Search

Supplementary Figures 1-9. Suppl. Fig. 1 contains quantification of Western blots. Suppl. Fig. 2 demonstrates the effect of PI3K/mTOR/Akt inhibitors on regulation of endogenous genes by Dex in CEM and Granta cells. Suppl. Fig. 3 shows cytotoxic effects of LY294002, Wortmannin and AZD8055 on CEM and Granta cells. Suppl. Fig. 4 demonstrates cytotoxic effects of LY294002, Wortmannin and AZD8055 on CEM cells, Granta cells and normal human monocytes. Suppl. Fig. 5 shows anti-lymphoma effect of Rapamycin and Dex in CEM and Granta cells. Suppl. Fig. 6 demonstrates the effect of LY294002, Rapamycin and Dex on animal body weight in xenograft study. Suppl. Fig. 7 shows anti-tumor effect of Dex, Rapa, LY294002 on Granta xenografts. Suppl. Fig. 8 demonstrates the effect of LY294002, Rapamycin and Dex on animal body weight in Dexamethasone-induced osteoporosis study. Suppl. Fig. 9 shows the data on Q-PCR analysis of Col1a1 and Col2a1 mRNA expression in bone tissue.

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.

Supplementary Tables 1-3. Suppl. Table 1 contains the list of REDD1 inhibitors identified by computational screen and selected for study. Suppl. Table 2 contains primer sets for Q-PCR analysis. Suppl. Table 3 contains IC50 values of WM, LY294002 and AZD8055 after 24 h of incubation.

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.

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-1β, 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.

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.

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.

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.

Dermal white adipose (dWAT) expansion is associated with important homeostatic and pathologic processes in skin. Even though mammalian target of Rapamycin (mTOR)/Akt signaling is important for adipogenesis, the role of regulated in development and DNA damage 1 (REDD1), a negative regulator of mTOR/Akt, is poorly understood. Loss of REDD1 in mice resulted in reduction of body mass, total fat, size of gonadal white adipose tissue (WAT) and interscapular brown adipose tissue (BAT). Interestingly, inguinal subcutaneous WAT and dWAT in REDD1 knockouts (KOs) were expanded compared to wild type (WT) mice. Size and number of mature adipocytes in dWAT were also increased in adult REDD1 KOs. This dWAT phenotype was established around postnatal (PN) day 18, and did not depend on the hair growth cycle. Surprisingly, adipocyte precursor cells (APCs) numbers were lower in REDD1 KO skin. In vitro analysis revealed increased differentiation of skin-derived REDD1 KO APCs 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 not previously reported REDD1-dependent mechanism of adipogenesis could be used to preferentially target skin-associated adipose tissue for therapeutic purposes.

Liver X receptor (LXR) agonists have the potential to alleviate obesity related diseases, particularly atherosclerosis. However, LXRs are transcriptional regulators that induce de novo lipogenesis and lipid accumulation in hepatocytes which represents a serious adverse effect. In this work, we sought to characterize the LXR agonist GW3965 effects on fatty acid (FA) and phospholipid (PL) remodelling and the correlation with gene expression in order to better understand the underlying effects leading to hepatic pathology upon LXR activation. Human primary hepatocytes treated for 48 h with GW3965 were analysed for changes in lipid metabolism gene expression by qPCR, variations in the FA profile was evaluated by GC-FID and in PL profiles using thin layer chromatography, ESI-MS and MS/MS analysis. Changes in cell membrane biochemical properties were studied using bilayer models generated with CHARMM-GUI. ELOLV6 and SCD1 mRNA increase was consistent with higher C16:1 and C18:1n9 at the expense of C16:0 and C18:0. The reduction of C18:2n6 and increase in C20:2n6 was in agreement with ELOVL5 upregulation. Phosphatydilethanolamine (PE) levels tended to decrease and phosphatidylinositol to increase; although differences did not reach significance, they correlated with changes in AGXT2L1, CDS1 and LPIN1 mRNA levels that were increased. The overall effect of GW3965 on PEs molecular profiles was an increase of long-chain polyunsaturated FA chains and a decrease of C16/C18 saturated and monounsaturated FAs chains. Additionally, PC (32:1) and PC (34:2) were decreased, and PC (36:1) and PC (34:1) were increased. AGXT2L1 is an enzyme with strict substrate specificity for phosphoethanolamine, which is converted into ammonia in GW3965-treated hepatocytes and could explain the PE reduction. In summary, LXR activation by GW3965 targets PE biosynthesis and FA elongation/desaturation, which tends to decrease PE in relation to total PL levels, and remodelling of PC and PE molecular species. We identified the human AGXT2L1 gene as induced by LXR activation by both synthetic and endogenous agonist treatment. The increase in acetaldehyde-induced oxidative stress, and in the lipid species identified have the potential to enhance the inflammatory process and impair membrane function. Future studies should focus on inhibition of AGXT2L1 activity with the aim of reverting the steatosis induced by LXR activation.

