Dataset Information

15-Lipoxygenase 1 interacts with phosphatidylethanolamine-binding protein to regulate MAPK signaling in human airway epithelial cells.

ABSTRACT: Epithelial 15-lipoxygenase 1 (15LO1) and activated ERK are increased in asthma despite modest elevations in IL-13. MAPK kinase (MEK)/ERK activation is regulated by interactions of Raf-1 with phosphatidylethanolamine-binding protein 1 (PEBP1). Epithelial 15LO1 generates intracellular 15-hydroxyeicosatetraenoic acid (15HETE) conjugated to phosphatidylethanolamine (PE) (15HETE-PE). We hypothesized that (i) 15LO1 and its product 15HETE-PE serve as signaling molecules interacting with PEBP1 to activate Raf-1/MEK/ERK and that (ii) this 15LO1-15HETE-PE-regulated ERK activation amplifies IL-4R? downstream pathways. Our results demonstrate that high epithelial 15LO1 levels correlate with ERK phosphorylation ex vivo. In vitro, IL-13 induces 15LO1, which preferentially binds to PEBP1, causing PEBP1 to dissociate from Raf-1 and activate ERK. Exogenous 15HETE-PE similarly induces dissociation of PEBP1 from Raf-1 independently of IL-13/15LO1. siRNA knockdown of 15LO1 decreases the dissociation of Raf-1 from PEBP1, and the resulting lower ERK activation leads to lower downstream IL-4R?-related gene expression. Identical protein-protein interactions are observed in endobronchial biopsies and fresh epithelial cells from asthmatics ex vivo. Colocalization of Raf-1 to PEBP1 is low in asthmatic tissue and cells compared with normals, whereas there is striking colocalization of 15LO1 with PEBP1 in asthma. Low 15LO1 levels in normals limit its colocalization with PEBP1. The results confirm a previously unknown signaling role for 15LO1 and its PE-conjugated eicosanoid product in human airway epithelial cells. This pathway enhances critical inflammatory pathways integral to asthma pathogenesis.

SUBMITTER: Zhao J

PROVIDER: S-EPMC3161579 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

15-Lipoxygenase 1 interacts with phosphatidylethanolamine-binding protein to regulate MAPK signaling in human airway epithelial cells.

Zhao Jinming J O'Donnell Valerie B VB Balzar Silvana S St Croix Claudette M CM Trudeau John B JB Wenzel Sally E SE

Proceedings of the National Academy of Sciences of the United States of America 20110809 34

Epithelial 15-lipoxygenase 1 (15LO1) and activated ERK are increased in asthma despite modest elevations in IL-13. MAPK kinase (MEK)/ERK activation is regulated by interactions of Raf-1 with phosphatidylethanolamine-binding protein 1 (PEBP1). Epithelial 15LO1 generates intracellular 15-hydroxyeicosatetraenoic acid (15HETE) conjugated to phosphatidylethanolamine (PE) (15HETE-PE). We hypothesized that (i) 15LO1 and its product 15HETE-PE serve as signaling molecules interacting with PEBP1 to activa ...[more]

