Ctrl, control; CO, croton oil; FA, fluocinolone acetonide; H&E, hematoxylin and eosin; ns, not significant; qPCR, quantitative PCR; Rapa, rapamycin; WB, western blotting; wks, weeks. Chronic topical treatment of mice with FA (every 72 hours for 2 weeks) induced severe skin atrophy (Figure 5c and d). target genes yet enhanced the repression of pro-proliferative and proinflammatory genes. Remarkably, rapamycin protected skin against glucocorticoid-induced atrophy but had no effect on the glucocorticoid anti-inflammatory activity in different in vivo models, suggesting the clinical potential of combining rapamycin with glucocorticoids for the treatment of inflammatory diseases. INTRODUCTION Glucocorticoids are among the most effective anti-inflammatory and anti-lymphoma drugs Felbamate (Lesovaya et al., 2015). Unfortunately, chronic treatment with glucocorticoids results in multiple metabolic and atrophic adverse effects that reflect glucocorticoid catabolic activity (De Bosscher et al., 2010; Lesovaya et al., 2015). Thus, there is a significant need for safer glucocorticoid receptor (GR)-targeted therapies. GR is a well-known transcription factor (TF). Upon hormone binding, GR translocates to the nucleus, where it regulates gene expression either by (i) transactivation via GR homodimer binding to glucocorticoid-responsive elements (GREs) or (ii) transrepression, which is frequently mediated via negative interaction between GR and other TFs, including proinflammatory NF-B (Lesovaya et al., 2015; Ramamoorthy and Cidlowski, 2013; Ratman et al., 2013). It is well accepted that GR transrepression plays an important role in the anti-inflammatory effects of glucocorticoids. In contrast, many adverse effects of steroids (glucose metabolism, steroid diabetes, osteoporosis, skin and muscle atrophy) strongly depend on GR transactivation (De Bosscher et al., 2010; Lesovaya et al., 2015; Schoepe et al., 2006). Even though some of the concepts in the GR field have been revised, it is still well accepted that selective GR activators that shift GR activity Rabbit Polyclonal to NXF1 toward transrepression have a better therapeutic index than classical glucocorticoids (Lesovaya et al., 2015). The alternative approach to safer GR-targeted therapies could be a combination of glucocorticoids with compounds that can protect tissues against their adverse effects. We used glucocorticoid-induced skin atrophy as a model for this proof-of-principle study. Skin atrophy, one of the major adverse effects of topical glucocorticoids, is characterized by a drastic hypoplasia of all skin compartments and a compromised skin barrier function (Schoepe et al., 2006; Woodbury and Kligman, 1992). Recently we identified REDD1, a negative regulator of mTOR/Akt signaling (Dennis et al., 2014; Ellisen, 2005; Shoshani et al., 2002), as a central Felbamate atrophogene in skin (Baida et al., 2015). REDD1 expression is activated by a variety of cellular stresses including hypoxia, depletion of growth factors, DNA damage, and glucocorticoids (Ellisen, 2005; Shimizu et al., 2011; Shoshani et al., 2002). We and others showed that REDD1 was strongly induced during steroid atrophy in skin and muscle and that REDD1 knockout animals were protected against steroid-induced skin atrophy and muscle waste (Baida et al., 2015; Britto et al., 2014; Wang et al., 2006). We discovered that lack of REDD1 did not alter the anti-inflammatory effects of glucocorticoids (Baida et al., 2015). We hypothesized that REDD1 inhibitors may act as anti-atrophogenes and could be combined with glucocorticoids for tissue protection. We used a drug repurposing approach and screened a connectivity map Felbamate (CMAP) database of transcriptional signatures induced by US Food and Drug Administration-approved and experimental drugs (Lamb et al., 2006) for their potential to reduce REDD1 expression. We identified several putative REDD1 inhibitors, including rapamycin. The potential of rapamycin to display anti-atrophogenic properties was unexpected, because it is a pharmacological REDD1 analog and a specific mTOR inhibitor (Li et al., 2014). The goals of this study were to test the effect of rapamycin on basal and glucocorticoid-induced REDD1 expression, its potential effects on GR function, and its effect on therapeutic (anti-inflammatory) and adverse (skin atrophy) effects of glucocorticoids. RESULTS Selection of rapamycin as a prospective REDD1 inhibitor Because pharmacological REDD1 inhibitors are not known, we used a modified connectivity mapping approach and screened a CMAP library representing molecular signatures of approximately 1,300 US Food and Drug Administration-approved and experimental drugs tested in human cancer cells to repurpose them for cancer treatment (Lamb et al., 2006). We selected compounds according to the number of CMAP experiments in which REDD1 was within the top 100 down-regulated genes in cells treated with these compounds (see Supplementary Table S1 online). We identified several putative REDD1 inhibitors, including rapamycin, which displayed consistent negative effects on REDD1 expression in more than 40 tests in multiple cell lines. Thus, we prioritized rapamycin as the top candidate for experimental validation. mTOR inhibitors rapamycin and.