Transcriptional effects of inhibiting epidermal growth factor receptor in keratinocytes

Background: Epidermal growth factor receptor (EGFR) activation is important in human epithelial malignancies, including cutaneous squamous cell carcinoma, lung, colon, pancreatic, and other cancers. Therapies targeting EGFR are currently used to treat such cancers, but one significant drawback to EGFR inhibitor therapies is the associated skin toxicity. This toxicity usually presents as papular or pustular folliculitis, dry skin with pruritus and hair and nail abnormalities. The side effects often limit the usefulness of EGFR inhibitors in cancer treatment. The transcriptional changes caused by EGFR inhibition in epidermal keratinocytes have not been extensively explored. Methods: To define the transcriptional changes caused by inhibition of EGFR in primary human epidermal keratinocytes, these cells were treated with Tyrphostin AG1478 and treated and control cultures were compared in parallel, using Affymetrix microarrays. Using meta-analysis approaches, the observed changes were integrated with a large set of already existing data on transcriptional profiling in epidermal keratinocytes. Results: We found that at the early time points, 1 hour and 4 hours after addition, AG1478 suppresses expression of genes associated with keratinocyte proliferation, attachment and motility. Apoptosis is facilitated by both induction of proapoptotic and suppression of antiapoptotic genes. Angiogenesis signals are suppressed as well. At late time points, 24 hours and 48 hours, EGFR inhibition induces mitochondrial activity and suppresses splicing and protein trafficking. Certain transcriptional effects of EGFR inhibition go against the transcriptional effects of retinoids. Surprisingly, at 48 hours, EGFR inhibition induces expression of markers of epidermal differentiation. Conclusion: Our results define the role of EGF receptor in human keratinocytes and the consequences of its inhibition

A Characteristic Subset of Psoriasis-Associated Genes Is Induced by Oncostatin-M in Reconstituted Epidermis

The pathological manifestations of psoriasis are orchestrated by many secreted proteins, but only a handful, tumor necrosis factor-alpha, IFN-γ 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

Transcriptional Profiling of Epidermal Keratinocytes: Comparison of Genes Expressed in Skin, Cultured Keratinocytes, and Reconstituted Epidermis, Using Large DNA Microarrays

Epidermal keratinocytes are complex cells that create a unique three-dimensional (3-D) structure, differentiate through a multistage process, and respond to extracellular stimuli from nearby cells. Consequently, keratinocytes express many genes, i.e., have a relatively large “transcriptome.” To determine which of the expressed genes are innate to keratinocytes, which are specific for the differentiation and 3-D architecture, and which are induced by other cell types, we compared the transcriptomes of skin from human subjects, differentiating 3-D reconstituted epidermis, cultured keratinocytes, and nonkeratinocyte cell types. Using large oligonucleotide microarrays, we analyzed five or more replicates of each, which yielded statistically consistent data and allowed identification of the differentially expressed genes. Epidermal keratinocytes, unlike other cells, express many proteases and protease inhibitors and genes that protect from UV light. Skin specifically expresses a higher number of receptors, secreted proteins, and transcription factors, perhaps influenced by the presence of nonkeratinocyte cell types. Surprisingly, mitochondrial proteins were significantly suppressed in skin, suggesting a low metabolic rate. Three-dimensional samples, skin and reconstituted epidermis, are similar to each other, expressing epidermal differentiation markers. Cultured keratinocytes express many cell-cycle and DNA replication genes, as well as integrins and extracellular matrix proteins. These results define innate, architecture-specific, and cell-type-regulated genes in epidermis