Figure 8. Signaling is blocked in keratinocytes derived from conditional c-Met mutant mice that are treated with HGF/SF, but not with TGF . (A) Western blot analysis of phospho Erk1/2, total Erk1/2, phospho-Akt, total Akt, phospho-Gab1, and phospho-PAK1/2 in keratinocytes derived from control and conditional c-Met mutant mice. Cells were stimulated with HGF/SF or TGFα for 0, 10, or 30 min. Note that Erk1/2, Akt, Gab1, and PAK1/2 are not activated (phosphorylated) in cultured keratinocytes from the conditional mutant mice after HGF/SF stimula- tion. (B) Quantiﬁcation of the phospho-PAK1/2 signal on Western blots shown in A, as assessed by pixel intensity. Discussion We show that c-Met and HGF/SF expression is induced in the HE of skin wounds, indicating that receptor and ligand may act in an autocrine manner during wound healing. We also report that c-Met is crucial for reepithelialization during wound closure in the skin and in cell culture; c-Met mutant keratino- cytes cannot contribute to the generation of the HE in vivo, and contribute inefficiently to the closure of scratch wounds in vitro. Analysis of cultured keratinocytes during the closure of scratch wounds indicates that the primary deficit of the mu- tant cells is their inability to proliferate and to migrate into the wounded area. HGF/SF and c-Met represent an autocrine system in wound healing In general, HGF/SF and c-Met are expressed in different cell types, although these may be closely apposed to allow an exchange of signals. For instance, HGF/SF is expressed primarily in mesen- chymal cells, whereas c-Met is generally expressed in nearby epi- thelia (Sonnenberg et al., 1993; Yang and Park, 1995; Birchmeier and Gherardi, 1998). Moreover, c-Met is expressed in the epithe- lial dermomyotome and in the migrating muscle progenitor cells that derive from it, whereas HGF/SF is expressed in mesenchymal cells close to the somites and along the route of the migrating cells (Birchmeier et al., 2003). In the cerebellum, granule cells express HGF/SF, whereas surrounding Bergmann glia cells express the c-Met receptor (Jung et al., 1994). In tumors, however, autocrine HGF/SF and c-Met signaling is frequently observed (Birchmeier et al., 2003). Our expression and genetic data indicate that, also in a normal physiological process (i.e., during healing of skin wounds), autocrine HGF/SF/c-Met signaling is required. HGF/SF and c-Met appear late during evolution and are only present in vertebrates (Birchmeier et al., 2003). In devel- opment, HGF/SF and c-Met control placentation and muscle precursor cell migration, which are processes that appear late in evolution. Genetic analyses demonstrate that c-Met is also impor- tant in regeneration of adult tissues, which we show here for skin repair after wounding, and which was previously demonstrated for liver regeneration (Borowiak et al., 2004; Huh et al., 2004). The conditional mutation of c-Met in the liver was introduced by the use of the inducible Mx-cre transgene, and a portion of the c-MET IN WOUND HEALING • CHMIELOWIEC ET AL. 159