DIFFERENTIATION OF CULTURED HUMAN EPIDERMAL-KERATINOCYTES AT HIGH CELL DENSITIES IS MEDIATED BY ENDOGENOUS ACTIVATION OF THE PROTEIN-KINASE-C SIGNALING PATHWAY

Normal human epidermal keratinocytes (NHEK) grown in serum-free medium on a plastic substrate spontaneously differentiate at high cell densi ties in vitro, Because protein kinase C (PKC) regulates murine keratin ocyte differentiation triggered by a variety of stimuli, we examined t he role of this signaling pathway in density-dependent activation of N HEK differentiation. Relative to subconfluent cultures, confluent NHEK expressed markedly higher levels of multiple differentiation markers assayed by immunoblotting, including keratin 1, loricrin, filaggrin, i nvolucrin, TG(K), and SPR-1. Expression of several of these markers co ntinued to increase for several days after cells reached confluency, T he total level of several PKC isoforms was not substantially altered i n NHEK harvested at different cell densities, based on immunoblotting; however, subcellular fractionation revealed that PKC alpha underwent a redistribution to the particulate fraction in confluent and postconf luent NHEK cultures, suggesting that this isozyme was activated under these conditions and may be involved in triggering the terminal differ entiation program. Supporting this concept, inhibition of PKC function using bryostatin 1 or GF 109203X blocked the induction of keratinocyt e differentiation markers at high cell densities. These data suggest t hat endogenous activation of PKC is responsible for cell density-media ted stimulation of NHEK differentiation, establishing a critical role for this pathway in regulating human as well as murine keratinocyte di fferentiation.