Localization of the triptolide-responsive element in the human IL-12p40 promoter. A, Putative and verified transcription factor binding sites in the −292/+108 p40 promoter region. The core binding sequences are in bold and uppercase, and positions are relative to the transcription initiation site (TIS), which is defined as −1. B, A series of 5′ deletion mutants of the full-length human IL-12p40 promoter-luciferase construct, which spans 3.3 kb upstream and 108-bp downstream of the TIS, were transfected transiently into RAW264.7. Cells were incubated for 15 h with (filled bars) or without 10 ng/ml triptolide (open bars), then stimulated with IFN-γ (15 h), followed by LPS (7 h). Cell lysates were assayed for luciferase activity. Data are summaries of four separate experiments. C, Rate of triptolide-induced inhibition of IL-12p40 promoter activities shown in B, expressed as 1-(triptolide-treated/nontreated). Results represent three independent experiments showing mean ± SE. D, Mutant promoter luciferase constructs of IL-12p40 promoter in the context of −292/+108 were transfected into RAW264.7. Luciferase activity was measured from cell lysates after stimulation of RAW264.7 cells with IFN-γ and LPS in the absence or presence of triptolide (15 h). E, Rate of triptolide-induced inhibition of IL-12p40 promoter activities shown in D. Results represent three independent experiments showing mean ± SE. *p < 0.05.

Effects of triptolide in C/EBPα knockout mice. A–C, Four- to 6-week-old WT and Mx1-cre/C/EBPα KO mice were injected with 250 μg poly(I:C) every other day for three doses in the first week. After 1–3 wk, in the treatment group, a dose of 1 mg/kg (body weight) of triptolide was administrated to mice by i. p. injection overnight, followed by stimulation of LPS (15 mg/kg) for 4 h. Proteins from splenocytes were analyzed for C/EBPα expression by Western blotting (A). Simultaneously, IL-12p40 levels in the serum were measured by ELISA (B). Each group had three mice. Results represent mean ± SE of the three mice. C, MPM from WT and Mx1-cre/C/EBPα KO mice were treated with different concentrations of poly(I:C) for 24 h, incubated with triptolide (filled bars) or without (open bars), followed by stimulation of LPS for 6 h. Total RNA was prepared, and analyzed for mRNA expression of C/EBPα and GAPDH by RT-PCR. Simultaneously, IL-12p40 levels in the supernatants were measured by ELISA (D). Results represent three independent experiments showing mean ± SE. *p < 0.05.

Regulation of MAPK and PI3K-AKT-GSK pathways by triptolide. A and B, Western blot analyses were performed to detect levels of phosphor-ERK1/2 (A) and phosphor-GSK-3β (B) in whole cell lysate prepared from MPM after incubation with or without triptolide, and stimulation with LPS. C, MPM were incubated for 15 h in the presence (filled bars) or absence (open bars) of triptolide, followed by 2 h treatment with p38 inhibitor (SB202190), ERK inhibitor (PD98059), and JNK inhibitor II, and subsequently cultured for an additional 6 h with LPS. Mouse IL-12p40 level in the supernatants was measured by ELISA. D, A similar protocol as that of C was used, except that cells were treated with the GSK-3β inhibitor LiCl or PD98059, or both. E, Same protocol was used as that of D. C/EBPα levels in whole cell lysate were determined by Western blotting. F, Same protocol as D was used. ChIP was performed to examine C/EBPα binding to the IL-12p40 promoter region in vivo. G–I, Western blot analyses were performed to detect phosphor-MEK1/2 (G), phosphor-AKT (H), and expression of MKP-1 and PTEN (I) in whole cell lysate. Results in C, D, and F represent three independent experiments showing mean ± SE. *p < 0.05.

Effects of triptolide on IL-12 gene expression in LPS-treated MPM and MoDC (A–D) MPM (A, B), and MoDCs (C, D) were incubated for 15 h with different concentrations of triptolide and subsequently cultured for an additional 6 h in the presence (filled bars) or absence (open bars) of 1 μg/ml LPS. Mouse IL-12p40 (A), mouse IL-12p70 (B), human IL-12p40 (C), human IL-12p70 (D), and mouse IL-23 (E) levels in the supernatants were measured by ELISA. Experiment shown is representative of three to four independent experiments with mean ± SE. *p < 0.05. F–H, Total RNA was isolated from 1 × 10⁶ MPM incubated with different concentrations of triptolide for 15 h with 1 μg/ml LPS (filled bars) or without (open bar) for an additional 3 h. Real-time PCR was performed to measure p40 (F), p35 (H) mRNA expression, and nascent transcripts for p40 (G). GAPDH mRNA was analyzed as an internal control. The experiment shown is representative of two independent experiments.

