Jinming Zhao

IL‐13 induced increases in nitrite levels are primarily driven by increases in inducible nitric oxide synthase as compared with effects on arginases in human primary bronchial epithelial cells

Background Exhaled nitric oxide is increased in asthma, but the mechanisms controlling its production, including the effects of T‐helper type 2 (Th2) cytokines, are poorly understood. In mouse and submerged human epithelial cells, Th2 cytokines inhibit expression of inducible nitric oxide synthase (iNOS). Arginases have been proposed to contribute to asthma pathogenesis by limiting the arginine substrate available to NOS enzymes, but expression of any of these enzymes has not been extensively studied in primary human cells.

Interleukin-13–induced MUC5AC Is Regulated by 15-Lipoxygenase 1 Pathway in Human Bronchial Epithelial Cells

RATIONALE 15-Lipoxygenase-1 (15LO1) and MUC5AC are highly expressed in asthmatic epithelial cells. IL-13 is known to induce 15LO1 and MUC5AC in human airway epithelial cells in vitro. Whether 15LO1 and/or its product 15-HETE modulate MUC5AC expression is unknown. OBJECTIVES To determine the expression of 15LO1 in freshly harvested epithelial cells from subjects with asthma and normal control subjects and to determine whether IL-13-induced 15LO1 expression and activation regulate MUC5AC expression in human bronchial epithelial cells in vitro. METHODS Human airway epithelial cells from subjects with asthma and normal subjects were evaluated ex vivo for 15LO1 and MUC5AC expression. The impact of 15LO1 on MUC5AC expression in vitro was analyzed by inhibiting 15LO1 through pharmacologic (PD146176) and siRNA approaches in human bronchial epithelial cells cultured under air-liquid interface. We analyzed 15 hydroxyeicosatetraenoic acid (15-HETE) by liquid chromatography/UV/mass spectrometry. MUC5AC and 15LO1 were analyzed by real-time RT-PCR, immunofluoresence, and Western blot. MEASUREMENTS AND MAIN RESULTS Epithelial 15LO1 expression increased with asthma severity (P < 0.0001). 15LO1 significantly correlated with MUC5AC ex vivo and in vitro. IL-13 increased 15LO1 expression and stimulated formation of two molecular species of 15-HETE esterified to phosphotidylethanolamine (15-HETE-PE). Inhibition of 15LO1 suppressed 15-HETE-PE and decreased MUC5AC expression in the presence of IL-13 stimulation. The addition of exogenous 15-HETE partially restored MUC5AC expression. CONCLUSIONS Epithelial 15LO1 expression increases with increasing asthma severity. IL-13 induction of 15-HETE-PE enhances MUC5AC expression in human airway epithelial cells. High levels of 15LO1 activity could contribute to the increases of MUC5AC observed in asthma.

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

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-4Rα 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-4Rα–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.

PEBP1 Wardens Ferroptosis by Enabling Lipoxygenase Generation of Lipid Death Signals

Ferroptosis is a form of programmed cell death that is pathogenic to several acute and chronic diseases and executed via oxygenation of polyunsaturated phosphatidylethanolamines (PE) by 15-lipoxygenases (15-LO) that normally use free polyunsaturated fatty acids as substrates. Mechanisms of the altered 15-LO substrate specificity are enigmatic. We sought a common ferroptosis regulator for 15LO. We discovered that PEBP1, a scaffold protein inhibitor of protein kinase cascades, complexes with two 15LO isoforms, 15LO1 and 15LO2, and changes their substrate competence to generate hydroperoxy-PE. Inadequate reduction of hydroperoxy-PE due to insufficiency or dysfunction of a selenoperoxidase, GPX4, leads to ferroptosis. We demonstrated the importance of PEBP1-dependent regulatory mechanisms of ferroptotic death in airway epithelial cells in asthma, kidney epithelial cells in renal failure, and cortical and hippocampal neurons in brain trauma. As master regulators of ferroptotic cell death with profound implications for human disease, PEBP1/15LO complexes represent a new target for drug discovery.

IL-13 desensitizes β2-adrenergic receptors in human airway epithelial cells through a 15-lipoxygenase/G protein receptor kinase 2 mechanism

BACKGROUND β2-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. OBJECTIVE We 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). METHODS HAECs 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. RESULTS Pretreatment 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. CONCLUSION IL-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.

Reply to Mabalirajan et al.: 15LO1 pathway activation—Are receptors important?

We appreciate the opportunity to respond to the comments of Mabalirajan et al. (1) on our recent article (2). Their three critical points are addressed below. [↵][1]1To whom correspondence should be addressed. E-mail: wenzelse{at}upmc.edu. [1]: #xref-corresp-1-1