Fig 1.
Oli-neuM cells express higher levels of MBP than Oli-neu cells.
(A) Oli-neu cells were plated in a 96-well plate, left to adhere for two days, treated with differentiation medium (DM) prior to treatment with 10μM Dexamethasone and/or transfected with the MRF-expressing construct (MyrfpCMV6) as indicated. After two days of growth cells were fixed and stained with an α-MBP antibody (panel MBP). Nuclei were stained with Hoechst (panel Hoechst). Scale bar: 10 μm. (B) Expression of MRF in Oli-neu cells and in Oli-neuM (Oli-neu cells stably expressing MRF) was measured by RT-PCR. The expression level in Oli-neu cells was arbitrarily set at 1. Data were acquired in triplicate and are presented as the mean ± SD. Statistical significance was analysed by a two-tailed Student's t test. (C) MBP protein levels were measured using the ScanR platform for acquisition and data analysis. Oli-neu or Oli-neuM cell lines were plated and treated as indicated in the text. Mean values ± SD of two independent experiments acquired in triplicate from the indicated cell line are plotted in the graph. The MBP protein level of the Oli-neu cell line was set arbitrarily at 100%. Statistical significance between dexamethasone-treated and untreated samples was analysed by a two-tailed Student's t test.
Fig 2.
The picture illustrates the flow and the results of screening re-profiling of the over 1,200 clinically approved compounds from Prestwick Chemicals, tested on the cell line Oli-neuM as described in Methods. MBP protein expression was plotted on a Microsoft Excel graph for all of the compounds at the three concentrations tested together with the control Dexamethasone. In the primary screen we considered as positive only those compounds that exceeded or matched the lowest MBP protein expression observed following Dexamethasone treatment. In the secondary screen we considered as hit compounds only those drugs that were able to induce MBP protein expression better than Dexamethasone at all three concentrations tested (1, 10, 25 μM). (Dexa = Dexamethasone).
Fig 3.
Glucocorticoids are potent myelin gene inducers.
(A) Glucocorticoid classification according to their activity on MBP in both the primary and secondary screens. Intensity scale: yellow colour (0) means no increase in MBP expression over Dexamethasone; green colour means MBP induction: the stronger the induction, the more intense the green colour is represented. Higher level of MBP induction has an intensity scale 3. Based on the intensity scale, glucocorticoids were divided into three classes (black brackets). (B) Dose response curves of all positive glucocorticoids, using Dexamethasone and Prednisone as controls. Ten dose-response curves were performed over a range of concentrations up to 25 μM as indicated in the text. Oli-neuM MBP protein levels were calculated by Olympus ScanR software analysis (more than 1,000 cells were analyzed for every condition). MBP protein levels were calculated as % increase compared to NT value. Data were acquired in triplicate (n = 2) and analyzed by nonlinear regression and fitted to a sigmoid dose-response using GraphPad Prism (GraphPad Software, Inc.). (C) Representative images of Oli-neuM untreated (DM+DMSO) or treated with 10 μM of the indicated drugs. Images were taken with a 20X objective of the Olympus ScanR platform (scale bar: 10 μm).
Fig 4.
Clobetasol and Halcinonide promote Smoothened activation.
(A) RT-PCR of MBP, PLP, CNP and MOG induced by 10 μM glucocorticoid treatment. All glucocorticoids significantly up-regulate all myelin genes tested with fold change induction above NT level (arbitrarily set as 1). Data acquired in triplicate are presented as the mean ± SD. Statistical significance was analyzed by a two-tailed Student's t test. * p<0.05, ** p≤0.01 for each gene vs. NT. MBP expression: Prednisone P < 0.0001, Medrysone, Fluticasone, Amcinonide, Clobetasol, Flurandrenolide, Dexamethasone P = 0.0016, Halcinonide P = 0.0012. PLP expression: Prednisone P = 0.0263, Medrysone P = 0.0255, Rimexolone, Dexamethasone P = 0.0001, Fluticasone, Clobetasol, Amcinonide P < 0.0001, Halcinonide P = 0.0008, Flurandrenolide P = 0.0175. CNP expression: Prednisone P = 0.0061, Medrysone, Rimexolone, Fluticasone, Amcinonide, Dexamethasone, Clobetasol, Flurandrenolide P < 0.0001, Halcinonide P = 0.0006. MOG expression: Medrysone, Rimexolone, Fluticasone, Amcinonide, Clobetasol, Flurandrenolide P < 0.0001, Dexamethasone P = 0.0006, Halcinonide P = 0.0026). (B) representative images and (C) quantification of MBP expression in Oli-neuM cells plated in 96-well plates containing DM for 48h and then treated with 10 μM of the indicated FGC with or without 150 nM Cyclopamine or 5 μM Itraconazole and further incubated for 48h prior to inspection. Images were taken with a 20X objective using the Olympus ScanR platform. More than 1,000 cells/sample were analysed. Data are plotted as the mean ± SD of three wells per analysis (n = 4). Statistical analysis was performed using one-way ANOVA single compound treatment and together with Itraconazole and Cyclopamine: Dexa vs Dexa+Itrac vs Dexa+Cycl = 0.3811; Predn vs Pren+Itrac vs Pred+Cycl = 0.2496: Clob vs Clob+Itrac vs Clob+Cycl = 0.0017; Halc vs Halc+Itrac vs Halc+Cycl = 0.0044. Dexa, Dexamethasone; Itrac, Itraconazole; Cycl, Cyclopamine; Predn, Prednisone; Clob, Clobetasol; Halc, Halcinonide. Scale bar: 10 μm.
Fig 5.
RxRγ expression is up-regulated by GCs.
(A) RT-PCR of RxRγ, Gli1 and NR3C1 expression. Data acquired were normalized using the GAPDH housekeeping gene and the NT level were arbitrarily set as 1. Data were acquired in triplicate and are presented as the mean ± SD. Statistical significance was analysed by a two-tailed Student's t test, comparing the fold change of each gene against its respective NT. * p<0.05, **p≤0.01 RxRγ expression: Prednisone P = 0.0106, Dexamethasone P = 0.0029, Clobetasol Fluticasone, Flurandrenolide, Amcinonide. P < 0.0001, Halcinonide P = 0.0007, Rimexolone P = 0.0008, Medrysone P = 0.0027. Gli1 expression: Halcinonide P < 0.0001, Amcinonide P = 0.0014. NR3C1 expression: Dexamethasone, Clobetasol, Fluticasone, Rimexolone, Halcinonide P < 0.0001, Medrysone. P = 0.0022, Flurandrenolide P = 0.0001, Amcinonide P = 0.0006. (B) RT-PCR of RxRγ and MBP expression induced by 10 μM of the indicated compounds with or without 1 μM UVI 3003 treatment. The UVI 3003 antagonist (+, where added) decreases RxRγ and MBP expression compared to Oli-neuM cells treated with glucocorticoid alone. Data acquired in triplicate (n = 2) were analyzed as described above. Statistical significance is shown for samples with (+) compared to samples without (-) UVI3003 for each treatment and were analysed by a two-tailed Student's t test. (C) Model of FGC mechanism of action on MBP expression in oligodendrocytes.