Synephrine and Its Derivative Compound A: Common and Specific Biological Effects.

This review is focused on synephrine, the principal phytochemical found in bitter orange and other medicinal plants and widely used as a dietary supplement for weight loss/body fat reduction. We examine different aspects of synephrine biology, delving into its established and potential molecular targets, as well as its mechanisms of action. We present an overview of the origin, chemical composition, receptors, and pharmacological properties of synephrine, including its anti-inflammatory and anti-cancer activity in various in vitro and animal models. Additionally, we conduct a comparative analysis of the molecular targets and effects of synephrine with those of its metabolite, selective glucocorticoid receptor agonist (SEGRA) Compound A (CpdA), which shares a similar chemical structure with synephrine. SEGRAs, including CpdA, have been extensively studied as glucocorticoid receptor activators that have a better benefit/risk profile than glucocorticoids due to their reduced adverse effects. We discuss the potential of synephrine usage as a template for the synthesis of new generation of non-steroidal SEGRAs. The review also provides insights into the safe pharmacological profile of synephrine.

Nutritional Sensor REDD1 in Cancer and Inflammation: Friend or Foe?

Regulated in Development and DNA Damage Response 1 (REDD1)/DNA Damage-Induced Transcript 4 (DDIT4) is an immediate early response gene activated by different stress conditions, including growth factor depletion, hypoxia, DNA damage, and stress hormones, i.e., glucocorticoids. The most known functions of REDD1 are the inhibition of proliferative signaling and the regulation of metabolism via the repression of the central regulator of these processes, the mammalian target of rapamycin (mTOR). The involvement of REDD1 in cell growth, apoptosis, metabolism, and oxidative stress implies its role in various pathological conditions, including cancer and inflammatory diseases. Recently, REDD1 was identified as one of the central genes mechanistically involved in undesirable atrophic effects induced by chronic topical and systemic glucocorticoids widely used for the treatment of blood cancer and inflammatory diseases. In this review, we discuss the role of REDD1 in the regulation of cell signaling and processes in normal and cancer cells, its involvement in the pathogenesis of different diseases, and the approach to safer glucocorticoid receptor (GR)-targeted therapies via a combination of glucocorticoids and REDD1 inhibitors to decrease the adverse atrophogenic effects of these steroids.

Roles Played by Stress-Induced Pathways in Driving Ethnic Heterogeneity for Inflammatory Skin Diseases.

Immune-mediated skin conditions (IMSCs) are a diverse group of autoimmune diseases associated with significant disease burden. Atopic dermatitis and psoriasis are among the most common IMSCs in the United States and have disproportionate impact on racial and ethnic minorities. African American patients are more likely to develop atopic dermatitis compared to their European American counterparts; and despite lower prevalence of psoriasis among this group, African American patients can suffer from more extensive disease involvement, significant post-inflammatory changes, and a decreased quality of life. While recent studies have been focused on understanding the heterogeneity underlying disease mechanisms and genetic factors at play, little emphasis has been put on the effect of psychosocial or psychological stress on immune pathways, and how these factors contribute to differences in clinical severity, prevalence, and treatment response across ethnic groups. In this review, we explore the heterogeneity of atopic dermatitis and psoriasis between African American and European American patients by summarizing epidemiological studies, addressing potential molecular and environmental factors, with a focus on the intersection between stress and inflammatory pathways.

The long winding road to the safer glucocorticoid receptor (GR) targeting therapies.

Glucocorticoids (Gcs) are widely used to treat inflammatory diseases and hematological malignancies, and despite the introduction of novel anti-inflammatory and anti-cancer biologics, the use of inexpensive and effective Gcs is expected to grow. Unfortunately, chronic treatment with Gcs results in multiple atrophic and metabolic side effects. Thus, the search for safer glucocorticoid receptor (GR)-targeted therapies that preserve therapeutic potential of Gcs but result in fewer adverse effects remains highly relevant. Development of selective GR agonists/modulators (SEGRAM) with reduced side effects, based on the concept of dissociation of GR transactivation and transrepression functions, resulted in limited success, and currently focus has shifted towards partial GR agonists. Additional approach is the identification and inhibition of genes associated with Gcs specific side effects. Others and we recently identified GR target genes REDD1 and FKBP51 as key mediators of Gcs-induced atrophy, and selected and validated candidate molecules for REDD1 blockage including PI3K/Akt/mTOR inhibitors. In this review, we summarized classic and contemporary approaches to safer GR-mediated therapies including unique concept of Gcs combination with REDD1 inhibitors. We discussed protective effects of REDD1 inhibitors against Gcs-induced atrophy in skin and bone and underlined the translational potential of this combination for further development of safer and effective Gcs-based therapies.

