Abstract
Acidic mammalian chitinase (AMCase) is expressed in an exaggerated fashion in epithelial cells at sites of pulmonary T helper cell type 2 inflammation and plays important roles in the pathogenesis of anti-parasite and asthma-like responses. However, the mechanisms that control epithelial cell AMCase secretion and its effector responses have not been adequately defined. To address these issues, we used in vivo and in vitro experimental systems to define the pathways of epithelial AMCase secretion and its epithelial regulatory effects. Here we demonstrate that, in murine T helper cell type 2 modeling systems, AMCase colocalizes with the epidermal growth factor receptor (EGFR) and ADAM17 (a membrane disintegrin and metallopeptidase 17) in lung epithelial cells. In vitro cotransfection experiments in A549 cells demonstrated that AMCase and EGFR physically interact with each other. Cotransfection of AMCase and EGFR also increased, whereas EGFR inhibition decreased AMCase secretion. Interestingly, AMCase secretion was not significantly altered by treatment with EGF but was significantly decreased when the upstream EGFR transactivator ADAM17 was inhibited. AMCase secretion was also decreased when the EGFR-downstream Ras was blocked. Transfected and recombinant AMCase induced epithelial cell production of CCL2, CCL17, and CXCL8. These studies demonstrate that lung epithelial cells secrete AMCase via an EGFR-dependent pathway that is activated by ADAM17 and mediates its effects via Ras. They also demonstrate that the AMCase that is secreted feeds back in an autocrine and/or paracrine fashion to stimulate pulmonary epithelial cell chemokine production.
Highlights
Epidermal growth factor receptor (EGFR) activation regulates epithelial cell activation, differentiation, proliferation, and survival (8 –10)
Given the important roles of ADAM17 and EGFR signaling in IL-13-mediated activation of epithelial cells and allergic asthma, we hypothesized that a ADAM17/EGFR pathway contributes to the secretion of acidic mammalian chitinase (AMCase) by lung epithelial cells
AMCase, EGFR, and ADAM17 Are Induced and Co-localize in Vivo at Sites of Th2 and Th2-Cytokine-mediated Airway Inflammation—To characterize and quantify intracellular AMCase expression in lung epithelial cells in vivo, we established a flow cytometric epithelial evaluation method based on light scatter parameters, negative expression of CD45, negative expression of CD31, and positive expression of cytokeratin (Fig. 1)
Summary
Th2, T helper cell type 2; AMCase, acidic mammalian chitinase; EGFR, epidermal growth factor receptor; TGF-␣, transforming growth factor-␣; IL, interleukin; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; PBS, phosphate-buffered saline; WT, wild type; OVA, ovalbumin. A number of studies have linked epithelial ADAM17 and EGFR signaling to IL-13 These studies demonstrated that IL-13 induces mucus metaplasia via an EGFRdependent pathway [17], ADAM17 plays a critical role in the regulation of MUC5AC mucin expression in cultured human airway epithelial cells [18], and IL-13 induces the proliferation of normal human bronchial epithelial cells via ADAM17-induced ectodomain shedding of TGF-␣ [19]. We hypothesized that secreted AMCase feeds back to regulate epithelial cell function To test these hypotheses, studies were undertaken to define the interactions of AMCase and EGFR and the role of the EGFR pathway in epithelial AMCase secretion. The effector responses of secreted AMCase were evaluated These studies demonstrate that respiratory epithelial cells secrete AMCase via a ADAM17/EGFR/Ras-dependent pathway. They highlight the ability of AMCase to stimulate epithelial chemokine elaboration
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