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IL-17 receptor signaling inhibits C/EBPbeta by sequential phosphorylation of the regulatory 2 domain.

ABSTRACT: Interleukin-17 (IL-17), the hallmark cytokine of T helper 17 (T(H)17) cells, signals through a distinct receptor subclass, yet little is known about the mechanisms involved. IL-17 activates the expression of target genes through the actions of the transcription factors nuclear factor kappaB (NF-kappaB), CAAT enhancer binding protein delta (C/EBPdelta), and C/EBPbeta. The adaptor proteins tumor necrosis factor receptor-associated factor 6 (TRAF6) and Act1 are upstream of NF-kappaB and C/EBPdelta, but the regulation of C/EBPbeta remains undefined. Here, we show that IL-17 signaling led to phosphorylation of two sites in the regulatory 2 domain of C/EBPbeta in a sequential, interdependent fashion. The first was rapid and dependent on extracellular signal-regulated kinase (ERK), whereas the second was dependent on the activity of glycogen synthase kinase 3beta (GSK-3beta). These pathways were mediated by distinct subdomains within IL-17 receptor A (IL-17RA). Whereas phosphorylation of threonine 188 (Thr188) was mediated by the previously identified SEF/IL-17R homology domain-Toll-IL-1R-like loop (SEFIR-TILL), phosphorylation of Thr179 occurred through a newly characterized motif located in the distal tail of IL-17RA. Phosphorylated C/EBPbeta mediated a negative signal, because blocking ERK and GSK-3beta increased expression of IL-17 target genes and a C/EBPbeta-Thr188 mutant enhanced activation of a C/EBP-dependent reporter. Overexpression of GSK-3beta inhibited IL-17-induced activation of a C/EBP-dependent reporter, and Thr179 of C/EBPbeta was not phosphorylated in GSK-3beta-deficient cells. Thus, IL-17 triggered the dual phosphorylation of C/EBPbeta, which inhibited the expression of proinflammatory genes. This detailed dissection is the first for the IL-17-mediated C/EBP pathway and the first known example of a negative signal mediated by IL-17RA.

SUBMITTER: Shen F

PROVIDER: S-EPMC2754870 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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IL-17 receptor signaling inhibits C/EBPbeta by sequential phosphorylation of the regulatory 2 domain.

Shen Fang F Li Nan N Gade Padmaja P Kalvakolanu Dhananjaya V DV Weibley Timothy T Doble Brad B Woodgett James R JR Wood Troy D TD Gaffen Sarah L SL

Science signaling 20090224 59

Interleukin-17 (IL-17), the hallmark cytokine of T helper 17 (T(H)17) cells, signals through a distinct receptor subclass, yet little is known about the mechanisms involved. IL-17 activates the expression of target genes through the actions of the transcription factors nuclear factor kappaB (NF-kappaB), CAAT enhancer binding protein delta (C/EBPdelta), and C/EBPbeta. The adaptor proteins tumor necrosis factor receptor-associated factor 6 (TRAF6) and Act1 are upstream of NF-kappaB and C/EBPdelta, ...[more]

PMID: 19244213

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Project description:The immunomodulatory and self-renewable features of human adipose mesenchymal stem cells (hAD-MSCs) mark their importance in regenerative medicine. Interleukin 23 (IL- 23) as a proinflammatory cytokine suppresses T regulatory cells (Treg) and promotes the response of T helper 17 (Th17) and T helper 1 (Th1) cells. This pathway starts inflammation and immunosuppression in several autoimmune diseases. The current study for producing recombinant IL- 23 decoy receptor (RIL- 23R) using hAD-MSCs as a good candidate for ex vivo cell-based gene therapy purposes reducing inflammation in autoimmune diseases. hAD-MSCs was isolated from lipoaspirate and then characterized by differentiation. RIL- 23R was designed and cloned into a pCDH-813A- 1 lentiviral vector. The transduction of hAD-MSCs was performed at MOI (multiplicity of infection) = 50 with pCDH- EFI ?- RIL- 23R- PGK copGFP. Expressions of RIL- 23R and octamer-binding transcription factor 4 (OCT- 4) were determined by real-time polymerase chain reaction (real time-PCR). Self-renewing properties were assayed with OCT- 4. Bioactivity of the designed RIL- 23R was evaluated by IL- 17 and IL- 10 expression of mouse splenocytes. Cell differentiation confirmed the true isolation of hAD-MSCs from lipoaspirate. Restriction of the enzyme digestion and sequencing verified the successful cloning of RIL- 23R in the CD813A-1 lentiviral vector. The green fluorescent protein (GFP) positive transduction rate was up to 90%, and real-time PCR showed the expression level of RIL-23R. Oct-4 had a similar expression pattern with nontransduced hAD-MSCs and transduced hAD-MSCs/ RIL-23R indicating that lentiviral vector did not affect hAD-MSCs characteristics. Downregulation of IL-17 and upregulation of IL-10 showed the correct activity of the engineered hAD-MSCs. The results showed that the transduced hAD-MSCs/ RIL- 23R, expressing IL-23 decoy receptor, can give a useful approach for a basic research on cell-based gene therapy for autoimmune disorders.

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IL-17 receptor signaling inhibits C/EBPbeta by sequential phosphorylation of the regulatory 2 domain.

Publications

IL-17 receptor signaling inhibits C/EBPbeta by sequential phosphorylation of the regulatory 2 domain.

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