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 regulates TNF-induced survival responses in MCE cell lines. (A) KSR...
KSR1 regulates TNF-induced survival responses in MCE cell lines. (A) KSR1–/– MCE cells were treated with TNF for 24 hours and fixed for TUNEL and DAPI staining. Apoptotic cell nuclei in TUNEL staining were labeled with FITC (indicated by arrowheads) and visualized using fluorescence microscopy (magnification, ×40). (B) The percentage of cells undergoing apoptosis following TNF treatment for the indicated times is shown from a representative experiment. KSR1–/– MCE cells (C) or KSR1–/– MCE cells transiently transfected with WT KSR1 or KI KSR1 (D) were treated with TNF or EGF, and cellular lysates were prepared for Western blot analysis with the indicated antibodies. KSR1 and its coprecipitated proteins from KSR1–/– MCE cells transiently transfected with vector, WT KSR1 or KI KSR1 were separated by SDS-PAGE and stained with colloidal blue. Raf-1 and MEK bound to KSR1 was determined by Western blot analysis with anti–Raf-1 or anti-MEK1/2 antibodies (E). Shown are representative data from 3 separate experiments.