Rationale ß2-AR is a prototypical G-protein coupled receptor (GPCR), which has a critical role in airway smooth muscle relaxation and bronchodilation. However, this receptor also plays a role in epithelial function, in particular controlling ciliary beat, serous stimulation, mucous secretion and lung fluid clearance. ?-AR desensitization as consequence of ß-agonist therapy can lead to diminish bronchodilator effects, but effects on epithelial cells are less clear. G-protein receptor kinases (GRK) play an important role in GPCR regulation by phosphorylating agonist-occupied receptors to promote their internalization and desensitization. GRK2 has been implicated in ß2-AR desensitization. However, little is known regarding the role or mechanisms by which adaptive immunity may regulate the ß2 receptor. Hypothesis IL-13 induces ß2-adrenergic receptor desensitization in human airways epithelial cell by inducing GRK2 phosphorylation, which leads to phosphorylation of the ß2-AR and promotion of receptor internalization and desensitization. Material and Methods Primary human bronchial epithelial cells (HBEC) from bronchial brushings of normal and asthmatic subjects were cultured in air-liquid interface (ALI). Cells were pretreated withwithout IL-13 (10ng/ml) for 48 hours and then stimulated or withwithout isoproterenol (ISO) (1 ?M) for 30 minutes prior to harvest. GRK2 and ß2-AR phosphorylation were detected by western blot on HBEC lysates. As the data were not normally distributed, the Kruskal-Wallis test for multiple comparisons was used, with Spearman's rho for correlations. The results were expressed as median and 25th -75th percentiles. Results IL-13 and ISO increased GRK2 phosphorylation (p=0.02 for IL-13 and p=0.04 for ISO). IL-13 and ISO also increased ß2-AR phosphorylation (p=0.03 for IL-13, p=0.06 for ISO). Interestingly, IL-13 stimulation in combination with ISO also increased GRK2 phosphorylation as compared to media control (p=0.03), but more importantly also tended to increase GRK2 phosphorylation as compared to treatment with either IL-13 alone (147% (69-200%, p=0.09) or ISO alone (143%(78-143%, p=0.3), although in small numbers this was not significant. Like GRK2, IL-13 stimulation in combination with ISO increased ß2-AR phosphorylation as compared to media control (p=0.01) or ISO alone (235% (0.93-5.16, p=0.03). ß2-AR phosphorylation also tended to increase with the combination of IL-13 and ISO as compared to IL-13 alone (129% (0.62-1.53%) compared to IL-13) although in small numbers this was not significant. Conclusion IL-13 appears to play a significant role in the regulation of ?2-AR desensitization through an effect on activation of GRK2. This effect augments the phosphorylation observed with a ß-agonist alone, which may alter epithelial responses to ß2-agonists in asthmatic patients
IL-13 induced beta2-adrenergic receptor (ß2-AR) desensitization in human airway epithelial cells.
Giusy Daniela Albano;Mirella Profita;
2013
Abstract
Rationale ß2-AR is a prototypical G-protein coupled receptor (GPCR), which has a critical role in airway smooth muscle relaxation and bronchodilation. However, this receptor also plays a role in epithelial function, in particular controlling ciliary beat, serous stimulation, mucous secretion and lung fluid clearance. ?-AR desensitization as consequence of ß-agonist therapy can lead to diminish bronchodilator effects, but effects on epithelial cells are less clear. G-protein receptor kinases (GRK) play an important role in GPCR regulation by phosphorylating agonist-occupied receptors to promote their internalization and desensitization. GRK2 has been implicated in ß2-AR desensitization. However, little is known regarding the role or mechanisms by which adaptive immunity may regulate the ß2 receptor. Hypothesis IL-13 induces ß2-adrenergic receptor desensitization in human airways epithelial cell by inducing GRK2 phosphorylation, which leads to phosphorylation of the ß2-AR and promotion of receptor internalization and desensitization. Material and Methods Primary human bronchial epithelial cells (HBEC) from bronchial brushings of normal and asthmatic subjects were cultured in air-liquid interface (ALI). Cells were pretreated withwithout IL-13 (10ng/ml) for 48 hours and then stimulated or withwithout isoproterenol (ISO) (1 ?M) for 30 minutes prior to harvest. GRK2 and ß2-AR phosphorylation were detected by western blot on HBEC lysates. As the data were not normally distributed, the Kruskal-Wallis test for multiple comparisons was used, with Spearman's rho for correlations. The results were expressed as median and 25th -75th percentiles. Results IL-13 and ISO increased GRK2 phosphorylation (p=0.02 for IL-13 and p=0.04 for ISO). IL-13 and ISO also increased ß2-AR phosphorylation (p=0.03 for IL-13, p=0.06 for ISO). Interestingly, IL-13 stimulation in combination with ISO also increased GRK2 phosphorylation as compared to media control (p=0.03), but more importantly also tended to increase GRK2 phosphorylation as compared to treatment with either IL-13 alone (147% (69-200%, p=0.09) or ISO alone (143%(78-143%, p=0.3), although in small numbers this was not significant. Like GRK2, IL-13 stimulation in combination with ISO increased ß2-AR phosphorylation as compared to media control (p=0.01) or ISO alone (235% (0.93-5.16, p=0.03). ß2-AR phosphorylation also tended to increase with the combination of IL-13 and ISO as compared to IL-13 alone (129% (0.62-1.53%) compared to IL-13) although in small numbers this was not significant. Conclusion IL-13 appears to play a significant role in the regulation of ?2-AR desensitization through an effect on activation of GRK2. This effect augments the phosphorylation observed with a ß-agonist alone, which may alter epithelial responses to ß2-agonists in asthmatic patientsI documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
