Jennifer B. Collins

Integrating phenotypic and expression profiles to map arsenic-response networks

BackgroundArsenic is a nonmutagenic carcinogen affecting millions of people. The cellular impact of this metalloid in Saccharomyces cerevisiae was determined by profiling global gene expression and sensitivity phenotypes. These data were then mapped to a metabolic network composed of all known biochemical reactions in yeast, as well as the yeast network of 20,985 protein-protein/protein-DNA interactions.ResultsWhile the expression data unveiled no significant nodes in the metabolic network, the regulatory network revealed several important nodes as centers of arsenic-induced activity. The highest-scoring proteins included Fhl1, Msn2, Msn4, Yap1, Cad1 (Yap2), Pre1, Hsf1 and Met31. Contrary to the gene-expression analyses, the phenotypic-profiling data mapped to the metabolic network. The two significant metabolic networks unveiled were shikimate, and serine, threonine and glutamate biosynthesis. We also carried out transcriptional profiling of specific deletion strains, confirming that the transcription factors Yap1, Arr1 (Yap8), and Rpn4 strongly mediate the cells adaptation to arsenic-induced stress but that Cad1 has negligible impact.ConclusionsBy integrating phenotypic and transcriptional profiling and mapping the data onto the metabolic and regulatory networks, we have shown that arsenic is likely to channel sulfur into glutathione for detoxification, leads to indirect oxidative stress by depleting glutathione pools, and alters protein turnover via arsenation of sulfhydryl groups on proteins. Furthermore, we show that phenotypically sensitive pathways are upstream of differentially expressed ones, indicating that transcriptional and phenotypic profiling implicate distinct, but related, pathways.

Human Glucocorticoid Receptor β Binds RU-486 and Is Transcriptionally Active

ABSTRACT Human glucocorticoid receptor (hGR) is expressed as two alternately spliced C-terminal isoforms, α and β. In contrast to the canonical hGRα, hGRβ is a nucleus-localized orphan receptor thought not to bind ligand and not to affect gene transcription other than by acting as a dominant negative to hGRα. Here we used confocal microscopy to examine the cellular localization of transiently expressed fluorescent protein-tagged hGRβ in COS-1 and U-2 OS cells. Surprisingly, yellow fluorescent protein (YFP)-hGRβ was predominantly located in the cytoplasm and translocated to the nucleus following application of the glucocorticoid antagonist RU-486. This effect of RU-486 was confirmed with transiently expressed wild-type hGRβ. Confocal microscopy of coexpressed YFP-hGRβ and cyan fluorescent protein-hGRα in COS-1 cells indicated that the receptors move into the nucleus independently. Using a ligand binding assay, we confirmed that hGRβ bound RU-486 but not the hGRα ligand dexamethasone. Examination of the cellular localization of YFP-hGRβ in response to a series of 57 related compounds indicated that RU-486 is thus far the only identified ligand that interacts with hGRβ. The selective interaction of RU-486 with hGRβ was also supported by molecular modeling and computational docking studies. Interestingly, microarray analysis indicates that hGRβ, expressed in the absence of hGRα, can regulate gene expression and furthermore that occupation of hGRβ with the antagonist RU-486 diminishes that capacity despite the lack of helix 12 in the ligand binding domain.

Selective regulation of bone cell apoptosis by translational isoforms of the glucocorticoid receptor.

ABSTRACT Glucocorticoids are widely used in the treatment of inflammatory and other diseases. However, high-dose or chronic administration often triggers troublesome side effects such as metabolic syndrome and osteoporosis. We recently described that one glucocorticoid receptor gene produces eight translational glucocorticoid receptor isoforms that have distinct gene-regulatory abilities. We show here that specific, but not all, glucocorticoid receptor isoforms induced apoptosis in human osteosarcoma U-2 OS bone cells. Whole human genome microarray analysis revealed that the majority of the glucocorticoid target genes were selectively regulated by specific glucocorticoid receptor isoforms. Real-time PCR experiments confirmed that proapoptotic enzymes necessary for cell death, granzyme A and caspase-6, were induced by specific glucocorticoid receptor isoforms. Chromatin immunoprecipitation assays further suggested that glucocorticoid receptor isoform-dependent induction of proapoptotic genes was likely due to selective coregulator recruitment and chromatin modification. Interestingly, the capabilities to transrepress proinflammatory genes were similar among glucocorticoid receptor isoforms. Together, these findings provide new evidence that translational glucocorticoid receptor isoforms can elicit distinct glucocorticoid responses and may be useful for the development of safe glucocorticoids with reduced side effects.

