Faculty Bibliography

BACKGROUND: Skin is our first line of defense against pathogenic microorganisms and the intimate contact between the epidermis and microbes has been well known. PURPOSES: Microbes that cause infection are associated with inflammatory dermatoses and exacerbate wound healing. It is therefore of vital importance to understand the intricacies of skin-microbiota interactions. However, until recently our knowledge and understanding was limited by being unable to deal with uncultivatable microorganisms, which constitute a large majority. BASIC PROCEDURES: Recent advances in DNA sequencing methodologies, analysis tools and affordability led to major breakthroughs in defining the cutaneous microbiome. MAIN FINDINGS: We now know that four phyla, Actinobacteria, Firmicytes, Proteobacteria and Bacteroidetes, constitute preponderance of skin bacteria, while Malassezia dominates the fungal microbiome. We know that there are some 300 different bacteria inhabiting our skin. We also know that there is remarkable interpersonal variation, that the microbiota change over time, that different body sites harbor specific microbial arrays and that microbiota characteristically change in skin diseases. PRINCIPAL CONCLUSIONS: The recent advances led to appreciation that microbes are, for the most part, our allies, useful and protective, and that with increased understanding we will be able to harness our cutaneous friends to maintain and promote our health.

Proline-rich protein tyrosine kinase 2 (Pyk2) is a member of focal adhesion kinase family. We studied Pyk2 role in cutaneous wound repair using Pyk2-null mice. We report that the rate of wound closure was delayed in Pyk2-null compared with control mice. To examine whether impaired wound healing of Pyk2-null mice was caused by a keratinocyte cell-autonomous defect, the capacities of primary keratinocytes from Pyk2-null and wild-type (WT) littermates to heal scratch wounds in vitro were compared. The rate of scratch wound repair by Pyk2-null keratinocytes was decreased compared with that by WT cells. Moreover, cultured human epidermal keratinocytes overexpressing dominant-negative mutant of Pyk2 failed to heal scratch wounds. Conversely, stimulation of Pyk2-dependent signaling via WT Pyk2 overexpression induced accelerated scratch wound closure, and was associated with increased expression of matrix metalloproteinases (MMPs) -1, -9, and -10. The Pyk2-stimulated increase in the rate of scratch wound repair was abolished by co-expression of dominant-negative mutant of Protein Kinase C delta (PKCdelta) and by GM-6001, a broad spectrum inhibitor of MMP activity. These results suggest that Pyk2 is essential for skin wound re-epithelialization both in vivo and in vitro, and that it regulates epidermal keratinocyte migration via a pathway that requires PKCdelta and MMP functions.

Because of its accessibility, skin has been among the first organs analyzed using DNA microarrays; psoriasis, melanomas, carcinomas, chronic wounds, and responses of epidermal keratinocytes in culture have been intensely investigated. Skin has everything: stem cells, differentiation, signaling, inflammation, hereditary diseases, etc. Here we provide step-by-step instructions for bioinformatics analysis of transcriptional profiling of skin. We also present methods for meta-analysis of transcription profiles from multiple contributors, available in public data repositories. Specifically, we describe the use of GCOS and RMAExpress programs for initial normalization and selection of differentially expressed genes and RankProd for meta-analysis of multiple related studies. We also describe DAVID and Lists2Networks programs for annotation of genes, and for statistically relevant identification of over- and underrepresented functional and biological categories in identified gene sets, as well as oPOSSUM for analysis of transcription factor binding sites in the promoter regions of gene sets. This work can serve as a primer for researchers embarking on skinomics, the comprehensive analysis of transcriptional changes in skin.

Epidermis, a continuously self-renewing and differentiating organ, produces a protective stratum corneum that shields us from external chemical, physical and microbial threats. Epidermal differentiation is a multi-step process regulated by influences, some unknown, others insufficiently explored. Detachment of keratinocytes from the basement membrane is one such pro-differentiation stimulus. Here, we define the transcriptional changes during differentiation, especially those caused by detachment from the substratum. Using comprehensive transcriptional profiling, we revisited the effects of detachment as a differentiation signal to keratinocytes. We identified the genes regulated by detachment, the corresponding ontological categories and, using metaanalysis, compared the genes and categories to those regulated by other pro-differentiating stimuli. We identified 762 genes overexpressed in suspended keratinocyte, including known and novel differentiation markers, and 1427 in attached cells, including basal layer markers. Detachment induced epidermis development, cornification and desmosomal genes, but also innate immunity, proliferation inhibitors, transcription regulators and MAPKs; conversely the attached cells overexpressed cell cycle, anchoring, motility, splicing and mitochondrial genes, and both positive and negative regulators of apoptosis. Metaanalysis identified which detachment-regulated categories overlap with those induced by suprabasal location in vivo, by reaching confluency in vitro, and by inhibition of JUN kinases. Attached and in vivo basal cells shared overexpression of mitochondrial components. Interestingly, melanosome trafficking components were also overexpressed in the attached and in vivo basal keratinocytes. These results suggest that specific pro-differentiation signals induce specific features of the keratinization process, which are in vivo orchestrated into harmonious epidermal homeostasis.

