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Item Death-Associated Protein Kinase Regulates Vascular Smooth Muscle Cell Signaling and Migration(2011-03-16) Blue, Emily Keller; Gallagher, Patricia J.; Elmendorf, Jeffrey S.; Herring, B. Paul; Rhodes, Simon J.; Thurmond, Debbie C.Cardiovascular disease is the number one cause of death for Americans. New treatments are needed for serious conditions like atherosclerosis, as it can lead to stroke and heart attack. Many types of cells contribute to the progression of cardiovascular disease, including smooth muscle cells that comprise the middle layers of arteries. Inappropriate growth and migration of smooth muscle cells into the lumen of arteries has been implicated in vascular diseases. Death associated protein kinase (DAPK) is a protein that has been found to regulate the survival and migration of cancer cells, but has not been well characterized in vascular cells. The objective of this work was to determine the signaling pathways that DAPK regulates in smooth muscle cells. These studies have focused on smooth muscle cells isolated from human coronary arteries (HCASM cells). We have determined that HCASM cells depleted of DAPK exhibit more rapid migration, showing that DAPK negatively regulates migration of vascular cells. Results from a focused RT-PCR array identified matrix metalloproteinase 9 (MMP9) as a gene that is increased in cells depleted of DAPK. MMP9 is an important enzyme that degrades collagen, a component of the extracellular matrix through which smooth muscle cells migrate during atherosclerosis. We found that DAPK regulates phosphorylation of the NF-kappa B transcription factor p65 at serine 536, a modification previously found to correlate with increased nuclear levels and activity of p65. In DAPK-depleted HCASM cells, there was more phosphorylation of p65, which causes increased MMP9 promoter activity. Additional experiments were conducted using transgenic mice in which the DAPK gene has been deleted. By studying these mice, we have determined that under some circumstances DAPK augments maximal MMP9 levels in mouse carotid arteries which have been injured by ligation surgery via other signaling pathways. MMP9 has been previously implicated as a protein that promotes vascular diseases such as atherosclerosis. Our research in identifying DAPK as a regulator of MMP9 expression identifies a new target for treatment of vascular diseases like atherosclerosis.Item Effect of Inhibition of S-Nitrosoglutathione Reductase on the NF-κB Pathway(2009-09-30T19:08:58Z) Fears, Sharry L.; Sanghani, Sonal P.; Sanghani, Paresh C.; Bosron, William F.S-nitrosoglutathione reductase (GSNOR) also known as glutathione- dependent formaldehyde dehydrogenase (FDH), is a zinc-dependent dehydrogenase. GSNOR oxidizes long chain alcohols to an aldehyde with the help of a molecule of NAD+. GSNOR was initially identified as FDH because of its role in the formaldehyde detoxification pathway. The only S-nitrosothiol (SNO) substrate recognized by GSNOR is GSNO. A transnitrosation reaction transfers NO from nitrosylated proteins or S-nitrosothiols (RSNO) to glutathione to form S-nitrosoglutathione. This GSNO is finally converted to glutathione disulfide (GSSG) by a two step mechanism. Cellular GSNO is a nitric oxide reservoir that can either transfer to or remove from the proteins a NO group. Reduction of GSNO by GSNOR depletes this reservoir and therefore indirectly regulates protein nitrosylation. GSNOR inhibitors which can increase the basal GSNO levels will be another potential therapy. Several GSNOR inhibitors were identified in our laboratory and the aim of this study was to understand their cellular effects. One of the experiments studied the effect of the compound on protein-SNO. The role of nitric oxide in regulation of NF-κB pathway is reviewed by Bove and van der Vliet. We focused on identification of nitrosylated proteins using protein specific antibodies. We identified nitrosylation of IKKβ. So the question raised was whether nitrosylation of IKKβ affects its activity. IKKβ is responsible for phosphorylation of IκBα and phosphorylation of IκBα results in its degradation and activation of NF-κB pathway. Therefore, we studied the phosphorylation of IκBα in the presence of inhibitor C3. Results showed a dose-dependent decrease of pIκB. So the next question was whether the phosphorylation of IKKβ was affected by nitrosylation. We did not detect any change in pIKKβ with different concentrations of C3. The decreased degradation of IκBα caused by C3 translated into decreased NF-κB activity as seen by a dose-dependent decrease in amounts of ICAM-1 with increasing C3 concentration. This data supports the premise that the activity of transcription factor NF-κB is suppressed by inhibiting GSNOR with compound C3Item Functional characterization of a competitive peptide antagonist of p65 in human macrophage-like cells suggests therapeutic potential for chronic inflammation(Dove Medical Press, 2014) Srinivasan, Mythily; Blackburn, Corinne; Lahiri, Debomoy K.; Department of Oral Pathology, Medicine and Radiology, IU School of DentistryGlucocorticoid-induced leucine zipper (GILZ) is a glucocorticoid responsive protein that links the nuclear factor-kappa B (NFκB) and the glucocorticoid signaling pathways. Functional and binding studies suggest that the proline-rich region at the carboxy terminus of GILZ binds the p65 subunit of NFκB and suppresses the immunoinflammatory response. A widely-used strategy in the discovery