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Blood, Vol. 109, Issue 4, 1584-1592, February 15, 2007
GSK-3 mediates differentiation and activation of proinflammatory dendritic cells
Blood Rodionova et al.
109: 1584
Supplemental materials for: Rodionova et al, Vol 9, Issue 4, 1584-1592
Files in this Data Supplement:
- Figure S1. Differential effects of LiCl and SB415286 on GSK-3 activity (PDF, 122 KB) -
Immature MoDCs were washed on days 5 to 7 of culture and resuspended in culture medium at a concentration of 2 to 3 × 105 cells/mL. Cells were either continued in GM-CSF and IL-4 (iDC) or activated with E coli (1:1000). LiCl (10 mM) or SB415286 (10 µM) concentration was added 20 minutes prior to activation or together with GM-CSF and IL-4. (A) Quantification of Western blots shown in Figure 2A. Quantification was performed using a Lumi-Imager F1 and the LumiAnalyst Version 3.1 for Windows Software.  -Actin was used to control for protein loading. Normalization of the results relative to the immature DC values was set as 1. Shown are the normalized means ± SD; n = 4. *P < .05; **P < .01. (B) Western blot analysis of DC lysates harvested 2 hours following activation and/or addition of LiCl or SB415286. Blot is representative of 3 experiments. Note: Enhanced -catenin expression is shown for both LiCl and SB415286, demonstrating inhibition of GSK-3 activity. In contrast, GSK-3-Ser21/9 phosphorylation is enhanced only by LiCl, suggesting additional effects of this inhibitor on the Akt-1 pathway.
- Figure S2. GSK-3 enhances IL-12p40, IL-6, and TNF-α, but not IL-10, secretion by monocytes activated with E coli (PDF, 29.4 KB) -
Monocytes were resuspended in culture medium at a concentration of 1 × 106/mL and directly exposed to E coli ± LiCl (10 mM) or ± SB415286 (10 µM). Supernatants were harvested after 36 hours. Absolute cytokine levels of E coli–activated monocytes: IL-12p70: 0 ± 0 ng/mL; IL-12p40: 0.77 ± 0.68 ng/mL; IL-6: 58.2 ± 24.1 ng/mL; IL-10: 2.2 ± 0.7 ng/mL; and TNF- : 3.2 ± 1.2 ng/mL. Shown are means ± SEM; n = 12. *P < .05, **P < .01 compared with activation without a GSK-3 inhibitor.
- Figure S3. Cytokine secretion and migration of MoDCs in the presence of the GSK-3 inhibitor azakenpaullone (PDF, 25.9 KB) -
Immature MoDCs were prepared as in Figures 2-3. Cells were either continued in GM-CSF and IL-4 (no inhibitor) or activated with live E coli (1:1) or a baby hamster kidney cell line transfected with CD40L (BHK-CD40L) at a 20:1 ratio of MoDCs/BHK-CD40L cells in the presence or absence of azakenpaullone (1 µM). (A) Cytokine levels in culture supernatants 36 hours following activation. Shown are means ± SEM; n = 4. (**P < .01 compared with activation without the inhibitor.) Absolute cytokine levels for BHK-CD40L in nanograms per milliliter: IL-12p70: 3.7 ± 1.1; IL-12p40: 90.5 ± 9.4; IL-6: 32.5 ± 7.8; IL-10: 4 ± 0.6; and TNF-: 19.5 ± 3.5. Absolute cytokine levels for E coli in nanograms per milliliter: IL-12p70: 1.1 ± 0.4; IL-12p40: 45.1 ± 7.8; IL-6: 871.1 ± 542.9; IL-10: 33 ± 2; and TNF-: 32.2 ± 1.4. (B) Migration toward CCL21 (40 nM) of MoDCs activated with E coli ± azakenpaullone (1 µM). Migration was tested in transwell assays with a pore size of 5 µm. Shown are means ± SEM of 5 experiments. **P < .01 compared with activation without the inhibitor.
- Figure S4. LiCl reduces up-regulation of maturation markers specifically on E coli–activated DCs (PDF, 129 KB) -
Immature MoDCs were prepared and activated in the presence or absence of LiCl (1 and 10 mM) as in Figures 2-3. Flow cytometry analyses were performed 36 to 48 hours after activation. Relative mean fluorescence intensity of MoDCs ± LiCl activated with (A) E coli or (B) BHK-CD40L ± the cAMP-analog Sp-5,6-DCl-cBIMPS (cBIMPS, 50 µM). Mean fluorescence values of CD80, CD86, HLA-A, -B, and -C, and HLA-DR are shown for MoDCs relative to MoDCs cultured in the absence of LiCl and cBIMP. Shown are means ± SD; n = 4 (FACS). *P < .05 compared with DCs not exposed to LiCl.
- Figure S5. Influence of SB415286 on phenotypic maturation of DCs (PDF, 18 KB) -
Immature MoDCs were prepared and activated in the presence or absence of SB415286 (10 µM) as in Figures 2-3. Cells were either continued in GM-CSF and IL-4 (immature DC, iDC) or activated with E coli or BHK-CD40L in the presence or absence of the cAMP analog cBIMPS (50 µM). Flow cytometric analyses were performed 36 to 48 hours after activation. Absolute MFIs of CD80, CD83, CD86, and CD14 are shown (means ± SD; n = 3).
Note that the GSK-3 inhibitor reduces but does not block up-regulation of CD80, CD83, and CD86 on E coli–activated DCs. In contrast, SB415286 reduces CD83 but not CD80/CD86 on DCs activated with CD40L.
- Figure S6. Inhibition of IL-12p70, TNF-α, and IL-10, but not IL-6 and IL-12p40, secretion by cAMP interaction with LiCl (PDF, 26.3 KB) -
Immature MoDCs were prepared and activated as in Figures 2-3. Cells were either continued in GM-CSF ± IL-4 (no activation) or activated with BHK-CD40L in the presence or absence of the cAMP-analog Sp-5,6-DCl-cBIMPS (cBIMPS, 50 µM) ± LiCl (10 mM). Absolute cytokine levels in culture supernatants were measured at 36 hours by ELISA. Shown are means ± SEM of 6 experiments. (*P < .05, **P < .01 compared with activation with the BHK-CD40L cell line in the absence of inhibitors or cBIMPS). Note: The inhibitory effect of LiCl on BHK-CD40L–induced IL-12p40 secretion is not observed in the presence of cBIMPS, although LiCl inhibits IL-6 secretion to a similar degree in both protocols.
- Figure S7. GSK-3 integrates activating and inhibitory pathways involved in IL-12p70 secretion (PDF, 24 KB) -
Schematic summary of factors influencing IL-12p70 secretion. Strength and persistence of extracellular activation stimuli together with a variety of extracellular factors induce both activating (eg, ERK1/2, p38K, other Ser/Thr kinases) and inhibitory (eg, Akt-1) intracellular pathways. Enhanced kinase activity results in Ser/Thr phosphorylation of proteins, which may become primed GSK-3 targets. Akt-1 phosphorylates the Ser21/9 moiety of GSK-3, which can bind the phospho-binding pocket of GSK-3 as an intramolecular pseudosubstrate. IL-12p70 production now depends on the outcome of competition of primed GSK-3 targets and the phospho-Ser21/9 moiety for access to the phospho-binding pocket of GSK-3.
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