Browsing by Author "Harris, Robert A. (Robert Allison), 1939-"

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    Effects of choline kinase activity on phospholipid metabolism and malignant phenotype of prostate cancer cells
    (2010-10) Bansal, Aditya; DeGrado, Timothy R.; Harris, Robert A. (Robert Allison), 1939-; Bosron, William F.; Klaunig, James E.
    High choline uptake and increased choline kinase activity have been reported in many cancers. This has motivated the use of choline as a biomarker for tumor imaging. Tumors in general are heterogeneous in nature with respect to oxygen tension. There are regions of hypoxia and normoxia that are expected to have different metabolism but regulation of choline metabolism under hypoxia is poorly understood. It is important to clarify the status of choline metabolism in hypoxic microenvironment as it will have an impact on potential of choline as a cancer biomarker. The primary goal was to determine the status of choline phosphorylation in hypoxic cancer cells and its effect on uptake of choline. This was examined by tracer studies in cancer cells exposed to hypoxia. It was observed that hypoxia universally inhibits choline uptake /phosphorylation in cancer cells. Decreased choline phosphorylation resulted in transient uptake of choline radiotracers in cultured cancer cells and 9L tumors suggesting potential problem in using choline as a biomarker for cancers in hypoxic microenvironment. To investigate the mechanism behind decrease in choline phosphorylation, steady state levels of choline metabolites were measured and choline kinase catalyzed choline phosphorylation step was found to be rate-limiting in PC-3 cells. This suggested that modulation in choline kinase levels can alter choline metabolism in hypoxic cancer cells. Expression and activity assays for choline kinase revealed that choline kinase expression is down-regulated in hypoxia. This regulation involved transcriptional level mediation by HIF1 at the conserved HRE7 site in choline kinase promoter. To further understand the importance of down-regulation of choline kinase in hypoxia, stable prostate cancer cell lines over-expressing choline kinase were generated. Effect of over-expression of choline kinase in hypoxia was evaluated in terms of malignant phenotypes like proliferation rate, anchorage independent growth and invasion potential. Both over-expression of choline kinase and hypoxia had a pronounced effect on malignant phenotypes of prostate cancer cells. Further study showed that increased choline kinase activity and hypoxic tumor microenvironment are important for progression of early-stage, androgen-dependent LNCaP prostate cancer cells but confer little survival advantage in undifferentiated, androgen-independent PC-3 prostate cancer cells.
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    Elucidating mechanisms that lead to persistent anxiety-like behavior in rats following repeated activation of corticotropin-releasing factor receptors in the basolateral amygdala
    (2012-03-16) Gaskins, Denise; Shekhar, Anantha, 1957-; Harris, Robert A. (Robert Allison), 1939-; Hingtgen, Cynthia M., 1966-; Truitt, William A.
    Anxiety disorders are estimated to impact 1 in 4 individuals within their lifetime. For some individuals, repeated episodes of the stress response leads to pathological anxiety and depression. The stress response is linked to increased levels of corticotropin-releasing factor (CRF) in the basolateral nucleus of the amygdala (BLA), a putative site for regulating anxiety and associative processes related to aversive emotional memories, and activation of CRF receptors in the BLA of rats produces anxiety-like behavior. Mimicking repeated episodes of the stress response, sub-anxiogenic doses of urocortin 1 (Ucn1), a CRF receptor agonist, are microinjected into the BLA of rats for five consecutive days, a procedure called priming. This results in 1) behavioral sensitization, such that a previously non-efficacious dose of Ucn1 will elicit anxiety-like response after the 3rd injection and 2) the development of a persistent anxiety-like phenotype that lasts at least five weeks after the last injection without any further treatment. Therefore, the purpose of this thesis was to identify mechanisms involved in the Ucn1-priming-induced anxiogenesis. The first a set of experiments revealed that the anxiety-like behavior was not due to aversive conditioning to the context or partner cues of the testing environment. Next, Ucn1-priming-induced gene expression changes in the BLA were identified: mRNA expression for Sst2, Sst4, Chrna4, Chrma4, and Gabrr1 was significantly reduced in Ucn1-primed compared to Vehicle-primed rats. Of these, Sst2 emerged as the primary receptor of interest. Subsequent studies found that antagonizing the Sstr2 resulted in anxiety-like behavior and activation of Sstr2 blocked acute Ucn1-induced anxiety-like responses. Furthermore, pretreatment with a Sstr2 agonist delayed the behavioral sensitization observed in Ucn1-induced priming but did not stop the development of persistent anxiety-like behavior or the Ucn1-priming-induced decrease in the Sstr2 mRNA. These results suggest that the decrease in Sstr2 mRNA is associated with the expression of persistent anxiety-like behavior but dissociated from the mechanisms causing the behavioral sensitization. Pharmacological studies confirmed that a reduced Sstr2 mediated effect in the BLA is likely to play a role in persistent anxiety and should be investigated further.
