Thus, EGFR activation is important for several downstream responses to HDE but is not the only mechanism for initiating HDE responses. Open in a separate window Figure 6. HDE stimulation of intracellular Ca2+ mobilization independent of EGFR activation. inhibitor AG1478. AG1478 and EGFR-neutralizing antibody reduced HDE-stimulated IL-6 ZSTK474 and IL-8 release by about half. Similar EGFR phosphorylation and requirement of EGFRs for maximal IL-6 and IL-8 release were found with primary isolates of human bronchial epithelial cells. Because HDE-stimulated IL-6 and IL-8 release involve the Ca2+Cdependent protein kinase C, we hypothesized that HDE would induce intracellular Ca2+ mobilization. ZSTK474 HDE exposure induced intracellular Ca2+ mobilization in Beas-2B cells and in primary cell isolates, but this response was neither mimicked by EGF nor inhibited by AG1478. Thus, HDE activates EGFRs and their downstream signaling, and EGFR activation is required for some but not all airway epithelial cell responses to HDE. model system for assessing the molecular mechanisms that may contribute to the human disease. Our laboratories have shown that an aqueous extract of Rabbit polyclonal to Shc.Shc1 IS an adaptor protein containing a SH2 domain and a PID domain within a PH domain-like fold.Three isoforms(p66, p52 and p46), produced by alternative initiation, variously regulate growth factor signaling, oncogenesis and apoptosis. this dust (hogbarn dust extract [HDE]) induces multiple effects in cultured human airway epithelial cells that are similar to those seen in patients, including release of IL-6 and IL-8 (11, 12), recruitment of neutrophils (9), and increased lymphocyte adherence (13). Several components of the cellular signaling responses to HDE have been established, in particular the sequential roles of protein kinase (PK)C- and tumor necrosis factor (TNF)- to facilitate IL-6 release, followed by additional activation of PKC-? to promote IL-8 release (12). The identity of any specific cell surface receptors that may detect active components of HDE and initiate the cellular responses to HDE has not been established. Epidermal growth factor receptors (EGFRs) are critical regulators of diverse functions of airway epithelial cells (14, 15). In addition to their well established role in lung cancer (16), EGFRs play important roles in mucus secretion (17), in IL-8 release (18), and in various other immune and inflammatory responses of these cells (19, 20). In particular, several inhaled agents have been shown to mediate effects on airway epithelial cells via EGFR transactivation, including bacterial lipopolysaccharide (21) and lipoteichoic acid (22), cigarette smoke (23), and various environmental and occupational dusts and particulates (24C27). Transactivation can occur by activation of cellular proteases that in turn release cell surface proligands to activate the EGFR extracellularly (18, 28) or by activation of cytosolic Src-family tyrosine kinases that directly phosphorylate the EGFR, leading to its ligand-independent intracellular activation (29, 30). The current studies tested whether EGFRs are activated upon exposure of airway epithelial cells to HDE and assessed whether EGFR activation is required for downstream cellular responses. Because of the importance of the Ca2+Cdependent PKC- isozyme for cytokine responses to HDE (12), the ability of HDE to stimulate Ca2+ mobilization was tested. The data show that HDE induces EGFR phosphorylation and that the tyrosine kinase activity of the activated EGFR is required for maximal IL-6 and IL-8 release in response to HDE. HDE also stimulates increases in intracellular Ca2+, but this response is not mimicked by EGF and is independent of EGFR activation by HDE. Thus, EGFRs are essential for some but not all of the cellular responses initiated upon exposure of airway epithelial cells to HDE. Portions of this work have been presented previously in meeting abstracts (31, 32). Materials and Methods Reagents The sources of all reagents are presented in the online supplement. Cell Culture All assays were conducted with the Beas-2B cell line or with primary isolates of human bronchial epithelial cells (hBECs). Details of cell isolation and culture are presented in the online supplement. Hogbarn Dust and ZSTK474 Extract Preparation and Properties The aqueous HDE used in these studies was prepared by dissolving 1 g of settled dust collected from regional swine confinement facilities in 10 ml of HEPES-buffered (pH 7.4) saline solution. This extract was centrifuged, and the supernate was filter sterilized and considered 100% HDE, as previously described (11, 33). This ZSTK474 HDE was used at 5% (vol/vol) unless indicated otherwise. Additional details on the properties of the dust used for these studies are presented in the online supplement. EGFR and ERK Phosphorylation Assays Details of procedures are presented in the online supplement. Briefly, cells were grown to confluence and starved overnight before being treated with HDE or with EGF or lysophosphatidic acid (LPA) for comparison, both in the absence or presence of inhibitors in some experiments. For EGFR phosphorylation assays, EGFRs were immunoprecipitated, electrophoresed, and then blotted for quantification of ZSTK474 total and phosphorylated EGFRs..
