Figure 1.
Occludin and Claudin-1 expressions exhibit a time of day-dependent variation in colonic epithelium, relying on normal Per2 activity.
(A) Real-time PCR analysis for Occludin, Zo-1, Claudin-1, -2, -3, -4, -7, and JAM mRNAs expression in the colon tissue samples obtained from the wild-type mice and mPer2m/m mice at the indicated time points (n = 8 per group). (B) Representative pictures of Western blot for Occludin, Claudin-1 and β-actin expression in colonic epithelial cell samples from wild-type and mPer2m/m mice obtained at the indicated time points (n = 3 per group). (C) The quantitative analysis of (B) (n = 3 per group). *p<0.05.
Figure 2.
Clock and Bmal1 bind to the E-box element in the promoter regions of Occludin and Claudin-1 genes and affect their transcriptional responses.
(A) CHiP assays for recruitments of Clock and Bmal1 to E-box elements in Occludin and Claudin-1 promoter region using colonic IECs obtained at the indicated time points from wild-type mice and mPer2m/m mice (n = 3 per group). Similar results were obtained in two independent experiments. (B) CHiP assays for recruitments of CLOCK and BMAL1 to E-box elements in Occludin and Claudin-1 promoter region using HCT116 cells (n = 3 per group). (C) Relative luciferase activity in HCT116 cells transfected with Occludin and Claudin-1 promoter reporter plasmids together with Clock and Bmal1 expression vectors or a control empty vector (n = 3 per group). *p<0.05.
Figure 3.
Colonic permeability is under the circadian control.
(A) Permeability to evans blue in vivo at the indicated time points from wild-type mice and mPer2m/m mice (n = 5 per group). (B) Permeability to FITC-dextran 4000 ex vivo obtained at the indicated time points from wild-type mice and mPer2m/m mice (n = 6 per group). (C) Permeability to HRP ex vivo obtained at the indicated time points from wild-type mice and mPer2m/m mice (n = 8 per group). *p<0.05.
Figure 4.
mPer2m/m mice show reduced susceptibility to DSS-induced colitis.
(A) Body weight changes in mice for 6 days after administration of 5% DSS. (B) Stool scores in mice for 6 days after administration of 5% DSS. (C) Colon length in mice on day 7 after administration of 5% DSS. (D) TNF-α and IL-6 levels in the colon tissue samples measured by ELISA. The samples were obtained from the mice on day 7 after administration of 5% DSS. The susceptibility to DSS-colitis in wild/mPer2m/m bone marrow chimeras mice was shown (E–H). (E) Body weight changes in mice 6 days after administration of 5% DSS. (F) Stool scores in mice 6 days after administration of 5% DSS. (G) Colon length in mice on day 7 after administration of 5% DSS. (H) IL-6 levels measured by ELISA in the colon tissue samples obtained from the mice on day 7 after the administration of 5% DSS. n = 8 per group, *p<0.05. The mice were sacrificed at ZT4 (10∶00 AM) in Fig. 5C, D, G and H.