AhR-E2F1-KGFR Signaling Is Involved In KGF-induced Intestinal Epithelial Cell Proliferation

Background:Interactions between intestinal epithelial cells and intraepithelial lymphocytes (IELs)play an important role in the growth and maintenance of the intestinal epithelium.Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family.KGF, which is expressed by gamma delta (y8) intraepithelial lymphocytes (IEL) in the mucosal layer, has an important role in promoting epithelial cell growth in a paracrine manner after interacting with the KGF receptor (KGFR) . Previously, we reported that exogenous KGF could protect the small intestine from ischemia-reperfusion injury (I/R) and radiation-induced intestinal damage by promoting intestinal epithelial cell proliferation .Studies have also reported that KGFR is abundantly expressed in the gastrointestinal tract,suggesting that the gut can both synthesize and respond to KGF .Recently, the aryl hydrocarbon receptor (AhR) has been reported to play an important role in KGF-induced regenerative growth in a zebrafish model. Fur 赛默飞 re,AhR expression could be regulated by FGF in murine 3T3 fibroblasts , which indicates that endogenous AhR might participate in FGF-mediated signaling.AhR is a DNA binding protein that belongs to the basic region-helix-loop-helix (bHLH)superfamily and is expressed by most cells. Unliganded AhR in the cytoplasmic compartment can form a stable complex with HSP90. However,when the ligand for AhR activates this transcription factor, the AhR-ligand complex then translocates to the nucleus and subsequently binds with AhR nuclear translocator (ARNT) to dioxin-responsive elements,leading to the transactivation of several genes that encode phase I and II xenobiotic metabolizing enzymes, such as cytochrome P450s . For several decades, the toxic effects mediated by AhR have been extensively studied. However,increasing evidence indicates that AhR may play an important role in the regulation of receptor-mediated signaling. For example, Lee et al. reported that Notchl is a downstream target of AhR based on microarray analysis of Roryt+ cells from wild-type and AhR-deficient mice. In addition, Qiu et al.suggested that the expression of IL-7Ra was reduced by AhR ablation,and Kiss et al. reported that the expression of cKit was markedly lower in Ahr -/- innate lymphoid cells (ILCs).Based on these findings, we hypothesized that endogenous AhR might affect the signals mediated by KGF through the regulation of KGFR expression.Methods:Firstly, adult C57BL/6J mice and C57BL/6J AhR -/- mice were randomized into four groups: Sham group,KGF group,AhR -/- + KGF group and AhR -/- group,each group included 6 mice. Mice were killed on day 5 after administration with KGF, and the small bowel was harvested for analysis. PCNAwas used to detect the EC proliferation. The changes of villus height, crypt depth, intestinal wet weight, content of RNA and protein were used to reflect intestinal changes. The histological changes were also performed by hematoxylin-eosin(HE) staining.Secondly,the expression of AhR in intestine of adult C57BL/6J mice and C57BL/6J AhR-/- mice was detected by western blot (WB). And the expression of KGFR in control group and AhRKO group was detected by western blot and Real-time PCR.Thirdly, LoVo cells were treated with siAhR, and were divided into three groups: control group, siNC group, siAhR group. AhR expression was evaluated by western blot, the KGFR expression was detected by western blot and Real-time PCR . Then , the expression of E2F1 in nuclear level and total protein level was detected by western blot.Fourthly, LoVo cells were treated with siE2F1, and were divided into three groups:control group,siNC group,siE2Fl group. The expression of E2F1 and KGFR were detected by western blot.Fifthly, LoVo cells were treated with KGF and/or siAhR, and were seperated into four groups: DMSO group, KGF group, KGF+siAhR group and siAhR group. The proliferation of LoVo cells was detected by trypan blue/hemocytometer and flow cytometric analyses.Results:1. KGF significantly increased the number of PCNA-positive cells in the KGF group compared with the control group(*P<0.05). However, knocking out AhR resulted in a reduced rate of KGF-induced epithelial cell proliferation. AhR knock-down led to a significant reduction in jejunal villus height compared with the KGF group. Crypt depth was also significantly reduced in the KGF + AhR -/- group. Intestinal wet weight and assessments of RNA and protein expression levels also indicated that AhR deficiency abolished the sensitivity of the intestine to KGF(*P<0.05).2. AhR expression was significantly knocked down in AhR knock-out mice. In physiological conditions, AhR knock-out significantly reduced the expression of KGFR(*P<0.05). Additionally, reduced levels of KGFR mRNA transcripts were also found by real-time PCR in AhR -/- epithelial cells (*P<0.05).3. AhR expression was significantly knocked down in LoVo cells transfected with siRNA specific for AhR(siAhR). Western blot results showed that reduced levels of KGFR protein expression in AhR-silenced cells. In accordance with the western blot results, down-regulated levels (0.46) of KGFR mRNA transcripts were also detected by real-time PCR in AhR-silenced epithelial cells. A significant reduction of E2F1 in the nucleus was detected by western blot in LoVo cells transfected with siRNA specific for AhR. But the protein of E2F1 showed no significant change in total protein level(*P<0.05).4. E2F1 expression was significantly knocked down in Lo Vo cells transfected with siRNA specific for E2F1(siE2F1). Western blot and real-time PCR results both showed reduced levels of KGFR expression in E2F1-silenced cells(*P<0.05).5. By using trypan blue/hemocytometer to count cell number,we found that in the KGF group, cell numbers were significantly increased compared with the other groups. And cell cycle analysis results performed by flow cytometric showed that KGF caused a reduction in the relative number of G0/G1 cells and an associated increase in S phase cells.Conclusion:1. AhR knock-out mice are not sensitized to KGF-induced intestinal epithelial cell proliferation.2. AhR deletion in intestinal epithelial cells induced the down-regulation of KGFR expression in vivo.3. KGFR expression in intestinal epithelial cells was regulated by AhR-E2F1 pathway in vivo.4. AhR knock-down LoVo cells were less sensitized to KGF stimulation induced cell proliferation in vitro.5. AhR-E2F1-KGFR signaling is involved in KGF-induced intestinal epithelial cell proliferation.