EGF and its receptor EGFR serve as a paradigm for signaling in cell, molecular and tumor biology. EGFR inhibitors, drugs targeting the intracellular kinase activity and antibodies targeting the extracellular ligand binding, are used to treat breast, lung, colon and other cancers. Nominally affecting the same target, inhibitors have different effects, suggesting that use of inhibitor combinations may provide beneficial in cancer treatment. To explore the specific and the common transcriptional effects of EGFR inhibitors, we present metaanalysis of 20 individual studies comprising 346 microarrays. We identified specific gene subsets regulated by kinase inhibitors, those regulated using antibodies and by suppressing EGFR expression using miR-7. Unreported before, the inhibitors prominently induce lysosome components. All inhibitors rely on related sets of transcription factors and protein kinases, both for transcriptional induction and suppression. However, we find that Gefitinib suppresses apoptosis inhibitors, while inducing cell-cycle inhibitors; conversely, Erlotinib suppresses cell-cycle and cell migration genes, while inducing proapoptotic genes. EGFR-targeting antibodies specifically suppress cell motility, developmental and differentiation processes, while inducing the contractile apparatus. miR-7, distinctively, suppresses cell-cycle genes, while inducing transcription machinery. These metaanalysis results suggest that different inhibitors have overlapping but quite distinct effects in target cells. Judicial use of EGFR-targeting combinations, i.e., simultaneous use of antibodies and multiple kinase inhibitors, may provide more effective cancer treatments with fewer side-effects and avoid development of resistance. We expect, moreover, that specific drug combination treatments can be fine-tuned to achieve specific, personalized results.

Easily accessible, skin was among the first targets analyzed using 'omics' and dermatology embraced the approaches very early. Microarrays have been used to define disease markers, identify transcriptional changes and even trace the course of treatment. Melanoma and psoriasis have been explored using microarrays. Particularly noteworthy is the multinational mapping of psoriasis susceptibility loci. The transcriptional changes in psoriasis have been identified using hundreds of biopsies. Epidermal keratinocytes have been studied because they respond to UV light, infections, inflammatory and immunomodulating cytokines, toxins and so on. Epidermal differentiation genes are being characterized and are expressed in human epidermal stem cells. Exciting discoveries defining human skin microbiomes have opened a new field of research with great medical potential. Specific to dermatology, the non-invasive skin sampling for microarray studies, using tape stripping, has been developed; it promises to advance dermatology toward 'omics' techniques directly applicable to the personalized medicine of the future.

Serum response factor (SRF) is a transcription factor that regulates the expression of growth-related immediate-early, cytoskeletal, and muscle-specific genes to control growth, differentiation, and cytoskeletal integrity in different cell types. To investigate the role for SRF in epidermal development and homeostasis, we conditionally knocked out SRF in epidermal keratinocytes. We report that SRF deletion disrupted epidermal barrier function leading to early postnatal lethality. Mice lacking SRF in epidermis displayed morphogenetic defects, including an eye-open-at-birth phenotype and lack of whiskers. SRF-null skin exhibited abnormal morphology, hyperplasia, aberrant expression of differentiation markers and transcriptional regulators, anomalous actin organization, enhanced inflammation, and retarded hair follicle (HF) development. Transcriptional profiling experiments uncovered profound molecular changes in SRF-null E17.5 epidermis and revealed that many previously identified SRF target CArG box-containing genes were markedly upregulated in SRF-null epidermis, indicating that SRF may function to repress transcription of a subset of its target genes in epidermis. Remarkably, when transplanted onto nude mice, engrafted SRF-null skin lacked hair but displayed normal epidermal architecture with proper expression of differentiation markers, suggesting that although keratinocyte SRF is essential for HF development, a cross-talk between SRF-null keratinocytes and the surrounding microenvironment is likely responsible for the barrier-deficient mutant epidermal phenotype.