Regulation of Mouse Small Heat Shock Protein αb-Crystallin Gene by Aryl Hydrocarbon Receptor

The stress-inducible small heat shock protein (shsp)/αB-crystallin gene is expressed highly in the lens and moderately in other tissues. Here we provide evidence that it is a target gene of the aryl hydrocarbon receptor (AhR) transcription factor. A sequence (−329/−323, CATGCGA) similar to the consensus xenobiotic responsive element (XRE), called here XRE-like, is present in the αBE2 region of αB-crystallin enhancer and can bind AhR in vitro and in vivo. αB-crystallin protein levels were reduced in retina, lens, cornea, heart, skeletal muscle and cultured muscle fibroblasts of AhR−/− mice; αB-crystallin mRNA levels were reduced in the eye, heart and skeletal muscle of AhR−/− mice. Increased AhR stimulated αB-crystallin expression in transfection experiments conducted in conjunction with the aryl hydrocarbon receptor nuclear translocator (ARNT) and decreased AhR reduced αB-crystallin expression. AhR effect on aB-crystallin promoter activity was cell-dependent in transfection experiments. AhR up-regulated αB-crystallin promoter activity in transfected HeLa, NIH3T3 and COS-7 cells in the absence of exogenously added ligand (TCDD), but had no effect on the αB-crystallin promoter in C2C12, CV-1 or Hepa-1 cells with or without TCDD. TCDD enhanced AhR-stimulated αB-crystallin promoter activity in transfected αTN4 cells. AhR could bind to an XRE-like site in the αB-crystallin enhancer in vitro and in vivo. Finally, site-specific mutagenesis experiments showed that the XRE-like motif was necessary for both basal and maximal AhR-induction of αB-crystallin promoter activity. Our data strongly suggest that AhR is a regulator of αB-crystallin gene expression and provide new avenues of research for the mechanism of tissue-specific αB-crystallin gene regulation under normal and physiologically stressed conditions

Studies on nucleoside and amino acid uptake and on RNA and protein synthesis by growing oocytes, unfertilized and fertilized

The principal theme of these investigations concerns the inhibited state of mature unfertilized sea urchin eggs with respect to uridine uptake and protein synthesis. Part I demonstrates that unfertilized-eggs are relatively impermeable to uridine. Fertilized eggs, however, develop during the first hour an energy-dependent, uptake mechanism for uridine accumulation. Labeled uridine assimilated by fertilized eggs is recovered as phosphorylated nucleosides, primarily triphosphates. Experiments support the idea that uridine penetration into sea urchin eggs depends upon the phosphorylation of the 5' carbon atom at the cell surface. Tests with puromycin show that protein synthesis is unnecessary for the generation of uridine uptake after fertilization. The evidence favors the view that uridine kinase is sequestered within the unfertilized egg and thus incapable of activity at the cell surface until after fertilization. Parts II and III use biochemical and autoradiographic methods to show that growing oocytes of sea urchins, in contrast to many other organisms, undergo considerable RNA and protein synthesis. Protein synthesis in isolated oocytes occurs throughout the germinal vesicle and cytoplasm and takes place on polyribosomes. RNA synthesis is localized in the nucleolus. Mature eggs, however, synthesize only little protein even in mixed suspensions with oocytes. Long-term maintenance of spawned female sea urchins, after but one injection of labeled uridine, produces ripe unfertilized eggs possessing highly radioactive RNA. The distribution of label in the extracted RNAs is 70-80% ribosomal, 10-20% heterogeneous, and 5-10% soluble. Part IV is an electron microscopic and biochemical examination of RNA-labeled mature unfertilized and fertilized eggs. The findings are correlated with the difference in protein synthesizing activity before and after fertilization. The results show that unfertilized eggs synthesize protein upon RNase-sensitive polyribosomes. The large increase in protein synthesis after fertilization occurs in association with the assembly of additional polyribosomes. Homogenates of unfertilized eggs also possess synthetically inactive, RNase-resistant, ribosomal aggregates. Evidence suggests that trypsin followed by RNase disperses the aggregates. Homogenates of fertilized eggs, however, contain very few RNase-resistant ribosomal aggregates. By forty minutes after fertilization, about 70% of the new protein synthesis can be attributed to the new polyribosomes. The weight of the evidence indicates that the remaining 30% of the stimulation of protein synthesis is due to the activation of "masked" polyribosomes. Appendix 1 shows that, for unfertilized and fertilized eggs, competition for uptake of amino acids occurs primarily among those belonging to the same charge group. Appendix 2 demonstrates that one amino acid can displace another of the same category from intact eggs both before and after fertilization. By combination of these facts, then, it is possible to achieve greater labeling of egg-proteins than has been previously realized

Preferential Transcription of Rabbit Aldh1a1 in the Cornea: Implication of Hypoxia-Related Pathways

Here we examine the molecular basis for the known preferential expression of rabbit aldehyde dehydrogenase class 1 (ALDH1A1) in the cornea. The rabbit Aldh1a1 promoter-firefly luciferase reporter transgene (−3519 to +43) was expressed preferentially in corneal cells in transfection tests and in transgenic mice, with an expression pattern resembling that of rabbit Aldh1a1. The 5′ flanking region of the rabbit Aldh1a1 gene resembled that in the human gene (60.2%) more closely than that in the mouse (46%) or rat (51.5%) genes. We detected three xenobiotic response elements (XREs) and one E-box consensus sequence in the rabbit Aldh1a1 upstream region; these elements are prevalent in other highly expressed corneal genes and can mediate stimulation by dioxin and repression by CoCl(2), which simulates hypoxia. The rabbit Aldh1a1 promoter was stimulated fourfold by dioxin in human hepatoma cells and repressed threefold by CoCl(2) treatment in rabbit corneal stromal and epithelial cells. Cotransfection, mutagenesis, and gel retardation experiments implicated the hypoxia-inducible factor 3α/aryl hydrocarbon nuclear translocator heterodimer for Aldh1a1 promoter activation via the XREs and stimulated by retinoic acid protein 13 for promoter repression via the E-box. These experiments suggest that XREs, E-boxes, and PAS domain/basic helix-loop-helix transcription factors (bHLH-PAS) contribute to preferential rabbit Aldh1a1 promoter activity in the cornea, implicating hypoxia-related pathways

RADIOACTIVE LABELING OF RNAs OF SEA URCHIN EGGS DURING OOGENESIS

Volume: 133Start Page: 229End Page: 24

Overexpression of Pax6 in Mouse Cornea Directly Alters Corneal Epithelial Cells: Changes in Immune Function, Vascularization, and Differentiation

The restricted overexpression of Pax6 in the mouse cornea combined with the abnormal corneal, but not lens, phenotype suggests that Pax6 is essential and functions autonomously in the cornea

INFLUENCE OF INDIVIDUAL AMINO ACIDS ON UPTAKE AND INCORPORATION OF VALINE, GLUTAMIC ACID AND ARGININE BY UNFERTILIZED AND FERTILIZED SEA URCHIN EGGS

Volume: 131Start Page: 204End Page: 21