ABSTRACT
Keratinocytes become migratory to heal wounds, during early neoplastic invasion, and when undergoing telomere-unrelated senescence in culture. All three settings are associated with expression of the cell cycle inhibitor p16INK4A (p16) and of the basement membrane protein laminin 5 (LN5). We have investigated cause-and-effect relationships among laminin 5, p16, hypermotility, and growth arrest. Plating primary human keratinocytes on the gamma2 precursor form of laminin 5 (LN5') immediately induced directional hypermotility at approximately 125 microm/hour, followed by p16 expression and growth arrest. Cells deficient in p16 and either p14ARF or p53 became hypermotile in response to LN5' but did not arrest growth. Plating on LN5' triggered smad nuclear translocation, and all LN5' effects were blocked by a transforming growth factor (TGF) beta receptor I (TGFbetaRI) kinase inhibitor. In contrast, plating cells on collagen I triggered a TGFbetaRI kinase-independent hypermotility unaccompanied by smad translocation or growth arrest. Plating on control surfaces with TGFbeta induced hypermotility after a 1-day lag time and growth arrest by a p16-independent mechanism. Keratinocytes serially cultured with TGFbetaRI kinase inhibitor exhibited an extended lifespan, and immortalization was facilitated following transduction to express the catalytic subunit of telomerase (TERT). These results reveal fundamental features of a keratinocyte hyper-motility/growth-arrest response that is activated in wound healing, tumor suppression, and during serial culture.
Subject(s)
Cell Adhesion Molecules/metabolism , Cell Culture Techniques/methods , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Keratinocytes/metabolism , Wound Healing , Cell Movement , Cell Proliferation , Cellular Senescence , Female , Humans , Male , Receptors, Transforming Growth Factor beta/metabolism , Tumor Suppressor Protein p14ARF/metabolism , KalininABSTRACT
The replicative lifespan of human keratinocytes in culture is restricted by a telomere-unrelated induction of p16INK4A (p16) and p14ARF. We have found that, in vivo, p16 is expressed by epidermal and oral keratinocytes at the migrating fronts of healing wounds and at the stromal interface of severely dysplastic and early invasive lesions and that such cells also invariably display increased expression of Laminin 5 (Lam5). In culture, p16 and Lam5 are coexpressed in keratinocytes at senescence, at the edges of wounds made in confluent cultures, and when cells are plated on dishes coated with the gamma2 precursor form of Lam5 (Lam5gamma2pre). Lam5/p16 coexpression in all three in vitro settings is associated with directional hypermotility and growth arrest. Hypermotility and growth arrest are uncoupled in p16- and p14ARF/p53-deficient keratinocytes and squamous cell carcinoma (SCC) cells; such cells become hypermotile is response to Lam5gamma2pre but do not growth arrest. Thus, the Lam5/p16 response is activated in normal wound healing, causing growth arrest of migratory keratinocytes that lead wound reepithelialization. This response also becomes activated at a critical stage of neoplastic progression, acting as a tumor suppressor mechanism. Rare premalignant cells that lose p16 remain motile and proliferative, thereby resulting in invasive growth as SCC.
