Faculty Bibliography

BACKGROUND: One challenge of systems biology is the integration of new data into the preexisting, and then re-interpretation of the integrated data. Here we use readily available metaanalysis computational methods to integrate new data on the transcriptomic effects of EGF in primary human epidermal keratinocytes with preexisting transcriptomics data in keratinocytes and in EGF-treated non-epidermal cell types. RESULTS: We find that EGF promotes keratinocyte proliferation, attachment and motility and, surprisingly, induces DUSPs that attenuate the EGF signal. Our metaanalysis identified overlapping effects of EGF with those of IL-1 and IFNgamma, activators of keratinocyte in inflammation and wound healing. We also identified the genes and pathways suppressed by EGF but induced by agents promoting epidermal differentiation. Metaanalysis comparison with the EGF effects in other cell types identified extensive similarities between responses in keratinocytes and in other epithelial cell types, but specific differences with the EGF effects in endothelial cells, and in transformed, oncogenic epithelial cell lines. CONCLUSIONS: This work defines the specific transcriptional effects of EGF on human epidermal keratinocytes. Our approach can serve as a suitable paradigm for integration of new omics data into preexisting databases and re-analysis of the integrated data sets.

Recent years witnessed the birth of bioinformatics technologies, which greatly advanced biological research. These 'omics' technologies address comprehensively the entire genome, transcriptome, proteome, microbiome etc. A large impetus in development of bioinformatics was the introduction of DNA microarrays for transcriptional profiling. Because of its accessibility, skin was among the first organs analyzed using DNA microarrays, and dermatology among the first medical disciplines to embrace the approach. Here, DNA microarray methodologies and their application in dermatology and skin biology are reviewed. The most studied disease has been, unsurprisingly, melanoma; markers of melanoma progression, metastatic potential and even melanoma markers in blood have been detected. The basal and squamous cell carcinomas have also been intensely studied. Psoriasis has been comprehensively explored using DNA microarrays, transcriptional changes correlated with genomic markers and several signaling pathways important in psoriasis have been identified. Atopic dermatitis, wound healing, keloids etc. have been analyzed using microarrays. Noninvasive skin sampling for microarray studies has been developed. Simultaneously, epidermal keratinocytes have been the subject of many skin biology studies because they respond to a rich variety of inflammatory and immunomodulating cytokines, hormones, vitamins, UV light, toxins and physical injury. The transcriptional changes occurring during epidermal differentiation and cornification have been identified and characterized. Recent studies identified the genes specifically expressed in human epidermal stem cells. As dermatology advances toward personalized medicine, microarrays and related 'omics' techniques will be directly applicable to the personalized dermatology practice of the future.

In the bi-directional signaling system comprising ephrins (EFNs) and ephrin receptors (Ephs), both EFNs and Ephs simultaneously function both as ligands and as receptors. Importantly, the EFN/Eph system is deregulated in human cancers and has been implicated in the metastatic processes because of its effects on the adhesion and migration of epithelial cells. The idiosyncratic function of Ephs, membrane-bound receptor kinases, as extracellular signaling ligands, has not been extensively studied. This prompted us to explore the transcriptional targets regulated by Ephs acting solely as ligands. To define the ligand function of EphB2 in human epidermal keratinocytes, we treated these cells with EphB2 as Fc-conjugate dimmers, which thus act exclusively as extracellular ligands. We compared the EphB2 and EFNA4 effects during a 48 h time course, using transcriptional profiling. We found that EphB2, acting as a ligand, promotes epidermal differentiation. For example, EphB2 induces expression of markers of epidermal differentiation, including keratins KRT1 and KRT10, SPRRs, desmosomal proteins and cell cycle inhibitors, while suppressing basal layer markers, integrins and cell cycle proteins. The effects of EphB2 are delayed relative to those of EFNA4. Unlike EFNA4, EphB2 did not induce lipid metabolism proteins, this particular aspect of epidermal differentiation seems not to be regulated by EphB2. Our results define the transcriptional targets of the reverse signaling by EphB2 acting exclusively as a ligand and begin to characterize this intriguing function of Ephs. J. Cell. Physiol. 227: 2330-2340, 2012. (c) 2011 Wiley Periodicals, Inc.