DNA double strand breaks activate a multi-functional genetic program in developing lymphocytes

DNA double-strand breaks are generated by genotoxic agents and by cellular endonucleases as intermediates of several important physiological processes. The cellular response to genotoxic DNA breaks includes the activation of transcriptional programs known primarily to regulate cell-cycle checkpoints and cell survival. DNA double-strand breaks are generated in all developing lymphocytes during the assembly of antigen receptor genes, a process that is essential for normal lymphocyte development. Here we show that in murine lymphocytes these physiological DNA breaks activate a broad transcriptional program. This program transcends the canonical DNA double-strand break response and includes many genes that regulate diverse cellular processes important for lymphocyte development. Moreover, the expression of several of these genes is regulated similarly in response to genotoxic DNA damage. Thus, physiological DNA double-strand breaks provide cues that can regulate cell-type-specific processes not directly involved in maintaining the integrity of the genome, and genotoxic DNA breaks could disrupt normal cellular functions by corrupting these processes.

Glycogen Synthase Kinase 3β-Mediated Serine Phosphorylation of the Human Glucocorticoid Receptor Redirects Gene Expression Profiles

ABSTRACT Aberrant glycogen synthase kinase 3β (GSK-3β) activity is associated with the progression of several pathological conditions such as diabetes, Alzheimers, and cancer. GSK-3β regulates cellular processes by directly phosphorylating metabolic enzymes and transcription factors. Here, we discovered a new target for GSK-3β phosphorylation: the human glucocorticoid receptor (GR). Glucocorticoid signaling is essential for life and regulates diverse biological functions from cell growth to metabolism to apoptosis. Specifically, we found hormone-dependent GR phosphorylation on serine 404 by GSK-3β. Cells expressing a GR that is incapable of GSK-3β phosphorylation had a redirection of the global transcriptional response to hormone, including the activation of additional signaling pathways, in part due to the altered ability of unphosphorylatable GR to recruit transcriptional cofactors CBP/p300 and the p65 (RelA) subunit of NF-κB. Furthermore, GSK-3β-mediated GR phosphorylation inhibited glucocorticoid-dependent NF-κB transrepression and attenuated the glucocorticoid-dependent cell death of osteoblasts. Collectively, our results describe a novel convergence point of the GSK-3β and the GR pathways, resulting in altered hormone-regulated signaling. Our results also provide a mechanism by which GSK-3β activity can dictate how cells will ultimately respond to glucocorticoids.

Sexually Dimorphic Actions of Glucocorticoids Provide a Link to Inflammatory Diseases with Gender Differences in Prevalence

