old mice relative to young adult mice, particularly in the external nucleus. We conclude that serotonin 2B receptors may play a role in the pathogenesis of age-related hearing loss.

Early G1 cyclin-dependent kinases as prognostic markers and potential therapeutic targets in esophageal adenocarcinoma

Purpose: Chromosomal gain at 7q21 is a frequent event in esophageal adenocarcinoma (EAC). However, this event has not been mapped with fine resolution in a large EAC cohort, and its association with clinical endpoints and functional relevance are unclear. Experimental Design: We used a cohort of 116 patients to fine map the 7q21 amplification using SNP microarrays. Prognostic significance and functional role of 7q21 amplification and its gene expression were explored. Results: Amplification of the 7q21 region was observed in 35% of tumors with a focal, minimal amplicon containing six genes. 7q21 amplification was associated with poor survival and analysis of gene expression identified cyclin-dependent kinase 6 (CDK6) as the only gene in the minimal amplicon whose expression was also associated with poor survival. A low-level amplification (10%) was observed at the 12q13 region containing the CDK6 homologue cyclin-dependent kinase 4 (CDK4). Both amplification and expression of CDK4 correlated with poor survival. A combined model of both CDK6 and CDK4 expressions is a superior predictor of survival than either alone. Specific knockdown of CDK4 and/or CDK6 by siRNAs shows that they are required for proliferation of EAC cells and that their function is additive. PD-0332991 targets the kinase activity of both molecules and suppresses proliferation and anchorage independence of EAC cells through activation of the pRB pathway. Conclusions: We suggest that CDK6 is the driver of 7q21 amplification and that both CDK4 and CDK6 are prognostic markers and bona fide oncogenes in EAC. Targeting these molecules may constitute a viable new therapy for this disease. Clin Cancer Res; 17(13); 4513–22. ©2011 AACR.

Age-related hearing loss: Aquaporin 4 gene expression changes in the mouse cochlea and auditory midbrain

Presbycusis -- age-related hearing loss, is the number one communication disorder, and one of the top three chronic medical conditions of our aged population. Aquaporins, particularly aquaporin 4 (Aqp4), are membrane proteins with important roles in water and ion flux across cell membranes, including cells of the inner ear and pathways of the brain used for hearing. To more fully understand the biological bases of presbycusis, 39 CBA mice, a well-studied animal model of presbycusis, underwent non-invasive hearing testing as a function of sound frequency (auditory brainstem response -- ABR thresholds, and distortion-product otoacoustic emission -- DPOAE magnitudes), and were clustered into four groups based on age and hearing ability. Aqp4 gene expression, as determined by genechip microarray analysis and quantitative real-time PCR, was compared to the young adult control group in the three older groups: middle aged with good hearing, old age with mild presbycusis, and old age with severe presbycusis. Linear regression and ANOVA showed statistically significant changes in Aqp4 gene expression and ABR and DPOAE hearing status in the cochlea and auditory midbrain -- inferior colliculus. Down-regulation in the cochlea was seen, and an initial down-, then up-regulation was discovered for the inferior colliculus Aqp4 expression. It is theorized that these changes in Aqp4 gene expression represent an age-related disruption of ion flux in the fluids of the cochlea that are responsible for ionic gradients underlying sound transduction in cochlear hair cells necessary for hearing. In regard to central auditory processing at the level of the auditory midbrain, aquaporin gene expression changes may affect neurotransmitter cycling involving supporting cells, thus impairing complex sound neural processing with age.

Gene Expression Changes for Antioxidants Pathways in the Mouse Cochlea: Relations to Age-related Hearing Deficits

Age-related hearing loss – presbycusis – is the number one neurodegenerative disorder and top communication deficit of our aged population. Like many aging disorders of the nervous system, damage from free radicals linked to production of reactive oxygen and/or nitrogen species (ROS and RNS, respectively) may play key roles in disease progression. The efficacy of the antioxidant systems, e.g., glutathione and thioredoxin, is an important factor in pathophysiology of the aging nervous system. In this investigation, relations between the expression of antioxidant-related genes in the auditory portion of the inner ear – cochlea, and age-related hearing loss was explored for CBA/CaJ mice. Forty mice were classified into four groups according to age and degree of hearing loss. Cochlear mRNA samples were collected and cDNA generated. Using Affymetrix® GeneChip, the expressions of 56 antioxidant-related gene probes were analyzed to estimate the differences in gene expression between the four subject groups. The expression of Glutathione peroxidase 6, Gpx6; Thioredoxin reductase 1, Txnrd1; Isocitrate dehydrogenase 1, Idh1; and Heat shock protein 1, Hspb1; were significantly different, or showed large fold-change differences between subject groups. The Gpx6, Txnrd1 and Hspb1 gene expression changes were validated using qPCR. The Gpx6 gene was upregulated while the Txnrd1 gene was downregulated with age/hearing loss. The Hspb1 gene was found to be downregulated in middle-aged animals as well as those with mild presbycusis, whereas it was upregulated in those with severe presbycusis. These results facilitate development of future interventions to predict, prevent or slow down the progression of presbycusis.

