Faculty Bibliography

DNA microarrays are capable of following the level of expression of, virtually, all genes in a human tissue. This has been employed to determine the aberrant gene expression profiles in many skin diseases, including ultraviolet light damage, inflammatory processes and cancers. Because of its accessibility, skin also served as one of the initial targets of basic research using DNA microarrays. Both the epidermis and dermis have been extensively investigated. Development of bed-side uses of DNA arrays, and the concomitant price reduction of the materials and methods of microarray analyses, holds great promise for improved diagnosis, treatment and prevention of dermatologic disorders.

OBJECTIVE: Adenosine regulates inflammation and tissue repair, and adenosine A2A receptors promote wound healing by stimulating collagen matrix production. We therefore examined whether adenosine A2A receptors contribute to the pathogenesis of dermal fibrosis. METHODS: Collagen production by primary human dermal fibroblasts was analyzed by real-time polymerase chain reaction, 14C-proline incorporation, and Sircol assay. Intracellular signaling for dermal collagen production was investigated using inhibitors of MEK-1 and by demonstration of ERK phosphorylation. In vivo effects were studied in a bleomycin-induced dermal fibrosis model using adenosine A2A receptor-deficient wild-type littermate mice, C57BL/6 mice, and mice treated with adenosine A2A receptor antagonist. Morphometric features and levels of hydroxyproline were determined as measures of dermal fibrosis. RESULTS: Adenosine A2A receptor occupancy promoted collagen production by primary human dermal fibroblasts, which was blocked by adenosine A2A, but not A1 or A2B, receptor antagonism. Adenosine A2A receptor ligation stimulated ERK phosphorylation, and A2A receptor-mediated collagen production by dermal fibroblasts was blocked by MEK-1 inhibitors. Adenosine A2A receptor-deficient and A2A receptor antagonist-treated mice were protected from developing bleomycin-induced dermal fibrosis. CONCLUSION: These results demonstrate that adenosine A2A receptors play an active role in the pathogenesis of dermal fibrosis and suggest a novel therapeutic target in the treatment and prevention of dermal fibrosis in diseases such as scleroderma

Lack of understanding of the molecular mechanisms and pathogenesis of impaired healing in chronic ulcers is a serious health issue that contributes to excessive limb amputations and mortality. Here we show that beta-catenin and its downstream targets in keratinocytes, c-myc, and keratins K6 and K16, play important roles in the development of chronic wounds. In contrast to normal epidermis, we observed a significant nuclear presence of beta-catenin and elevated c-myc expression at the nonhealing wound edge of chronic ulcers from 10 patients. In vitro studies indicated that stabilization of nuclear beta-catenin inhibited wound healing and keratinocyte migration by blocking epidermal growth factor response, inducing c-myc and repressing the K6/K16 keratins (cytoskeletal components important for migration). The molecular mechanism of K6/K16 repression involved beta-catenin and arginine methyltransferase (CARM-1) acting as co-repressors of glucocorticoid receptor monomers. We conclude that activation of the beta-catenin/c-myc pathway(s) contributes to impaired healing by inhibiting keratinocyte migration and altering their differentiation. The presence of activated beta-catenin and c-myc in the epidermis of chronic wounds may serve as a molecular marker of impaired healing and may provide future targets for therapeutic intervention

Transcriptional regulation by nuclear receptors occurs through complex interactions that involve DNA response elements, co-activators/co-repressors, and histone modifying enzymes. Very little is known about how molecular interplay of these components may determine tissue specificity of hormone action. We have shown previously that retinoic acid (RA) and thyroid hormone (T3) repress transcription of a specific group of epidermal keratin genes through a novel mechanism that utilizes receptors homodimers. In this paper, we have analyzed the epidermal specificity of RA/T3 action by testing the role of co-repressors and co-activators in regulation of epidermal genes. Using transient co-transfections, northern blots, antisense oligonucleotides, and a histone deacetylase (HDAC) inhibitor, trichostatin A, we found that in the context of specific keratin RE (KRE), co-activators and histone acetylase become co-repressors of the RA/T3 receptors in the presence of their respective ligands. Conversely, co-repressors and HDAC become co-activators of unliganded T3Ralpha. The receptor-co-activator interaction is intact and occurs through the NR-box. Therefore, the role of co-activator is to associate with liganded receptors whereas the KRE-receptor interaction determines specific transcriptional signal, in this case repression. This novel molecular mechanism of transcriptional repression conveys how RA and T3 target specific groups of epidermal genes, thus exerting intrinsic tissue specificity

The field of epidermal stem cells has dramatically advanced in the last decade, leading to a better understanding of the molecular factors, signalling pathways and cellular events that identify and characterize stem cells, thus revealing their immense potential for therapeutic use. Furthermore, multipotent epidermal stem cells present the major advantage of easy accessibility with the discovery of their specific location within the bulge of the hair follicle. This review focuses on the most recent findings on epidermal stem cells, and their potential role in initial epidermal commitment, differentiation and wound healing processes in the skin

