The central hypothesis of this proposal is that, in the Tumor Necrosis Factor-alpha (TNF)-activated pulmonary endothelium, reactive nitrogen species (RNS), such as nitric oxide (NO) and peroxynitrite (ONOO-) modulate the prolonged (greater than or equal to 4 hrs.) activation of Protein Kinase C-alpha (PKC-alpha), at least in part, by stimulating activator protein-1 (AP-1)-mediated transcription of PKCalpha. The pathogenetic sequence begins with RNS (either ONOO-, NO or both) activating guanylate cyclase, resulting in the generation of cGMP. The stimulated cGMP-PKG pathway phosphorylates the AP-1 subcomponents cFos and/or cJun, leading to AP-1 activation. The activated AP- 1 induces transcription of the PKCalpha gene. The increased transcription results in increased translation of PKCalpha mRNA into C Kinase protein, promoting the prolonged activation of PKC, with subsequent well-studied injurious effects on the pulmonary endothelium.
The specific aims of the current proposal are: (1) to study whether TNF induces transcription of the pulmonary endothelial PKCalpha gene, (2) to determine the roles of RNS generation and AP-1 activation in the TNF induction of message for pulmonary endothelial PKCalpha, (3) to investigate whether the cGMP-PKG-mediated phosphorylation induced by TNF and RNS activates AP-1, and (4) to probe the roles of RNS, AP-1 and PKCalpha message in the prolonged activation of pulmonary endothelial PKCalpha induced by TNF. The hypothesis will be tested using human and bovine pulmonary microvessel endothelial cells. TNF-induced PKC-alpha transcription is studied using Nuclear Run-On and Northern Blot of cytosolic RNA. AP-1 activation is investigated using the Electrophoretic Mobility Shift Assay. Transfection with AP-1 reporter plasmids is used to verify role of RNS in TNF-induced AP-1 activation in gene regulation. Antisense oligonucleotides (anti-cJun, anti-PKC-alpha) is used to verify the AP-1-mediated maintenance of TNF-induced PKC-alpha transcription and activity. The role of RNS and PKG is investigated using (1) direct assay and (2) agonists and antagonists of RNS and PKG. Synthesis of PKC-alpha is studied using immunoprecipitation and Western Blot of 35S-labeled PKC, and activity of PKC is assessed by PKC translocation and phosphorylation of PKC-alpha specific substrate.
| Johnson, Arnold; Neumann, Paul H; Peng, Jianya et al. (2015) The intracerebroventricular injection of rimonabant inhibits systemic lipopolysaccharide-induced lung inflammation. J Neuroimmunol 286:16-24 |
| Neumann, Paul; Alsaffar, Hiba; Gertzberg, Nancy et al. (2013) Inhibition of GSK3?/? promotes increased pulmonary endothelial permeability to albumin by reactive oxygen/nitrogen species. Pulm Pharmacol Ther 26:685-92 |
| Barton-Pai, Amy; Feleder, Carlos; Johnson, Arnold (2011) Tumor necrosis factor-? induces increased lung vascular permeability: a role for GSK3?/?. Eur J Pharmacol 657:159-66 |
| Johnson, Arnold (2009) TNF-induced activation of pulmonary microvessel endothelial cells: a role for GSK3beta. Am J Physiol Lung Cell Mol Physiol 296:L700-9 |
| Gertzberg, Nancy; Gurnani, Tina; Neumann, Paul et al. (2007) Tumor necrosis factor-alpha causes barrier dysfunction mediated by tyrosine198 and tyrosine218 in beta-actin. Am J Physiol Lung Cell Mol Physiol 293:L1219-29 |
| Neumann, Paul; Gertzberg, Nancy; Vaughan, Erin et al. (2006) Peroxynitrite mediates TNF-alpha-induced endothelial barrier dysfunction and nitration of actin. Am J Physiol Lung Cell Mol Physiol 290:L674-L684 |
| Huang, Fei; Subbaiah, Papasani V; Holian, Oksana et al. (2005) Lysophosphatidylcholine increases endothelial permeability: role of PKCalpha and RhoA cross talk. Am J Physiol Lung Cell Mol Physiol 289:L176-85 |
| Neumann, Paul; Gertzberg, Nancy; Johnson, Arnold (2004) TNF-alpha induces a decrease in eNOS promoter activity. Am J Physiol Lung Cell Mol Physiol 286:L452-9 |
| Gertzberg, Nancy; Neumann, Paul; Rizzo, Victor et al. (2004) NAD(P)H oxidase mediates the endothelial barrier dysfunction induced by TNF-alpha. Am J Physiol Lung Cell Mol Physiol 286:L37-48 |
| Siflinger-Birnboim, Alma; Johnson, Arnold (2003) Protein kinase C modulates pulmonary endothelial permeability: a paradigm for acute lung injury. Am J Physiol Lung Cell Mol Physiol 284:L435-51 |
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