Glucocorticoids are more effective at counteracting inflammation in males than in females. Gender Bias for Glucocorticoids The release of glucocorticoids from adrenal glands during times of stress activates transcription of genes encoding factors that suppress inflammation. Synthetic glucocorticoids, such as dexamethasone, are widely prescribed for inflammatory conditions. Because several diseases with inflammatory components show gender-specific differences in prevalence, Duma et al. investigated whether glucocorticoid responses were also gender-biased. Microarray analysis of messenger RNA abundance indicated that dexamethasone treatment of male and female rats increased the number of genes with gender-specific differences in liver expression. In male rat liver, more genes implicated in inflammatory disorders showed dexamethasone-induced alterations (usually decreases) in expression than did genes in female rat liver. In rats injected with lipopolysaccharide (LPS) to trigger systemic inflammation and subsequently treated with dexamethasone, more males than females survived. Male rats produced lower concentrations of inflammatory factors in response to dexamethasone treatment, and ovariectomy improved the survival of female rats after LPS challenge and dexamethasone treatment. The accompanying Perspective by Chrousos discusses the evolutionary context for gender-specific differences in responses to stress, inflammation, and glucocorticoids. Males and females show differences in the prevalence of many major diseases that have important inflammatory components to their etiology. These gender-specific diseases, which include autoimmune diseases, hepatocellular carcinoma, diabetes, and osteoporosis, are largely considered to reflect the actions of sex hormones on the susceptibility to inflammatory stimuli. However, inflammation reflects a balance between pro- and anti-inflammatory signals, and investigation of gender-specific responses to the latter has been neglected. Glucocorticoids are the primary physiological anti-inflammatory hormones in mammals, and synthetic derivatives of these hormones are prescribed as anti-inflammatory agents, irrespective of patient gender. We explored the possibility that sexually dimorphic actions of glucocorticoid regulation of gene expression may contribute to the dimorphic basis of inflammatory disease by evaluating the rat liver, a classic glucocorticoid-responsive organ. Surprisingly, glucocorticoid administration expanded the set of hepatic sexually dimorphic genes. Eight distinct patterns of glucocorticoid-regulated gene expression were identified, which included sex-specific genes. Our experiments also defined specific genes with altered expression in response to glucocorticoid treatment in both sexes, but in opposite directions. Pathway analysis identified sex-specific glucocorticoid-regulated gene expression in several canonical pathways involved in susceptibility to and progression of diseases with gender differences in prevalence. Moreover, a comparison of the number of genes involved in inflammatory disorders between sexes revealed 84 additional glucocorticoid-responsive genes in the male, suggesting that the anti-inflammatory actions of glucocorticoids are more effective in males. These gender-specific actions of glucocorticoids in liver were substantiated in vivo with a sepsis model of systemic inflammation.

Farnesol-induced apoptosis in human lung carcinoma cells is coupled to the endoplasmic reticulum stress response.

Farnesol (FOH) and other isoprenoid alcohols induce apoptosis in various carcinoma cells and inhibit tumorigenesis in several in vivo models. However, the mechanisms by which they mediate their effects are not yet fully understood. In this study, we show that FOH is an effective inducer of apoptosis in several lung carcinoma cells, including H460. This induction is associated with activation of several caspases and cleavage of poly(ADP-ribose) polymerase (PARP). To obtain insight into the mechanism involved in FOH-induced apoptosis, we compared the gene expression profiles of FOH-treated and control H460 cells by microarray analysis. This analysis revealed that many genes implicated in endoplasmic reticulum (ER) stress signaling, including ATF3, DDIT3, HERPUD1, HSPA5, XBP1, PDIA4, and PHLDA1, were highly up-regulated within 4 h of FOH treatment, suggesting that FOH-induced apoptosis involves an ER stress response. This was supported by observations showing that treatment with FOH induces splicing of XBP1 mRNA and phosphorylation of eIF2alpha. FOH induces activation of several mitogen-activated protein kinase (MAPK) pathways, including p38, MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK, and c-jun NH2-terminal kinase (JNK). Inhibition of MEK1/2 by U0126 inhibited the induction of ER stress response genes. In addition, knockdown of the MEK1/2 and JNK1/2 expression by short interfering RNA (siRNA) effectively inhibited the cleavage of caspase-3 and PARP and apoptosis induced by FOH. However, only MEK1/2 siRNAs inhibited the induction of ER stress-related genes, XBP1 mRNA splicing, and eIF2alpha phosphorylation. Our results show that FOH-induced apoptosis is coupled to ER stress and that activation of MEK1/2 is an early upstream event in the FOH-induced ER stress signaling cascade.

