Previous work has shown that the epidermal growth factor receptor (EGFR) tyrosine kinase moiety provides protection to normal human keratinocytes against apoptosis. This protection is, at least in part, due to EGFRdependent expression of the antiapoptotic Bcl-2 family member, Bcl-x L . Here we focused on intracellular signaling pathways relevant to keratinocyte survival and/or Bcl-x L expression. By using pharmacological inhibitors and dominant negative expression constructs, we observed that phosphatidylinositol 3-kinase/AKT and phospholipase C␥/protein kinase C␣ activation were required for keratinocyte survival independently of EGFR activation or Bcl-x L expression. By contrast, MEK activity required EGFR activation and, as shown by use of the MEK inhibitor PD98059 and a dominant negative MEK construct, was necessary for Bcl-x L expression and survival. Consistent with an earlier study, blocking SRC kinase activities similarly led to down-regulation of Bcl-x L protein expression and impaired keratinocyte survival. In conclusion, our results demonstrate that EGFR-dependent MEK activity contributes to both Bcl-x L expression and survival of normal keratinocytes. Other signaling pathways (i.e. phosphatidylinositol 3-kinase/AKT and phospholipase C␥/protein kinase C␣) are obligatory to keratinocyte survival but not to Bcl-x L expression, and control of these pathways by EGFR activation is not rate-limiting to normal keratinocyte survival.

Normal epithelial cells undergo apoptosis when they are denied contact with the extracellular matrix, in a process termed "anoikis." Conversely, malignant epithelial cells typically acquire anchorage independence, i.e., the capacity to survive and grow in the absence of matrix interaction. Here we asked the question whether anoikis is affected by signaling through the EGF receptor (EGFR). We focused on the EGFR because EGFR signaling is frequently deregulated in malignant epithelial cells. We demonstrate that EGFR activation markedly alleviated the requirement of matrix engagement for survival of primary and immortalized human keratinocytes in suspension culture. Protection of epithelial cells through EGFR activation against anoikis was associated with and required sustained MAPK phosphorylation during the early phase of suspension culture. Interestingly, high levels of MAPK phosphorylation were not only required for EGFR-mediated protection against anoikis but also occurred as a consequence of caspase activation at later stages of suspension culture. These results demonstrate that EGFR activation contributes to anchorage-independent epithelial cell survival and identify MAPK activation as an important mechanism in this process. INTRODUCTIONNormal epithelial cells require contact with extracellular matrix components to survive. In the absence of matrix attachment, these cells die exhibiting molecular characteristics of programmed cell death or apoptosis (Meredith et al., 1993;Frisch and Francis, 1994). This form of cell death has been termed anoikis (Frisch and Francis, 1994) and is likely to preclude dissemination of normal epithelial cells to inappropriate sites. Malignant transformation by oncogenic forms of Ras and Src confers resistance of cultured epithelial cells to anoikis (Frisch and Francis, 1994;Rosen et al., 2000). Activation of proto-oncogenic forms of Ras and Src in epithelial cells occurs not only during integrin engagement but also in response to soluble growth factors, including ligands for the EGF receptor (EGFR) (Luttrell et al., 1994;Walker et al., 1998). Our previous work has implicated the EGFR in protecting normal human keratinocytes against apoptosis of adherent cells in vitro (Rodeck et al., 1997a,b;Jost et al., 1999). Together, these findings led us to investigate whether signaling from the EGFR may also affect survival of human keratinocytes in the absence of matrix engagement. This is an important question because effective growth factor receptor signaling has been shown in fibroblasts to be contingent on adhesion receptor signaling (Lin et al., 1997;Renshaw et al., 1997;Bottazzi et al., 1999;Roovers et al., 1999). Thus, it may be argued that EGFR activation is not likely to affect survival of cells in forced suspension culture.By contrast, we demonstrate here that activation of the EGFR by endogenous and exogenous ligands substantially delayed death of normal human keratinocytes held in forced suspension. We describe that EGFR-dependent protection of keratinocytes against anoikis req...

Cell-substratum adhesion is an essential requirement for survival of human neonatal keratinocytes in vitro. Similarly, activation of the epidermal growth factor receptor (EGF-R) has recently been implicated not only in cell cycle progression but also in survival of normal keratinocytes. The mechanisms by which either cell-substratum adhesion or EGF-R activation protect keratinocytes from programmed cell death are poorly understood. Here we describe that blockade of the EGF-R and inhibition of substratum adhesion share a common downstream event, the down-regulation of the cell death protector Bcl-x L . Expression of Bcl-x L protein was down-regulated during forced suspension culture of keratinocytes, concurrent with large-scale apoptosis. Similarly, EGF-R blockade was accompanied by down-regulation of Bcl-x L steady-state mRNA and protein levels to an extent comparable to that observed in forced suspension culture. However, down-regulation of Bcl-x L expression by EGF-R blockade was not accompanied by apoptosis; in this case, a second signal, generated by passaging, was required to induce rapid and large-scale apoptosis. These findings are consistent with the conclusions that (i) Bcl-x L represents a shared molecular target for signaling through cell-substrate adhesion receptors and the EGF-R, and (ii) reduced levels of Bcl-x L expression through EGF-R blockade lower the tolerance of keratinocytes for cell death signals generated by cellular stress.