To elucidate the biological role of Stat3 in the skin, conditional gene targeting using the Cre-loxP system was performed as germline Stat3 ablation leads to embryonic lethality. K5-Cre;Stat3 flox/-transgenic mice, whose epidermal and follicular keratinocytes lack functional Stat3, were viable and the development of epidermis and hair follicles appeared normal. However, hair cycle and wound healing processes were severely compromised. Furthermore, mutant mice expressed sparse hair and developed spontaneously occurring ulcers with age. Growth factor-dependent in vitro migration of Stat3-disrupted keratinocytes was impaired despite normal proliferative responses. We therefore conclude that Stat3 plays a crucial role in transducing a signal required for migration but not for proliferation of keratinocytes, and that Stat3 is essential for skin remodeling, including hair cycle and wound healing.

Glycosylphosphatidylinositol (GPI)-anchored proteins are widely distributed on plasma membranes of eukaryotes. More than 50 GPI-anchored proteins have been shown to be spatiotemporally expressed in mice with a deficiency of GPI-anchor biosynthesis that causes embryonic lethality. Here, we examine the functional roles of GPIanchored proteins in mouse skin using the Cre-loxP recombination system. We disrupted the Pig-a gene, an X-linked gene essential for GPI-anchor biosynthesis, in skin. The Cre-mediated Pig-a disruption occurred in skin at almost 100% efficiency in male mice bearing two identically orientated loxP sites within the Pig-a gene. Expression of GPIanchored proteins was completely absent in the skin of these mice. The skin of such mutants looked wrinkled and more scaly than that of wild-type mice. Furthermore, histological examination of mutant mice showed that the epidermal horny layer was tightly packed and thickened. Electron microscopy showed that the intercellular space was narrow and there were many small vesicles embedded in the intercellular space that were not observed in equivalent wild-type mouse skin preparations. Mutant mice died within a few days after birth, suggesting that Pig-a function is essential for proper skin differentiation and maintenance.

Ras acts with other proteins to induce neoplasia. By itself, however, strong Ras signaling can suppress proliferation of normal cells. In primary epidermal cells, we found that oncogenic Ras transiently decreases cyclin-dependent kinase (CDK) 4 expression in association with cell cycle arrest in G1 phase. CDK4 co-expression circumvents Ras growth suppression and induces invasive human neoplasia resembling squamous cell carcinoma. Tumorigenesis is dependent on CDK4 kinase function, with cyclin D1 required but not sufficient for this process. In facilitating escape from G1 growth restraints, Ras and CDK4 alter the composition of cyclin D and cyclin E complexes and promote resistance to growth inhibition by INK4 cyclin-dependent kinase inhibitors. These data identify a new role for oncogenic Ras in CDK4 regulation and highlight the functional importance of CDK4 suppression in preventing uncontrolled growth.