Showing papers by "David A. Pearce published in 1999"

Epithelial sodium channel regulated by aldosterone-induced protein sgk.

Abstract: Sodium homeostasis in terrestrial and freshwater vertebrates is controlled by the corticosteroid hormones, principally aldosterone, which stimulate electrogenic Na+ absorption in tight epithelia. Although aldosterone is known to increase apical membrane Na+ permeability in target cells through changes in gene transcription, the mechanistic basis of this effect remains poorly understood. The predominant early effect of aldosterone is to increase the activity of the epithelial sodium channel (ENaC), although ENaC mRNA and protein levels do not change initially. Rather, the open probability and/or number of channels in the apical membrane are greatly increased by unknown modulators. To identify hormone-stimulated gene products that modulate ENaC activity, a subtracted cDNA library was generated from A6 cells, a stable cell line of renal distal nephron origin, and the effect of candidates on ENaC activity was tested in a coexpression assay. We report here the identification of sgk (serum and glucocorticoid-regulated kinase), a member of the serine–threonine kinase family, as an aldosterone-induced regulator of ENaC activity. sgk mRNA and protein were strongly and rapidly hormone stimulated both in A6 cells and in rat kidney. Furthermore, sgk stimulated ENaC activity approximately 7-fold when they were coexpressed in Xenopus laevis oocytes. These data suggest that sgk plays a central role in aldosterone regulation of Na+ absorption and thus in the control of extracellular fluid volume, blood pressure, and sodium homeostasis.

Action of BTN1, the yeast orthologue of the gene mutated in Batten disease.

Abstract: Neuronal ceroid-lipofuscinoses (NCL) are autosomal recessive disorders that form the most common group of progressive neurodegenerative diseases in children, with an incidence as high as 1 in 12,500 live births, and with approximately 440,000 carriers in the United States1,2. Disease progression is characterized by a decline in mental abilities, increased severity of untreatable seizures, blindness, loss of motor skills and premature death. The CLN3 gene, which is responsible for Batten disease, has been positionally cloned3. The yeast gene, denoted BTN1, encodes a non-essential protein that is 39% identical and 59% similar to human CLN3 ( ref. 4). Strains lacking Btn1p, btn1-Δ, are resistant to D-(-)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP) in a pH-dependent manner5. This phenotype was complemented by expression of human CLN3, demonstrating that yeast Btn1p and human CLN3 share the same function5. Here, we report that btn1-Δ yeast strains have an abnormally acidic vacuolar pH in the early phases of growth. Furthermore, DNA microarray analysis of BTN1 and btn1-Δ strains revealed differential expression of two genes, with at least one, HSP30, involved in pH control. Because Btn1p is located in the vacuole, we suggest that Batten disease is caused by a defect in vacuolar (lysosomal) pH control. Our findings draw parallels between fundamental biological processes in yeast and previously observed characteristics of neurodegeneration in humans.

Distinct glucocorticoid receptor transcriptional regulatory surfaces mediate the cytotoxic and cytostatic effects of glucocorticoids.

Abstract: Glucocorticoids act through the glucocorticoid receptor (GR), which can function as a transcriptional activator or repressor, to elicit cytostatic and cytotoxic effects in a variety of cells. The molecular mechanisms regulating these events and the target genes affected by the activated receptor remain largely undefined. Using cultured human osteosarcoma cells as a model for the GR antiproliferative effect, we demonstrate that in U20S cells, GR activation leads to irreversible growth inhibition, apoptosis, and repression of Bcl2. This cytotoxic effect is mediated by GR's transcriptional repression function, since transactivation-deficient mutants and ligands still bring about apoptosis and Bcl2 down-regulation. In contrast, the antiproliferative effect of GR in SAOS2 cells is reversible, does not result in apoptosis or repression of Bcl2, and is a function of the receptor's ability to stimulate transcription. Thus, the cytotoxic versus cytostatic outcome of glucocorticoid treatment is cell context dependent. Interestingly, the cytostatic effect of glucocorticoids in SAOS2 cells involves multiple GR activation surfaces. GR mutants and ligands that disrupt individual transcriptional activation functions (activation function 1 [AF-1] and AF-2) or receptor dimerization fail to fully inhibit cellular proliferation and, remarkably, discriminate between the targets of GR's cytostatic action, the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1). Induction of p21(Cip1) is agonist dependent and requires AF-2 but not AF-1 or GR dimerization. In contrast, induction of p27(Kip1) is agonist independent, does not require AF-2 or AF-1, but depends on GR dimerization. Our findings indicate that multiple GR transcriptional regulatory mechanisms that employ distinct receptor surfaces are used to evoke either the cytostatic or cytotoxic response to glucocorticoids.

