P0618 DEFICIENCY OF KINASE SUPPRESSOR OF RAS (KSR) PROMOTES TUMOR NECROSIS FACTOR (TNF)-INDUCED APOPTOSIS DURING INTESTINAL INFLAMMATION

Abstract

Introduction: The homeostatic balance between proliferation and apoptosis is essential for the intestinal epithelium to function as a physiological barrier. TNF regulates this balance through signal transduction pathways including KSR, a 97 kDa Ser/Thr kinase that determines survival of cultured intestinal epithelial cells. We recently reported that reduced KSR expression exacerbates colitis in TNF-dependent mouse colitis. Therefore, these studies were designed to test the hypothesis that KSR is a regulatory molecule for TNF-mediated intestinal epithelial homeostasis in vivo. Methods: KSR wild-type (wt, KSR+/+) or deficient (KSR−/−) mice were injected with TNF (104 U/mouse) or PBS as control. Paraffin-embedded colon sections were studied for apoptosis using In Situ Oligo Ligation kit and active caspase-3 staining. Activation of signaling proteins was determined on colon mucosal lysates by Western blot analysis or tissue sections by immunohistochemistry. KSR−/− mouse colon epithelial (MCE) cells with or without transfection with wt KSR or kinase inactive (ki) were treated with TNF (100 ng/ml) and studied for apoptosis and activation of signal transduction pathways. IL-10 deficient (IL-10−/−) mice were investigated to determine inflammation-dependent KSR kinase activity. Results: KSR expression in colon was primarily localized to epithelial cells where apoptosis and caspase-3 activity were increased up to 3-fold by TNF in the KSR−/− compared to wt mice. Furthermore, TNF stimulated apoptosis in KSR−/−MCE cells, but not KSR+/+MCE cells. Loss of KSR blocked TNF-activated anti-apoptotic signals, ERK1/2 MAP kinase, Akt/PKB, NF-kappaB activation and cIAP2 production in KSR−/−colon epithelial cells both in vivo and in vitro. Cell survival and anti-apoptotic signal trans-duction pathways were restored by expression of wt KSR, but not ki KSR. KSR recovered from inflamed IL-10−/− colon mucosa showed increased kinase activity towards Raf-1 that was reversed by phosphatase treatment of KSR. Conclusion: TNF and mucosal inflammation regulate intestinal epithelial cell fate in vivo through activation of anti-apoptotic signals that require KSR. Thus, KSR plays a protective role in intestinal epithelial cells during inflammatory processes with implications for cell survival regulation and inflammatory bowel diseases.