CHARACTERIZATION OF APOPTOTIC MACHINERY CONTROLLING CELL DEATH IN EPIDERMIS.

Ironically, the functional integrity of epidermis is dependent on a “planned cell death” pathway by which keratinocytes (KCs) undergo terminal differentiation, and then die producing comeocytes. To explore the molecular basis for crucial steps regulating epidermal KC cell death, normal human skin was examined by immunostaining and Western blot analysis. Fractionating KCs by state of maturation using discontinuous Percoll gradients followed by immunoblotting confirmed immunohistology results in which KCs in the upper level strata – including the granular cell layer expressed high levels of TRAIL and its 2 death receptors (DRs), but not decoy receptors (DcRs), whereas low and mid level KCs expressed 2 DcRs for TRAIL. While caspases 8 and 3 were detected equally throughout all cell layers, caspase 14 was preferentially expressed in upper layer KCs. To establish a cause and effect relationship amongst these mediators of cell death, submerged living epidermal equivalents (EEs) containing stratified, multi-layered but relatively undifferentiated KCs grown on a permeable membrane were raised to an air/liquid interface (A/L-I), which triggers KC terminal differentiation/corneogenesis. Western blot analysis of proteins extracted from submerged EEs versus EEs raised to A/L-I for 2 days or 5 days revealed a 5-10 fold increase in TRAIL levels, accompanied by increased TRAIL DRs and decreased DcRs. Moreover, upon raising EEs to A/L-I triggers degradation (activation) of caspases 14, 8 and 3. Pre-incubation of submerged EEs with a cocktail of caspase inhibitors (CIs) blocked these biochemical reactions and subsequent KC terminal differentiation / cell death. To further explore a role for TRAIL as a relevant death inducing ligand in normal epidermis, cultured KCs were grown to confluence and exposed to either TNF- a (10^3 U/ml), soluble FasL (20 ng/ml), or LZ-TRAIL (100 ng/ml). Only TRAIL could induce rapid and prominent apoptosis of KC monolayers, which was blocked by CIs. Moreover, TRAIL induced greater apoptosis in confluent versus non-confluent cultures which correlated to enhanced DR and decreased DcR cell surface expression (determined by FACS analysis), which occurred in KCs upon reaching a state of confluency. These results indicate that TRAIL together with specific effector caspases play key roles as physiological mediators of KC cell death in normal human skin.

Apoptosis, Caspase-14, Caspase-3, Caspase-8, Corneocytes, Corneogenesis, Death receptors (DRs), Decoy receptors (DcRs), EpiDerm, EpiDerm-201, TRAIL