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 subsequent gene expression in human skin organ culture. Our preliminary data indicate that MPs are required for autocrine ERK activation in normal human keratinocytes (NHK). They also demonstrate that MP inhibitors block proliferation and wound-induced migration of NHK, and that different ligands appear to be involved in LPA stimulation, autocrine proliferation, and wound-induced migration of NHK. Together, these data indicate that autocrine keratinocyte (KC) signaling requires context-dependent cleavage of membrane-bound ErbB ligands by MPs. The MP(s) involved in transactivating ErbB receptors in skin and KCs have not yet been elucidated. 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 EGF receptors in mammary epithelial cells. Our preliminary data indicate that ADAM10 is abundantly expressed in NHK, and that expression of the active form of ADAM10 correlates with EGF-independent migration and ERK activation. 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:
Specific 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), and (b) to elucidate the context-dependent functions of the various autocrine ErbB ligands identified in Aim la.
Specific Aim 2 determines whether and in which contexts ADAM10 is involved in the shedding of ErbB ligands in human KCs.