Novel approach to select genes from RMA normalized microarray data using functional hearing tests in aging mice

UNLABELLED Presbycusis - age-related hearing loss - is the number one communicative disorder and one of the top three chronic medical condition of our aged population. High-throughput technologies potentially can be used to identify differentially expressed genes that may be better diagnostic and therapeutic targets for sensory and neural disorders. Here we analyzed gene expression for a set of GABA receptors in the cochlea of aging CBA mice using the Affymetrix GeneChip MOE430A. Functional phenotypic hearing measures were made, including auditory brainstem response (ABR) thresholds and distortion-product otoacoustic emission (DPOAE) amplitudes (four age groups). Four specific criteria were used to assess gene expression changes from RMA normalized microarray data (40 replicates). Linear regression models were used to fit the neurophysiological hearing measurements to probe-set expression profiles. These data were first subjected to one-way ANOVA, and then linear regression was performed. In addition, the log signal ratio was converted to fold change, and selected gene expression changes were confirmed by relative real-time PCR. MAJOR FINDINGS expression of GABA-A receptor subunit alpha6 was upregulated with age and hearing loss, whereas subunit alpha1 was repressed. In addition, GABA-A receptor associated protein like-1 and GABA-A receptor associated protein like-2 were strongly downregulated with age and hearing impairment. Lastly, gene expression measures were correlated with pathway/network relationships relevant to the inner ear using Pathway Architect, to identify key pathways consistent with the gene expression changes observed.

Bile exposure inhibits expression of squamous differentiation genes in human esophageal epithelial cells.

Objective:This study aimed to identify pathways and cellular processes that are modulated by exposure of normal esophageal cells to bile and acid. Background:Barretts esophagus most likely develops as a response of esophageal stem cells to the abnormal reflux environment. Although insights into the underlying molecular mechanisms are slowly emerging, much of the metaplastic process remains unknown. Methods:We performed a global analysis of gene expression in normal squamous esophageal cells in response to bile or acid exposure. Differentially expressed genes were classified into major biological functions using pathway analysis and interaction network software. Array data were verified by quantitative PCR and western blot both in vitro and in human esophageal biopsies. Results:Bile modulated expression of 202 genes, and acid modulated expression of 103 genes. Genes involved in squamous differentiation formed the largest functional group (n = 45) all of which were downregulated by bile exposure. This included genes such as involucrin (IVL), keratinocyte differentiation-associated protein (KRTDAP), grainyhead-like 1 (GRHL1), and desmoglein1 (DSG1) the downregulation of which was confirmed by quantitative PCR and western blot. Bile also caused expression changes in genes involved in cell adhesion, DNA repair, oxidative stress, cell cycle, Wnt signaling, and lipid metabolism. Analysis of human esophageal biopsies demonstrated greatly reduced expression of IVL, KRTDAP, DSG1, and GRHL1 in metaplastic compared to squamous epithelia. Conclusions:We report for the first time that bile inhibits the squamous differentiation program of esophageal epithelial cells. This, coordinated with induction of genes driving intestinal differentiation, may be required for the development of Barretts esophagus.

A novel high-throughput analysis approach: immune response-related genes are upregulated in age-related hearing loss

correspondence: robert D Frisina Department of chemical and Biomedical engineering, 4202 e Fowler Avenue, enB 118, University of south Florida, Tampa FL 33620, UsA Tel +1 813 974 4394 Fax +1 813 974 3651 email rfrisina@usf.edu Background: Presbycusis is defined as the hearing loss that occurs with aging. Genes that are responsible for this clinical condition have still not been identified. The present study explores gene discovery for age-related hearing loss using the CBA/CaJ mouse model of age-related hearing loss. Methods and results: In the present investigation, in addition to a gene-based analysis, a molecular pathway analysis was performed to evaluate differences in gene expression in 15 aged mice with age-related hearing loss, and 25 young to middle-aged mice with normal hearing. Using three different statistical approaches, the gene-based analysis revealed four common probe sets, ie, Ctss (Cathespin), Csnk (casein kappa), Mpeg1 (macrophage-expressed gene 1), and Clecsf12 (C-type calcium-dependent carbohydrate recognition domain lectin, superfamily member 12) that are significantly upregulated with age and hearing loss in the mouse cochlea (inner ear). Relative real-time polymerase chain reaction was used for quantitative, sensitivity analysis of gene transcription changes and confirmed the gene microarray results. Three strategies for pathway analysis were consistent with these gene expression discoveries, and revealed a common immune response pathway. More specifically, the analysis suggests that B cell-mediated humoral immune function plays an important role in the underlying etiology of presbycusis, similar to certain other neurodegenerative diseases. Other top pathways from this analysis included those involving dendritic antigen-presenting cells, carbohydrate binding, G-protein coupled receptor binding, and epithelial-to-mesenchymal transition pathways. Conclusion: Combining microarray gene discovery, polymerase chain reaction, and multiple pathway analyses revealed insights into immune system involvement in the progression of age-related hearing loss.