Crohn disease (CD) is a multifactorial disease of unknown etiology characterized by a chronic inflammation of the entire gastrointestinal tract, most commonly occurring in the distal ileum and colon [1]. Immune, genetic, and environmental factors are thought to contribute to CD [2]. Cytokine tumor necrosis factor (TNF)-a contributes substantially to the pathology of Crohn disease, a role that is highlighted by the responsiveness of the disease to TNF-a blocking agents [3].

Signaling by the Wnt family of secreted lipoproteins plays a central role in embryogenesis and tissue homeostasis [4,5]. Abnormal Wnt/b-catenin signaling is associated with many diseases in humans, including cancer and osteoporosis [5,6]. b-catenin is a fundamental component of the Wnt signaling pathway, which acts as a coactivator through its ability to recruit components that promote chromatin remodeling and the transcriptional process [7]. Dickkopf-1 (DKK-1) is a secreted glycoprotein that has been shown to act as a potent inhibitor of the canonical Wnt/b-catenin signaling pathway [8,9]. DKK-1 plays essential roles in many biological processes, ranging from the induction of anterior mesoderm formation and head development during embryogenesis to bone formation and bone mass regulation in adult organisms [10]. However, relatively little is known about the localization of Wnt signaling components and the importance of DKK-1 within the intestine.

In this study, we explored the potential roles of DKK-1 and b-catenin in CD and evaluated the effects of a TNF-a inhibitor (infliximab) on Wnt signaling in patients with CD.

A monoclonal immunoglobulin G1 chimeric antibody directed against TNF-a (infliximab) was administered to CD patients by intravenous infusion at a dose of 5 mg/kg at weeks 0, 2, and 6, and this course was repeated every 8 weeks for 10 months thereafter.

Colonic mucosal specimens from all 21 CD patients and 14 healthy controls were assessed for mRNA expressions of DKK-1 and b-catenin. Colonoscopy was performed for all patients by a single pediatric gastroenterologist, who collected one or two additional biopsies within close proximity of the area in which biopsies were taken for routine histology. In patients with Crohn disease, mucosal biopsies were performed before infliximab therapy and after eight courses of infliximab therapy. The specimens were frozen and stored at -80°C for RNA isolation.

Total RNA for DKK-1 and b-catenin from the frozen colonic tissue was obtained using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. The amount and purity of the obtained RNA was determined using a ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). Reverse transcription was performed using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). cDNA was prepared from 1 µg of mRNA with oligo/dT according to the manufacturer’s instructions. Real-time PCR was constructed using commercially available assays for the DKK-1 gene (assay ID Hs00183740_mL, Genbank accession number NM_012242.2; Applied Biosystems, Foster City, CA, USA), b-catenin (assay ID Hs01076483_mL, Genbank accession number NM_001904.3), and human endogenous control GAPDH (assay ID Hs99999905_mL, Genbank accession number NM_002046.3) in combination with the TaqMan Universal PCR Master Mix (Applied Biosystems). Then, PCR was performed in a 7900HT real-time PCR system (Applied Biosystems). Comparative analyses of each gene were performed using computer programs SDS 2.3 and RQ 2.1 (Applied Biosystems). The relative gene expressions (RQ) were calculated using the 2_ÑÑCT method.

Statistical analyses: Statistical analyses were performed using the Mann-Whitney U-test for unpaired samples and the Wilcoxon signed-rank test for paired samples using SPSS 24.0 (SPSS, Chicago, IL, USA). Correlations between DKK-1 or b-catenin expression from CD patients and disease activity were determined by simple linear regression. Values of P<0.05 were considered statistically significant.

Results

The enrolled children included 21 with Crohn disease (16 males) and 14 healthy controls (12 males). The mean age of the healthy controls was patients with Crohn disease did not differ significantly [15.9 (1.4) vs 14.4 (2.1) years]. After one year of infliximab treatment, disease activity according to PCDAI score, and from ESR and C-reactive protein declined significantly (Table I).

TABLE I	Baseline Characteristics and Clinical Outcomes

	Controls	Patients (n=21)
	(n=14)	Before IFX Tx	After IFX Tx
Albumin	4.7 (0.2)	3.9  (0.4)	4.5 (0.3)
ESR (mm/hr)	11.1 (11.8)	61.9 (31.0)	16.1  (14.2)
CRP (mg/dL)	0.6  (1.8)	2.1 ( 2.2)	0.2  (0.3)
PCDAI score		35.2  (10.8)	4.2  (4.9)
IFX Tx: infliximab therapy; ESR: Erythrocyte sedimentation rate; CRP: C-reactive protein; PCDAI: Pediatric Crohn Disease Activity Index.

