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NF k B – What is it and What’s the deal with radicals?

NF k B – What is it and What’s the deal with radicals?. The Virtual Free Radical School. Emily Ho, Ph.D Linus Pauling Institute Scientist Department of Nutrition and Food Management Oregon State University 117 Milam Hall Corvallis, OR 97331

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NF k B – What is it and What’s the deal with radicals?

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  1. NFkB – What is it and What’s the deal with radicals? The Virtual Free Radical School Emily Ho, Ph.D Linus Pauling Institute Scientist Department of Nutrition and Food Management Oregon State University 117 Milam Hall Corvallis, OR 97331 emily.ho@oregonstate.edu (Tel) 541-737-9559; (Fax) 541-737-6914 NFB 9/2002 SFRBM EducationProgram Emily Ho 1

  2. Cytotoxicity of ROS O2•¯ DNA oxidation Fe/Cu H2O2 HO• Protein oxidation Lipid oxidation More recently, the role of ROS as a signal molecules has gained increasing attention. The cytotoxicity of ROS may be associated with the ability of ROS to signal distinct pathways, such as the NFkB pathway, to induce pathology. NFB 9/2002 SFRBM EducationProgram Emily Ho 2

  3. What is NFkB? • First discovered by Baltimore & Sen as a B cell specific nuclear protein that binds to a site in the immunoglobulin k light chain gene enhancer (Cell 47:921-928, 1986) • NFkB comprises a family of transcription factors that are involved in regulating a large number of genes related to immune function, inflammation, apoptosis and cell proliferation. • Mammalian cells have 5 distinct NFkB subunits based on a highly conserved 300 amino acid dimerization domain called the rel homology domain. • Several different combinations of subunits in the cytoplasm, the most common being a heterodimer of p50/p65 (Rel A) and the inhibitory IkBa inhibitory subunit. NFB 9/2002 SFRBM EducationProgram Emily Ho 3

  4. Family of NFkB and Inhibitory kB (IkB) proteins Nat.Rev.Cancer 2:301-310, 2002 NFB 9/2002 SFRBM EducationProgram Emily Ho 4

  5. Genes Regulated by NFkB Nat.Rev.Cancer 2:301-310, 2002 NFB 9/2002 SFRBM EducationProgram Emily Ho 5

  6. Function of NFkB • Key mediator of a variety of cellular responses • Immune and inflammatory response • Cell proliferation and survival • Protecting cells from undergoing apoptosis in response to DNA damage or cytokine treatment Many chronic disease states have been associated with aberrant activation of NFkB, and several therapeutic strategies targeting NFkB activation have been considered for the treatment of inflammation and cancer. NFB 9/2002 SFRBM EducationProgram Emily Ho 6

  7. Rheumatoid arthritis Atherosclerosis Vascular dysfunction Multiple sclerosis Neurodegenerative disorders Inflammatory bowel disease H. pylori-associated gastritis Systemic inflammatory response syndrome And the list is growing… Autoimmune thyroid disease Cystic fibrosis Diabetes Aging Macular degeneration HIV/AIDS Cancer Septic shock Disorders associated with aberrant NFkB activation NFB 9/2002 SFRBM EducationProgram Emily Ho 7

  8. B cell lymphoma Hodgkin’s disease T-cell lymphoma Acute lymphoblastic leukemia Breast Liver Thyroid Prostate Melanoma Head and neck SCC Colon Multiple myeloma Ovarian Bladder Lung Tumors that express constitutively active NFkB Leukemia16:1053-1068, 2002 NFB 9/2002 SFRBM EducationProgram Emily Ho 8

  9. Factors that induce NFkB Reactive Oxygen Species (ROS) Leukemia16:1053-1068, 2002 NFB 9/2002 SFRBM EducationProgram Emily Ho 9

  10. Activation of NFkB • NFkB is normally found in its inactive form in the cytosol as the heterodimer p50/65 bound to its inhibitory unit IkBa • In response to extracellular inducers, such as ROS, the IkBa kinase complex is activated and IkBa becomes phosphorylated at serines 32 and 36, and leads to ubiquination at lysines 21 and 22 • This leads to degradation of IkBa subunit by the 26S proteosome • Degradation of the inhibitory subunit, releases the p50/p65 complex, allowing the complex to translocate from the cytoplasm to the nucleus • In the nucleus, the transcription factor binds to a consensus sequence (5’-GGGACTTTC-3’) and activates gene expression NFB 9/2002 SFRBM EducationProgram Emily Ho 10

  11. Activating Stimuli (ROS) Inflammatory/Immune Proteins IkB kinase/NEMO/IKAP P p50 IkBa IkBa IkBa p65 p65 p50 Degradation mRNA CYTOPLASM p65 p50 NUCLEUS Transcription p65 p50 Translocation Target genes NFB 9/2002 SFRBM EducationProgram Emily Ho 11

  12. Role of ROS and Redox Status in NFkB activation • Many factors that activate NFkB also produce ROS. • Hypoxia/reoxygenation and oxidants (such as hydrogen peroxide) have been shown to induce NFkB activation in some cell types. • Inhibition or overexpression of enzymes that affect intracellular ROS can modulate activation of NFkB. • Antioxidant supplementation can block NFkB activation. • The DNA binding domain needs to be in reduced form, (especially cysteine 62) in vitro, to bind to its responsive elements. • Redox regulation may be dual-fold: reducing conditions can block IkB degradation but can enhance transcriptional activity by enhancing its ability to bind in the nucleus NFB 9/2002 SFRBM EducationProgram Emily Ho 12

  13. p65 p65 p65 p65 p65 p65 p65 p65 p50 p50 p50 p50 p50 p50 p50 p50 IkB Modulation of NFkB Activation by Antioxidants Reactive Oxygen Species (ROS) Inactiveform - Antioxidants IkB Kinase IkB Ubiquitination Proteolysis P NFkB IkB NUCLEUS Degradation Antioxidants + Active form Nuclear translocation target genes DNA binding domain NFB 9/2002 SFRBM EducationProgram Emily Ho 13

  14. Free Radic Biol Med 25:346-361 NFB 9/2002 SFRBM EducationProgram Emily Ho 14

  15. Some Considerations… • Activation of NFkB by hydrogen peroxide may be cell specific. • Not all activation pathways require oxidative stress as a component • Lipid peroxides may be important in activation in some cell types • Antioxidants may inhibit NFkB activity through mechanisms distinct from redox regulation • However, in certain cases, oxidative stress is a potent activator of NFkB and has an important role in regulating cell survival and immune response. NFB 9/2002 SFRBM EducationProgram Emily Ho 15

  16. Summary • NFkB plays an important role in regulating immune and inflammatory response, apoptosis and cell survival. • ROS and redox status plays an important role in NFkB activation in some cases. • Several steps in the activation cascade may be affected by redox status, including IKK complex phosphorylation and DNA binding. • Antioxidants may be an effective strategy in modulating excess NFkB activation in chronic inflammatory states and cancer. NFB 9/2002 SFRBM EducationProgram Emily Ho 16

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