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Translational Control of Ceruloplasmin Gene Expression: Beyond the IRE

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Translational Control of Ceruloplasmin Gene Expression: Beyond the IRE MAZUMDER ET AL. Biol Res 39, 2006, 59-66 59 Biol Res 39: 59-66, 2006 BR Translational control of ceruloplasmin gene expression: Beyond the IRE BARSANJIT MAZUMDER1, PRABHA SAMPATH2 and PAUL L. FOX3 1Department of Biology, Cleveland State University, Cleveland, OH, 2Department of Pathology, University of Washington, Seattle, WA, USA, and 3Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA ABSTRACT Translational control is a common regulatory mechanism for the expression of iron-related proteins. For example, three enzymes involved in erythrocyte development are regulated by three different control mechanisms: globin synthesis is modulated by heme-regulated translational inhibitor; erythroid 5- aminolevulinate synthase translation is inhibited by binding of the iron regulatory protein to the iron response element in the 5’-untranslated region (UTR); and 15-lipoxygenase is regulated by specific proteins binding to the 3’-UTR. Ceruloplasmin (Cp) is a multi-functional, copper protein made primarily by the liver and by activated macrophages. Cp has important roles in iron homeostasis and in inflammation. Its role in iron metabolism was originally proposed because of its ferroxidase activity and because of its ability to stimulate iron loading into apo-transferrin and iron efflux from liver. We have shown that Cp mRNA is induced by interferon (IFN)-γ in U937 monocytic cells, but synthesis of Cp protein is halted by translational silencing. The silencing mechanism requires binding of a cytosolic inhibitor complex, IFN-Gamma-Activated Inhibitor of Translation (GAIT), to a specific GAIT element in the Cp 3’-UTR. Here, we describe our studies that define and characterize the GAIT element and elucidate the specific trans-acting proteins that bind the GAIT element. Our experiments describe a new mechanism of translational control of an iron-related protein and may shed light on the role that macrophage-derived Cp plays at the intersection of iron homeostasis and inflammation. Key terms: Translational control, ceruloplasmin, iron homeostasis, GAIT element, inflammation Translational control of expression of control mechanisms of two types: global proteins of iron metabolism control in which synthesis of many or most proteins is regulated and transcript-selective Actively translating mRNAs usually consist control in which the synthesis of one (or of (starting from the 5’-end) an m7GpppN several) protein(s) is regulated. Global cap, a 5’-UTR, the coding region, a 3’-UTR, protein synthesis is commonly inhibited by and a poly(A) tail. Translation is initiated by phosphorylation and reversible inactivation a sequence of events initiated by binding of of a critical translation factor, eukaryotic a cap-binding complex, recruitment of the translation initiation factor 2α (eIF2α). Four 43S pre-initiation complex (containing the regulatable kinases are known to 40S ribosomal subunit), scanning of the 43S phosphorylate eIF2α and inhibit global complex to the AUG initiation codon, and protein synthesis: heme-regulated joining of the 60S large ribosomal subunit to translational inhibitor (HRI), which is form a translation-competent 80S ribosome activated by heme or iron deficiency; (Gebauer and Hentze, 2004). The rate of double-stranded RNA-regulated protein translation may be regulated by secondary kinase (PKR), which is activated by viral Correspondence: Paul L. Fox, Ph.D., Professor of Molecular Medicine, Department of Cell Biology / NC10, The Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA, Tel.: (1-216) 444-8053, Fax: (1-216) 444-9404, E-mail: [email protected] Received: March 31, 2005. Accepted: April 17, 2005. 60 MAZUMDER ET AL. Biol Res 39, 2006, 59-66 infection or double-stranded RNA; PKR-like this translational control has been endoplasmic reticulum (ER) kinase (PERK), investigated in detail. Iron deficiency which is activated by unfolded proteins in induces the binding of an iron regulatory the ER; and GCN2, which is activated by protein (IRP) to an iron-responsive element amino acid deficiency (Dever, 2002). In (IRE) in the 5’-untranslated region (UTR) most cases of transcript-selective regulation, of the mRNA. The IRE is a small stem-loop translational control is dictated by binding of structure that lacks the thermodynamic an RNA-binding protein to a cis-acting stability on its own to block translation but, structural element in the 5’- or 3’-UTR of in conjunction with the IRP, blocks the the target transcript. In eukaryotic cells, association of the 43S translation pre- initiation is the usual regulated step, and initiation complex (which contains the protein/mRNA interactions generally small ribosomal subunit) to the IRE- repress, rather than activate, translation containing target mRNA (Gray and Hentze, (Standart and Jackson, 1994). 