RESUMEN
PURPOSE: To identify lens epithelial genes with altered expression levels in age-related human cataract. METHODS: Epithelia from age-related cataracts and from normal lenses were microdissected and RNA was extracted. RNAs were compared for gene expression differences by RT-PCR differential display. Transcripts exhibiting altered levels of gene expression were cloned and identified by sequencing. The expression levels of identified clones were confirmed by semiquantitative RT-PCR with three separately isolated RNA preparations. Specific primers were designed and used to examine the mRNA levels of other genes important in protein synthesis. RESULTS: Numerous transcripts exhibited altered levels of gene expression. One transcript exhibiting a decreased level of expression in cataract compared with normal lenses was identified as encoding ribosomal protein L21. Three additional ribosomal proteins, L15, L13a, and L7a, also exhibited decreased expression in cataract compared with normal human lenses. By contrast, the levels of elongation factor (EF)-1alpha1 and eucaryotic initiation factor (eIF)-4E remained unchanged. CONCLUSIONS: The results provide evidence that human age-related cataract is associated with decreased expression of L21 and other ribosomal proteins. The results suggest that modulation of protein synthesis and/or other functions mediated by ribosomal proteins is associated with age-related cataract.
Asunto(s)
Envejecimiento/metabolismo , Catarata/metabolismo , Cristalino/metabolismo , Proteínas Ribosómicas/metabolismo , Anciano , Secuencia de Bases , Células Epiteliales/metabolismo , Femenino , Amplificación de Genes , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , ARN/aislamiento & purificación , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Ribosómicas/genéticaRESUMEN
PURPOSE: The Emory mouse is a well-characterized model for age-onset cataract. The purpose of the present study was to identify differentially expressed genes between pre- and postcataract Emory mouse lenses. METHODS: Eyes were extracted from Emory mice at 3 weeks (precataract) and 7.5 months (postcataract) of age, and lenses were dissected. Lens RNA was compared for gene expression differences by RT-PCR differential display, and transcripts exhibiting altered levels of gene expression were cloned and identified by sequencing. The levels of two transcripts were further evaluated by RT-PCR in 3-week- and 7.5-month-old lenses and the remainder of the eye. The same transcripts were also measured in lenses from three non-Emory mouse strains (FVB/N, 129Sv, and CD1) ages 4 weeks to 11.5 months. RESULTS: Three transcripts were identified as exhibiting altered levels of gene expression between 3-week- and 7.5-month-old Emory mouse lenses. These encoded alphaA-crystallin (decreased), betaA3/A1-crystallin (decreased), and adhesion-related kinase (ARK) receptor tyrosine kinase (increased). Decreased alphaA-crystallin and increased ARK expression were not detected in lenses isolated from three non-Emory mouse strains of similar age. Increased expression of ARK was not detected between 3-week- and 7.5-month-old Emory mouse eye nonlens tissues. CONCLUSIONS: The present data confirm that expression of the alphaA-crystallin gene is decreased in cataract in the Emory mouse lens relative to age-matched control lenses and they provide evidence for cataract- and lens-specific upregulation of the ARK receptor tyrosine kinase in the Emory mouse.
Asunto(s)
Catarata/enzimología , Regulación Enzimológica de la Expresión Génica , Cristalino/enzimología , Proteínas Oncogénicas , Proteínas Tirosina Quinasas Receptoras/genética , Envejecimiento/fisiología , Animales , Clonación Molecular , Cristalinas/genética , Cristalinas/metabolismo , Modelos Animales de Enfermedad , Ratones , Proteínas Proto-Oncogénicas , ARN/aislamiento & purificación , ARN Mensajero/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba , Tirosina Quinasa del Receptor AxlRESUMEN
PURPOSE: To identify those metallothionein and alpha-crystallin/small heat-shock genes induced by toxic metals in human lens cells and to evaluate the levels of these metals between young and aged human lenses. METHODS: Human SRA01/04 and primary human lens epithelial cells were cultured and exposed to Cd(2+), Cu(2+), and Zn(2+). The levels of lens metallothioneins (Ig, If, Ih, Ie, and IIa) and alpha-crystallin/small heat-shock (alphaA-crystallin, alphaB-crystallin, and HSP27) genes were analyzed by semiquantitative and quantitative competitive RT-PCR. The content of aluminum, cadmium, calcium, chromium, copper, iron, lead, magnesium, manganese, nickel, potassium, sodium, and zinc in young (mean, 32.8 years), middle-aged (mean, 52.3 years), and old (mean, 70.5 years) human lenses was analyzed by inductively coupled plasma-emission spectroscopy. RESULTS: Lens metallothioneins (Ig, If, Ih, Ie, and IIa) and alpha-crystallin/small heat-shock genes (alphaA-crystallin, alphaB-crystallin, and HSP27) were differentially induced by specific metals in SRA01/04 human lens epithelial cells. Cd(2+) and Zn(2+), but not Cu(2+), induced the metallothioneins, whereas Cd(2+) and Cu(2+), but not Zn(2+), induced alphaB-crystallin and HSP27. alphaA-crystallin was induced by Cu(2+) only. Similar responses of the metallothionein IIa gene were detected in identically treated primary human lens epithelial cells. Cd(2+) and Zn(2+) induced metallothionein IIa to five times higher levels than metallothionein Ig. Of 13 different metals, only iron was altered, exhibiting an 81% decrease in old versus young lenses. CONCLUSIONS: Induction of metallothioneins and alpha-crystallin/small heat shock proteins by different metals indicates the presence of metal-specific lens regulatory pathways that are likely to be involved in protection against metal-associated stresses.
