Establishing an effective epidermal barrier requires a series of coordinated molecular events involving keratinocytes (KCs) within a stratified epithelium. Epidermal maturation depends on convergence of pathways involving components of NF-kB and peroxisome proliferator activated receptor (PPAR) signaling systems that promote terminal differentiation and production of a stratum corneum. The Notch-1 receptor and its ligand Delta-1 have been proposed by others to participate in early events in KC differentiation. Here, we establish differential expression patterns for several Notch receptors and ligands in normal human skin. These immunolocalization findings, together with functional studies demonstrating increased levels of Notch ligand/receptors occurring during the onset of differentiation, prompted use of a soluble Notch ligand, a peptide derived from the most conspicuously expressed ligand in skin, Jagged-1. Exposing submerged KC monolayers to this peptide (JAG-1) in co-presence of elevated calcium ion concentration, produced stratification with loricrinexpression.Usingaliving humanepidermal equivalent (EE) model system, when submerged cultures were raised to an air/liquid interface to generate a fully mature epidermis, activation of Notch signaling was detected. Addition of JAG-1 peptide to submerged EEs was sufficient to induce epidermal maturation. Moreover, a soluble decoy Notch inhibitor prevented such differentiation and corneogenesis in human EEs exposed to either an air/liquid interface or to the JAG-1 peptide. In KC monolayers, addition of JAG-1 peptide induced IKKa mediated NF-kB activation, as well as increased PPARg expression. Immunoprecipitation/Western blot analysis revealed a physical association between the p65 subunit of NFkB and PPARg. These results indicate that activation of Notch signaling is necessary for maturation of human epidermis, and activation by a soluble Notch ligand is sufficient to trigger complete KC differentiation including cornification.

The elimination of ultraviolet (UV) radiation-damaged keratinocytes via apoptosis is an important mechanism for the protection of the skin from sunlight, an ubiquitous environmental carcinogen. Due to the pleiotropic nature of UV radiation, the molecular mechanisms of UV-induced apoptosis are poorly understood. Protein kinase C (PKC) is a family of enzymes critically involved in the regulation of differentiation in the epidermis, and is associated with the induction of apoptosis by ionizing radiation in other cell types. In normal human keratinocytes, the induction of apoptosis by UV exposure correlated with generation of the catalytic domain of PKC␦ in the soluble fraction. In contrast, phorbol ester 12-O-tetradecanoylphorbol-13-acetate caused translocation of PKC␦ from the soluble to the particulate fraction without inducing apoptosis. The effect of UV radiation on PKC␦ was isoform specific, as UV exposure did not stimulate the cleavage, or effect the subcellular distribution of any other PKC isoform. The soluble, catalytic domain of PKC␦ induced by UV exposure was associated with an increase in soluble PKC␦ activity. Proteases of the caspase family are activated during UV-induced apoptosis. Inhibition of caspases blocked the UV-induced cleavage of PKC␦ and apoptosis. In addition, inhibition of PKC activity specifically inhibited UV-induced apoptosis of keratinocytes, without affecting the G 0/ G 1 cell cycle block induced by UV exposure. These results indicate that PKC activation is involved in the UV-induced death effector pathway of keratinocytes undergoing apoptosis, and defines a novel role for this enzyme in epidermal homeostasis.

Skin provides an attractive organ system for exploring coordinated regulation of keratinocyte (KC) proliferation, differentiation, senescence, and apoptosis. Our main objective was to determine whether various types of cell cycle arrest confer resistance to apoptosis. We postulated that KC cell cycle and cell death programs are tightly regulated to ensure epidermal homeostasis. In this report, simultaneous expression of cyclin-dependent kinase inhibitors (p15, p16, p21, and p27), a marker of early differentiation (keratin 1), mediators of apoptosis (caspases 3 and 8), and NF-B were analyzed in three types of KCs. By comparing the response of proliferating, senescent, and immortalized KCs (HaCaT cells) to antiproliferative agents followed by UV exposure, we observed: 1) Normal KCs follow different pathways to abrupt cell cycle arrest; 2) KCs undergoing spontaneous replicative senescence or confluency predominantly express p16; 3) Abruptly induced growth arrest, confluency, and senescence pathways are associated with resistance to apoptosis; 4) The death-defying phenotype of KCs does not require early differentiation; 5) NF-B is one regulator of resistance to apoptosis; and 6) HaCaT cells have undetectable p16 protein (hypermethylation of the promoter), dysfunctional NF-B, and diminished capacity to respond to antiproliferative treatments, and they remain highly sensitive to apoptosis with cleavage of caspases 3 and 8. These data indicate that KCs (but not HaCaT cells) undergoing abruptly induced cell cycle arrest or senescence become resistant to apoptosis requiring properly regulated activation of NF-B but not early differentiation.

The desmosome is a highly organized plasma membrane domain that couples intermediate filaments to the plasma membrane at regions of cell–cell adhesion. Desmosomes contain two classes of cadherins, desmogleins, and desmocollins, that bind to the cytoplasmic protein plakoglobin. Desmoplakin is a desmosomal component that plays a critical role in linking intermediate filament networks to the desmosomal plaque, and the amino-terminal domain of desmoplakin targets desmoplakin to the desmosome. However, the desmosomal protein(s) that bind the amino-terminal domain of desmoplakin have not been identified. To determine if the desmosomal cadherins and plakoglobin interact with the amino-terminal domain of desmoplakin, these proteins were co-expressed in L-cell fibroblasts, cells that do not normally express desmosomal components. When expressed in L-cells, the desmosomal cadherins and plakoglobin exhibited a diffuse distribution. However, in the presence of an amino-terminal desmoplakin polypeptide (DP-NTP), the desmosomal cadherins and plakoglobin were observed in punctate clusters that also contained DP-NTP. In addition, plakoglobin and DP-NTP were recruited to cell–cell interfaces in L-cells co-expressing a chimeric cadherin with the E-cadherin extracellular domain and the desmoglein-1 cytoplasmic domain, and these cells formed structures that were ultrastructurally similar to the outer plaque of the desmosome. In transient expression experiments in COS cells, the recruitment of DP-NTP to cell borders by the chimera required co-expression of plakoglobin. Plakoglobin and DP-NTP co-immunoprecipitated when extracted from L-cells, and yeast two hybrid analysis indicated that DP-NTP binds directly to plakoglobin but not Dsg1. These results identify a role for desmoplakin in organizing the desmosomal cadherin–plakoglobin complex and provide new insights into the hierarchy of protein interactions that occur in the desmosomal plaque.

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