Inflammation is implicated in the pathogenesis of many diseases. In the lung, some of the examples include asthma, bronchitis, sarcoidosis, and a variety of disorders that ultimately lead to the loss of lung function. Monocytes are important components during the initiation and resolution of the innate immune response. Circulating monocytes normally live 24-48 hrs and in the absence of a survival signal undergo spontaneous apoptosis. In the presence of stimulatory factors, monocytes survive and differentiate into macrophages, which live up to 3 months. Thus, the understanding of the molecular mechanisms that control the apoptotic proteins is essential to manipulate the accumulation of monocyte/macrophages at inflammatory sites. Caspase-3 is a key executioner of the apoptotic pathway in monocytes. We demonstrated that stimulatory stimulus, such as lipopolysaccharide (LPS), induces monocyte survival by blocking the activation of caspase-3. Our preliminary studies demonstrate that caspase-3 is a phosphoprotein that associates and is phosphorylated by a member of the protein kinase C family (PKC). Our results suggest that phosphorylation induces caspase-3 activity and promotes apoptosis. The caspase-3-associated-kinase activity is modulated during monocyte life span and by inflammatory stimuli. The goal of this proposal is to understand the mechanisms that regulate apoptosis in monocytes. Here, we propose in Aim 1 to determine the biological role of caspase-3 phosphorylation. For this purpose, we will determine first the number and location of the phosphorylated sites on caspase-3 in vivo and in vitro. Next, using in vivo labeled caspase-3 and caspase-3 mutants we will elucidate whether phosphorylation affects the activation or the activity of caspase-3. Next, we will determine the role the caspase-3 phosphorylation in apoptosis.
In Aim 2, we propose to identify the kinase/s that are responsible for caspase-3 phosphorylation in vivo using siRNA and animal models. We propose in Aim 3, to investigate whether the phosphorylation changes the localization of caspase-3 and to study whether the localization changes during apoptosis. Finally, in Aim 4 we propose to determine the mechanisms by which caspase-3 phosphorylation and the caspase-3-associated-kinases regulate stimulation,differentiation, and life span of monocytes. Altogether, these studies should advance our understandingof the basic mechanisms that regulate apoptosis and help identifying new therapeutic targets to selectively induced apoptosis of inflammatory cells. Furthermore, it is our goal that the studies proposed here will contribute to improve the treatments for inflammation.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
3R01HL075040-02S1
Application #
7425139
Study Section
Lung Cellular, Molecular, and Immunobiology Study Section (LCMI)
Program Officer
Reynolds, Herbert Y
Project Start
2006-02-09
Project End
2011-01-31
Budget Start
2007-02-01
Budget End
2008-01-31
Support Year
2
Fiscal Year
2007
Total Cost
$28,893
Indirect Cost
Name
Ohio State University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
Cardenas, Horacio; Arango, Daniel; Nicholas, Courtney et al. (2016) Dietary Apigenin Exerts Immune-Regulatory Activity in Vivo by Reducing NF-?B Activity, Halting Leukocyte Infiltration and Restoring Normal Metabolic Function. Int J Mol Sci 17:323
Malavez, Yadira; Voss, Oliver H; Gonzalez-Mejia, Martha Elba et al. (2015) Distinct contribution of protein kinase C? and protein kinase C? in the lifespan and immune response of human blood monocyte subpopulations. Immunology 144:611-20
Arango, Daniel; Diosa-Toro, Mayra; Rojas-Hernandez, Laura S et al. (2015) Dietary apigenin reduces LPS-induced expression of miR-155 restoring immune balance during inflammation. Mol Nutr Food Res 59:763-72
Duarte, Silvia; Arango, Daniel; Parihar, Arti et al. (2013) Apigenin protects endothelial cells from lipopolysaccharide (LPS)-induced inflammation by decreasing caspase-3 activation and modulating mitochondrial function. Int J Mol Sci 14:17664-79
Arango, Daniel; Morohashi, Kengo; Yilmaz, Alper et al. (2013) Molecular basis for the action of a dietary flavonoid revealed by the comprehensive identification of apigenin human targets. Proc Natl Acad Sci U S A 110:E2153-62
Das, Amlan; Gopalakrishnan, Bhavani; Voss, Oliver H et al. (2012) Inhibition of ROS-induced apoptosis in endothelial cells by nitrone spin traps via induction of phase II enzymes and suppression of mitochondria-dependent pro-apoptotic signaling. Biochem Pharmacol 84:486-97
Henkels, Karen M; Frondorf, Kathleen; Gonzalez-Mejia, M Elba et al. (2011) IL-8-induced neutrophil chemotaxis is mediated by Janus kinase 3 (JAK3). FEBS Lett 585:159-66
Gonzalez-Mejia, M E; Voss, O H; Murnan, E J et al. (2010) Apigenin-induced apoptosis of leukemia cells is mediated by a bimodal and differentially regulated residue-specific phosphorylation of heat-shock protein-27. Cell Death Dis 1:e64
Parihar, Arti; Eubank, Timothy D; Doseff, Andrea I (2010) Monocytes and macrophages regulate immunity through dynamic networks of survival and cell death. J Innate Immun 2:204-15
Wood, Karen L; Nunley, David R; Moffatt-Bruce, Susan et al. (2010) The role of heat shock protein 27 in bronchiolitis obliterans syndrome after lung transplantation. J Heart Lung Transplant 29:786-91

Showing the most recent 10 out of 17 publications