Glucocorticoids (GCs) have a long history of use as therapeutic agents for numerous skin diseases. Surprisingly, their specific molecular effects are largely unknown. To characterize GC action in epidermis, we compared the transcriptional profiles of primary human keratinocytes untreated and treated with dexamethasone (DEX) for 1, 4, 24, 48, and 72 h using large scale microarray analyses. The majority of genes were found to be regulated only after 24 h and remained regulated throughout treatment. In addition to regulation of the expected pro-inflammatory genes, we found that GCs regulate cell fate, tissue remodeling, cell motility, differentiation, and metabolism. GCs suppress the expression of essentially all IFN␥-regulated genes, including IFN␥ receptor and STAT-1, an effect that was previously unknown. GCs also block STAT-1 activation and nuclear translocation. Unexpectedly, GCs induce the expression of anti-apoptotic genes and repress pro-apoptotic ones, preventing UVinduced keratinocyte apoptosis. Consequently, treatment with GCs blocked UV-induced apoptosis of keratinocytes. GCs have profound effect on wound healing by inhibiting cell motility and the expression of the proangiogenic factor, vascular endothelial growth factor. They play an important role in tissue remodeling and scar formation by suppressing the expression of TGF␤1 and -2 and MMP1, -2, -9, and -10 and inducing TIMP-2. Finally, GCs promote terminal epidermal differentiation while simultaneously inhibiting early stage differentiation. These results provide new insights into the beneficial and adverse effects of GCs in the epidermis, defining the participating genes and mechanisms that coordinate the cellular responses important for GC-based therapies.

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.

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 hours, 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.

Farnesyl pyrophosphate (FPP), a key intermediate in the mevalonate pathway and protein farnesylation, can act as an agonist for several nuclear hormone receptors. Here we show a novel mechanism by which FPP inhibits wound healing acting as an agonist for glucocorticoid receptor (GR). Elevation of endogenous FPP by the squalene synthetase inhibitor zaragozic acid A (ZGA) or addition of FPP to the cell culture medium results in activation and nuclear translocation of the GR, a known wound healing inhibitor. We used functional studies to evaluate the effects of FPP on wound healing. Both FPP and ZGA inhibited keratinocyte migration and epithelialization in vitro and ex vivo. These effects were independent of farnesylation and indicate that modulation of FPP levels in skin may be beneficial for wound healing. FPP inhibition of keratinocyte migration and wound healing proceeds, in part, by repression of the keratin 6 gene. Furthermore, we show that the 3-hydroxy-3-methylglutaryl-CoA-reductase inhibitor mevastatin, which blocks FPP formation, not only promotes epithelialization in acute wounds but also reverses the effect of ZGA on activation of the GR and inhibition of epithelialization. We conclude that FPP inhibits wound healing by acting as a GR agonist. Of special interest is that FPP is naturally present in cells prior to glucocorticoid synthesis and that FPP levels can be further altered by the statins. Therefore, our findings may provide a better understanding of the pleiotropic effects of statins as well as molecular mechanisms by which they may accelerate wound healing.