1994). Control of expression at the level of Control of 15-lipoxygenase (15-LOX) translation is a pervasive feature of metal- expression represents the third distinct containing and metal-related proteins. In fact, translational control mechanism involving several major mechanisms of translational erythrocyte homeostasis. 15-LOX is control were discovered in studies of these responsible for red cell mitochondrial proteins. Iron deficiency has long been known membrane breakdown, and its translation is to inhibit total (global) protein synthesis inhibited specifically in erythroid precursor specifically in reticulocytes. This event is cells. The silencing mechanism involves partly responsible for the hypochromic binding of a complex of heterogeneous (hemoglobin-deficient) erythrocytes in iron- nuclear ribonucleoproteins (hnRNP) K and deficiency in humans. As mentioned above, E1 to the differentiation control element protein synthesis is blocked by activation of (DICE) in the 3’-UTR of 15-LOX (Ostareck HRI, a heme-regulated eIF2α kinase (Crosby et al., 1997). Interaction of the regulatory et al., 1994). Phosphorylation of eIF2α blocks complex with the 3’-UTR prevents joining of the formation of the ternary complex (eIF2, the 60S ribosomal subunit at the AUG codon GTP, and the charged initiator tRNA Met- near the distant 5’-UTR of the transcript Met tRNAi ) required for initiation of protein (Ostareck et al., 2001). hnRNP K is activated synthesis in all eukaryotic cells. The cell by ERK-mediated phosphorylation, which specificity of the inhibition results from the results in cytoplasmic accumulation and relatively specific localization of HRI in inhibition of 15-LOX translation (Habelhah et reticulocytes. Since globin mRNA is the al., 2001). hnRNP K is inactivated by src- major transcript in these cells, global mediated phosphorylation, which then inhibition of protein synthesis causes a permits 15-LOX translation and breakdown dramatic decrease in globin synthesis and of mitochondrial (and other) membranes hemoglobin formation, the hallmark of iron- required during late stages of reticulocyte deficiency anemia. maturation (Ostareck-Lederer et al., 2002). In the best-studied example of transcript-specific translational control, Ceruloplasmin and its Role in Iron synthesis of multiple proteins of iron Homeostasis and Inflammation metabolism is regulated at the level of translation (Klausner et al., 1993). Included Ceruloplasmin (Cp) is an acute-phase plasma among these proteins are erythroid 5- protein made by liver hepatocytes and by aminolevulinate synthase (eLAS), the rate- activated monocyte/macrophages. It is a 132 limiting enzyme in heme biosynthesis; kDa monomer with amino acid sequence ferroportin and divalent metal transporter 1 homology to Factors V and VIII of the (DMT1), two key iron transporters; and coagulation cascade and to hephaestin, a ferritin, the cellular iron storage protein and recently discovered copper protein involved the original protein in the family in intestinal iron absorption (Vulpe et al., (Pantopoulos, 2004). The mechanism of 1999). Cp contains 6-7 copper atoms per MAZUMDER ET AL. Biol Res 39, 2006, 59-66 61 molecule and accounts for 95% of the major source of Cp in inflammatory sites. circulating copper in healthy adults. The In our laboratory, we have investigated the mean, unevoked concentration in human “all-or-none” induction of Cp mRNA and serum is approximately 300 µg/ml. The protein by interferon (IFN)-γ in human physiological function of Cp is uncertain, monocytic U937 cells (Mazumder et al., but roles in bactericidal activity, 1997). Surprisingly, Cp protein synthesis coagulation, iron homeostasis, vascular stopped abruptly and almost completely relaxation, defense against oxidant stress, after about 16 h of IFN-γ treatment, even in and lipoprotein oxidation have been the presence of abundant transcript, a result proposed (see Musci, 2001, for review). An consistent with translational silencing elevated level of plasma Cp has been shown (Mazumder et al., 1997). Regulation at the to be a risk factor for cardiovascular level of translation was confirmed by pulse- diseases, including atherosclerosis and labeling experiments and by IFN-g- myocardial infarction (Fox et al., 2000). mediated release of ribosomes from the Cp Cp is an important regulator of iron transcript, the latter finding consistent with metabolism in vitro and in vivo. It is the an inhibition of translation-initiation principal plasma enzyme that catalyzes iron (Mazumder and Fox, 1999). IFN-γ did not oxidation, and is required for efficient iron inhibit total protein synthesis indicating a loading into
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