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Inhibition of TLR4 signaling by TRAM-derived decoy peptides in vitro and in vivo.

ABSTRACT: Toll/IL-1R (TIR) domain-containing adapter-inducing IFN-? (TRIF)-related adapter molecule (TRAM) serves as a bridging adapter that enables recruitment of TRIF to activated TLR4 and thereby mediates the induction of TRIF-dependent cytokines. A library of cell-permeating decoy peptides derived from TRAM TIR domain has been screened for the ability of individual peptides to inhibit TLR4 signaling in primary murine macrophages. Peptides derived from TRAM TIR BB loop (TM4) and C helix (TM6) inhibited the LPS-induced activation of MyD88-dependent and TRIF-dependent cytokines, as well as MAPK activation. TM4 and TM6 did not block macrophage activation induced by TLR2, TLR9, or retinoic acid-inducible gene 1-like receptor agonists. Both TM4 and TM6 blocked coimmunoprecipitation of TRAM and TLR4 ectopically expressed in HEK293T cells. Both peptides also blocked the LPS-induced recruitment of MyD88 to TLR4 in primary murine macrophages. In vivo examination of TRAM-derived peptides demonstrated that all peptides that were inhibitory in vitro profoundly suppressed systemic inflammatory response elicited in mice by a sublethal LPS dose, and protected mice against a lethal LPS challenge. This research identifies novel TLR inhibitors effective in vitro and in vivo and validates the approach taken in this study as a rational way for development of signaling inhibitors and lead therapeutics.

SUBMITTER: Piao W

PROVIDER: S-EPMC3578136 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Inhibition of TLR4 signaling by TRAM-derived decoy peptides in vitro and in vivo.

Piao Wenji W Vogel Stefanie N SN Toshchakov Vladimir Y VY

Journal of immunology (Baltimore, Md. : 1950) 20130123 5

Toll/IL-1R (TIR) domain-containing adapter-inducing IFN-β (TRIF)-related adapter molecule (TRAM) serves as a bridging adapter that enables recruitment of TRIF to activated TLR4 and thereby mediates the induction of TRIF-dependent cytokines. A library of cell-permeating decoy peptides derived from TRAM TIR domain has been screened for the ability of individual peptides to inhibit TLR4 signaling in primary murine macrophages. Peptides derived from TRAM TIR BB loop (TM4) and C helix (TM6) inhibited ...[more]

PMID: 23345333

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Project description:BackgroundPeriosteum plays critical roles in de novo bone formation and fracture repair. Wnt16 has been regarded as a key regulator in periosteum bone formation. However, the role of Wnt16 in periosteum derived cells (PDCs) osteogenic differentiation remains unclear. The study goal is to uncover whether and how Wnt16 acts on the osteogenesis of PDCs.MethodsWe detected the variation of Wnt16 mRNA expression in PDCs, which were isolated from mouse femur and identified by flow cytometry, cultured in osteogenic medium for 14 days, then knocked down and over-expressed Wnt16 in PDCs to analysis its effects in osteogenesis. Further, we seeded PDCs (Wnt16 over-expressed/vector) in β-tricalcium phosphate cubes, and transplanted this complex into a critical size calvarial defect. Lastly, we used immunofluorescence, Topflash and NFAT luciferase reporter assay to study the possible downstream signaling pathway of Wnt16.ResultsWnt16 mRNA expression showed an increasing trend in PDCs under osteogenic induction for 14 days. Wnt16 shRNA reduced mRNA expression of Runx2, collage type I (Col-1) and osteocalcin (OCN) after 7 days of osteogenic induction, as well as alizarin red staining intensity after 21days. Wnt16 also increased the mRNA expression of Runx2 and OCN and the protein production of Runx2 and Col-1 after 2 days of osteogenic stimulation. In the orthotopic transplantation assay, more bone volume, trabecula number and less trabecula space were found in Wnt16 over-expressed group. Besides, in the newly formed tissue Brdu positive area was smaller and Col-1 was larger in Wnt16 over-expressed group compared to the control group. Finally, Wnt16 upregulated CTNNB1/β-catenin expression and its nuclear translocation in PDCs, also increased Topflash reporter luciferase activity. By contrast, Wnt16 failed to increase NFAT reporter luciferase activity.ConclusionTogether, Wnt16 plays a positive role in regulating PDCs osteogenesis, and Wnt16 may have a potential use in improving bone regeneration.

Dataset Information

Inhibition of TLR4 signaling by TRAM-derived decoy peptides in vitro and in vivo.

Publications

Inhibition of TLR4 signaling by TRAM-derived decoy peptides in vitro and in vivo.

Similar Datasets

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