Microbiota-Dependent Induction of Colonic Cyp27b1 Is Associated With Colonic Inflammation: Implications of Locally Produced 1,25-Dihydroxyvitamin D3 in Inflammatory Regulation in the Colon

Abstract

Our recent studies demonstrated that intestinal epithelial vitamin D receptor (VDR) signaling plays a critical role in regulating colonic inflammation by protecting epithelial barrier integrity. Epithelial VDR is downregulated in colitis, but how mucosal inflammation affects local 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] production is unknown. Here we showed that cytochrome P450 27b1 (Cyp27b1), a cytochrome P450 enzyme necessary for 1,25(OH)2D3 biosynthesis, is highly induced in colonic mucosa in inflammatory response. Although VDR is reduced in colon biopsies from patients with ulcerative colitis, Cyp27b1 is markedly upregulated in these samples. Colon mucosal Cyp27b1 was also markedly induced in an experimental colitis mouse model, and this local Cyp27b1 induction and colonic inflammation required the presence of commensal bacteria. Vitamin D deficiency further exaggerated colonic Cyp27b1 induction and aggravated colonic inflammation in mice. In HCT116 cells, lipopolysaccharide or tumor necrosis factor-α treatment induced Cyp27b1 in time- and dose-dependent manners, and the induced Cyp27b1 was enzymatically active. The inflammation-induced upregulation of Cyp27b1 was mediated by nuclear factor κB. Collectively these data suggest that induction of colonic epithelial Cyp27b1, which is expected to increase local production of 1,25(OH)2D3, is a protective mechanism that partially compensates for the downregulation of epithelial VDR during colonic inflammation. Increased local 1,25(OH)2D3 maintains 1,25(OH)2D3-VDR signaling to protect the mucosal barrier and reduce colonic inflammation.

Figure 1.

Cyp27b1 is markedly upregulated in colonic biopsies from patients with ulcerative colitis. (A) Western blot analyses of biopsies of inflamed lesion (L) and adjacent normal tissues (N) from seven patients with ulcerative colitis using the antibodies indicated. (B) Relative protein levels in the lesions compared with the normal tissues, determined by densitometric quantitation of protein bands on the blots. *P < 0.05, ***P < 0.001 vs normal; n = 7. (C) Immunostaining with anti–TNF-α antibody. Note TNF-α–positive cells in the lamina propria of the lesion sections. Magnification ×100. (D) Immunostaining with anti-Cyp27b1 antibody. Arrows indicate examples of Cyp27b1-negative (left panel) and Cyp27b1-positive (right panel) epithelial cells. Magnification ×400.

Figure 2.

Induction of colon Cyp27b1 in TNBS colitis model. (A) Western blot analyses of colonic mucosal VDR and Cyp27b1 in control and TNBS-treated mice on day 3. (B) Densitometric semiquantitation of the Western blot data. *P < 0.05, ***P < 0.001 vs corresponding control; n = 4–5. (C) Real-time RT-PCR quantitation of Vdr, Cyp27b1, Cyp24a1, Tlr4, and Cd14 transcripts in colonic mucosa from four groups of mice: control, TNBS-treated, paricalcitol-treated, TNBS + paricalcitol (Pari). *P < 0.05; ***P < 0.001 vs the rest; ^#P < 0.05 vs control and paricalcitol or TNBS. n = 5 in each group.

Figure 3.

Microbiota are necessary for the induction of colon Cyp27b1 in colonic inflammation. (A) Body weight changes in five groups of mice from 0 to 3 days after TNBS instillation: control (Ctrl), AIMD, TNBS, AIMD + TNBS, and AIMD + LPS + TNBS. *P < 0.05, ** P < 0.01, *** P < 0.001 vs Ctrl; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs AIMD + TNBS; n = 5 in each group. (B) Clinical score and (C) histological score of the colonic sections, from TNBS, AIMD + TNBS, and AIMD + LPS + TNBS mice; *P < 0.05, **P < 0.01 vs the rest. (D) Representative hematoxylin and eosin sections of distal colons from the indicated groups of mice. Magnification, ×400. (E) Real-time RT-PCR quantitation of Vdr, Cyp27b1, Cyp24a1, Tnfa, Il1b, Il6, and Ifng transcripts in colonic mucosa from these five groups of mice. *P < 0.05, **P < 0.01, ***P < 0.001 vs Ctrl; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs AIMD + TNBS. (F) Western blot analysis of colonic mucosal VDR, Cyp27b1, MLCK, p-MLC, and PUMA in the indicated groups of mice.

