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PAK1 modulates a PPAR?/NF-?B cascade in intestinal inflammation.

ABSTRACT: P21-activated kinases (PAKs) are multifunctional effectors of Rho GTPases with both kinase and scaffolding activity. Here, we investigated the effects of inflammation on PAK1 signaling and its role in colitis-driven carcinogenesis. PAK1 and p-PAK1 (Thr423) were assessed by immunohistochemistry, immunofluorescence, and Western blot. C57BL6/J wildtype mice were treated with a single intraperitoneal TNF? injection. Small intestinal organoids from these mice and from PAK1-KO mice were cultured with TNF?. NF-?B and PPAR? were analyzed upon PAK1 overexpression and silencing for transcriptional/translational regulation. PAK1 expression and activation was increased on the luminal intestinal epithelial surface in inflammatory bowel disease and colitis-associated cancer. PAK1 was phosphorylated upon treatment with IFN?, IL-1?, and TNF?. In vivo, mice administered with TNF? showed increased p-PAK1 in intestinal villi, which was associated with nuclear p65 and NF-?B activation. p65 nuclear translocation downstream of TNF? was strongly inhibited in PAK1-KO small intestinal organoids. PAK1 overexpression induced a PAK1-p65 interaction as visualized by co-immunoprecipitation, nuclear translocation, and increased NF-?B transactivation, all of which were impeded by kinase-dead PAK1. Moreover, PAK1 overexpression downregulated PPAR? and mesalamine recovered PPAR? through PAK1 inhibition. On the other hand PAK1 silencing inhibited NF-?B, which was recovered using BADGE, a PPAR? antagonist. Altogether these data demonstrate that PAK1 overexpression and activation in inflammation and colitis-associated cancer promote NF-?B activity via suppression of PPAR? in intestinal epithelial cells.

SUBMITTER: Dammann K

PROVIDER: S-EPMC4576212 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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PAK1 modulates a PPARγ/NF-κB cascade in intestinal inflammation.

Dammann Kyle K Khare Vineeta V Lang Michaela M Claudel Thierry T Harpain Felix F Granofszky Nicolas N Evstatiev Rayko R Williams Jonathan M JM Pritchard D Mark DM Watson Alastair A Gasche Christoph C

Biochimica et biophysica acta 20150531 10 Pt A

P21-activated kinases (PAKs) are multifunctional effectors of Rho GTPases with both kinase and scaffolding activity. Here, we investigated the effects of inflammation on PAK1 signaling and its role in colitis-driven carcinogenesis. PAK1 and p-PAK1 (Thr423) were assessed by immunohistochemistry, immunofluorescence, and Western blot. C57BL6/J wildtype mice were treated with a single intraperitoneal TNFα injection. Small intestinal organoids from these mice and from PAK1-KO mice were cultured with ...[more]

PMID: 26036343

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Project description:BackgroundDocosahexaenoic acid (DHA) and DHA-derived lipid mediators have recently been shown to possess anti-inflammatory and pro-resolving properties. In fact, DHA can down-regulate lipolysaccharide (LPS)-induced activation of NF-κB via a PPARγ-dependent pathway. We sought to investigate the effects of the novel DHA-derived mediator resolvin D1 (RvD1) on LPS-induced acute lung injury and to determine whether these effects occur via a PPARγ-dependent pathway.MethodsBALB/c mice aged 6-8 weeks were randomly divided into seven groups: two control groups receiving saline or RvD1 (600 ng) without LPS; a control group receiving LPS only; an experimental group receiving RvD1 (300 ng) or RvD1 (600 ng), followed by LPS; a group receiving the PPARγ antagonist GW9662; and a group receiving GW9662, then RvD1 (600 ng) and finally LPS. LPS (50 μM) and saline were administered intratracheally. RvD1 was injected intravenously 24 h and 30 min before LPS, while GW9662 was injected intravenously 30 min before RvD1. Mice were killed at 6, 12, and 24 h. Samples of bronchoalveolar lavage fluid (BALF) were analyzed for cell counts and cytokine analysis. Lung tissues were collected for histology, Western blotting and electrophoretic mobility shift assays (EMSAs).ResultsAt all three time points, groups receiving either dose of RvD1 followed by LPS had significantly lower total leukocyte counts and levels of TNF-α and IL-6 levels in BALF than did the group given only LPS. RvD1 markedly attenuated LPS-induced lung inflammation at 24 h, based on hematoxylin-eosin staining of histology sections. RvD1 activated PPARγ and suppressed IκBα degradation and NF-κB p65 nuclear translocation, based on Western blots and EMSAs. The PPARγ inhibitor GW9662 partially reversed RvD1-induced suppression of IκBα degradation and p65 nuclear translocation.ConclusionsThese results suggest that RvD1 may attenuate lung inflammation of LPS-induced acute lung injury by suppressing NF-κB activation through a mechanism partly dependent on PPARγ activation.

Project description:Background & aimsp21-activated kinase-1 (PAK1) belongs to a family of serine-threonine kinases and contributes to cellular pathways such as nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), and Wingless-related integration site(Wnt)/β-catenin, all of which are involved in intestinal homeostasis. Overexpression of PAK1 is linked to inflammatory bowel disease as well as colitis-associated cancer (CAC), and similarly was observed in interleukin (IL)10 knockout (KO) mice, a model of colitis and CAC. Here, we tested the effects of PAK1 deletion on intestinal inflammation and carcinogenesis in IL10 KO mice.MethodsIL10/PAK1 double-knockout (DKO) mice were generated and development of colitis and CAC was analyzed. Large intestines were measured and prepared for histology or RNA isolation. Swiss rolls were stained with H&E and periodic acid-Schiff. Co-immunoprecipitation and immunofluorescence were performed using intestinal organoids, SW480, and normal human colon epithelial cells 1CT.ResultsWhen compared with IL10 KO mice, DKOs showed longer colons and prolonged crypts, despite having higher inflammation and numbers of dysplasia. Crypt hyperproliferation was associated with Notch1 activation and diminished crypt differentiation, indicated by a reduction of goblet cells. Gene expression analysis indicated up-regulation of the Notch1 target hairy and enhancer of split-1 and the stem cell receptor leucin-rich repeat-containing G-protein-coupled receptor 5 in DKO mice. Interestingly, the stem cell marker olfactomedin-4 was present in colonic tissue. Increased β-catenin messenger RNA and cytoplasmic accumulation indicated aberrant Wnt signaling. Co-localization and direct interaction of Notch1 and PAK1 was found in colon epithelial cells. Notch1 activation abrogated this effect whereas silencing of PAK1 led to Notch1 activation.ConclusionsPAK1 contributes to the regulation of crypt homeostasis under inflammatory conditions by controlling Notch1. This identifies a novel PAK1-Notch1 axis in intestinal pathophysiology of inflammatory bowel disease and CAC.

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PAK1 modulates a PPAR?/NF-?B cascade in intestinal inflammation.

Publications

PAK1 modulates a PPARγ/NF-κB cascade in intestinal inflammation.

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