Regulation of C/EBPα and β binding to IL-12p40 promoter by triptolide in vitro and in vivo. A, EMSA was performed to examine the DNA-protein interaction at the composite C/EBP/AP-1 site in the IL-12p40 promoter in vitro using nuclear extracts prepared from MPM after incubation with or without triptolide, and stimulation with LPS (3 h). The five discernible complexes are labeled I–V. B, Supershift EMSA was performed to identify the components of the C/EBP complexes. The nuclear extracts used in this procedure were from LPS-stimulated MPM treated with or without triptolide. C, Triptolide regulates C/EBP binding to the p40 promoter in vivo. After incubation with or without triptolide, and stimulation with LPS (3 h), MPM were crosslinked by formaldehyde treatment and lysed. Cell lysates were subjected to immunoprecipitation with control IgG or anti-C/EBPβ (upper) or anti-C/EBPα (lower). Input DNA and DNA recovered from the immunoprecipitation were amplified by real-time PCR with primers spanning the C/EBP/AP-1-binding site in the IL-12p40 promoter. D–F, Western blotting was performed to detect the endogenous C/EBPα (D), C/EBPβ (E), and C/EBPδ (F) expression levels in 3 h LPS-stimulated MPM treated with or without triptolide, in the nucleus and cytoplasm. To aid in the appropriate identification of the various isoforms of endogenous C/EBP recombinant C/EBPs were loaded onto the same blots. Expression of α-tubulin was analyzed as a nuclear loading control whereas GAPDH expression was used as a loading control for whole cell lysate. *p < 0.05.

Regulation of transcription and phosphorylation of C/EBPα by triptolide. A, MPM were incubated for 15 h with (dotted line) or without (solid line) 10 ng/ml triptolide and subsequently cultured for different times in the presence of 1 μg/ml LPS or absence (gray line and gray dotted line). B, A mouse C/EBPα promoter-luciferase construct, which spans 809-bp upstream and 187-bp downstream of the transcription initiation site, was transfected transiently into RAW264.7 cells. Cells were incubated for 15 h with (filled bars) or without 10 ng/ml triptolide (open bars), then stimulated with or without IFN-γ (15 h), followed by LPS (7 h). Cell lysates were assayed for luciferase activity. Data are summaries of four separate experiments. C, Western blotting was performed to detect Ser21- and Thr222/226-phosphorylation of C/EBPα in MPM after incubation with or without triptolide, and stimulation with LPS (1 h). D, Stably expressed siRNA against C/EBPα efficiently inhibited expression of the endogenous C/EBPα at both mRNA (left) and protein (right) levels in RAW264.7 cells. E, Mouse C/EBPα promoter-luciferase construct was cotransfected with WT or various phosphorylation mutants of C/EBPα expression vector into RAW264.7 cells depleted of endogenous C/EBPα by stable siRNA expression. Cell lysates were assayed for luciferase activity 36 h after transfection. F, IL-12p40 luciferase reporter was cotransfected with WT or various phosphorylation mutants of C/EBPα expression vector into RAW264.7 cells depleted of endogenous C/EBPα. Luciferase activity was measured from cell lysates after stimulation of RAW264.7 cells with IFN-γ and LPS in the presence or absence of triptolide (15 h). G, Western blot analysis of recombinant C/EBPα and phosphorylation mutants expressed in transfected RAW264.7 cells that had been depleted of the endogenous C/EBPα via siRNA, then stimulated with IFN-γ/LPS in the presence of triptolide. Expression of GAPDH was analyzed as a loading control. All transfection results represent three independent experiments showing mean ± SE. *p < 0.05.

Model for regulation of IL-12p40 production by triptolide. Triptolide augments TLR4-mediated activation of ERK1/2 and GSK-3β by interfering with the expression of phosphatases MKP-1 and PTEN. ERK1/2 and GSK-3β enhance the phosphorylation of C/EBPα, and its expression because of autoregulation. C/EBPα functions as an inhibitor of p40 transcription via a yet to be elucidated mechanism.