Wound healing in its complexity depends on the concerted activity of many signaling pathways. Here, we analyzed how the simultaneous presence of glucocorticoids (GC), retinoic acid (RA) and epidermal growth factor (EGF) affect wound healing at the molecular, cellular and tissue levels. We found that GC inhibit wound healing by inhibiting keratinocyte migration, whereas RA does not. Furthermore, GC block EGF-mediated migration, whereas RA does not. On the molecular level, these compounds target expression of one of the earliest markers of wound healing, cytoskeletal components, keratins K6 and K16. Both GC and RA repress their transcription, whereas EGF induces it. Interestingly, the GC inhibition is mediated by a repressosome complex consisting of four monomers of the GC receptor, beta-catenin and coactivator-associated-arginine-methyltransferase-1. GC are dominant, EGF cannot rescue GC-mediated inhibition. Pre-treatment of keratinocytes with GC shifts the balance towards the repressosome, allowing for dominant inhibition of K6 even in the presence of EGF or c-fos/c-jun. Although RA receptor gamma and glucocorticoid receptor bind to the same response element repressing transcription of keratins K6/K16, RA receptor interacts with the components of the EGF-enhanceosome (co-activators: glucocorticoid-receptor-interactive protein-1(GRIP-1)/steroid-receptors coactivator-1 (SRC-1)) without breaking it. Consequently, RA has a co-dominant effect with EGF: when present simultaneously, their effects balance each other. When keratinocytes are pre-treated with mitogen-activated protein kinase (MAPK) inhibitor, thus blocking EGF, the balance is shifted towards the RA repression. Similar to clinical findings, pre-treatment of keratinocytes with RA blocks GC-mediated inhibition. In summary, our results identify complex molecular mechanisms through which RA alleviates GC-mediated inhibition of wound healing

Many of the limitations in treatment of chronic wounds are based on lack of knowledge of the molecular mechanism(s) of wound healing. Furthermore, diagnostic tools in wound healing are still primarily macroscopic, visual, and histologic. Thus, by understanding mechanisms of wound healing at a molecular level, new treatments can be designed, prevention programs developed, and a better understanding of current treatments provided. The ability to methodically analyze the expression patterns of thousands of genes simultaneously allows for identification of groups of molecular defects that lead to chronic inhibition of the wound-healing process. Gene array technology is having a major impact on the field of wound healing and has the potential to profoundly affect the way we understand the pathogenesis, diagnosis, prevention, and treatment of chronic wounds. Currently, gene array technology is used in the field of chronic wound healing to (1) understand the pathogenesis of pressure ulcers and venous ulcers, (2) understand the pathogenesis of diabetic foot ulcers, including the role that neuropathy may play in delayed healing of diabetic foot ulcers, and (3) determine the mechanism(s) of established and new local treatments, that is, pharmacogenomics for pressure ulcers and diabetic foot ulcers

Nuclear hormone receptor coregulator (NRC) is a 2,063-amino-acid coregulator of nuclear hormone receptors and other transcription factors (e.g., c-Fos, c-Jun, and NF-kappaB). We and others have generated C57BL/6-129S6 hybrid (C57/129) NRC(+/-) mice that appear outwardly normal and grow and reproduce. In contrast, homozygous deletion of the NRC gene is embryonic lethal. NRC(-/-) embryos are always smaller than NRC(+/+) embryos, and NRC(-/-) embryos die between 8.5 and 12.5 days postcoitus (dpc), suggesting that NRC has a pleotrophic effect on growth. To study this, we derived mouse embryonic fibroblasts (MEFs) from 12.5-dpc embryos, which revealed that NRC(-/-) MEFs exhibit a high rate of apoptosis. Furthermore, a small interfering RNA that targets mouse NRC leads to enhanced apoptosis of wild-type MEFs. The finding that C57/129 NRC(+/-) mice exhibit no apparent phenotype prompted us to develop 129S6 NRC(+/-) mice, since the phenotype(s) of certain gene deletions may be strain dependent. In contrast with C57/129 NRC(+/-) females, 20% of 129S6 NRC(+/-) females are infertile while 80% are hypofertile. The 129S6 NRC(+/-) males produce offspring when crossed with wild-type 129S6 females, although fertility is reduced. The 129S6 NRC(+/-) mice tend to be stunted in their growth compared with their wild-type littermates and exhibit increased postnatal mortality. Lastly, both C57/129 NRC(+/-) and 129S6 NRC(+/-) mice exhibit a spontaneous wound healing defect, indicating that NRC plays an important role in that process. Our findings reveal that NRC is a coregulator that controls many cellular and physiologic processes ranging from growth and development to reproduction and wound repair