Tumor necrosis factor (TNF) is a multifunctional cytokine secreted primarily by mitogen-activated macrophages. The biological properties of TNF include modulation of differentiation, promotion of inflammation, chemoattraction of neutrophils and macrophages, cytotoxicity towards certain cells and induction of expression of genes involved in immune function and inflammation (such as IL-1 and Il-6). Expression of TNF is associated with endotoxic shock, with inflammatory manifestations associated with rheumatoid arthritis, with cachexia associated with cancer and AIDS and with other diseases. The ability of TNF to induce specific gene expression is controlled (at least in part) by its ability to induced the DNA-binding activity of pre-existing transcription factor known as NF-kB. NF-kB binds to regulatory elements in genes such as Il-1, IL-6, MHC classI, IL-2 and TNFalpha itself and stimulates transcription. Classic NF-kB is composed of a heterodimer of a 50kD and 65kD subunit, both of which are related to the product of the c-rel proto-oncogene. Interestingly, the 50kD subunit is derived by proteolytic processing from a precursor protein known as IkB. In vitro studies have shown the phosphorylation of IB likely plays a role in release of IkB from NF-kB. We recently identified a cDNA encoding one form of IkB (IkBalpha/MAD-3) and demonstrated that it contains multiple copies of the so-called ankyrin/SWI6 repeats. Our data demonstrate that TNFalpha induces the rapid in vivo phosphorylation of IkBalpha. Furthermore, we have shown that IkBa is rapidly lost format he TNF-treated cells and two forms of NF-kB associated with IkB concomitantly appear in the nucleus. Within 1-2 hrs, the normal levels of IkBalpha have returned to the TNF-treated cells. Intriguingly, okadaic acid (an inhibitor of type 1 and type 2A phosphatases) induced a similar response to that induced by TNF (the apparent phosphorylation and loss of IkBalpha). In this NIH application we propose to: (1) determine the molecular basis of the phosphorylation of IkBalpha induced b TNF (2) the rapid resynthesis of IkBalpha following release of NF-kB (experiments focus n the promoter of the IkBalpha gene (4) characterize the signal transduction pathways involved in the phosphorylation of IkB, determining whether inactivation of a phosphatase is involved in the process (5) determine whether such a phosphatase is a target of oxygen radicals which are known to induce NF-kB, (6) assay the precursor forms of NF-kB and another inhibitor Bcl-3 to determine if they are also targets of TNF, and (7) characterize two TNF responsive elements, one in the c-jun promoter and one in the IL-8 promoter. These experiments will lead to the enhancement of our knowledge of the mechanisms of action of TNF and of the transduction pathways involved in its signaling.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI035098-05
Application #
2633542
Study Section
Allergy and Immunology Study Section (ALY)
Project Start
1994-01-01
Project End
1998-12-31
Budget Start
1998-01-01
Budget End
1998-12-31
Support Year
5
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Hsia, Hung-Ching; Hutti, Jessica E; Baldwin, Albert S (2017) Cytosolic DNA Promotes Signal Transducer and Activator of Transcription 3 (STAT3) Phosphorylation by TANK-binding Kinase 1 (TBK1) to Restrain STAT3 Activity. J Biol Chem 292:5405-5417
Hsia, Hung-Ching; Stopford, Charles M; Zhang, Zhigang et al. (2017) Signal transducer and activator of transcription 3 (Stat3) regulates host defense and protects mice against herpes simplex virus-1 (HSV-1) infection. J Leukoc Biol 101:1053-1064
Bradford, Jennifer W; Baldwin, Albert S (2014) IKK/nuclear factor-kappaB and oncogenesis: roles in tumor-initiating cells and in the tumor microenvironment. Adv Cancer Res 121:125-145
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Stein, Sarah J; Baldwin, Albert S (2013) Deletion of the NF-?B subunit p65/RelA in the hematopoietic compartment leads to defects in hematopoietic stem cell function. Blood 121:5015-24
Hutti, Jessica E; Porter, Melissa A; Cheely, Adam W et al. (2012) Development of a high-throughput assay for identifying inhibitors of TBK1 and IKK?. PLoS One 7:e41494
Awwad, Yousef; Geng, Tao; Baldwin, Albert S et al. (2012) Observing single cell NF-?B dynamics under stimulant concentration gradient. Anal Chem 84:1224-8
Hutti, Jessica E; Pfefferle, Adam D; Russell, Sean C et al. (2012) Oncogenic PI3K mutations lead to NF-ýýB-dependent cytokine expression following growth factor deprivation. Cancer Res 72:3260-9
Comb, William C; Hutti, Jessica E; Cogswell, Patricia et al. (2012) p85ýý SH2 domain phosphorylation by IKK promotes feedback inhibition of PI3K and Akt in response to cellular starvation. Mol Cell 45:719-30

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