Toxicity of copper, cobalt, and nickel salts is dependent on histidine metabolism in the yeast Saccharomyces cerevisiae.

Abstract: The pH-dependent inhibition of 22 metal salts have been systematically investigated for the yeast Saccharomyces cerevisiae We have established that the inhibition of growth by Cu, Co, or Ni salts is markedly enhanced by histidine auxotrophy and by increasing the pH of the medium Each of the his1-his7 mutant strains were unable to grow in the presence of elevated levels of Cu, Co, or Ni at nearly neutral pHs, in contrast to His+ strains, which grew under these conditions The Cu, Co, or Ni inhibition was reversed by the addition of histidine to the medium Deletion of the high-affinity histidine permease Hip1p in His− strains resulted in even greater sensitivity to Cu, Co, and Ni and the requirement of an even higher level of histidine to reverse the inhibition These results suggest that intracellular histidine, most likely in the vacuole, diminishes the pH-dependent toxicity of Cu, Co, and Ni Furthermore, the toxicity of many salts is exacerbated in strains with a defective vacuolar H+-ATPase, which abolishes the ability of yeast to maintain an acidic vacuole, a compartment known to sequester metal compounds We suggest that the accumulation of histidine in the vacuole is a normal process used to detoxify Cu, Co, and Ni

Phenotypic reversal of the btn1 defects in yeast by chloroquine: a yeast model for Batten disease.

Abstract: BTN1 of Saccharomyces cerevisiae encodes an ortholog of CLN3, the human Batten disease gene. We have reported previously that deletion of BTN1, btn1-Δ, resulted in a pH-dependent resistance to d-(−)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP). This phenotype was caused by btn1-Δ strains having an elevated ability to acidify growth medium through an elevated activity of the plasma membrane H+-ATPase, resulting from a decreased vacuolar pH during early growth. We have determined that growing btn1-Δ strains in the presence of chloroquine reverses the resistance to ANP, decreases the rate of medium acidification, decreases the activity of plasma membrane H+-ATPase, and elevates vacuolar pH. However, an additional effect of this phenotypic reversal is that activity of plasma membrane H+-ATPase is decreased further and vacuolar pH is increased further as btn1-Δ strains continue to grow. This phenotypic reversal of btn1-Δ can be considered for developing a therapy for Batten disease.

Hereditary spastic paraplegia: mitochondrial metalloproteases of yeast.

Abstract: Hereditary spastic paraplegia (HSP) is a genetically heterogenous group of inherited neurodegenerative disorders. Recently, an autosomal recessive form of HSP was mapped to 16q24.3, and subsequently the defective gene associated to HSP was identified and designated SPG7. The SPG7 gene product was predicted to encode a protein of 795 amino acids, and is called paraplegin. Paraplegin is highly homologous to a class of well studied yeast ATP-dependent zinc metalloproteases, which show 55%, 55% and 52% identity, respectively, to Afg3p, Rca1p and Yme1p. Mutation of either Afg3p, Rca1p or Yme1p in yeast results in pleiotropic effects with regard to growth, respiration and, particularly, in the assembly and/or degradation of more than one mitochondrial protein complex. Taking into account the homology of paraplegin to these yeast ATP-dependent zinc metalloproteases and what is known about their function, allows us to speculate as to what function paraplegin plays in normal individuals.

DNA microarray analysis of strains lacking BTN1 , the yeast ortholog of the human Batten disease gene

Abstract: DNA microarray analysis of strains lacking BTN1 , the yeast ortholog of the human Batten disease gene