Kinase suppressor of Ras-1 protects intestinal epithelium from cytokine-mediated apoptosis during inflammation
Fang Yan, Sutha K. John, Guinn Wilson, David S. Jones, M. Kay Washington, D. Brent Polk
Fang Yan, Sutha K. John, Guinn Wilson, David S. Jones, M. Kay Washington, D. Brent Polk
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Article Cell biology

Kinase suppressor of Ras-1 protects intestinal epithelium from cytokine-mediated apoptosis during inflammation

Abstract

TNF plays a pathogenic role in inflammatory bowel diseases (IBDs), which are characterized by altered cytokine production and increased intestinal epithelial cell apoptosis. In vitro studies suggest that kinase suppressor of Ras-1 (KSR1) is an essential regulatory kinase for TNF-stimulated survival pathways in intestinal epithelial cell lines. Here we use a KSR1-deficient mouse model to study the role of KSR1 in regulating intestinal cell fate during cytokine-mediated inflammation. We show that KSR1 and its target signaling pathways are activated in inflamed colon mucosa. Loss of KSR1 increases susceptibility to chronic colitis and TNF-induced apoptosis in the intestinal epithelial cell. Furthermore, disruption of KSR1 expression enhances TNF-induced apoptosis in mouse colon epithelial cells and is associated with a failure to activate antiapoptotic signals including Raf-1/MEK/ERK, NF-κB, and Akt/protein kinase B. These effects are reversed by WT, but not kinase-inactive, KSR1. We conclude that KSR1 has an essential protective role in the intestinal epithelial cell during inflammation through activation of cell survival pathways.

Authors

Fang Yan, Sutha K. John, Guinn Wilson, David S. Jones, M. Kay Washington, D. Brent Polk

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KSR1 is activated in inflamed colon mucosa. (A) H&E staining of para...
KSR1 is activated in inflamed colon mucosa. (A) H&E staining of paraffin-embedded colon tissues prepared from IL-10+/+ (BALB/c) or IL-10–/– mice before developing overt signs of colitis (8-week-old mice) or with rectal prolapse (12-week-old mice) showing characteristic epithelial ulceration (magnification, ×20). (B) Colon mucosal lysates were prepared from the respective mice for Western blot analysis with indicated antibodies. KSR1 (C, E, and F) or Raf-1 (D) was immunoprecipitated from colon mucosal lysates using anti-KSR1 or anti–Raf-1 antibodies, respectively, and subjected to Western blot analysis to detect phosphorylation levels using anti–P-Thr or anti–P-Ser antibodies. Using a 2-stage in vitro kinase assay (E and F), immunoprecipitated KSR1 was incubated first with recombinant Raf-1, and then Raf-1 was incubated with KI MEK1 as substrate as detailed in Methods. Phosphorylation of Raf-1 or MEK1 was determined using anti–P-Thr or anti–P-MEK1/2 antibodies, respectively. Immunoprecipitated KSR1 was treated with calf intestinal alkaline phosphatase (PTase) prior to incubation with Raf-1 (F), as indicated. The results shown here are from 2 representatives of 5 mice. WBA, Western blot analysis.