Intestinal calcium absorption is a critical step in the maintenance of calcium homeostasis [Fleet, 2006]. It occurs by both paracellular and transcellular pathways [Bronner, 2003; Hoenderop et al., 2005]. The transcellular calcium absorption pathway is an active process that is regulated by the active form of vitamin D, 1,25 dihydroxyvitamin D3 (1,25(OH)2D3). 1,25 (OH)2D3 increases efficiency of transcellular Calcium absorption by increasing the abundance of critical proteins: calbindin D9k (CaBPD9k), the transient receptor vanelloid family member 6 (TRPV6), and the plasma membrane calcium ATPase 1b (PMCA1b) [Fleet et al., 2002; Song et al., 2003b; Fleet, 2006]. The actions of 1,25(OH)2D3 are mediated by the vitamin D receptor (VDR), a transcription factor that belongs to nuclear hormone receptor superfamily [Haussler et al., 1998]. The deletion of VDR in the enterocyte results in a 70% reduction in intestinal calcium absorption efficiency that is coincident with a significant reduction in CaBPD9k, TRPV6, and PMCA1b gene expression [Van Cromphaut et al., 2001; Song et al., 2003a].Previous research shows that binding of 1,25(OH)2D3 to VDR in the cytoplasm of cells stimulates heterodimerization of VDR with RXR and the redistribution of the VDR-RXR- hormone complex to the nucleus [Barsony et al., 1990; Michigami et al., 1999; Racz and Barsony, 1999; Sunn et al., 2001]. The nuclear import of the VDR-RXR-hormone complex is active [Racz and Barsony, 1999;Miyauchi et al., 2005], enhanced by 1,25(OH)2D3 treatment [Michigami et al., 1999; Racz and Barsony, 1999; Sunn et al., 2001], dependent upon the presence of intact nuclear localization sequences (NLSs) in both VDR and RXR [Prufer et al., 2000], and requires various importins [Miyauchi et al., 2005; Yasmin et al., 2005]. Most of the studies examining the mechanism of VDR nuclear import have been conducted in COS-7 kidney cells (characterized by low expression of endogenous VDR) and some characteristics of VDR import have been confirmed in cells from bone, skin, or kidney. In contrast the impact of 1,25(OH)2D3 treatment on VDR distribution has not been examined in absorptive enterocytes, a primary target cell of 1,25(OH)2D3 action. In this article we have examined the nucleocytoplasmic trafficking of VDR receptor in proliferating and differentiated Caco-2 cells. Caco-2 cells are an intestinal cell line that spontaneously differentiates and recapitulates many of the features of the absorptive epithelial cell of the small intestine, including vitamin D regulated intestinal calcium absorption [Giuliano and Wood, 1991; Fleet et al., 2002]. We find that VDR nuclear import occurs under basal conditions and that import is accelerated by 1,25(OH)2D3. This process is not influenced by the state of cellular differentiation. Our data also suggest that 1,25(OH)2D3-induced nuclear import is balanced by nuclear export in Caco-2 cells. In addition, our data are consistent with an essential role for nuclear import of VDR in the genomic responses of enterocytes to 1,25(OH)2D3.

We have shown that 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) down-regulates renin expression. To explore the molecular mechanism, we analyzed the mouse Ren-1c gene promoter by luciferase reporter assays. Deletion analysis revealed two DNA fragments from -2,725 to -2,647 (distal fragment) and from -117 to +6 (proximal fragment) that are sufficient to mediate the repression. Mutation of the cAMP response element (CRE) in the distal fragment blunted forskolin stimulation as well as 1,25(OH)(2)D(3) inhibition of the transcriptional activity, suggesting the involvement of CRE in 1,25(OH)(2)D(3)-induced suppression. EMSA revealed that 1,25(OH)(2)D(3) markedly inhibited nuclear protein binding to the CRE in the promoter. ChIP and GST pull-down assays demonstrated that liganded VDR blocked the binding of CREB to the CRE by directly interacting with CREB with the ligand-binding domain, and the VDR-mediated repression can be rescued by CREB, CBP, or p300 overexpression. These data indicate that 1,25(OH)(2)D(3) suppresses renin gene expression at least in part by blocking the formation of CRE-CREB-CBP complex.

Calbindin D9k(CaBP) is critical for intestinal calcium absorption; its in vivo expression is restricted to differentiated enterocytes of the small intestine. Our goal was to identify factors controlling the transcriptional regulation of this gene in the human intestine. Both the natural gene and a 4600-bp promoter construct were strongly regulated by differentiation (>100-fold) but not by treatment with 1,25(OH)2vitamin D (