Transcriptome Analysis Reveals Intrinsic Proinflammatory Signaling in Healthy African American Skin.

Differences in the morphology and physiology of darkly pigmented skin compared with those of lightly pigmented skin are well-recognized. There are also disparities in the prevalence and clinical features for many inflammatory skin diseases, including atopic dermatitis and psoriasis; however, the underlying mechanisms are largely unknown. We compared the baseline gene expression in full-thickness skin biopsies from healthy individuals self-reporting as African American (AA) or as White non-Hispanic (WNH). Extensively validated RNA-sequencing analysis identified 570 differentially expressed genes in AA skin, including Igs and their receptors such as FCER1G; proinflammatory genes such as TNFα and IL32; and epidermal differentiation cluster and keratin genes. Differentially expressed genes were functionally enriched for inflammatory responses, keratinization, and cornified envelope formation. RNA-sequencing analysis of three-dimensional human skin equivalents made from AA and WNH primary keratinocytes revealed 360 differentially expressed genes (some shared with skin) that were enriched by similar functions. AA human skin equivalents appeared more responsive to TNF-α proinflammatory effects. Finally, AA-specific differentially expressed genes in the skin and human skin equivalents significantly overlapped with molecular signatures of skin in patients with atopic dermatitis and psoriasis. Overall, these findings suggest the existence of intrinsic proinflammatory circuits in AA keratinocytes/skin that may account for disease disparities and will help to build a foundation for the development of targeted skin disease prevention.

Early Stress-Response Gene REDD1 Controls Oxazolone-Induced Allergic Contact Dermatitis.

REDD1 is an energy sensor and stress-induced mTOR inhibitor. Recently, its novel role in linking metabolism and inflammation/immune responses has emerged. In this study, we assessed the role of REDD1 in murine oxazolone-induced allergic contact dermatitis (ACD), a T cell-dependent model with features of human ACD. A variety of immune indices, including edema, cellular infiltration, inflammatory gene expression, and glucocorticoid response, were compared in Redd1 knockout (KO) and isogenic (C57BL/6 × 129)F1 wild-type mice after sensitization and subsequent ear challenge with oxazolone. Despite relatively normal thymic profiles and similar T cell populations in the lymph nodes of naive Redd1 KO mice, early T cell expansion and cytokine production were profoundly impaired after sensitization. Surprisingly, higher steady-state populations of CD4+ and CD8+ T cells, as well as macrophages (CD45+/Ly-6G-/CD11b+), dendritic cells (CD45+/Ly-6G-/CD11c+), neutrophils (CD45+/Ly-6G+/CD11b+), and innate lymphoid cells (CD45+/Lineage-/IL-7Ra+/ST2+/c-Kit+), were observed in the ears of naive Redd1 KO mice. Upon challenge, ear edema, T cell, macrophage, neutrophil, and dendritic cell infiltration into the ear was significantly reduced in Redd1 KO animals. Accordingly, we observed significantly lower induction of IFN-γ, IL-4, and other cytokines as well as proinflammatory factors, including TSLP, IL-33, IL-1ß, IL-6, and TNF-α, in challenged ears of Redd1 KO mice. The response to glucocorticoid treatment was also diminished. Taken together, these data establish REDD1 as an essential immune modulator that influences both the initiation of ACD disease, by driving naive T cell activation, and the effector phase, by promoting immune cell trafficking in T cell-mediated skin inflammation.

Anti-Proliferative Effects of Compound A and Its Effect in Combination with Cisplatin in Cholangiocarcinoma Cells.

BACKGROUND: Cholangiocarcinoma (CCA) is a fatal cancer with high resistance to anticancer drugs.  The development of new drugs or compounds to be used alone or in combination with currently available chemotherapeutic agents to improve the treatment of CCA is needed. Compound A (CpdA), which is a small plant-derived glucocorticoid receptor modulator, strongly inhibited the growth and survival of several cancers.  However, the effect of CpdA on cholangiocarcinoma has not been elucidated. The aim of this study was to investigate the effect of CpdA on CCA. METHODS: Cytotoxicity of CpdA was tested in primary cells including peripheral blood mononuclear cells (PBMCs), fibroblasts, and human umbilical vein endothelial cells (HUVECs), as well as on CCA cell lines (KKU-100, KKU-055, and KKU-213) was examined. Cell cycle distribution and IL-6 expression was assessed by flow cytometry and real-time polymerase chain reaction, respectively.  The effect of combination CpdA and cisplatin was evaluated by cell viability assay. RESULTS: CpdA significantly inhibited cell cycle at G1 phase in CCA cell lines, and reduced IL-6 mRNA expression.  However, combination CpdA and cisplatin did not enhance the inhibitory effect. TGFßR-II expression was increased in CCA cells after the combination treatment. CONCLUSIONS: These results indicate the potential of CpdA for CCA treatment. However, combination treatment with CpdA and cisplatin increased CCA cell survival. The molecular mechanism is likely attributable to promotes cell survival via the TGFßR-II signaling pathway. The combination of CpdA with other anticancer drugs for CCA treatment should be further examined.

A Novel Approach to Safer Glucocorticoid Receptor-Targeted Anti-lymphoma Therapy via REDD1 (Regulated in Development and DNA Damage 1) Inhibition.

Glucocorticoids are widely used for therapy of hematologic malignancies. Unfortunately, chronic treatment with glucocorticoids commonly leads to adverse effects including skin and muscle atrophy and osteoporosis. We found recently that REDD1 (regulated in development and DNA damage 1) plays central role in steroid atrophy. Here, we tested whether REDD1 suppression makes glucocorticoid-based therapy of blood cancer safer. Unexpectedly, approximately 50% of top putative REDD1 inhibitors selected by bioinformatics screening of Library of Integrated Network-Based Cellular Signatures database (LINCS) were PI3K/Akt/mTOR inhibitors. We selected Wortmannin, LY294002, and AZD8055 for our studies and showed that they blocked basal and glucocorticoid-induced REDD1 expression. Moreover, all PI3K/mTOR/Akt inhibitors modified glucocorticoid receptor function shifting it toward therapeutically important transrepression. PI3K/Akt/mTOR inhibitors enhanced anti-lymphoma effects of Dexamethasone in vitro and in vivo, in lymphoma xenograft model. The therapeutic effects of PI3K inhibitor+Dexamethasone combinations ranged from cooperative to synergistic, especially in case of LY294002 and Rapamycin, used as a previously characterized reference REDD1 inhibitor. We found that coadministration of LY294002 or Rapamycin with Dexamethasone protected skin against Dexamethasone-induced atrophy, and normalized RANKL/OPG ratio indicating a reduction of Dexamethasone-induced osteoporosis. Together, our results provide foundation for further development of safer and more effective glucocorticoid-based combination therapy of hematologic malignancies using PI3K/Akt/mTOR inhibitors.

Sexual dimorphism in atrophic effects of topical glucocorticoids is driven by differential regulation of atrophogene REDD1 in male and female skin.

Topical glucocorticoids, well-known anti-inflammatory drugs, induce multiple adverse effects, including skin atrophy. The sex-specific effects of systemic glucocorticoids are known, but sexual dimorphism of therapeutic and side effects of topical steroids has not been studied. We report here that female and male mice were equally sensitive to the anti-inflammatory effect of glucocorticoid fluocinolone acetonide (FA) in ear edema test. At the same time, females were more sensitive to FA-induced skin atrophy. We recently reported that REDD1 (regulated in development and DNA damage 1) plays central role in steroid atrophy. We found that REDD1 was more efficiently activated by FA in females, and that REDD1 knockout significantly protected female but not male mice from skin atrophy. Studies using human keratinocytes revealed that both estradiol and FA induced REDD1 mRNA/protein expression, and cooperated when they were combined at low doses. Chromatin immunoprecipitation analysis confirmed that REDD1 is an estrogen receptor (ER) target gene with multiple estrogen response elements in its promoter. Moreover, experiments with GR and ER inhibitors suggested that REDD1 induction by these hormones was interdependent on functional activity of both receptors. Overall, our results are important for the development of safer GR-targeted therapies suited for female and male dermatological patients.

Regulated in Development and DNA Damage Responses 1 Prevents Dermal Adipocyte Differentiation and Is Required for Hair Cycle-Dependent Dermal Adipose Expansion.

Dermal white adipose tissue (dWAT) expansion is associated with important homeostatic and pathologic processes in skin. Even though mTOR/protein kinase B signaling is important for adipogenesis, the role of regulated development of DNA damage responses 1 (REDD1), a negative regulator of mTOR/protein kinase B, is poorly understood. Loss of REDD1 in mice resulted in reduction of body mass, total fat, size of gonadal white adipose tissue, and interscapular brown adipose tissue. Inguinal subcutaneous white adipose tissue and dWAT in REDD1 knockouts were expanded compared with wild type mice. Size and number of mature adipocytes in dWAT were also increased in adult REDD1 knockouts. This dWAT phenotype was established around postnatal day 18 and did not depend on the hair growth cycle. Numbers of adipocyte precursor cells were lower in REDD1 knockout skin. In vitro analysis revealed increased differentiation of skin-derived REDD1 knockout adipocyte precursor cells as indicated by higher lipid accumulation and increased adipogenic marker expression. 3T3L1 cells overexpressing REDD1 had decreased sensitivity to differentiation. Overall, our findings indicate that REDD1 silencing induced expansion of dWAT through hypertrophy and hyperplasia. This REDD1-dependent mechanism of adipogenesis could be used to preferentially target skin-associated adipose tissue for therapeutic purposes.

Transcriptomic Network Interactions in Human Skin Treated with Topical Glucocorticoid Clobetasol Propionate.

Glucocorticoids are the most frequently used anti-inflammatory drugs in dermatology. However, the molecular signature of glucocorticoids and their receptor in human skin is largely unknown. Our validated bioinformatics analysis of human skin transcriptome induced by topical glucocorticoid clobetasol propionate (CBP) in healthy volunteers identified numerous unreported glucocorticoid-responsive genes, including over a thousand noncoding RNAs. We observed sexual and racial dimorphism in the CBP response including a shift toward IFN-α/IFN-γ and IL-6/Jak/Signal transducer and activator of transcription (STAT) 3 signaling in female skin; and a larger response to CBP in African-American skin. Weighted gene coexpression network analysis unveiled a dense skin network of 41 transcription factors including circadian Kruppel-like factor 9 (KLF9), and ∼260 of their target genes enriched for functional pathways representative of the entire CBP transcriptome. Using keratinocytes with Kruppel-like factor 9 knockdown, we revealed a feedforward loop in glucocorticoid receptor signaling, previously unreported. Interestingly, many of the CBP-regulated transcription factors were involved in the control of development, metabolism, circadian clock; and 80% of them were associated with skin aging showing similarities between glucocorticoid-treated and aged skin. Overall, these findings indicate that glucocorticoid receptor acts as an important regulator of gene expression in skin-both at the transcriptional and posttranscriptional level-via multiple mechanisms including regulation of noncoding RNAs and multiple core transcription factors.

PI3K inhibitors protect against glucocorticoid-induced skin atrophy.

BACKGROUND: Skin atrophy is a major adverse effect of topical glucocorticoids. We recently reported that REDD1 (regulated in development and DNA damage 1) and FKBP51 (FK506 binding protein 5), negative regulators of mTOR/Akt signaling, are induced by glucocorticoids in mouse and human skin and are central drivers of steroid skin atrophy. Thus, we hypothesized that REDD1/FKBP51 inhibitors could protect skin against catabolic effects of glucocorticoids. METHODS: Using drug repurposing approach, we screened LINCS library (http://lincsproject.org/LINCS/) to identify repressors of REDD1/FKBP51 expression. Candidate compounds were tested for their ability to inhibit glucocorticoid-induced REDD1/FKBP51 expression in human primary/immortalized keratinocytes and in mouse skin. Reporter gene expression, microarray, and chromatin immunoprecipitation were employed to evaluate effect of these inhibitors on the glucocorticoid receptor (GR) signaling. FINDINGS: Bioinformatics analysis unexpectedly identified phosphoinositide-3-kinase (PI3K)/mTOR/Akt inhibitors as a pharmacological class of REDD1/FKBP51 repressors. Selected PI3K/mTOR/Akt inhibitors-Wortmannin (WM), LY294002, AZD8055, and two others indeed blocked REDD1/FKBP51expression in human keratinocytes. PI3K/mTOR/Akt inhibitors also modified global effect of glucocorticoids on trascriptome, shifting it towards therapeutically important transrepression; negatively impacted GR phosphorylation; nuclear translocation; and GR loading on REDD1/FKBP51 gene promoters. Further, topical application of LY294002 together with glucocorticoid fluocinolone acetonide (FA) protected mice against FA-induced proliferative block and skin atrophy but did not alter the anti-inflammatory activity of FA in ear edema test. INTERPRETATION: Our results built a strong foundation for development of safer GR-targeted therapies for inflammatory skin diseases using combination of glucocorticoids with PI3K/mTOR/Akt inhibitors. FUND: Work is supported by NIH grants R01GM112945, R01AI125366, and HESI-THRIVE foundation.

Deletion of the glucocorticoid receptor chaperone FKBP51 prevents glucocorticoid-induced skin atrophy.

FKBP51 (FK506-binding protein 51) is a known co-chaperone and regulator of the glucocorticoid receptor (GR), which usually attenuates its activity. FKBP51 is one of the major GR target genes in skin, but its role in clinical effects of glucocorticoids is not known. Here, we used FKBP51 knockout (KO) mice to determine FKBP51's role in the major adverse effect of topical glucocorticoids, skin atrophy. Unexpectedly, we found that all skin compartments (epidermis, dermis, dermal adipose and CD34+ stem cells) in FKBP51 KO animals were much more resistant to glucocorticoid-induced hypoplasia. Furthermore, despite the absence of inhibitory FKBP51, the basal level of expression and glucocorticoid activation of GR target genes were not increased in FKBP51 KO skin or CRISPR/Cas9-edited FKBP51 KO HaCaT human keratinocytes. FKBP51 is known to negatively regulate Akt and mTOR. We found a significant increase in AktSer473 and mTORSer2448 phosphorylation and downstream pro-growth signaling in FKBP51-deficient keratinocytes in vivo and in vitro. As Akt/mTOR-GR crosstalk is usually negative in skin, our results suggest that Akt/mTOR activation could be responsible for the lack of increased GR function and resistance of FKBP51 KO mice to the steroid-induced skin atrophy.

Rapamycin Modulates Glucocorticoid Receptor Function, Blocks Atrophogene REDD1, and Protects Skin from Steroid Atrophy.

Glucocorticoids have excellent therapeutic properties; however, they cause significant adverse atrophogenic effects. The mTORC1 inhibitor REDD1 has been recently identified as a key mediator of glucocorticoid-induced atrophy. We performed computational screening of a connectivity map database to identify putative REDD1 inhibitors. The top selected candidates included rapamycin, which was unexpected because it inhibits pro-proliferative mTOR signaling. Indeed, rapamycin inhibited REDD1 induction by glucocorticoids dexamethasone, clobetasol propionate, and fluocinolone acetonide in keratinocytes, lymphoid cells, and mouse skin. We also showed blunting of glucocorticoid-induced REDD1 induction by either catalytic inhibitor of mTORC1/2 (OSI-027) or genetic inhibition of mTORC1, highlighting role of mTOR in glucocorticoid receptor signaling. Moreover, rapamycin inhibited glucocorticoid receptor phosphorylation, nuclear translocation, and loading on glucocorticoid-responsive elements in REDD1 promoter. Using microarrays, we quantified a global effect of rapamycin on gene expression regulation by fluocinolone acetonide in human keratinocytes. Rapamycin inhibited activation of glucocorticoid receptor 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.

Endogenous Glucocorticoid Deficiency in Psoriasis Promotes Inflammation and Abnormal Differentiation.

The factors involved in maintaining a localized inflammatory state in psoriatic skin remain poorly understood. Here, we demonstrate through metabolomic and transcriptomic profiling marked suppression of glucocorticoid biosynthesis in the epidermis of psoriatic skin leading to localized deficiency of cortisol. Utilizing a 3D human epidermis model, we demonstrate that glucocorticoid biosynthesis is suppressed by proinflammatory cytokines and that glucocorticoid deficiency promotes inflammatory responses in keratinocytes. Finally, we show in vitro and in vivo that treatment with topical glucocorticoids leads to rapid restoration of glucocorticoid biosynthesis gene expression coincident with normalization of epidermal differentiation and suppression of inflammatory responses. Taken together, our data suggest that localized glucocorticoid deficiency in psoriatic skin interferes with epidermal differentiation and promotes a sustained and localized inflammatory response. This may shed new light on the mechanism of action of topical steroids, and demonstrates the critical role of endogenous steroid in maintaining both inflammatory and differentiation homeostasis in the epidermis.

Important role of kallikrein 6 for the development of keratinocyte proliferative resistance to topical glucocorticoids.

One of the major adverse effects of topical glucocorticoids is cutaneous atrophy often followed by development of resistance to steroids (tachyphylaxis). Previously we showed that after two weeks, interfollicular mouse keratinocytes acquired resistance to anti-proliferative effects of glucocorticoid fluocinolone acetonide (FA). One of the top genes activated by FA during tachyphylaxis was Klk6 encoding kallikrein-related peptidase 6, known to enhance keratinocyte proliferation. KLK6 was also strongly induced by chronic glucocorticoids in human skin. Double immunostaining showed that KLK6+ keratinocytes, localized in suprabasal layer of mouse skin, were frequently adjacent to proliferating 5-bromo-2'-deoxyuridine-positive basal keratinocytes. We used KLK6 knockout (KO) mice to evaluate KLK6 role in skin regeneration after steroid-induced atrophy. KLK6 KOs had thinner epidermis and decreased keratinocyte proliferation. The keratinocytes in wild type and KLK6 KO epidermis were equally sensitive to acute anti-proliferative effect of FA. However, the development of proliferative resistance during chronic treatment was reduced in KO epidermis. This was not due to the changes in glucocorticoid receptor (GR) expression or function as GR protein level and induction of GR-target genes were similar in wild type and KLK6 KO skin. Overall, these results suggest a novel mechanism of epidermal regeneration after glucocorticoid-induced atrophy via KLK6 activation.

N-bromotaurine surrogates for loss of antiproliferative response and enhances cisplatin efficacy in cancer cells with impaired glucocorticoid receptor.

Glucocorticoids (GCs) are frequently used in anticancer combination regimens; however, their continuous use adds selective pressure on cancer cells to develop GC-resistance via impairment of the glucocorticoid receptor (GR), therefore creating a need for GC-alternatives. Based on the drug repurposing approach and the commonalities between inflammation and neoplasia, drugs that are either in late-stage clinical trials and/or already marketed for GC-refractory inflammatory diseases could be evaluated as GC-substitutes in the context of cancer. Advantageously, unlike new molecular entities currently being de novo developed to restore GC-responsiveness of cancer cells, such drugs have documented safety and efficacy profile, which overall simplifies their introduction in clinical cancer trials. In this study, we estimated the potential of a well-established, multistage, cell line-based, mouse skin carcinogenesis model to be exploited as an initial screening tool for unveiling covert GC-substitutes. First, we categorized the cell lines of this model to GC-sensitive and GC-resistant, in correlation with their corresponding GR status, localization, and functionality. We found that GC-resistance starts in papilloma stages, due to a dysfunctional GR, which is overexpressed, DNA binding-competent, but transactivation-incompetent in papilloma, squamous, and spindle stages of the model. Then, aided by this tool, we evaluated the ability of N-bromotaurine, a naturally occurring, small-molecule, nonsteroid anti-inflammatory drug which is under consideration for use interchangeably/in replacement to GCs in skin inflammations, to restore antiproliferative response of GC-resistant cancer cells. Unlike GCs, N-bromotaurine inhibited cell-cycle progression in GC-resistant cancer cells and efficiently synergized with cisplatin, thus indicating a potential to be exploited instead of GCs against cancer.

Discovery of Compound A--a selective activator of the glucocorticoid receptor with anti-inflammatory and anti-cancer activity.

