Purpose: Asthma is a chronic inflammatory lung disease. The heterogeneity of this disease has led to explore new possibilities understanding the molecular mechanisms underlying the worsening of the disease. Airway smooth muscle (ASM) cells play a key role in the development of this disease. A crucial process occurring during asthma is airway remodeling, as evidenced by increased airway smooth muscle mass leading to an increase in airway narrowing in lungs. This has been supported by several studies including our lab. Asthmatic ASM cells exhibit a significantly higher proliferation rate compared to non-asthmatic at basal and following mitogen stimulation. Complications associated with remodeling are irreversible structural change in airways ultimately causing patients to have poorer clinical management. Irreversible ASM proliferation is a risk for increased morbidity and mortality. Little is known about the mechanisms involved in the proliferation of ASM in asthmatic airways. Thus, our study focuses on exploring the mechanisms and signaling pathways which could potentially involve increased ASM proliferation in asthmatic airway remodeling.
Several studies have shown elevated levels of a vasoconstrictor, Endothelin-1 (ET-1) in asthmatics. ET-1 has been shown to function as a mitogen. To explore the role of ET-1 as a mitogen we investigated cell proliferation signaling by the Receptor Tyrosine Kinase, EGFR, and the G-protein coupled Endothelin Receptor (ETR).
We hypothesized ET-1 activates the ETR which cross talks with the EGF Receptor via a key player Src and triggers a series of events for ASM cell proliferation by Mitogen-Activated Protein Kinase (MAPK) signaling pathway. The signaling proposes ET-1 to be a mitogenic stimulus which transactivates EGF Receptor at Tyr 1068 in the cytoplasmic domain of the receptor. The transactivation is mediated by activated Src, a secondary messenger further activating a group of enzymes called Matrix metalloproteinase (MMPs) during signal transduction. MMPs cleave the pro-Heparin binding EGF like growth factor (pro-HBEGF) to HB-EGF, a soluble protein ligand which then binds to the EGF receptor and phosphorylates at Tyr1068 to initiate the cascade for MAPK signaling leading to ASM cell proliferation. The investigation of this proposed mechanism is carried out in primary human asthmatic airway smooth muscle cells.
Methods: Cell culture: Diseased (asthmatic) and normal human bronchial airway smooth muscle cells were grown to 80-85 % confluency and serum starved for 24 hours to arrest their growth. The cells were incubated in 37 ºC in 95% O2 and 5% CO2 in Smooth Muscle Basal medium enriched with supplements Epidermal Growth Factor (0.5 ng/ml), Basic Fibroblast Growth Factor (2ng/ml), Fetal Calf Serum (0.05 ml/ml), Insulin (5 μg/ml) and Gentamycin.
Western blot: A time course on the effect of ET-1 phosphorylation of the EGFR (Tyr1068) in both cell lines was determined. Cells were treated in the absence or presence of ET-1 (10 nM) for 2 min, 5 min, 15 min, 30 min, 60 min, 2h and 6h. Cell lysates were collected, analyzed by BCA assay for protein concentration, and prepared in reducing sample buffer. A fixed concentration of protein (20 ug) was run on
SDS-PAGE, followed by immunoblotting with Phospho-EGF Receptor Tyr1068 (D7A5) XP® Rabbit mAb at concentration 1:1000 in blocking buffer. Protein expression of Tyr1068 was normalized against GAPDH (D16H11) XP Rabbit mAb (HRP conjugate) at concentration 1:3000 in blocking buffer.
Cell proliferation: BrdU Cell proliferation assay was performed. Cells were grown in 96 well plates at a density of 4000-4500 cells/well for 48 hours till they reached 85-90% confluency. The cells were then serum starved for 24 h. ASM cells were then pretreated with several inhibitors: Src (PP1, PP2), MMPs (ONO-4817) for 30 minutes or EGF Blocking peptide (Human EGF Neutralizing (D8A1) Rabbit mAb) for 1 hour and then treated again with these inhibitors in presence of ET-1 for 48 hours. BrdU reagent was incorporated after 24 h of treatment. Thereafter, the BrdU incorporation was determined via a colorimetric reaction and absorbance detected using a plate reader.
Results: Phosphorylation of EGFR Tyr1068 by ET-1 showed differential activation profiles. In asthmatic ASM, Tyr1068 phosphorylation was increased significantly at 2 and 5 min compared to normal ASM (p<0.01). A slower onset of phosphorylation on this residue was observed in normal ASM with a significant increase at 30 min compared to asthmatic ASM (p<0.01). For asthmatic ASM, there is 2-, 3- and 7-fold decrease in cell proliferation in presence of ET-1 and Src inhibitors, PP1, PP2 or PP1 and PP2, respectively, compared to ET-1 alone (P=0.0002). A significant reduction in ASM cell proliferation was observed in presence of ONO-4817 peptides (p<0.0001) and EGF Blocking peptide (p<0.05).
Conclusion: ET-1 initiates cross talk between ETR and EGFR by phosphorylating Tyr1068 for downstream mitogenic signaling. Phosphorylation of the EGFR is regulated by Src and MMPs suggesting ligand independent and triple-membrane-passing-signal mechanisms operating in asthmatic ASM. Activation of both of these signaling mechanisms may be lead to airway remodeling in asthmatic ASM.
Alice Gardner– Professor of Pharmacology & Toxicology, MCPHS University, Worcester, Massachusetts