KLF4 activates NFκB signaling and esophageal epithelial inflammation via the Rho-related GTP-binding protein RHOF

Abstract

Understanding the regulatory mechanisms within esophageal epithelia is essential to gain insight into the pathogenesis of esophageal diseases, which are among the leading causes of morbidity and mortality throughout the world. The zinc-finger transcription factor Krüppel-like factor (KLF4) is implicated in a large number of cellular processes, such as proliferation, differentiation, and inflammation in esophageal epithelia. In murine esophageal epithelia, Klf4 overexpression causes chronic inflammation which is mediated by activation of NFκB signaling downstream of KLF4, and this esophageal inflammation produces epithelial hyperplasia and subsequent esophageal squamous cell cancer. Yet, while NFκB activation clearly promotes esophageal inflammation, the mechanisms by which NFκB signaling is activated in esophageal diseases are not well understood. Here, we demonstrate that the Rho-related GTP-binding protein RHOF is activated by KLF4 in esophageal keratinocytes, leading to the induction of NFκB signaling. Moreover, RHOF is required for NFκB activation by KLF4 in esophageal keratinocytes and is also important for esophageal keratinocyte proliferation and migration. Finally, we find that RHOF is upregulated in eosinophilic esophagitis, an important esophageal inflammatory disease in humans. Thus, RHOF activation of NFκB in esophageal keratinocytes provides a potentially important and clinically-relevant mechanism for esophageal inflammation and inflammation-mediated esophageal squamous cell cancer.

Fig 1. KLF4 activates NFκB signaling in esophageal epithelial cells.

(A) By qPCR, Ikk1, Ikk2, and Ikk3 expression was increased in primary esophageal keratinocytes from mice overexpressing Klf4 (ED-L2/Klf4 mice) compared to keratinocytes from control mice. (B) By qPCR, expression of Ikk1, Ikk2, and Ikk3 was reduced in primary esophageal keratinocytes from mice with loss of Klf4 (ED-L2/Cre;Klf4^loxp/loxp mice) compared to control keratinocytes from Klf4^loxp/loxp mice without Cre. (C) Compared to control keratinocytes from Klf4^loxp/loxp mice without Cre, primary esophageal keratinocytes from ED-L2/Cre;Klf4^loxp/loxp mice had less phosphorylated p65 on Western blots but no change in total p65. (D). Compared to a scrambled shRNA control (shSCR), shRNA directed against KLF4 reduced p65 phosphorylation in primary human esophageal keratinocytes, as seen on Western blot.

Fig 2. KLF4 increases RhoF expression and activity in esophageal epithelial cells.

(A) By qPCR, Klf4, RhoF, and the guanine-exchange factor Arhgef17 were significantly decreased in esophageal epithelial cells from mice with Klf4 deletion, compared to cells from control mice. (B) When Klf4 was increased in esophageal epithelial cells from mice with Klf4 overexpression, RhoF and Arhgef17 were increased on qPCR, compared to control cells. (C) Esophageal epithelial cells from mice with Klf4 overexpression also demonstrated an increase in the amount of activated RHOF, indicated on a RHOF activation assay. (D) Staining for RHOF (brown) was low in esophageal epithelia of control mice and increased markedly in mice with Klf4 overexpression. Magnification = 100x.

Fig 3. RHOF is upregulated in inflammation and activates pro-inflammatory cytokines.

(A) By qPCR, expression of the NFκB activators Ikk1, Ikk2, and Ikk3 decreased when RhoF was knocked down by shRNA in primary esophageal epithelial cells from wild-type mice, compared to similar cells infected with scrambled controls (shScr). (B) Compared to cells with shScr, primary mouse esophageal epithelial cells with shRNA against RhoF also had reduced expression of pro-inflammatory TNFα, IL-1α, CXCL5, and G-CSF. (C) When human primary esophageal epithelial cells were grown in organotypic culture with PBMCs that were stimulated with IL-2, IL-7, and IL-15, RHOF levels increased markedly within epithelial cells, compared to cells grown without PBMCs.

Fig 4. Knockdown of RhoF prevents KLF4-mediated activation NFκB.

(A) By Western blot, RhoF knockdown with shRNA blocked p65 phosphorylation in primary esophageal epithelial cells from mice with Klf4 overexpression. (B) By qPCR, RhoF knockdown in mouse esophageal epithelial cells with Klf4 overexpression reduced Ikk1 and Ikk2 expression (*p≤0.05). (C) KLF4 upregulates RHOF and increases RHOF activation in esophageal epithelia, leading to NFκB activation, inflammation, and cancer.

Fig 5. RHOF is upregulated in human EoE.

(A) Primary human esophageal epithelial cells stimulated with IL-4, IL-13, or TGF-β, cytokines that are physiologically relevant for human EoE, had significant increases in RHOF expression by qPCR, compared to unstimulated cells. (B) By immunohistochemistry, RHOF levels increased dramatically in EoE compared to normal esophageal epithelia.