Abstract
Ras association domain family protein 1A (RASSF1A) is a tumor suppressor gene silenced in cancer. Here we report that RASSF1A is a novel regulator of intestinal inflammation as Rassf1a+/−, Rassf1a−/− and an intestinal epithelial cell specific knockout mouse (Rassf1a IEC-KO) rapidly became sick following dextran sulphate sodium (DSS) administration, a chemical inducer of colitis. Rassf1a knockout mice displayed clinical symptoms of inflammatory bowel disease including: increased intestinal permeability, enhanced cytokine/chemokine production, elevated nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) activity, elevated colonic cell death and epithelial cell injury. Furthermore, epithelial restitution/repair was inhibited in DSS-treated Rassf1a−/− mice with reduction of several makers of proliferation including Yes associated protein (YAP)-driven proliferation. Surprisingly, tyrosine phosphorylation of YAP was detected which coincided with increased nuclear p73 association, Bax-driven epithelial cell death and p53 accumulation resulting in enhanced apoptosis and poor survival of DSS-treated Rassf1a knockout mice. We can inhibit these events and promote the survival of DSS-treated Rassf1a knockout mice with intraperitoneal injection of the c-Abl and c-Abl related protein tyrosine kinase inhibitor, imatinib/gleevec. However, p53 accumulation was not inhibited by imatinib/gleevec in the Rassf1a−/− background which revealed the importance of p53-dependent cell death during intestinal inflammation. These observations suggest that tyrosine phosphorylation of YAP (to drive p73 association and up-regulation of pro-apoptotic genes such as Bax) and accumulation of p53 are consequences of inflammation-induced injury in DSS-treated Rassf1a−/− mice. Mechanistically, we can detect robust associations of RASSF1A with membrane proximal Toll-like receptor (TLR) components to suggest that RASSF1A may function to interfere and restrict TLR-driven activation of NFκB. Failure to restrict NFκB resulted in the inflammation-induced DNA damage driven tyrosine phosphorylation of YAP, subsequent p53 accumulation and loss of intestinal epithelial homeostasis.
Highlights
The Ras association domain family protein 1A (RASSF1A) is frequently lost in human cancers by promoter-specific methylation [1]
The present study argues that RASSF1A is an important regulatory element to restrict nuclear factor kappa B (NFkB) activity
We have demonstrated that dextran sulphate sodium (DSS)-treated Rassf1a2/2 and Rassf1aIEC-KO mice have elevated NFkB activity
Summary
The Ras association domain family protein 1A (RASSF1A) is frequently lost in human cancers by promoter-specific methylation [1]. The loss of RASSF1A in the context of the APCmin/+ mice (Rassf1a2/2APCmin/+ mice) has been reported to result in increased b-catenin nuclear localization, intestinal adenomas and poorer survival than single knockout littermate controls [11] These data would suggest the importance of the early loss of RASSF1A during premalignant formation of adenocarcinomas that may lead to increased malignant transformation and tumorigenesis. Song et al (2012) demonstrated that RASSF2 can associate with the IKK complex to inhibit NFkB [20] and several reports indicate that RASSF1C and 8 may influence the function of the Wnt/b-catenin pathways, pathways important for the appearance of intestinal tumorigenesis [19,21] It is currently unknown if RASSF1A can influence NFkB activation via TNF-R1 or TLR pathways. Restricting NFkB-dependent inflammation and tyrosine phosphorylation of YAP will maintain epithelial homeostasis, promote epithelial restitution and allow recovery from inflammation induced injury
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