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

Abstract

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.

Keywords: 15-lipoxygenase 1; G protein receptor kinase 2; IL-13; asthma; cAMP; phosphatidylethanolamine binding protein 1; β(2)-adrenergic receptor.

Figure 1

IL-13 pretreatment decreases cAMP release in response to Isoproterenol stimulation. ALI-cultured primary BAECs were pretreated with either ISO (30 min) or IL-13 (48 hr) followed by stimulation with ISO for 10 min. Cells were harvested for cAMP levels by ELISA. (a). ISO stimulation for 10 min induced high cAMP levels (415±84 SEM)), which were inhibited by ISO (30min) pretreatment (350±77(SEM)). (b). IL-13 pretreatment for 48 hrs inhibited cAMP release induced by ISO for 10min alone (267± 76 (SEM)).

Figure 2

IL-13 and ISO induce β2AR and GRK2 phosphorylation in primary BAECs. ALI-cultured BAECs were stimulated with ISO (30 min) or IL-13 (48 hr). Total protein was harvested for β2AR and GRK2 phosphorylation by Western blot and densitometry analysis. (a) Both IL-13 (1.06±0.13) and ISO (1.01±0.13) significantly increased β2AR phosphorylation compared to media alone (0.75±0.11). (b) Both IL-13 (0.88±0.27) and ISO (0.69±0.16) significantly increased GRK2 phosphorylation compared to media alone (0.39±0.08).

Figure 3

IL-13 suppresses GRK2 binding to PEBP1 and enhances 15LO1 binding to PEBP1 in response to ISO in vitro. BAECs in the presence or absence of IL-13 (48 hr) were stimulated with ISO for 10 min. Total protein was harvested for Western blot/IP. Cells were also fixed for IF staining. (a) GRK2 bound with PEBP1 in response to ISO while pretreatment with IL-13 markedly decreased binding of GRK2 to PEBP1 and increased 15LO1 binding to PEBP1. (b) Densitometry analysis of Western blot/IP. (C) Confocal analysis showing that ISO treatment increased the colocalization (yellow) of GRK2 (green) with PEBP1 (red) as compared to control. This colocalization was less in the presence of IL-13. In contrast, 15LO1 (green) was highly expressed and colocalized/bound (yellow) to PEBP1 in the presence of IL-13 and not affected by ISO.

Figure 4

ALOX15 siRNA knock-down of 15LO1 expression restored ISO induced cAMP generation in BAECs. ALI-cultured cells transfected with ALOX15 siRNA or scramble siRNA were stimulated with IL-13 for 48 h + ISO 10 min. Total protein was harvested for Western blot and ELISA analysis. Data are presented as the mean of duplicates. (a) ALOX15 siRNA knocked down 15LO1 protein and decreased β2AR phosphorylation induced by IL-13/ISO stimulation. (b) ALOX15 siRNA knock-down restored ISO (10 min) induced cAMP generation in cells treated with IL-13 for 48 hours (244±102 SEM) as compared to scramble siRNA (196±91 SEM).

Figure 5

β2AR and GRK2 phosphorylation positively correlate with 15LO1 expression in primary human airway epithelial cells ex vivo. (a) Total protein from freshly brushed airway epithelial cells was harvested and β2AR and GRK2 phosphorylation measured by Western blot. Densitometry analysis was performed. pβ2AR, pGRK2 and 15LO1 were normalized to GAPDH and reported as arbitrary units. (b) pβ2AR positively correlated with pGRK2. (c) phosphorylated GRK2 and (d) pβ2AR levels in 15LO1-Hi group were higher as compared to 15LO1-Lo group. Abbreviations: HC=Healthy control, MA=mild asthma/no ICS, MAC=mild-moderate+ICS, and SA=Severe Asthma.

Figure 6

15LO1 binding to PEBP1 is associated with less GRK2 binding to PEBP1 in asthmatic cells and tissue as compared to health controls. Total protein from freshly brushed airway epithelial cells was harvested for Western blot/IP analysis. Lung tissue from asthmatic and healthy control subjects were fixed for Immunofluorescent-confocal microscopy studies. (a) Higher levels of 15LO1 binding to PEBP1 were associated with lower GRK2 binding in freshly brushed epithelial cells. (b) Densitometry analysis of Western blot/IP. (c) Expression 15LO1 and its colocalization with PEBP1 was higher in an asthmatic subject as compared to the control donor lung. In contrast, GRK2 colocalization with PEBP1 in the airway epithelium was lower in the asthmatic subject as compared to a healthy control.