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    The mechanisms regulating the transcription factor ATF5 and its function in the integrated stress response
    (2010-11) Zhou, Donghui; Wek, Ronald C.; Harris, Robert A. (Robert Allison), 1939-; Quilliam, Lawrence; Morral, Nuria
    Phosphorylation of eukaryotic initiation factor 2 (eIF2) is an important mechanism regulating global and gene-specific translation during different environmental stresses. Repressed global translation by eIF2 phosphorylation allows for cells to conserve resources and elicit a program of gene expression to alleviate stress-induced injury. Central to this gene expression program is eIF2 phosphorylation induction of preferential translation of ATF4. ATF4 is a transcriptional activator of genes involved in stress remediation, a pathway referred to as the Integrated Stress Response (ISR). We investigated whether there are additional transcription factors whose translational expression is regulated by eIF2 kinases. We found that the expression of the transcriptional regulator ATF5 is enhanced in response to many different stresses, including endoplasmic reticulum stress, arsenite exposure, and proteasome inhibition, by a mechanism requiring eIF2 phosphorylation. ATF5 is regulated by translational control as illustrated by the preferential association of ATF5 mRNA with large polyribosomes in response to stress. ATF5 translational control involves two upstream open reading frames (uORFs) located in the 5′-leader of the ATF5 mRNA, a feature shared with ATF4. Mutational analyses of the 5′-leader of ATF5 mRNA fused to a luciferase reporter suggests that the 5′-proximal uORF1 is positive-acting, allowing scanning ribosomes to reinitiate translation of a downstream ORF. During non-stressed conditions, when eIF2 phosphorylation is low, ribosomes reinitiate translation at the next ORF, the inhibitory uORF2. Phosphorylation of eIF2 during stress delays translation reinitiation, allowing scanning ribosomes to bypass uORF2, and instead translate the ATF5 coding region. In addition to translational control, ATF5 mRNA and protein levels are significantly reduced in mouse embryo fibroblasts deleted for ATF4, or its target gene, the transcriptional factor CHOP. This suggests that ISR transcriptional mechanisms also contribute to ATF5 expression. To address the function of ATF5 in the ISR, we employed a shRNA knock-down strategy and our analysis suggests that ATF5 promotes apoptosis under stress conditions via caspase-dependent mechanisms. Given the well-characterized role of CHOP in the promotion of apoptosis, this study suggests that there is an ATF4-CHOP-ATF5 signaling axis in the ISR that can determine cell survival during different environmental stresses.
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    Pdx-1 modulates endoplasmic reticulum calcium homeostasis in the islet β cell via transcriptional enhancement of SERCA2b
    (2014-12) Johnson, Justin Sean; Evans-Molina, Carmella; Elmendorf, Jeffrey S.; Harris, Robert A. (Robert Allison), 1939-; Mirmira, Raghavendra G.