Figure 4.

Vitamin D deficiency exaggerates colonic Cyp27b1 induction in TNBS model. (A) Serum 25-hydroxyvitamin D₃ levels in four groups of mice: S-VitD, D-VitD, S-VitD + TNBS, D-VitD + TNBS; **P < 0.01 vs S-VitD control (Ctrl). (B) Body weight changes after TNBS instillation in the indicated groups of mice. *P < 0.05, **P < 0.01, ***P < 0.001 vs S-VitD Ctrl. (C) Clinical score from S-VitD + TNBS, D-VitD + TNBS mice and (D) histological score of the colonic sections from S-VitD + TNBS, D-VitD + TNBS mice; *P < 0.05. n = 5 in each group. (E) Representative hematoxylin and eosin sections of distal colons from these four groups of mice. Magnification, ×100. (F) Real-time RT-PCR quantitation of Tnfa, Ifng, Il1b, Il6, Il17, Il12, and Mcp1 transcripts in colonic mucosa from the indicated groups of mice. **P < 0.01, ***P < 0.001 vs S-VitD Ctrl. (G) Western blot analysis of colonic mucosal VDR, Cyp27b1, MLCK, p-MLC, and PUMA in these four groups of mice.

Figure 5.

LPS induces Cyp27b1 in HCT116 cells. (A) Time course of LPS effects on Vdr, Cyp27b1, Cyp24a1, Tlr4, and Cd14 transcripts in HCT116 cells. (B) Dose-dependent effects of LPS on Vdr, Cyp27b1, Cyp24a1, Tlr4, and Cd14 transcripts in HCT116 cells. *P < 0.05, **P < 0.01 vs corresponding 0 hour or 0 ng/mL. (C) Time-dependent changes in TLR4, VDR, and Cyp27b1 proteins in HCT116 after LPS treatment. (D) Time course curve of TLR4, VDR, and Cyp27b1 proteins in HCT116 after LPS treatment. Pearson correlation coefficient (r) and P values for each curve are presented. (E–G) Assessment of Cyp27b1 activity to convert 25(OH)D₃ to 1,25(OH)₂D₃ in HCT116 cells. HCT116 cells were treated with LPS in the presence of 1,25(OH)₂D₃ or 25(OH)D₃ for 16 hours, and (E) Tnfa transcript was measured by RT-PCR; or (F) after the cells were transfected with a scramble or Cyp27b1-specific siRNA to knock down Cyp27b1, (G) a similar experiment was performed.

Figure 6.

TNF-α induces Cyp27b1 in HCT116 cells. (A) Dose-dependent effects of TNF-α on VDR and Cyp27b1 proteins in HCT116 cells. (B) Relative VDR and Cyp27b1 protein levels plotted against TNF-α dose. (C) Time course of LPS effects on VDR and Cyp27b1 proteins in HCT116 cells. (D) Relative VDR and Cyp27b1 protein levels plotted against TNF-α treatment time. Pearson correlation coefficient (r) and P values for each curve are presented.

Figure 7.

Cyp27b1 induction by LPS or TNF-α is mediated by NF-κB. HCT116 cells were treated with (A, C) 100 ng/mL LPS or (B, D)100 ng/mL TNF-α overnight in the presence or absence of (A, B) 20 ng/mL BAY 11-7082 or (C, D) 20 ng/mL T-5224 as indicated. Cyp27b1 protein levels were measured by Western blotting. (E) HCT116 cells were transfected with an IKKβ-expressing plasmid, followed by BAY 11-7082 treatment for 24 hours before assessing Cyp27b1 protein levels. (F) HCT116 cells were treated with LPS (100 ng/mL) overnight in the presence or absence of neutralizing anti–TNF-α antibody (4 μg/mL). VDR, Cyp27b1, MLCK, and TLR4 proteins were assessed by Western blotting after these treatments.