of peptide drugs involves exploitation of the complementary surfaces of naturally occurring binding partners. Previously, we observed that a synthetic peptide (GILZ-P) derived from the proline-rich region of GILZ bound activated p65 and ameliorated experimental encephalomyelitis. Here we characterize the secondary structure of GILZ-P by circular dichroic analysis. GILZ-P adopts an extended polyproline type II helical conformation consistent with the structural conformation commonly observed in interfaces of transient intermolecular interactions. To determine the potential application of GILZ-P in humans, we evaluated the toxicity and efficacy of the peptide drug in mature human macrophage-like THP-1 cells. Treatment with GILZ-P at a wide range of concentrations commonly used for peptide drugs was nontoxic as determined by cell viability and apoptosis assays. Functionally, GILZ-P suppressed proliferation and glutamate secretion by activated macrophages by inhibiting nuclear translocation of p65. Collectively, our data suggest that the GILZ-P has therapeutic potential in chronic CNS diseases where persistent inflammation leads to neurodegeneration such as multiple sclerosis and Alzheimer's disease.Item NF-κB-dependent and -independent epigenetic modulation using the novel anti-cancer agent DMAPT(Nature Publishing Group, 2015-01-22) Nakshatri, H.; Appaiah, H. N.; Anjanappa, M.; Gilley, D.; Tanaka, H.; Badve, Sunil; Crooks, P. A.; Mathews, W.; Sweeney, C.; Bhat-Nakshatri, P.; Department of Surgery, IU School of MedicineThe transcription factor nuclear factor-kappaB (NF-κB) is constitutively active in several cancers and is a target of therapeutic development. We recently developed dimethylaminoparthenolide (DMAPT), a clinical grade water-soluble analog of parthenolide, as a potent inhibitor of NF-κB and demonstrated in vitro and in vivo anti-tumor activities in multiple cancers. In this study, we show DMAPT is an epigenetic modulator functioning in an NF-κB-dependent and -independent manner. DMAPT-mediated NF-κB inhibition resulted in elevated histone H3K36 trimethylation (H3K36me3), which could be recapitulated through genetic ablation of the p65 subunit of NF-κB or inhibitor-of-kappaB alpha super-repressor overexpression. DMAPT treatment and p65 ablation increased the levels of H3K36 trimethylases NSD1 (KMT3B) and SETD2 (KMT3A), suggesting that NF-κB directly represses their expression and that lower H3K36me3 is an epigenetic marker of constitutive NF-κB activity. Overexpression of a constitutively active p65 subunit of NF-κB reduced NSD1 and H3K36me3 levels. NSD1 is essential for DMAPT-induced expression of pro-apoptotic BIM, indicating a functional link between epigenetic modification and gene expression. Interestingly, we observed enhanced H4K20 trimethylation and induction of H4K20 trimethylase KMT5C in DMAPT-treated cells independent of NF-κB inhibition. These results add KMT5C to the list NF-κB-independent epigenetic targets of parthenolide, which include previously described histone deacetylase 1 (HDAC-1) and DNA methyltransferase 1. As NSD1 and SETD2 are known tumor suppressors and loss of H4K20 trimethylation is an early event in cancer progression, which contributes to genomic instability, we propose DMAPT as a potent pharmacologic agent that can reverse NF-κB-dependent and -independent cancer-specific epigenetic abnormalities.Item A proteasome-resistant fragment of NIK mediates oncogenic NF-κB signaling in schwannomas(Oxford University Press, 2019-02-15) Gehlhausen, Jeffrey R.; Hawley, Eric; Wahle, Benjamin Mark; He, Yongzheng; Edwards, Donna; Rhodes, Steven D.; Lajiness, Jacquelyn D.; Staser, Karl; Chen, Shi; Yang, Xianlin; Yuan, Jin; Li, Xiaohong; Jiang, Li; Smith, Abbi; Bessler, Waylan; Sandusky, George; Stemmer-Rachamimov, Anat; Stuhlmiller, Timothy J.; Angus, Steven P.; Johnson, Gary L.; Nalepa, Grzegorz; Yates, Charles W.; Clapp, D. Wade; Park, Su-Jung; Pediatrics, School of MedicineSchwannomas are common, highly morbid and medically untreatable tumors that can arise in patients with germ line as well as somatic mutations in neurofibromatosis type 2 (NF2). These mutations most commonly result in the loss of function of the NF2-encoded protein, Merlin. Little is known about how Merlin functions endogenously as a tumor suppressor and how its loss leads to oncogenic transformation in Schwann cells (SCs). Here, we identify nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-inducing kinase (NIK) as a potential drug target driving NF-κB signaling and Merlin-deficient schwannoma genesis. Using a genomic approach to profile aberrant tumor signaling pathways, we describe multiple upregulated NF-κB signaling elements in human and murine schwannomas, leading us to identify a caspase-cleaved, proteasome-resistant NIK kinase domain fragment that amplifies pathogenic NF-κB signaling. Lentiviral-mediated transduction of this NIK fragment into normal SCs promotes proliferation, survival, and adhesion while inducing schwannoma formation in a novel in vivo orthotopic transplant model. Furthermore, we describe an NF-κB-potentiated hepatocyte growth factor (HGF) to MET proto-oncogene receptor tyrosine kinase (c-Met) autocrine feed-forward loop promoting SC proliferation. These innovative studies identify a novel signaling axis underlying schwannoma formation, revealing new and potentially druggable schwannoma vulnerabilities with future therapeutic potential.Item Regulation of the hepatitis B virus core promoter by NF-kappa B(1995) Quets, Anne Trudy