Products of arachidonic acid (AA), eicosanoids, are well-established mediators of inflammation. The production of AA by phospholipases is the initial rate-limiting step in eicosanoid biosynthesis, and the major phospholipase that regulates eicosanoid synthesis in response to inflammatory agonists is group IVA cytosolic phospholipase A2 (cPLA2a). Our laboratory first discovered that ceramide-1-phosphate (C1P) is a potent activator of cPLA2a both in vitro and in cells. In our previous funding cycle, the PI's laboratory demonstrated that CERK and its product, C1P, are required for the activation of cPLA2a, and are major regulators of eicosanoid synthesis in cells. Our laboratory has now begun to study CERK in depth since CERK is a possible target for anti-inflammatory therapeutics. Preliminary results demonstrate that CERK is phosphorylated in response to inflammatory agonists (e.g. IL-1?). These results also demonstrated that phosphorylation of Ser424 regulates both the enzymatic activity of CERK as well as eicosanoid synthesis in cells. Other preliminary results from our laboratory demonstrate that CERK specifically binds and is activated by phosphatidic acid (PA). Based on these data, we hypothesize that CERK is activated by phosphorylation and interaction with the lipid co-factor, PA, in response to inflammatory mediators. Our preliminary results also suggest that lipid phosphate phosphatase-3 (LPP-3), a member of the LPP family, is responsible for the catalytic degradation of C1P. Therefore, building upon our previous work and these data, we hypothesize that C1P is catabolized by LPP-3 to effectively "shut-down" prolonged activation of eicosanoid synthesis in response to inflammatory agonists. Lastly, CERK inhibitors (e.g. multiple siRNAs) are effective in blocking eicosanoid synthesis in response to inflammatory agonists in tissue culture systems. Therefore, one of our central hypotheses is that C1P produced from the phosphorylation of ceramide by CERK is an important in vivo mediator of eicosanoid synthesis induced by inflammatory phenotypes via activation of cPLA2a. To validate our above hypotheses, we will: 1) Determine the role of lipid cofactors in regulating CERK in response to inflammatory agonists;2) Determine the role of phosphorylation in regulating CERK in response to inflammatory agonists;3) To determine the catabolic regulation of C1P in response to inflammatory cytokines;and 4) Determine the role of CERK in regulating eicosanoid synthesis in vivo. *Significance: Since almost nothing is known about the regulation of this enzyme critical for eicosanoid synthesis, we predict that these studies will produce great insights into the regulation of this enzyme as well as identify additional upstream mediators of eicosanoid synthesis. These studies will also define a role of CERK in mediating inflammatory responses in vivo. This cannot be understated because CERK will be defined as a therapeutic target for modulation of eicosanoid synthesis. Indeed, the proposed studies will generate insights into novel aspects of signal transduction in inflammation perhaps leading to new therapeutics for asthma, thrombosis, ATH, and Alzheimer's disease.

Public Health Relevance

These studies will produce great insights into the regulation of ceramide kinase as well as identify additional upstream mediators of eicosanoid synthesis. These studies will also largely define a role of CERK and its product, C1P, in mediating inflammatory responses in vivo. This cannot be understated because CERK will be defined as a therapeutic target for modulation of eicosanoid synthesis. Thus, this grant application has the high potential (HIGH IMPACT) for establishing a tangible new factor that impinges on the important inflammatory mediators, eicosanoids. Indeed, the proposed studies will generate significant insights into novel aspects of signal transduction in inflammation perhaps leading to new therapeutics for asthma/AHR, sepsis/pulmonary infection, thrombosis, cancer metastasis, inflammation, ATH, and Alzheimer's disease.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL072925-08
Application #
8212379
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (90))
Program Officer
Sarkar, Rita
Project Start
2003-04-01
Project End
2014-01-31
Budget Start
2012-02-01
Budget End
2014-01-31
Support Year
8
Fiscal Year
2012
Total Cost
$551,203
Indirect Cost
$178,703
Name
Virginia Commonwealth University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
105300446
City
Richmond
State
VA
Country
United States
Zip Code
23298
Mohammed, Bassem M; Fisher, Bernard J; Huynh, Quoc K et al. (2014) Resolution of sterile inflammation: role for vitamin C. Mediators Inflamm 2014:173403
van den Elsen, Lieke W J; Spijkers, Léon J A; van den Akker, Rob F P et al. (2014) Dietary fish oil improves endothelial function and lowers blood pressure via suppression of sphingolipid-mediated contractions in spontaneously hypertensive rats. J Hypertens 32:1050-8; discussion 1058
Dhall, Sandeep; Do, Danh; Garcia, Monika et al. (2014) A novel model of chronic wounds: importance of redox imbalance and biofilm-forming bacteria for establishment of chronicity. PLoS One 9:e109848
Gomez-Arroyo, Jose; Mizuno, Shiro; Szczepanek, Karol et al. (2013) Metabolic gene remodeling and mitochondrial dysfunction in failing right ventricular hypertrophy secondary to pulmonary arterial hypertension. Circ Heart Fail 6:136-44
Mietla, Jennifer A; Wijesinghe, Dayanjan S; Hoeferlin, L Alexis et al. (2013) Characterization of eicosanoid synthesis in a genetic ablation model of ceramide kinase. J Lipid Res 54:1834-47
Ward, Katherine E; Bhardwaj, Nitin; Vora, Mohsin et al. (2013) The molecular basis of ceramide-1-phosphate recognition by C2 domains. J Lipid Res 54:636-48
Simanshu, Dhirendra K; Kamlekar, Ravi Kanth; Wijesinghe, Dayanjan S et al. (2013) Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids. Nature 500:463-7
Shultz, Jacqueline C; Vu, Ngoc; Shultz, Michael D et al. (2012) The Proto-oncogene PKCýý regulates the alternative splicing of Bcl-x pre-mRNA. Mol Cancer Res 10:660-9
Shultz, Jacqueline C; Chalfant, Charles E (2012) The flip-flop HuR: part of the problem or the solution in fighting cancer? J Clin Invest 122:16-9
Shultz, Jacqueline C; Goehe, Rachel W; Murudkar, Charuta S et al. (2011) SRSF1 regulates the alternative splicing of caspase 9 via a novel intronic splicing enhancer affecting the chemotherapeutic sensitivity of non-small cell lung cancer cells. Mol Cancer Res 9:889-900

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