PI3K/Akt and Wnt/β-catenin Signaling Cross-regulate NF-κB Signaling in TNF-α-induced Human Lgr5⁺ Intestinal Stem Cells

Abstract

Background/Aim: Intestinal stem cells (ISCs) are responsible for intestinal proliferation, differentiation, and neoplasia, and also play a crucial role in inflammation. Thus, it is important to investigate the effect of TNF-α on the activities of NF-κB, PI3K/Akt, and Wnt/β-catenin signaling pathways. Materials and Methods: The Lgr5⁺ intestinal cells were isolated using fluorescence-activated cell sorting from NCM460 spheroid cells, and the potential molecular mechanisms were investigated via short hairpin RNA (shRNA) transfection or the use of an inhibitor. Results: The Lgr5⁺ cells were termed ISCs because of the higher expression of stem cell genes, including Sox2, Nanog, Oct4, Lgr5, and CD133. The Lgr5⁺ ISCs had a higher proliferation capacity, invasive ability, and drug resistance to 5-fluorouracil, as well as higher expression levels of anti-apoptotic proteins but lower expression levels of pro-apoptotic proteins, compared with Lgr5~ cells. The PI3K/Akt and Wnt/β-catenin pathways were triggered by the TNF-α-induced activation of NF-κB signaling. Notably, when p65 expression was knocked-down via shRNA transfection in Lgr5⁺ ISCs, the TNF-α-induced activation of the NF-κB, PI3K/Akt, and Wnt/β-catenin pathways were reversed. The same effect was observed with regards to β-catenin shRNA transfection. Moreover, the Akt inhibitor MK2206 inhibited the TNF-α-induced activation of the PI3K/Akt pathway, as well as the NF-κB and Wnt/β-catenin pathways. Conclusion: PI3K/Akt and Wnt/β-catenin signaling cross-regulate NF-κB signaling in TNF-α-induced human Lgr5⁺ ISCs.

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