PMID: 21831839

Similar Datasets

Project description:Backgroundβ2-Adrenergic receptor (β2AR) agonists are critical treatments for asthma. However, receptor desensitization can lead to loss of therapeutic effects. Although desensitization to repeated use of β2-agonists is well studied, type 2 inflammation could also affect β2AR function.ObjectiveWe sought to evaluate the effect of the type 2 cytokine IL-13 on β2AR desensitization in human airway epithelial cells (HAECs) and determine whether 15-lipoxygenase-1 (15LO1) binding with phosphatidylethanolamine-binding protein 1 (PEBP1) contributes to desensitization through release of G protein receptor kinase 2 (GRK2).MethodsHAECs in air-liquid interface culture with or without IL-13 (48 hours) or isoproterenol hydrochloride (ISO; 30 minutes) pretreatment were stimulated with ISO (10 minutes). Cyclic adenosine 3, 5-monophosphate (cAMP) levels were measured using ELISA, and β2AR and GRK2 phosphorylation was measured using Western blotting. Short interfering RNA was used for 15LO1 knockdown. Interactions of GRK2, PEBP1, and 15LO1 were detected by means of immunoprecipitation/Western blotting and immunofluorescence. HAECs and airway tissue from control subjects and asthmatic patients were evaluated for I5LO1, PEBP1, and GRK2.ResultsPretreatment with ISO or IL-13 decreased ISO-induced cAMP generation compared with ISO for 10 minutes alone paralleled by increases in β2AR and GRK2 phosphorylation. GRK2 associated with PEBP1 after 10 minutes of ISO in association with low phosphorylated GRK2 (pGRK2) levels. In contrast, in the presence of IL-13 plus ISO (10 minutes), binding of GRK2 to PEBP1 decreased, whereas 15LO1 binding and pGRK2 levels increased. 15LO1 knockdown restored ISO-induced cAMP generation. These findings were recapitulated in freshly brushed HAECs from cells and tissue of asthmatic patients.ConclusionIL-13 treatment of HAECs leads to β2AR desensitization, which involves 15LO1/PEBP1 interactions to free GRK2, and allows it to phosphorylate (and desensitize) β2ARs, suggesting that the beneficial effects of β2-agonists could be blunted in patients with type 2 associated asthma.

Project description:Allosteric regulation of human lipoxygenase (hLO) activity has recently been implicated in the cellular biology of prostate cancer. In the current work, we present isotope effect, pH, and substrate inhibitor data of epithelial 15-hLO-2, which probe the allosteric effects on its mechanistic behavior. The Dk(cat)/KM for 15-hLO-2, with AA and LA as substrate, is large indicating hydrogen atom abstraction is the principle rate-determining step, involving a tunneling mechanism for both substrates. For AA, there are multiple rate determining steps (RDS) at both high and low temperatures, with both diffusion and hydrogen bonding rearrangements contributing at high temperature, but only hydrogen bonding rearrangements contributing at low temperature. The observed kinetic dependency on the hydrogen bonding rearrangement is eliminated upon addition of the allosteric effector, 13-(S)-hydroxyoctadecadienoic acid (13-HODE), and no allosteric effects were seen on diffusion or hydrogen atom abstraction. The (k(cat)/KM)AA/(k(cat)/KM)LA ratio was observed to have a pH dependence, which was fit with a titration curve (pKa = 7.7), suggesting the protonation of a histidine residue, which could hydrogen bond with the carboxylate of 13-HODE. Assuming this interaction, 13-HODE was docked to the solvent exposed histidines of a 15-hLO-2 homology model and found to bind well with H627, suggesting a potential location for the allosteric site. Utilizing d31-LA as an inhibitor, it was demonstrated that the binding of d31-LA to the allosteric site changes the conformation of 15-hLO-2 such that the affinity for substrate increases. This result suggests that allosteric binding locks the enzyme into a catalytically competent state, which facilitates binding of LA and decreases the (k(cat)/KM)AA/(k(cat)/KM)LA ratio. Finally, the magnitude of the 13-HODE KD for 15-hLO-2 is over 200-fold lower than that of 13-HODE for 15-hLO-1, changing the substrate specificity of 15-hLO-2 to 1.9. This would alter the LO product distribution and increase the production of the pro-tumorigenic, 13-HODE, possibly representing a pro-tumorigenic feedback loop for 13-HODE and 15-hLO-2.

Dataset Information

15-Lipoxygenase 1 interacts with phosphatidylethanolamine-binding protein to regulate MAPK signaling in human airway epithelial cells.

Publications

15-Lipoxygenase 1 interacts with phosphatidylethanolamine-binding protein to regulate MAPK signaling in human airway epithelial cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets