Gleb Baida

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.

Selective Activator of the Glucocorticoid Receptor Compound A Dissociates Therapeutic and Atrophogenic Effects of Glucocorticoid Receptor Signaling in Skin

Background: Glucocorticoids are effective anti-inflammatory drugs widely used in dermatology and for the treatment of blood cancer patients. Unfortunately, chronic treatment with glucocorticoids results in serious metabolic and atrophogenic adverse effects including skin atrophy. Glucocorticoids act via the glucocorticoid receptor (GR), a transcription factor that causes either gene transactivation (TA) or transrepression (TR). Compound A (CpdA), a novel non-steroidal GR ligand, does not promote GR dimerization and TA, retains anti-inflammatory potential but induces fewer metabolic side effects compared to classical glucocorticoids when used systemically. As topical effects of CpdA have not been well studied, this work goal was to compare the anti-inflammatory and side effects of topical CpdA and glucocorticoids and to assess their effect on GR TA and TR in keratinocytes. Methods: We used murine immortalized keratinocytes and F1 C57BlxDBA mice. Effect of glucocorticoid fluocinolone acetonide (FA) and CpdA on gene expression in keratinocytes in vitro and in vivo was evaluated by reverse transcription-PCR. The anti-inflammatory effects were assessed in the model of tumor promoter 12-O-tertradecanoyl-acetate (TPA)-induced dermatitis and in croton oil-induced ear edema test. Skin atrophy was assessed by analysis of epidermal thickness, keratinocyte proliferation, subcutaneous adipose hypoplasia, and dermal changes after chronic treatment with FA and CpdA. Results: In mouse keratinocytes in vitro and in vivo, CpdA did not activate GR-dependent genes but mimicked closely the inhibitory effect of glucocorticoid FA on the expression of inflammatory cytokines and matrix metalloproteinases. When applied topically, CpdA inhibited TPA-induced skin inflammation and hyperplasia. Unlike glucocorticoids, CpdA itself did not induce skin atrophy which correlated with lack of induction of atrophogene regulated in development and DNA damage response 1 (REDD1) causatively involved in skin and muscle steroid-induced atrophy. Conclusions: Overall, our results suggest that CpdA and its derivatives represent novel promising class of anti-inflammatory compounds with reduced topical side effects.

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 FKBP51s 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.

Abstract 549: Role of the glucocorticoid receptor in skin carcinogenesis and stem cell maintenance: Implication for the use of selective GR activators (SEGRA) as anti-cancer drugs

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Glucocorticoids are important physiological and pharmacological regulators of epidermal proliferation, differentiation and skin tumorigenesis. The glucocorticoid effects are mediated via the glucocorticoid receptor (GR), a transcription factor that regulates genes expression by DNA-binding dependent transactivation, and DNA-binding independent transrepression through negative interaction with other transcription factors. Our previous studies demonstrated that keratin5.GR transgenic animals are resistant to skin carcinogenesis. We also found that GR diminishes the number of follicular epithelial stem cells (SC), reduces their proliferative and survival potential and affects the expression of follicular SC “signature” genes. To further explore the tumor suppressor role of GR in skin carcinogenesis we generated skin-specific GR knockout animals by breeding of GRflox/flox animals with Keratin5.Cre recombinase animals in C57Bl genetic background. Skin-specific GR KO (Cre/GRfl/fl) mice do not have gross skin or hair phenotype. However, the detailed analysis of keratinocyte markers revealed decreased expression of some intermediate/late markers of differentiation including Keratin 10 and loricrin. We also found that Cre/GRfl/fl mice are more sensitive to the effects of UV irradiation and tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The ongoing DMBA/TPA skin carcinogenesis experiment showed that Cre/GRfl/fl mice are more prone to skin carcinogenesis: they have higher tumor incidence and tumor multiplicity compared to GRfl/fl littermates. The analysis of global effect of the GR overexpression and GR silencing on gene expression in follicular epithelial SC, basal keratinocytes, and mouse skin tumors 1) revealed important GR targets in keratinocytes including STAT3 and REDD1 - the major regulator of mTOR; 2) provided an unexpected evidence that gene transrepression by GR plays critical role in the maintenance of SC and in inhibition of skin carcinogenesis. It is well understood that therapeutic effects of glucocorticoids are mediated by GR transreprssion, while many adverse metabolic effects are mediated by GR transactivation. Thus, our findings are important in light of drug discovery programs focused on the development of selective GR acivators (SEGRA) that preferentially induce GR transrepression. Some of SEGRA entered preclinical studies or clinical trials as anti-inflammatory GR ligands with reduced side effects. Our results suggest that SEGRA could be also used for skin cancer prevention/treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 549. doi:1538-7445.AM2012-549