AKT1 provides an essential survival signal required for differentiation and stratification of primary human keratinocytes.

Keratinocyte differentiation and stratification are complex processes involving multiple signaling pathways, which convert a basal proliferative cell into an inviable rigid squame. Loss of attachment to the basement membrane triggers keratinocyte differentiation, while in other epithelial cells, detachment from the extracellular matrix leads to rapid programmed cell death or anoikis. The potential role of AKT in providing a survival signal necessary for stratification and differentiation of primary human keratinocytes was investigated. AKT activity increased during keratinocyte differentiation and was attributed to the specific activation of AKT1 and AKT2. Targeted reduction of AKT1 expression, but not AKT2, by RNA interference resulted in an abnormal epidermis in organotypic skin cultures with a thin parabasal region and a pronounced but disorganized cornified layer. This abnormal stratification was due to significant cell death in the suprabasal layers and was alleviated by caspase inhibition. Normal expression patterns of both early and late markers of keratinocyte differentiation were also disrupted, producing a poorly developed stratum corneum.

E7 abolishes raf-induced arrest via mislocalization of p21(Cip1).

The cellular response to oncogenic Ras depends upon the presence or absence of cooperating mutations. In the absence of immortalizing oncogenes or genetic lesions, activation of the Ras/Raf pathway results in a p21(Cip1)-dependent cellular arrest. The human papillomavirus oncoprotein E7 transforms primary cells in cooperation with Ras and abolishes p21(Cip1)-mediated growth arrest in the presence of various antimitogenic signals. Here we have utilized a conditional Raf molecule to investigate the effects of E7 on p21(Cip1) function in the context of Raf-induced cellular arrest. E7 bypassed Raf-induced arrest and alleviated inhibition of cyclin E-CDK2 without suppressing Raf-specific synthesis of p21(Cip1) or derepressing p21(Cip1)-associated CDK2 complexes. Activation of Raf led to nuclear accumulation of p21(Cip1), and we provide evidence that this effect is mediated by inhibition of Akt, a regulator of p21(Cip1) localization. Loss of Akt activity appears to be an important event in the cellular arrest associated with Raf-induction, since maintenance of Akt activity was necessary and sufficient to bypass Raf-induced arrest. In agreement, expression of E7 sustained Akt activity and reduced nuclear accumulation of p21(Cip1), resulting in decreased association between p21(Cip1) and cyclin E-CDK2. Taken together, these data suggest that E7 inhibits p21(Cip1) function in the context of Raf signaling by altering Raf-Akt antagonism and preventing the proper subcellular localization of p21(Cip1). We propose that E7 elicits a proliferative response to Raf signaling by targeting p21(Cip1) function via a novel mechanism.