Faculty Bibliography

In epidermal differentiation basal keratinocytes detach from the basement membrane, stop proliferating, and express a new set of structural proteins and enzymes, which results in an impermeable protein/lipid barrier that protects us. To define the transcriptional changes essential for this process, we purified large quantities of basal and suprabasal cells from human epidermis, using the expression of beta4 integrin as the discriminating factor. The expected expression differences in cytoskeletal, cell cycle and adhesion genes confirmed the effective separation of the cell populations. Using DNA microarray chips, we comprehensively identify the differences in genes expressed in basal and differentiating layers of the epidermis, including the ECM components produced by the basal cells, the proteases in both the basal and suprabasal cells, and the lipid and steroid metabolism enzymes in suprabasal cells responsible for the permeability barrier. We identified the signaling pathways specific for the two populations, and found two previously unknown paracrine and one juxtacrine signaling pathway operating between the basal and suprabasal cells. Furthermore, using specific expression signatures, we identified a new set of late differentiation markers and mapped their chromosomal loci, as well as a new set of melanocyte-specific markers. The data represent a quantum jump in understanding the mechanisms of epidermal differentiation

Interleukin-12 (IL-12) is a proinflammatory and immunomodulatory cytokine that plays a critical role it in innate and adaptive immunity by inducing production of interferon-gamma and other cytokines. IL-12 was shown to block the ultraviolet light-induced immunosuppression, important in cancer immunosurveillance, cutaneous allergies and inflammation. To characterize the molecular effects of IL-12 in epidermis we used large DNA microarrays and defined the transcriptional changes in human epidermal keratinocytes 1 h, 4 h, 24 h, and 48 h after treatment with IL-12, as well as in cells treated with both IL-12 and UV light. In keratinocytes, IL-12 activates STAT3 and STAT4; surprisingly, despite activating these transcription factors, the transcriptional effects of IL-12 did not rise above background levels. However, pre-treatment of keratinocytes with IL-12 strongly modulated the transcriptional effects of UV. Pre-treatment with IL-12 enhanced the UV-mediated regulation of 20 and antagonized the regulation of 263 genes. IL-12 enhanced the induction of cytokines by UV. IL-12 antagonized the suppression of cytoskeletal, junctional, metabolic, mitochondrial, and extracellular matrix proteins, while antagonizing the induction of certain signaling proteins and RNA processing enzymes. We conclude that in the epidermis, IL-12 interferes with a specific subset of transcriptional effects of UV irradiation

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

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.Journal of Investigative Dermatology advance online publication, 17 August 2006; doi:10.1038/sj.jid.5700461

Because of its accessibility, skin has been among the first organs analyzed using DNA microarrays. Skin cancers, melanomas, and basal and squamous cell carcinomas have been intensely investigated because they are very frequent and can be fatal. Psoriasis, one of the most common human inflammatory diseases, has been studied comprehensively using DNA microarrays. In addition, epidermal keratinocytes have been the target of many studies because they respond to a rich variety of inflammatory and immunomodulating cytokines, hormones, vitamins, ultraviolet (UV) light, toxins, and physical injury. Because of the ethical considerations, the effects of harmful or dangerous agents on skin have been studied using artificial skin substitutes. Transcriptional mechanisms that regulate epidermal differentiation and cornification have begun to yield their mysteries, and very exciting recent studies identified the genes specifically expressed in epidermal stem cells. Thus, skin has everything: stem cells, differentiation, signaling, inflammation, diseases, and cancer. All these exciting facets of skin have been explored using DNA microarrays. Researchers in skin biology and dermatology were among the first to implement this technology and we expect that they will continue to generate exciting and useful new knowledge