Both ephrins (EFNs) and their receptors (Ephs) are membrane-bound, restricting their interactions to the sites of direct cell-to-cell interfaces. They are widely expressed, often co-expressed, and regulate developmental processes, cell adhesion, motility, survival, proliferation, and differentiation. Both tumor suppressor and oncogene activities are ascribed to EFNs and Ephs in various contexts. A major conundrum regarding the EFN/Eph system concerns their large number and functional redundancy given the promiscuous cross-activation of ligands and receptors and the overlapping intracellular signaling pathways. To address this issue, we treated human epidermal keratinocytes with five EFNAs individually and defined the transcriptional responses in the cells. We found that a large set of genes is coregulated by all EFNAs. However, although the responses to EFNA3, EFNA4, and EFNA5 are identical, the responses to EFNA1 and EFNA2 are characteristic and distinctive. All EFNAs induce epidermal differentiation markers and suppress cell adhesion genes, especially integrins. Ontological analysis showed that all EFNAs induce cornification and keratin genes while suppressing wound healing-associated, signaling, receptor, and extracellular matrix-associated genes. Transcriptional targets of AP1 are selectively suppressed by EFNAs. EFNA1 and EFNA2, but not the EFNA3, EFNA4, EFNA5 cluster, regulate the members of the ubiquitin-associated proteolysis genes. EFNA1 specifically induces collagen production. Our results demonstrate that the EFN-Eph interactions in the epidermis, although promiscuous, are not redundant but specific. This suggests that different members of the EFN/Eph system have specific, clearly demarcated functions

TGFbeta is important in inflammation, angiogenesis, re-epithelialization and connective tissue regeneration during wound healing. We analyzed components of TGFbeta signaling pathway in biopsies from ten patients with non-healing venous ulcers (VUs). Using comparative genomics of transcriptional profiles of VUs and TGFbeta treated keratinocytes, we found deregulation of TGFbeta target genes in VUs. Using qPCR and immunohistochemistry, we found suppression of TGFbetaRI, RII and RIII and complete absence of phosphorylated Smad2 (pSmad2) in VUs epidermis. In contrast, pSmad2 was induced in the cells of migrating epithelial tongue of acute wounds. TGFbeta inducible transcription factors (GADD45beta, ATF3 and ZFP36L1) were suppressed in VUs. Likewise, genes suppressed by TGFbeta (FABP5, CSTA and S100A8) were induced in non-healing VUs. An inhibitor of Smad signaling, Smad7 was also down-regulated in VUs. We conclude that TGFbeta signaling is functionally blocked in VUs by down-regulation of TGFbeta receptors and attenuation of Smad signaling resulting in deregulation of TGFbeta target genes and consequent hyperproliferation. These data suggest that application of exogenous TGFbeta may not be beneficial treatment for VUs

Because of its accessibility, skin has been among the first organs analyzed using DNA microarrays; psoriasis, melanomas, carcinomas, chronic wound biopsies, and epidermal keratinocytes in culture have been intensely investigated. Skin has everything: stem cells, differentiation, signaling, inflammation, diseases, cancer, etc. Here we provide step-by-step instructions for bioinformatics analysis of transcriptional profiling of skin. Specifically, we describe the use of GCOS and RMA programs for initial normalization and selection of differentially expressed genes, DAVID and LOLA programs for annotation of genes, and statistically relevant identification of over- and under-represented functional and biological categories in identified gene sets, L2L and Venn diagrams for comparing multiple lists of genes, and oPOSSUM for identification of statistically over-represented transcription factor binding sites in the promoter regions of gene sets. The work can be a primer for researchers embarking on skinomics, the comprehensive analysis of transcriptional changes in the skin

Retinoids (RA) have been used as therapeutic agents for numerous skin diseases, from psoriasis to acne and wrinkles. While RA is known to inhibit keratinocyte differentiation, the molecular effects of RA in epidermis have not been comprehensively defined. To identify the transcriptional targets of RA in primary human epidermal keratinocytes, we compared the transcriptional profiles of cells grown in the presence or absence of all-trans retinoic acid for 1, 4, 24, 48, and 72 h, using large DNA microarrays. As expected, RA suppresses the protein markers of cornification; however the genes responsible for biosynthesis of epidermal lipids, long-chain fatty acids, cholesterol, and sphingolipids, are also suppressed. Importantly, the pathways of RA synthesis, esterification and metabolism are activated by RA; therefore, RA regulates its own bioavailability. Unexpectedly, RA regulates many genes associated with the cell cycle and programmed cell death. This led us to reveal novel effects of RA on keratinocyte proliferation and apoptosis. The response to RA is very fast: 315 genes were regulated already after 1 h. More than one-third of RA-regulated genes function in signal transduction and regulation of transcription. Using in silico analysis, we identified a set of over-represented transcription factor binding sites in the RA-regulated genes. Many psoriasis-related genes are regulated by RA, some induced, others suppressed. These results comprehensively document the transcriptional changes caused by RA in keratinocytes, add new insights into the molecular mechanism influenced by RA in the epidermis and demonstrate the hypothesis-generating power of DNA microarray analysis

Keratinocyte-specific Pten-null mice revealed distinct phenotypes, including epidermal and sebaceous gland hyperplasia. To determine the candidate genes that contribute to their phenotypes, we analyzed a comprehensive gene expression of Pten-null keratinocytes using microarray technology. Consequently, it was demonstrated that the most induced gene was adipocyte-specific fatty acid-binding protein (FABP4). Collectively, it is conceivable that the FABP4 pathway mediates the sebaceous gland hyperplasia in keratinocyte-specific Pten-null mice