    Diabetes mellitus affects an estimated 285 million people worldwide, and a central component of diabetes pathophysiology is diminished pancreatic islet beta cell function resulting in the inability to manage blood glucose effectively. The beta cell is a highly specialized metabolic factory that possesses a number of specialized characteristics, chief among these a highly developed endoplasmic reticulum (ER). The sarco endoplasmic reticulum Ca2+ ATPase 2b (SERCA2b) pump maintains a steep Ca2+ gradient between the cytosol and ER lumen, and while the Pancreatic and duodenal homeobox 1 (Pdx-1) transcription factor is known to play an indispensable role in beta cell development and function, recent data also implicate Pdx-1 in the maintenance of ER health. Our data demonstrates that a decrease of beta cell Pdx-1 occurs in parallel with decreased SERCA2b expression in models of diabetes, while in silico analysis of the SERCA2b promoter reveals multiple putative Pdx-1 binding sites. We hypothesized that Pdx-1 loss under inflammatory and diabetic conditions leads to decreased SERCA2b with concomitant alterations in ER health. To test this, siRNA-mediated knockdown of Pdx-1 was performed in INS-1 cells. Results revealed reduced SERCA2b expression and decreased ER Ca2+, which was measured using an ER-targeted D4ER adenovirus and fluorescence lifetime imaging microscopy. Co-transfection of human Pdx-1 with a reporter fused to the human SERCA2 promoter increased luciferase activity three-fold relative to the empty vector control, and direct binding of Pdx-1 to the proximal SERCA2 promoter was confirmed by chromatin immunoprecipitation. To determine whether restoration of SERCA2b could rescue ER stress induced by Pdx-1 loss, Pdx1+/- mice were fed high fat diet for 8 weeks. Isolated islets from these mice demonstrated increased expression of spliced Xbp1, signifying ER stress, while subsequent SERCA2b overexpression in isolated islets reduced spliced Xbp1 levels to that of wild-type controls. These results identify SERCA2b as a direct transcriptional target of Pdx-1 and define a novel role for altered ER Ca2+ regulation in Pdx-1 deficient states.
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    REGULATION OF CHOP TRANSLATION IN RESPONSE TO eIF2 PHOSPHORYLATION AND ITS ROLE IN CELL FATE
    (2012-05) Palam, Lakshmi Reddy; Wek, Ronald C.; Herring, Brian P.; Harris, Robert A. (Robert Allison), 1939-; Skalnik, David G.
    In response to different environmental stresses, phosphorylation of eukaryotic initiation factor-2 (eIF2) rapidly reduces protein synthesis, which lowers energy expenditure and facilitates reprogramming of gene expression to remediate stress damage. Central to the changes in gene expression, eIF2 phosphorylation also enhances translation of ATF4, a transcriptional activator of genes subject to the Integrated Stress Response (ISR). The ISR increases the expression of genes important for alleviating stress, or alternatively triggering apoptosis. One ISR target gene encodes the transcriptional regulator CHOP whose accumulation is critical for stress-induced apoptosis. In this dissertation research, I show that eIF2 phosphorylation induces preferential translation of CHOP by a mechanism involving a single upstream ORF (uORF) located in the 5’-leader of the CHOP mRNA. In the absence of stress and low eIF2 phosphorylation, translation of the uORF serves as a barrier that prevents translation of the downstream CHOP coding region. Enhanced eIF2 phosphorylation during stress facilitates ribosome bypass of the uORF, and instead results in the translation of CHOP. Stable cell lines were also constructed that express CHOP transcript containing the wild type uORF or deleted for the uORF and each were analyzed for expression changes in response to the different stress conditions. Increased CHOP levels due to the absence of inhibitory uORF sensitized the cells to stress-induced apoptosis when compared to the cells that express CHOP mRNA containing the wild type uORF. This new mechanism of translational control explains how expression of CHOP and the fate of cells are tightly linked to the levels of phosphorylated eIF2 and stress damage.
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    The role of pyruvate dehydrogenase kinase in glucose and ketone body metabolism
    (2012-07) Rahimi, Yasmeen; Harris, Robert A. (Robert Allison), 1939-; Considine, Robert V.; Roach, Peter J.; Wek, Ronald C.
