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

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

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

Using high-density oligonucleotide arrays, we measured expression of >12,000 genes in surgical excisions of invasive human squamous cell carcinomas (SCCs) versus site-matched control skin. This analysis defined >1,900 genes with altered expression in SCCs that were statistically different from controls. As SCCs are composed of epithelial cells, which are both hyperplastic and invasive, we sought to define gene sets associated with these biologic processes by comparing gene expression to psoriasis vulgaris, which is a condition of benign keratinocyte hyperplasia without invasiveness or pre-malignant potential. Through this analysis, we found genes that were commonly upregulated in both conditions and unique genes with increased expression in SCCs. Differential gene regulation in these two conditions was confirmed by real-time reverse transcription-PCR and immunohistochemistry. We found that benign hyperplasia is associated with upregulation of genes including DEFB4 (defensin B4), SERPINB3 (serine proteinase inhibitor, member 3), STAT1 (signal transducer and activator of transcription 1), K16 (keratin 16), CEACAMs (carcinoembryonic antigen-related cell adhesion molecules), and WNT 5A (wingless-type MMTV integration site family, member 5A). WNT receptor frizzled homolog 6 (FZD6) and prostaglandin-metabolizing enzyme hydroxyprostaglandin dehydrogenase were increased in SCC alone. Growth factor pleiotrophin (PTN) was expressed at higher levels in non-tumor-bearing skin adjacent to excised SCC. SCC was further characterized by upregulation of matrix metalloproteinases 1, 10, and 13, cathepsin L2, cystatin E/M as well as STAT3 and microseminoprotein, beta (MSMB), and downregulation of inducible nitric oxide synthase, granzyme B, CD8, and CD83. The current study defines a unique gene expression signature for cutaneous SCC in humans and suggests potential roles for WNT, FZD, and PTN in the pathogenesis of SCC

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