Asunto(s)
Envejecimiento/fisiología , Proteínas de Choque Térmico/biosíntesis , Cristalino/efectos de los fármacos , Metalotioneína/biosíntesis , Metales/farmacología , alfa-Cristalinas/biosíntesis , Adulto , Anciano , Cadmio/farmacología , Células Cultivadas , Cobre/farmacología , Células Epiteliales/química , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Proteínas de Choque Térmico/genética , Humanos , Cristalino/química , Cristalino/metabolismo , Metalotioneína/genética , Metales/análisis , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Zinc/farmacología , alfa-Cristalinas/genéticaRESUMEN
PURPOSE: Oxidative stress (OS) is believed to be a major contributor to age-related cataract and other age-related diseases. METHODS: cDNA microarrays were used to identify the spectrum and range of genes with transcript levels that are altered in response to acute H(2)O(2)-induced OS in human lens epithelial (HLE) cells. HLE cells were treated with 50 microM H(2)O(2) for 1 hour in the absence of serum, followed by a return to complete medium. RNAs were prepared from treated and untreated cells at 0, 1, 2, and 8 hours after H(2)O(2) treatment. RESULTS: The data showed 1171 genes that were significantly up- and downregulated in response to H(2)O(2) treatment. Several functional subcategories of genes were identified, including those encoding DNA repair proteins, antioxidant defense enzymes, molecular chaperones, protein biosynthesis enzymes, and trafficking and degradation proteins. Differential expression of selected genes was confirmed at the level of RNA and/or protein. Many of the identified genes (e.g., glutathione S-transferase [MGST2], thioredoxin reductase beta, and peroxiredoxin 2) have been identified as participants in OS responses in the lens and other systems. Some genes induced by OS in the current study (e.g., oxygen regulated protein [ORP150] and heat shock protein [HSP40]) are better known to respond to other forms of stress. Two genes (receptor tyrosine kinase [AXL/ARK] and protein phosphatase 2A) are known to be differentially expressed in cataract. Most of the genes point to a novel pathways associated with OS. CONCLUSIONS: The present data provide a global perspective on those genes that respond to acute OS, point to novel genes and pathways associated with OS, and set the groundwork for understanding the functions of OS-related genes in lens protection and disease.
Asunto(s)
Cristalinas/metabolismo , Células Epiteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/toxicidad , Cristalino/efectos de los fármacos , Estrés Oxidativo , Apoptosis , Western Blotting , Técnicas de Cultivo de Célula , Supervivencia Celular , Cartilla de ADN/química , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica , Humanos , Cristalino/citología , Cristalino/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
PURPOSE: Age-related cataract is a multi-factorial disease with a poorly understood etiology. Numerous studies provide evidence that the human eye lens has evolved specific regulatory and protective systems to ameliorate lens damage associated with cataract. Other studies suggest that the presence of cataract is associated with the altered expression of specific genes including metallothionein IIa, osteonectin, transglutaminase 2, betaig-h3, multiple ribosomal proteins, ADAM9, and protein phosphatase 2A. Here, we sought to identify further gene expression changes that are associated with cataract and to cluster the identified genes into specific biological pathways. METHODS: Oligonucleotide microarray hybridization was used to analyze the full complement of gene expression differences between lens epithelia isolated from human age-related cataract relative to clear lenses. The expression levels of a subset of the identified genes were further evaluated by semi-quantitative RT-PCR. The identified genes were functionally clustered into specific categories and the probability of over-representation of each category was determined using the computer program EASE. RESULTS: 412 transcripts were observed to be increased and 919 transcripts were observed to be decreased by 2 fold or more in lens epithelia isolated from age-related cataract relative to clear lenses. Of these, 74 were increased and 241 were decreased at the 5 fold level or greater. Seventeen genes selected for further confirmation exhibited similar trends in expression when examined by RT-PCR using both the original and separately prepared clear and cataract RNA populations. Functional clustering of the identified genes using the EASE bioinformatics software package revealed that, among others, transcripts increased in cataract are associated with transcriptional control, chromosomal organization, ionic and cytoplasmic transport, and extracellular matrix components while transcripts decreased in cataract are associated with protein synthesis, defense against oxidative stress, heat-shock/chaperone activity, structural components of the lens, and cell cycle control. CONCLUSIONS: These data suggest that cataract is associated with multiple previously identified and novel changes in lens epithelial gene expression and they point to numerous pathways likely to play important roles in lens protection, maintenance, and age-related cataract.