Abstract 137: PAK1 modulates a PPARγ/NF-κB cascade in intestinal inflammation

Abstract

Abstract Background & Aims: 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 in inflammatory bowel diseases (IBD) including Crohn's disease (CD), ulcerative colitis (UC), and its role in colitis-associated carcinogenesis (CAC). Methods: PAK1 and p-PAK1 (Thr423) were assessed by immunohistochemistry (IHC), immunofluorescence, and Western blot. C57BL6/J wildtype mice were treated with a single i.p. TNFα injection. Small intestinal organoids from these mice and from PAK1-KO mice were cultured with TNFα. NF-κB and PPARγ were analyzed upon PAK1 overexpression and silencing. All data are mean±SD. Results: IHC revealed PAK1 expression increased from 2.8±1.6 in normal mucosa to 5.2±1.4 in CD, 6.0±1.7 in UC, and 8.8±1.0 in CAC. In UC, PAK1 activation increased at the luminal intestinal epithelial surface from 1.3±1.4 to 5.0±2.3. In primary colon epithelial cells, PAK1 was phosphorylated upon treatment with IFNγ, IL-1β, and TNFα. In vivo, mice administered with TNFα showed increased p-PAK1 from 4.3±1.0 to 9.9±0.1 in intestinal villi, which co-localized with NF-κB activation. Indeed, p65 nuclear translocation downstream of TNFα was strongly inhibited in PAK1-KO small intestinal organoids. PAK1 overexpression induced a PAK1-p65 interaction as visualized by co-immunoprecipitation, nuclear p65 translocation, and increased NF-κB transactivation four fold, all of which were impeded upon kinase-dead PAK1. Also PAK1 overexpression downregulated PPARγ mRNA levels from 1 to 0.4±0.1 and mesalamine recovered PPARγ to 0.8±0.04 through PAK1 inhibition. On the other hand PAK1 silencing inhibited NF-κB transactivation from 1 to 0.1±0.02, which was recovered upon the PPARγ antagonist BADGE to 0.5±0.1. Conclusions: Altogether these data demonstrate that PAK1 overexpression and activation in IBD and CAC promote NF-κB activity via suppression of PPARγ in intestinal epithelial cells. Citation Format: Kyle Dammann, Vineeta Khare, Michaela Lang, Thierry Claudel, Felix Harpain, Nicolas Granofszky, Rayko Evstatiev, Jonathan M. Williams, D. Mark Pritchard, Alastair Watson, Christoph Gasche. PAK1 modulates a PPARγ/NF-κB cascade in intestinal inflammation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 137. doi:10.1158/1538-7445.AM2015-137