RTCB deficiency triggers colitis in mice by influencing the NF-κB and Wnt/β-catenin signaling pathways

Abstract

RNA terminal phosphorylase B (RTCB) has been shown to play a significant role in multiple physiological processes. However, the specific role of RTCB in the mouse colon remains unclear. In this study, we employ a conditional knockout mouse model to investigate the effects of RTCB depletion on the colon and the potential molecular mechanisms. We assess the efficiency and phenotype of Rtcb knockout using PCR, western blot analysis, histological staining, and immunohistochemistry. Compared with the control mice, the Rtcb-knockout mice exhibit compromised colonic barrier integrity and prominent inflammatory cell infiltration. In the colonic tissues of Rtcb-knockout mice, the protein levels of TNF-α, IL-8, and p-p65 are increased, whereas the levels of IKKβ and IκBα are decreased. Moreover, the level of GSK3β is increased, whereas the levels of Wnt3a, β-catenin, and LGR5 are decreased. Collectively, our findings unveil a close association between RTCB and colonic tissue homeostasis and demonstrate that RTCB deficiency can lead to dysregulation of both the NF-κB and Wnt/β-catenin signaling pathways in colonic cells.

Keywords: LGR5; NF-κB; RTCB; Wnt/β-catenin; colitis.

Figure 1

Growth changes in mice after TAM treatment (A) Body weight changes of the Rtcbf/f and Rtcbf/f; UBC-CreERT2 mice after TAM treatment (n=3). (B) Survival rates of the Rtcbf/f and Rtcbf/f; UBC-CreERT2 mice after TAM treatment (n=9).

Figure 2

Experimental design and efficiency evaluation of TAM treatment (A) Experimental design. Mice were injected intraperitoneally with TAM for 5 consecutive days (D1 to D5). Body weight was measured daily until D9. Tissue collection was performed on D9. (B) PCR analysis of genomic DNA from different tissues of the TAM-treated Rtcbf/f and Rtcb f/f;UBC-CreERT2 mice using loxP5 and Frt2 primers (n=3). (C,D) Western blot analysis of RTCB in the colon tissues of TAM-treated Rtcbf /f (control) and Rtcbf /f;UBC-CreERT2 mice (n=3). Data are expressed as the mean± SEM. *P<0.05.

Figure 3

Histological analysis of the mouse colons after TAM treatment on D9 (A) Comparison of colon length between the TAM-treated Rtcbf/ f and Rtcbf/f ;UBC-CreERT2 mice (n=3). (B) Representative H&E staining of colon sections from the TAM-treated Rtcbf /f and Rtcbf/ f;UBC-CreERT2 mice (n=3). (C) Representative immunohistochemical staining of E-cadherin in colon sections from TAM-treated Rtcbf/f and Rtcbf/f; UBC-CreERT2 mice (n=3). (D) Representative immunohistochemical staining of E-cadherin and Alcian blue in colon sections from TAM-treated Rtcbf/f and Rtcbf/f; UBC-CreERT2 mice (n=3). Scale bar: 50 μm.

Figure 4

Analysis of colonic inflammation in mice after TAM treatment on D9 Western blot analysis of cleaved caspase 3 (A,B), IL-8 (C,D), and TNF-α (E,F) in the colon tissues of TAM-treated Rtcbf/f (control) and Rtcbf/f ;UBC-CreERT2 mice (n=3). Data are expressed as the mean±SEM. *P<0.05, ***P<0.001.

Figure 5

Analysis of the IRE1α-XBP1 signaling pathway in colon tissues after TAM treatment on D9 (A, B) Western blot analysis of XBP1-S in the colon tissues of TAM-treated Rtcb f/f (control) and Rtcb f/f;UBC-CreERT2 mice (n=3). (C) Survival rate of the Xbp1f/f and Xbp1f/f; UBC-CreERT2 mice after TAM treatment (n=3). (D) Body weight changes of the Xbp1f/f and Xbp1f/f; UBC-CreERT2 mice after TAM treatment (n=3). (E, F) Western blot analysis of XBP1-S expression in the colon tissues of TAM-treated Xbp1 f/f (control) and Xbp1 f/f;UBC-CreERT2 mice (n=3). (G) Comparison of colon length between TAM-treated Xbp1 f/f and Xbp1f /f;UBC-CreERT2 mice (n=3). (H) Representative H&E staining of colon sections from TAM-treated Xbp1f/f and Xbp1 f/f;UBC-CreERT2 mice (n=3). Scale bar: 50 μm. (I) Representative immunohistochemical staining of E-cadherin in colon sections from TAM-treated Xbp1 f/f and Xbp1f /f;UBC-CreERT2 mice (n=3). Scale bar: 50 μm. (J) Representative immunohistochemical staining of E-cadherin and Alcian blue in colon sections from TAM-treated Xbp1 f/f and Xbp1f /f;UBC-CreERT2 mice (n=3). Scale bar: 50 μm. Data are expressed as the mean±SEM. *P<0.05. ns, no significance.

Figure 6

Analysis of the NF-κB signaling pathway in colon tissues after TAM treatment on D9 Western blot analysis of IKKβ (A,B), IκBα (C,D), and p-p65 (E) in the colon tissues of TAM-treated Rtcbf/f (control) and Rtcbf/f; UBC-CreERT2 mice (n=3). Data are expressed as the mean±SEM. **P<0.01.

Figure 7

Analysis of the Wnt/β-catenin signaling pathway in colon tissues after TAM treatment on D9 Western blot analysis of Wnt3a (A,B), GSK3β (C,D), β-catenin (E,F), and LGR5 (G,H) in the colon tissues of TAM-treated Rtcbf/f (control) and Rtcbf/f ;UBC-CreERT2 mice (n=3). Data are expressed as the mean±SEM. *P<0.05, **P<0.01.