Abstract
We showed previously that treatment of human airway smooth muscle cells and lung fibroblasts with lysophosphatidic acid (LPA) increases the binding of epidermal growth factor (EGF) to EGF receptors (EGFRs). The purpose of this study was to determine whether LPA also regulates EGFR binding in airway epithelial cells. Airway epithelial cells were incubated in the absence or presence of 10 μM LPA for increasing times, and binding of 125I-EGF to intact cells on ice was measured. Exposure to LPA for only 15 min caused a 30 to 70% decrease in EGFR binding in a dose-dependent manner, depending on the cell line. This decrease in binding was sustained to at least 18 h in BEAS-2B and primary human bronchial epithelial cells. In contrast, the LPA-induced decrease in binding reversed rapidly in two lung cancer epithelial cell lines, H292 and A549, returning to control levels within 3 h. LPA increased phosphorylation of the EGFR in BEAS-2B cells, and this phosphorylation was inhibited by both 4-(3′-chloroanilino)-6,7-dimethoxy-quinazoline (AG1478; EGFR tyrosine kinase inhibitor) and N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide (GM6001; matrix metalloproteinase inhibitor) but not by CRM197 (heparin-binding EGF inhibitor). AG1478 and GM6001 also inhibited the LPA-induced decrease in EGFR binding but only by 50%, suggesting only partial involvement of EGFR transactivation in the decrease in EGFR binding. In summary, LPA stimulates a decrease in EGFR binding in airway epithelial cells that is sustained in normal cells but that rapidly reverses in cancer cells. LPA-induced transactivation of EGFRs occurs and contributes to the decrease in EGFR binding, but additional pathway(s) may also be involved.
Footnotes
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This work was supported by American Heart Fellowship 0415367Z and a Skala Fellowship (to K.M.K.) and GlaxoSmithKline Grant 100909 and Nebraska Department of Health and Human Services Grant 2007-39 (to M.L.T.).
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Portions of this work were published in abstract form: Kassel KM, Parker SM, Danforth BL, Lanik AD, Mihaljevic JA, Meeks AS, Schulte NA, and Toews ML (2005) Lysophosphatidic acid decreases epidermal growth factor receptor binding in airway epithelial cells. FASEB J19:A527 (presented at Experimental Biology 2005, San Diego, CA) and Toews ML, Parker SM, and Kassel KM (2006) Signaling pathways for lysophosphatidic acid (LPA) inhibition of epidermal growth factor receptor (EGFR) binding in lung epithelial cells. FASEB J20:A695 (presented at Experimental Biology 2006, San Francisco, CA). Portions of this work were published as part of K.M.K.'s dissertation: Kassel KM (2007) Regulation of epidermal growth factor receptors and mitogenic signaling by lysophosphatidic acid and β2 adrenergic receptors in airway cells. Doctoral dissertation, University of Nebraska Medical Center, Omaha, NE.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.120584.
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ABBREVIATIONS: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; HASM, human airway smooth muscle; LPA, lysophosphatidic acid; ether LPA, 1-O-octadecyl-2-hydroxy-sn-glycero-3-phosphate; hBEC, human bronchial epithelial cell; MMP, matrix metalloproteinase; GPCR, G protein-coupled receptor; FBS, fetal bovine serum; Pen/Strep, penicillin/streptomycin; AG1478, 4-(3′-chloroanilino)-6,7-dimethoxy-quinazoline; GM6001, N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; S1P, sphingosine-1-phosphate; LPC, lysophosphatidylcholine; PA, phosphatidic acid; HB, heparin-binding; CTL, control (vehicle).
- Received January 25, 2007.
- Accepted July 18, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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