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

Abstract

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.

Figure 1.. Pharmacological or genetic inhibition of mTOR blocks glucocorticoid-induced REDD1 expression.

(a–c) HaCaT, NHEK, and CEM cells were pretreated with rapamycin (1 μmol/L × 6 hours) and treated with solvent (control) or glucocorticoid FA (1 μmol/L) for 24 hours. (d, e) HaCaT cells were pretreated with varying doses of rapamycin (as indicated) for 6 hours, followed by treatment with glucocorticoids: (d) dexamethasone or (e) CBP for 24 hours. (f) shRaptor-HaCaT cells with genetically knocked down raptor and control pLKO.1-HaCaT cells were treated with either DMSO or FA(1 μmol/L) for 24 hours. (g, h) HaCaT cells were pretreated with OSI-027 (1 μmol/L × 6 hours) and treated with solvent (control) or glucocorticoid FA (1 μmol/L) for 24 hours. Expressions of REDD1 and raptor and activity of mTOR substrates were monitored using Western blotting. Tubulin was used as the loading control. Quantitative PCR results for REDD1 expression were normalized to the housekeeping gene RPL27 and presented as fold change compared with control. The mean ± standard deviation was calculated for three individual RNA samples/condition. ^aStatistically significant difference (P < 0.001) compared with control (DMSO). ^bStatistically significant difference (P < 0.001) compared with FA and where reflected. CBP, clobetasol propionate; FA, fluocinolone acetonide; M, mol/L; NHEK, normal human epidermal keratinocyte; NS, not significant compared with control (DMSO) in shRaptor-HaCaT cells; p-, phosphorylated; qPCR, quantitative PCR; Rapa, rapamycin; sh, short hairpin; WB, Western blotting.

Figure 2.. Rapamycin inhibits GR transactivation, blocks NF-κB, and enhances GR transrepression.

(a–d) Luciferase assay. (a) HaCaT-GRE.Luc, (b) HaCaT-NF-κB.Luc, (c) CEM-GRE.Luc, and (d) CEM-NF-κB.Luc cells were pretreated with solvent or rapamycin (1 μmol/L × 6 hours) and treated with solvent or FA (1 μmol/L) for 8 hours (n = 3). Luciferase induction is presented as mean ± standard deviation. ^aStatistically significant difference (P<0.001) compared with control, ^bStatistically significant difference (P<0.001) compared with FA. (e, f) Effect of rapamycin on NF-κB activation. HaCaT cells treated with solvent or rapamycin (1 μmol/L × 24 hours) were stimulated with TNF-α (50 ng/ml × 15 minutes). The levels of (e) phosphorylated and non-phosphorylated p65 and (f) IκB-α were analyzed by Western blotting (n = 3). GAPDH and lamin B served as loading controls. CE, cytoplasmic; Ctrl, control; FA, fluocinolone acetonide; GR, glucocorticoid receptor; GRE, glucocorticoid responsive element; NE, nuclear; Rapa, rapamycin; WB, western blotting; WC, whole cell.

Figure 3.. Illumina array analysis of rapamycin effect on glucocorticoid-induced differential gene expression.

(a) HaCaT cells treated with vehicle (control), rapamycin (1 μmol/L, 24 hours), FA (1 μmol/L, 24 hours) and FA plus rapamycin. Total RNA was extracted from cells in two independent experiments and used for analysis of gene expression by HT-12 Illumina (San Diego, CA) microarray. x-axis, experimental groups; y-axis, boxplot of the top 150 most up- and down-regulated genes (based on log₂ fold changes, logFC). Boxplot encompasses the first and third quartiles of logFC. Horizontal black lines indicate mean. Genes validated via quantitative PCR are labeled. *For up-regulated genes (FA + rapa vs. FA): t statistic = −4.24, P-value = 3.8 × 10⁻⁵; **For down-regulated genes (FA + rapa vs. FA): t statistic = −3.42; P-value = 7.9 10⁻⁵. (b) Array validation by quantitative PCR. a, statistically significant difference (P < 0.001) as compared to control (DMSO); b, statistically significant difference (P < 0.001) as compared to FA; C, control; FA, fluocinolone acetonide; Rapa, rapamycin.

Figure 4.. Rapamycin inhibits GR phosphorylation, nuclear translocation, and binding to GREs in REDD1 promoter.

(a, b) Western blot analysis of GR expression in (a) HaCaT and (b) CEM cells treated with rapamycin (1 μmol/L). (c–f) Western blot and immunofluorescence analysis of GR translocation and phosphorylation in (c, e) HaCaT and (d, f) CEM cells pretreated with rapamycin (1 μmol/L × 6 hours) and treated with FA (1 μmol/L). GAPDH and HDAC1 served as protein loading controls. Scale bars = 10 μm. (g) GR loading on seven GREs in REDD1 promoter was assessed in HaCaT cells treated as described by ChIP. Statistically significant difference (P<0.001) compared with ^acontrol and ^bFA. Data are mean × standard deviation, n = 3. ChIP, chromatin immunoprecipitation; Ctrl, control; FA, fluocinolone acetonide; GR, glucocorticoid receptor; GRE, glucocorticoid responsive element; h, hour; min, minute; p-, phosphorylation.

Figure 5.. Rapamycin protects skin against steroid-induced atrophy.

(a, b) Mice were treated with solvent or FA (2 μg) with or without rapamycin (0.5 mg) for 8 hours. RNA and proteins were extracted from epidermis. Rpl27 gene/protein served as loading control. Data are mean ± standard deviation, n = 3. (c, d) Skin atrophy. Animals were treated as described, every 72 hours for 2 weeks. (c) H&E staining. Scale bar = 20 μm. (d) Epidermal thickness fold change, % to control. Data are mean ± standard deviation, n = 30. (a–c) Statistically significant difference (P < 0.001) compared with ^acontrol and ^bFA. (e) Ear edema induced by croton oil after pretreatment with solvent, rapamycin or FA ± rapamycin. Ear punch weight (inflammation readout) is presented as fold change versus control. Data are mean ± standard deviation for six punches/condition. Statistically significant difference (P < 0.001) compared with ^acontrol and ^bcroton oil. 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.

Figure 6.. Modulation of GR-REDD1-mTOR crosstalk signaling to prevent glucocorticoid-induced atrophy.

(a) mTOR signaling plays a pro-proliferative role in cells.(b) Glucocorticoids activate GR, thus augmenting REDD1 (mTOR inhibitor/potent atrophogene) expression, leading to skin atrophy. *REDD1 acts as an atrophogenein skin and is causatively involved in glucocorticoid-induced atrophy (Baida et al., 2015). (c) Rapamycin inhibits REDD1 induction by GR and inhibits mTOR-GR crosstalk, resulting in reduced GR activity, alleviating atrophic phenotypes. GC, glucocorticoid; GR, glucocorticoid receptor; GRE, glucocorticoid responsive element; sh, short hairpin.