Altered Gene Expression and DNA Damage in Peripheral Blood Cells from Friedreich's Ataxia Patients: Cellular Model of Pathology

The neurodegenerative disease Friedreichs ataxia (FRDA) is the most common autosomal-recessively inherited ataxia and is caused by a GAA triplet repeat expansion in the first intron of the frataxin gene. In this disease, transcription of frataxin, a mitochondrial protein involved in iron homeostasis, is impaired, resulting in a significant reduction in mRNA and protein levels. Global gene expression analysis was performed in peripheral blood samples from FRDA patients as compared to controls, which suggested altered expression patterns pertaining to genotoxic stress. We then confirmed the presence of genotoxic DNA damage by using a gene-specific quantitative PCR assay and discovered an increase in both mitochondrial and nuclear DNA damage in the blood of these patients (p<0.0001, respectively). Additionally, frataxin mRNA levels correlated with age of onset of disease and displayed unique sets of gene alterations involved in immune response, oxidative phosphorylation, and protein synthesis. Many of the key pathways observed by transcription profiling were downregulated, and we believe these data suggest that patients with prolonged frataxin deficiency undergo a systemic survival response to chronic genotoxic stress and consequent DNA damage detectable in blood. In conclusion, our results yield insight into the nature and progression of FRDA, as well as possible therapeutic approaches. Furthermore, the identification of potential biomarkers, including the DNA damage found in peripheral blood, may have predictive value in future clinical trials.

Voluntary exercise protects hippocampal neurons from trimethyltin injury: Possible role of interleukin-6 to modulate tumor necrosis factor receptor-mediated neurotoxicity

In the periphery, exercise induces interleukin (IL)-6 to downregulate tumor necrosis factor (TNF), elevate interleukin-1 receptor antagonist (IL-1RA), decreasing inflammation. Exercise also offers neuroprotection and facilitates brain repair. IL-6 production in the hippocampus following exercise suggests the potential of a similar protective role as in the periphery to down-regulate TNFα and inflammation. Using a chemical-induced model of hippocampal dentate granule cell death (trimethyltin, TMT 2.4 mg/kg, ip) dependent upon TNF receptor signaling, we demonstrate neuroprotection in mice with 2 weeks access to running wheel. Exercise attenuated neuronal death and diminished elevations in TNFα, TNF receptor 1, myeloid differentiation primary response gene (MyD) 88, transforming growth factor β, chemokine (C-C motif) ligand 2 (CCL2), and CCL3. Elevated mRNA levels for IL-1α, IL-1RA, occurred with injury and protection. mRNA and protein levels of IL-6 and neuronal expression of IL-6 receptor α, were elevated with injury and protection. Microarray pathway analysis supported an up-regulation of TNFα cell death signaling pathways with TMT and inhibition by exercise. IL-6 pathway recruitment occurred in both conditions. IL-6 downstream signal events differed in the level of STAT3 activation. Exercise did not increase mRNA levels of brain derived neurotrophic factor, nerve growth factor, or glial derived neurotrophic factor. In IL-6 deficient mice, exercise did not attenuate TMT-induced tremor and a diminished level of neuroprotection was observed. These data suggest a contributory role for IL-6 induced by exercise for neuroprotection in the CNS similar to that seen in the periphery.

Krüppel-Like Zinc Finger Protein Glis2 Is Essential for the Maintenance of Normal Renal Functions

ABSTRACT To obtain insight into the physiological functions of the Krüppel-like zinc finger protein Gli-similar 2 (Glis2), mice deficient in Glis2 expression were generated. Glis2 mutant (Glis2mut) mice exhibit significantly shorter life spans than do littermate wild-type (WT) mice due to the development of progressive chronic kidney disease with features resembling nephronophthisis. Glis2mut mice develop severe renal atrophy involving increased cell death and basement membrane thickening in the proximal convoluted tubules. This development is accompanied by infiltration of lymphocytic inflammatory cells and interstitial/glomerular fibrosis. The severity of the fibrosis, inflammatory infiltrates, and glomerular and tubular changes progresses with age. Blood urea nitrogen and creatinine increase, and Glis2mut mice develop proteinuria and ultimately die prematurely of renal failure. A comparison of the gene expression profiles of kidneys from 25-day-old/60-day-old WT and Glis2mut mice by microarray analysis showed increased expressions of many genes involved in immune responses/inflammation and fibrosis/tissue remodeling in kidneys of Glis2mut mice, including several cytokines and adhesion and extracellular matrix proteins. Our data demonstrate that a deficiency in Glis2 expression leads to tubular atrophy and progressive fibrosis, similar to nephronophthisis, that ultimately results in renal failure. Our study indicates that Glis2 plays a critical role in the maintenance of normal kidney architecture and functions.