Glucocorticoids are among the most effective anti-inflammatory drugs, and are widely used for cancer therapy. Unfortunately, chronic treatment with glucocorticoids results in multiple side effects. Thus, there was an intensive search for selective glucocorticoid receptor (GR) activators (SEGRA), which retain therapeutic potential of glucocorticoids, but with fewer adverse effects. GR regulates gene expression by transactivation (TA), by binding as homodimer to gene promoters, or transrepression (TR), via diverse mechanisms including negative interaction between monomeric GR and other transcription factors. It is well accepted that metabolic and atrophogenic effects of glucocorticoids are mediated by GR TA. Here we summarized the results of extensive international collaboration that led to discovery and characterization of Compound A (CpdA), a unique SEGRA with a proven "dissociating" GR ligand profile, preventing GR dimerization and shifting GR activity towards TR both in vitro and in vivo. We outlined here the unusual story of compound's discovery, and presented a comprehensive overview of CpdA ligand properties, its anti-inflammatory effects in numerous animal models of inflammation and autoimmune diseases, as well as its anti-cancer effects. Finally, we presented mechanistic analysis of CpdA and glucocorticoid effects in skin, muscle, bone, and regulation of glucose and fat metabolism to explain decreased CpdA side effects compared to glucocorticoids. Overall, the results obtained by our and other laboratories underline translational potential of CpdA and its derivatives for treatment of inflammation, autoimmune diseases and cancer.

REDD1 functions at the crossroads between the therapeutic and adverse effects of topical glucocorticoids.

Cutaneous atrophy is the major adverse effect of topical glucocorticoids; however, its molecular mechanisms are poorly understood. Here, we identify stress-inducible mTOR inhibitor REDD1 (regulated in development and DNA damage response 1) as a major molecular target of glucocorticoids, which mediates cutaneous atrophy. In REDD1 knockout (KO) mice, all skin compartments (epidermis, dermis, subcutaneous fat), epidermal stem, and progenitor cells were protected from atrophic effects of glucocorticoids. Moreover, REDD1 knockdown resulted in similar consequences in organotypic raft cultures of primary human keratinocytes. Expression profiling revealed that gene activation by glucocorticoids was strongly altered in REDD1 KO epidermis. In contrast, the down-regulation of genes involved in anti-inflammatory glucocorticoid response was strikingly similar in wild-type and REDD1 KO mice. Integrative bioinformatics analysis of our and published gene array data revealed similar changes of gene expression in epidermis and in muscle undergoing glucocorticoid-dependent and glucocorticoid-independent atrophy. Importantly, the lack of REDD1 did not diminish the anti-inflammatory effects of glucocorticoids in preclinical model. Our findings suggest that combining steroids with REDD1 inhibitors may yield a novel, safer glucocorticoid-based therapies.

Asymmetric expression of connexins between luminal epithelial- and myoepithelial- cells is essential for contractile function of the mammary gland.

Intercellular communication is essential for glandular functions and tissue homeostasis. Gap junctions couple cells homotypically and heterotypically and co-ordinate reciprocal responses between the different cell types. Connexins (Cxs) are the main mammalian gap junction proteins, and the distribution of some Cx subtypes in the heterotypic gap junctions is not symmetrical; in the murine mammary gland, Cx26, Cx30 and Cx32 are expressed only in the luminal epithelial cells and Cx43 is expressed only in myoepithelial cells. Expression of all four Cxs peaks during late pregnancy and throughout lactation suggesting essential roles for these proteins in the functional secretory activity of the gland. Transgenic (Tg) mice over-expressing Cx26 driven by keratin 5 promoter had an unexpected mammary phenotype: the mothers were unable to feed their pups to weaning age leading to litter starvation and demise in early to mid-lactation. The mammary gland of K5-Cx26 female mice developed normally and produced normal levels of milk protein, suggesting a defect in delivery rather than milk production. Because the mammary gland of K5-Cx26 mothers contained excessive milk, we hypothesized that the defect may be in an inability to eject the milk. Using ex vivo three-dimensional mammary organoid cultures, we showed that tissues isolated from wild-type FVB females contracted upon treatment with oxytocin, whereas, organoids from Tg mice failed to do so. Unexpectedly, we found that ectopic expression of Cx26 in myoepithelial cells altered the expression of endogenous Cx43 resulting in impaired gap junction communication, demonstrated by defective dye coupling in mammary epithelial cells of Tg mice. Inhibition of gap junction communication or knock-down of Cx43 in organoids from wild-type mice impaired contraction in response to oxytocin, recapitulating the observations from the mammary glands of Tg mice. We conclude that Cx26 acts as a trans-dominant negative for Cx43 function in myoepithelial cells, highlighting the importance of cell type-specific expression of Cxs for optimal contractile function of the mammary myoepithelium.