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

In inflamed tissue, normal signal transduction pathways are altered by extracellular signals. For example, the JNK pathway is activated in psoriatic skin, which makes it an attractive target for treatment. To define comprehensively the JNK-regulated genes in human epidermal keratinocytes, we compared the transcriptional profiles of control and JNK inhibitor-treated keratinocytes, using DNA microarrays. We identified the differentially expressed genes 1, 4, 24, and 48 h after the treatment with SP600125. Surprisingly, the inhibition of JNK in keratinocyte cultures in vitro induces virtually all aspects of epidermal differentiation in vivo: transcription of cornification markers, inhibition of motility, withdrawal from the cell cycle, stratification, and even production of cornified envelopes. The inhibition of JNK also induces the production of enzymes of lipid and steroid metabolism, proteins of the diacylglycerol and inositol phosphate pathways, mitochondrial proteins, histones, and DNA repair enzymes, which have not been associated with differentiation previously. Simultaneously, basal cell markers, including integrins, hemidesmosome and extracellular matrix components, are suppressed. Promoter analysis of regulated genes finds that the binding sites for the forkhead family of transcription factors are over-represented in the SP600125-induced genes and c-Fos sites in the suppressed genes. The JNK-induced proliferation appears to be secondary to inhibition of differentiation. The results indicate that the inhibition of JNK in epidermal keratinocytes is sufficient to initiate their differentiation program and suggest that augmenting JNK activity could be used to delay cornification and enhance wound healing, whereas attenuating it could be a differentiation therapy-based approach for treating psoriasis.

Identification of tumor necrosis factor ␣ (TNF␣) as the key agent in inflammatory disorders led to new therapies specifically targeting TNF␣ and avoiding many side effects of earlier anti-inflammatory drugs. However, because of the wide spectrum of systems affected by TNF␣, drugs targeting TNF␣ have a potential risk of delaying wound healing, secondary infections, and cancer. Indeed, increased risks of tuberculosis and carcinogenesis have been reported as side effects after anti-TNF␣ therapy. TNF␣ regulates many processes (e.g. immune response, cell cycle, and apoptosis) through several signal transduction pathways that convey the TNF␣ signals to the nucleus. Hypothesizing that specific TNF␣-dependent pathways control specific processes and that inhibition of a specific pathway may yield even more precisely targeted therapies, we used oligonucleotide microarrays and parthenolide, an NF-B-specific inhibitor, to identify the NF-B-dependent set of the TNF␣-regulated genes in human epidermal keratinocytes. Expression of ϳ40% of all TNF␣-regulated genes depends on NF-B; 17% are regulated early (1-4 h post-treatment), and 23% are regulated late (24 -48 h). Cytokines and apoptosisrelated and cornification proteins belong to the "early" NF-B-dependent group, and antigen presentation proteins belong to the "late" group, whereas most cell cycle, RNA-processing, and metabolic enzymes are not NF-Bdependent. Therefore, inflammation, immunomodulation, apoptosis, and differentiation are on the NF-B pathway, and cell cycle, metabolism, and RNA processing are not. Most early genes contain consensus NF-B binding sites in their promoter DNA and are, presumably, directly regulated by NF-B, except, curiously, the cornification markers. Using siRNA silencing, we identified cFLIP/CFLAR as an essential NF-B-dependent antiapoptotic gene. The results confirm our hypothesis, suggesting that inhibiting a specific TNF␣-dependent signaling pathway may inhibit a specific TNF␣-regulated process, leaving others unaffected. This could lead to more specific anti-inflammatory agents that are both more effective and safer.

The pathological manifestations of psoriasis are orchestrated by many secreted proteins, but only a handful, tumor necrosis factor-alpha, IFN-gamma and IL-1, have been studied in great detail. Oncostatin-M (OsM) has also been found in psoriatic skin and we hypothesized that it makes a unique and characteristic contribution to the psoriatic processes. To define in-depth the molecular effects of OsM in epidermis, we used high-density DNA microarrays for transcriptional profiling of OsM-treated human skin equivalents. We identified 374 unambiguously OsM-regulated genes, out of 22,000 probed. OsM suppressed the expression of the "classical" epidermal differentiation markers, but strongly and specifically induced the S100A proteins. Cytoskeletal and complement proteins, proteases, and their inhibitors were also induced by OsM. Interestingly, a large set of genes was induced by OsM at early time points but suppressed later; these genes are known regulatory targets of IFN and thus provide a nexus between the OsM and IFN pathways. OsM induces IL-4 and suppresses the T-helper 1-type and IL-1-responsive signals, potentially attenuating the psoriatic pathology. The data suggest that OsM plays a unique role in psoriasis, different from all other, more thoroughly studied cytokines.