    The expression of pyruvate dehydrogenase kinase (PDK) 2 and 4 are increased in the fasted state to inactivate the pyruvate dehydrogenase complex (PDC) by phosphorylation to conserve substrates for glucose production. To assess the importance of PDK2 and PDK4 in regulation of the PDC to maintain glucose homeostasis, PDK2 knockout (KO), PDK4 KO, and PDK2/PDK4 double knockout (DKO) mice were generated. PDK2 deficiency caused higher PDC activity and lower blood glucose levels in the fed state while PDK4 deficiency caused similar effects in the fasting state. DKO intensified these effects in both states. PDK2 deficiency had no effect on glucose tolerance, PDK4 deficiency produced a modest effect, but DKO caused a marked improvement, lowered insulin levels, and increased insulin sensitivity. However, the DKO mice were more sensitive than wild-type mice to long term fasting, succumbing to hypoglycemia, ketoacidosis, and hypothermia. Stable isotope flux analysis indicated that hypoglycemia was due to a reduced rate of gluconeogenesis. We hypothesized that hyperglycemia would be prevented in DKO mice fed a high saturated fat diet for 30 weeks. As expected, DKO mice fed a high fat diet had improved glucose tolerance, decreased adiposity, and were euglycemic due to reduction in the rate of gluconeogenesis. Like chow fed DKO mice, high fat fed DKO mice were unusually sensitive to fasting because of ketoacidosis and hypothermia. PDK deficiency resulted in greater PDC activity which limited the availability of pyruvate for oxaloacetate synthesis. Low oxaloacetate resulted in overproduction of ketone bodies by the liver and inhibition of ketone body and fatty acid oxidation by peripheral tissues, culminating in ketoacidosis and hypothermia. Furthermore, when fed a ketogenic diet consisting of low carbohydrate and high fat, DKO mice also exhibited hypothermia, ketoacidosis, and hypoglycemia. The findings establish that PDK2 is more important in the fed state, PDK4 is more important in the fasted state, survival during long term fasting depends upon regulation of the PDC by both PDK2 and PDK4, and that the PDKs are important for the regulation of glucose and ketone body metabolism.
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    Starch-binding domain-containing protein 1: a novel participant in glycogen metabolism
    (2011-06) Jiang, Sixin; Roach, Peter J.; DePaoli-Roach, Anna A.; Harris, Robert A. (Robert Allison), 1939-; Morral, Nuria
    Glycogen, a branched polymer of glucose, acts as an intracellular carbon and energy reserve in many tissues and cell types. The breakdown of glycogen by hormonally regulated degradation involving the coordinated action of glycogen phosphorylase and debranching enzyme has been well studied. However, the importance of lysosomal disposal of glycogen has been underscored by a glycogen storage disorder, Pompe disease. This disease destroys tissues by over-accumulating glycogen in lysosomes due to a genetic defect in the lysosomal acid α-glucosidase. Details of the intracellular trafficking of glycogen are not well understood. Starch-binding domain-containing protein 1 (Stbd1) is a protein of previously unknown function with predicted hydrophobic N-terminus and C-terminal CBM20 carbohydrate binding domain. The protein is highly expressed in the liver and muscle, the major repositories of glycogen. Stbd1 binds to glycogen in vitro and in vivo with a preference for less branched and more phosphorylated polysaccharides. In animal models, the protein level of Stbd1 correlates with the genetic depletion of glycogen. Endogenous Stbd1 is found in perinuclear compartments in cultured mouse and rat cells. When over-expressed in cells, Stbd1 accumulates and coincides with glycogen and GABARAPL1, the autophagy protein. They form enlarged perinuclear structures which are abolished by removing the hydrophobic N-terminus of Stbd1. Stbd1, with point mutations in the CBM20 domain, retains the perinuclear localization but without concentration of glycogen in this compartment. In cells that are stably over-expressing glycogen synthase, glycogen exists as large perinuclear deposits, where Stbd1 can also be present. Removing glucose from the culture leads to a breakdown of the massive glycogen accumulation into numerous smaller and scattered deposits which are still positive for Stbd1. Furthermore, the autophagy protein GABARAPL1 co-immunoprecipates and co-localizes with Stbd1 when co-expressed in cells. Point mutation or deletion of the autophagy protein interacting region on Stbd1 eliminates the interaction and co-localization with GABARAPL1 but not the characteristic perinuclear distribution of Stbd1. We propose that Stbd1 is involved in glycogen metabolism. In particular, it participates in the vesicular transfer of glycogen to the lysosome with the recruitment of autophagy related proteins GABARAPL1 and/or GABARAP, as these vesicles mature prior to lysosomal fusion.