Substantial evidence implicates EGF-like growth factors (GFs) in promoting the survival, proliferation, migration and invasiveness of normal and malignant epithelial cells. Several different signaling pathways have been shown to promote shedding of membrane-bound EGF-like GFs. Our published data demonstrate that metalloproteinases (MPs) are a major stimulus for ErbB signaling, ERK activation and gene expression in skin organ culture. Our preliminary data indicate that MPs are required for autocrine ERK activation, proliferation and wound-induced migration of NHK, and that different ligands appear to be involved in different cellular responses. Together, these data indicate that autocrine keratinocyte (KC) signaling requires context-dependent cleavage of membrane-bound ErbB ligands by MPs. Several members of the ADAM family are strongly implicated in ectodomain processing of various membrane-bound proteins. ADAM10 was recently shown to mediate the basal as well as G protein-coupled receptors (GPCR)- stimulated activation of ErbB1 in epithelial cells. Our preliminary data indicate that ADAM10 is abundantly expressed in NHK, that expression of the active form of ADAM 10 correlates with EGF-independent migration and ERK activation, and that overexpression of ADAM10 promotes cleavage of heparin-binding EGF-like growth factor (HB-EGF). We also find that overexpression of dominant negative (dn)ADAM10 leads to KC cell death in the context of KC reattachment after trypsinization. Based on these observations, we hypothesize that KCs utilize ADAM10 to catalyze shedding of one or more transmembrane EGF-like GF precursors in a context-dependent manner, leading to stimulus-specific KC responses. To test this hypothesis, we propose the following specific aims:
Aim 1 : To (a) determine which ErbB receptor ligands are shed from KC via MP-dependent proteolysis in three cellular contexts (LPA stimulation, autocrine proliferation, and wound-induced migration);(b) compare the patterns of ErbB ligands released by cultured KCs vs. skin organ cultures, and (c) to elucidate the context-dependent functions of the various autocrine ErbB ligands identified in Aims 1a &b.
Aim 2 : To determine whether and in which contexts ADAM10 is involved in the shedding of ErbB ligands in human KCs.
Aim 3 : To characterize the mechanisms whereby ADAM-10 elicits cellular responses in human KCs.
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