Fig. 1 Expression of Dickkopf-1 in peripheral blood. (a) The serum levels of Dickkopf-1 were significantly higher in patients with Crohn disease than in healthy controls (P=0.003) and were decreased in Crohn disease patients treated with infliximab (P=0.026). (b) Serum Dickkopf-1 levels were correlated with levels of ESR (r=0.527, P=0.025) in patients with Crohn disease.

The plasma level of DKK-1 in CD patients before infliximab therapy was significantly higher than that of healthy controls (Fig. 1a, P=0.003). After infliximab therapy, the level of DKK-1 in CD patients was decreased compared to that before therapy (Fig. 1a, P=0.03). Correlations were found between DKK-1 in plasma from children with Crohn disease and ESR (r=0.527, P=0.025), CRP (r=0.502, P=0.034), albumin (r=0.363, P=0.021) and PCDAI (r=0.462, P=0.054) (Fig. 1b).

DKK-1 mRNA expression in the colonic mucosa was higher in patients than in controls [2.18 (1.69) vs 1.60 (1.48)] and decreased after infliximab treatment [2.18 (1.69) vs 1.83 (2.32)]. b-catenin expression in the colonic mucosa was lower in patients than in controls [0.69 (1.19) vs 1.02 (1.25)] and increased after infliximab treatment [0.69 (1.19) vs 0.92 (0.71)]. However, there were no significant differences between each groups before and after infliximab therapy or compared to normal controls. There were no correlations between DKK-1 or b-catenin in the colonic mucosa from children with Crohn disease and disease activity.

Since Wnt signaling is known to cause cancer and malformations, many animal models are now in use to assess the pathogenesis of various diseases and the toxicity of therapeutic agents. Recently, a number of diseases have been associated with abnormalities in Wnt signaling, including adipogenesis [15], schizophrenia [16], and Alzheimer disease [17], as well as cancer and developmental difficulties. Therefore, the role of Wnt signaling as a pathogenic process in various diseases has been examined in the context of efforts to identify the role of Wnt signaling in cancer. DKK-1 is a downstream target gene for Wnt/b-catenin signaling, and has been shown to regulate Wnt signaling through negative feedback [18,19]. Inflammatory stimuli such as TNF-a induce DKK-1 release in various cells [20]. Inflammatory bowel disease can lead to chronic relapsing inflammation of the gastrointestinal tract. The Wnt antagonist DKK-1 is induced by inflammatory cytokines and that exacerbates intestinal inflammation. However, little is known about the localization of Wnt signaling components and the importance of DKK-1 in inflammatory bowel disease, especially CD.

In this study, we found that DKK-1 was inhibited by TNF-a inhibition in CD patients. The plasma level of DKK-1 in CD patients before infliximab therapy was significantly higher than that of healthy controls. After infliximab therapy, the level of DKK-1 in CD patients was decreased compared to that before therapy. Correlations were also found between DKK-1 in plasma from CD patients and inflammatory marker or disease activity. Inhibition of DKK-1 may increase the transcriptional activity and survival signaling pathway of b-catenin, thereby promoting epithelial cell proliferation and wound repair. These results suggest that the Wnt signaling activator can be used to maintain the regeneration of gut epithelium, homeostasis, and to treat CD.

The number of patients was relatively low, which requires a cautious interpretation of the results. Preventive and therapeutic Wnt/b-catenin activation led to a significant improvement of CD. Future in vitro and in vivo studies will certainly provide more insights into the principles underlying decreased Wnt/b-catenin signaling in CD, and whether therapeutic Wnt/b-catenin activation will present as a future therapeutic tool in this chronic disease.

Since DKK-1, as a part of the canonical Wnt/b-Catenin pathway, has already been shown to be involved in pathological processes such as cell migration and invasion, this observation also supports the hypothesis that DKK-1 might be involved in the pathogenesis of CD patients. DKK-1 may be an important mediator in the pathophysiology of CD, and DKK-1 level may be a good marker for predicting the inflammation and prognosis of CD patients. Our findings warrant further research examining the potential of DKK-1 as a therapeutic agent of CD.