Expression of REG family genes in human inflammatory bowel diseases and its regulation

Abstract

The pathophysiology of inflammatory bowel disease (IBD) reflects a balance between mucosal injury and reparative mechanisms. Some regenerating gene (Reg) family members have been reported to be expressed in Crohn's disease (CD) and ulcerative colitis (UC) and to be involved as proliferative mucosal factors in IBD. However, expression of all REG family genes in IBD is still unclear. Here, we analyzed expression of all REG family genes (REG Iα, REG Iβ, REG III, HIP/PAP, and REG IV) in biopsy specimens of UC and CD by real-time RT-PCR. REG Iα, REG Iβ, and REG IV genes were overexpressed in CD samples. REG IV gene was also overexpressed in UC samples. We further analyzed the expression mechanisms of REG Iα, REG Iβ, and REG IV genes in human colon cells. The expression of REG Iα was significantly induced by IL-6 or IL-22, and REG Iβ was induced by IL-22. Deletion analyses revealed that three regions (- 220 to - 211, - 179 to - 156, and - 146 to - 130) in REG Iα and the region (- 274 to- 260) in REG Iβ promoter were responsible for the activation by IL-22/IL-6. The promoters contain consensus transcription factor binding sequences for MZF1, RTEF1/TEAD4, and STAT3 in REG Iα, and HLTF/FOXN2F in REG Iβ, respectively. The introduction of siRNAs for MZF1, RTEF1/TEAD4, STAT3, and HLTF/FOXN2F abolished the transcription of REG Iα and REG Iβ. The gene activation mechanisms of REG Iα/REG Iβ may play a role in colon mucosal regeneration in IBD.

Keywords: CD, Crohn's disease; CDX2, caudal-type homeobox transcription factor 2; Celiac disease; Crohn's disease; FOXN2, forkhead box protein N2; GATA6, GATA DNA-binding protein 6; HLTF, helicase-like transcription factor; IBD, inflammatory bowel disease; IL, interleukin; MZF1, myeloid zinc finger 1; REG family genes; REG, regenerating gene; RTEF1, related transcriptional enhancer factor-1; SOCS3, suppressors of the cytokine signaling 3; STAT3, signal transducer and activator of transcription 3; TEAD4, TEA Domain transcription Factor 4; Transcription; UC, ulcerative colitis; Ulcerative colitis; siRNA, small interfering RNA.

Graphical abstract

Fig. 1

Expression of the REG family mRNAs in CD and UC colons. Expression of REG Iα (A), REG Iβ (B), REG III (C), HIP/PAP (D), and REG IV (E) was measured by real-time RT-PCR. The mRNA levels of REG Iα, REG Iβ, and REG IV in the CD colon were significantly higher than those of the control (P = 0.0002, 0.0011, and 0.0036, respectively). The mRNA level of REG IV in the UC colon was higher than that of the control (P = 0.0083). The mRNA levels of REG III and HIP/PAP were not different among control, CD, and UC. Data are expressed as mean ± SE for each group (n = 44 (Control), 49 (CD), and 39 (UC)). The statistical analyses were performed using Student's t-test.

Fig. 2

Transcriptional activation of REG Iα by IL-22 and IL-6 via MZF1, RTEF1, and STAT3. Promoter activities of REG Iα in LS-174T (A) and HT-29 cells (B) stimulated with IL-6, IL-8, IL-17A, IL-22, TNFα, HGF, bFGF, or EGF were measured. The statistical analyses were performed using Student's t-test. Deletion analysis of REG Iα stimulated by IL-22 (C) and IL-6 (D). Human LS-174T cells were transfected with constructs containing various deletion mutants of REG Iα promoter. Constructs listed on ordinate are numbered according to their 5’ terminus in the REG Iα promoter. The transfected cells were stimulated with IL-22 (20 ng/mL) (C) or IL-6 (20 ng/mL) (D), after which the luciferase activities were measured. The diagram represents fold increase of luciferase activities to the untreated cells. All data are represented as the mean ± SE of the samples (n = 4). The statistical analyses were performed using Student's t-test against no addition. Effects of siRNA transfection on IL-22-induced increase of REG Iα mRNA in LS-174T cells (E). After siRNA was introduced, LS-174T human colon epithelial cells were stimulated with IL-22 (20 ng/mL). The expression of REG Iα (E) mRNA was measured by real-time RT-PCR using β-actin as an endogenous control. Data are expressed as mean fold (vs no addition) ± SE for each group (n = 4). The statistical analyses were performed using Student's t-test.

Fig. 3

Transcriptional activation of REG Iβ by IL-22 via HLTF. (A) Promoter activities of REG Iβ in LS-174T cells stimulated with IL-6, IL-8, IL-17A, IL-22, TNFα, HGF, bFGF, or EGF were measured. The statistical analyses were performed using Student's t-test. (B) Deletion analysis of REG Iβ promoter. Human LS-174T cells were transfected with constructs containing various deletion mutants of REG Iβ promoter. Constructs listed on ordinate are numbered according to their 5’ terminus in the REG Iβ promoter. The transfected cells were stimulated with IL-22 (20 ng/mL), after which the luciferase activities were measured. The diagram represents fold increase of luciferase activities to the untreated cells. All data are represented as the mean ± SE of the samples (n = 4). The statistical analyses were performed using Student's t-test against no addition. (C) Effects of siRNA transfection on IL-22-induced increase of REG Iβ mRNA in LS-174T cells. After siRNA was introduced, LS-174T human colon epithelial cells were stimulated with IL-22 (20 ng/mL). The expression of REG Iβ mRNA was measured by real-time RT-PCR using β-actin as an endogenous control. Data are expressed as mean fold (vs no addition) ± SE for each group (n = 4). The statistical analyses were performed using Student's t-test.

Fig. 4

Requirement of GATA6 in REG IV expression. (A) Promoter activities of REG IV in LS-174T cells stimulated with IL-6, IL-8, IL-17A, IL-22, TNFα, HGF, bFGF, or EGF were measured. The statistical analyses were performed using Student's t-test. (B) Effects of siRNA transfection on TNFα-induced decrease of REG IV mRNA. After siRNA was introduced, LS-174T human colon epithelial cells were stimulated with TNFα (20 ng/mL). The expression of REG IV mRNA was measured by real-time RT-PCR using β-actin as an endogenous control. Data are expressed as mean fold (vs no addition) ± SE for each group (n = 4). The statistical analyses were performed using Student's t-test.