Identification of tumor necrosis factor-α (TNFα) as the key agent in inflammatory disorders, e.g. rheumatoid arthritis, Crohn's disease, and psoriasis, led to TNFα-targeting therapies, which, although avoiding many of the side-effects of previous drugs, nonetheless causes other side-effects, including secondary infections and cancer. By controlling gene expression, TNFα orchestrates the cutaneous responses to environmental damage and inflammation. To define TNFα action in epidermis, we compared the transcriptional profiles of normal human keratinocytes untreated and treated with TNFα for 1, 4, 24, and 48 h by using oligonucleotide microarrays. We found that TNFα regulates not only immune and inflammatory responses but also tissue remodeling, cell motility, cell cycle, and apoptosis. Specifically, TNFα regulates innate immunity and inflammation by inducing a characteristic large set of chemokines, including newly identified TNFα targets, that attract neutrophils, macrophages, and skin-specific memory T-cells. This implicates TNFα in the pathogenesis of psoriasis, fixed drug eruption, atopic and allergic contact dermatitis. TNFα promotes tissue repair by inducing basement membrane components and collagen-degrading proteases. Unexpectedly, TNFα induces actin cytoskeleton regulators and integrins, enhancing keratinocyte motility and attachment, effects not previously associated with TNFα. Also unanticipated was the influence of TNFα upon keratinocyte cell fate by regulating cell-cycle and apoptosis-associated genes. Therefore, TNFα initiates not only the initiation of inflammation and responses to injury, but also the subsequent epidermal repair. The results provide new insights into the harmful and beneficial TNFα effects and define the mechanisms and genes that achieve these outcomes, both of which are important for TNFα-targeted therapies.

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Glucocorticoids (GCs) have a long history of use as therapeutic agents for numerous skin diseases. Surprisingly, their specific molecular effects are largely unknown. To characterize GC action in epidermis, we compared the transcriptional profiles of primary human keratinocytes untreated and treated with dexamethasone (DEX) for 1, 4, 24, 48, and 72 h using large scale microarray analyses. The majority of genes were found to be regulated only after 24 h and remained regulated throughout treatment. In addition to regulation of the expected pro-inflammatory genes, we found that GCs regulate cell fate, tissue remodeling, cell motility, differentiation, and metabolism. GCs suppress the expression of essentially all IFNγ-regulated genes, including IFNγ receptor and STAT-1, an effect that was previously unknown. GCs also block STAT-1 activation and nuclear translocation. Unexpectedly, GCs induce the expression of anti-apoptotic genes and repress pro-apoptotic ones, preventing UV-induced keratinocyte apoptosis. Consequently, treatment with GCs blocked UV-induced apoptosis of keratinocytes. GCs have profound effect on wound healing by inhibiting cell motility and the expression of the proangiogenic factor, vascular endothelial growth factor. They play an important role in tissue remodeling and scar formation by suppressing the expression of TGFβ1 and -2 and MMP1, -2, -9, and -10 and inducing TIMP-2. Finally, GCs promote terminal epidermal differentiation while simultaneously inhibiting early stage differentiation. These results provide new insights into the beneficial and adverse effects of GCs in the epidermis, defining the participating genes and mechanisms that coordinate the cellular responses important for GC-based therapies.

Epidermal injury results in activation of keratinocytes which produce and respond to growth factors and cytokines and become migratory. Activated keratinocytes express a specific pair of keratin proteins, K6 and K16, distinct from the keratins in the healthy epidermis. Keratinocytes can be activated, for example, by binding of the appropriate ligands to the epidermal growth factor receptor (EGFR). We have analyzed the effects of EGFR activation on keratin gene transcription by transfecting DNAs containing keratin promoters linked to a reporter gene into primary cultures of human epidermal keratinocytes in the presence or absence of EGF or transforming growth factor alpha (TGF alpha), two growth factors that activate EGFR. The activation of EGFR had no effect on the promoters of simple epithelial, basal-layer-specific, or differentiation-specific keratins. In contrast, the expression of K6 and K16 was strongly and specifically induced. A 20-bp DNA segment of the K16 gene promoter conveyed the EGF regulation, functioned in a heterologous construct, and therefore constituted an EGF-responsive element. A nuclear protein specifically bound to this element and to the analogous sequence of the K6 promoter. Thus, EGF specifically induces K6 and K16, markers of activated keratinocytes, via nuclear proteins that bind to EGF-responsive elements in the promoters of these keratin genes.