CYP24A1 Participates In The Regulation Of Wnt Signaling Pathway By Inflammatory Factors

Part 1:The Mechanism of CYP24A1 Upregulating Wnt Signaling Pathway Induced by Proinflammatory CytokinesPurposeThe CYP24A1 gene encodes a vitamin D3 catabolic enzyme.CYP24A1 plays a prominent role in the extra-renal autocrine/paracrine vitamin D system and affected local concentrate of 1α,25-(OH)2D3,which is a repressor of Wnt/β-catenin signaling pathway.The expression level of CYP24A1 has been found to be significantly higher in CRC tissues and the distribution of CYP24A1 SNPs are related to the risk of ulcerative colitis.Inflammatory cytokines IL-6 and TNF-a and NF-κB pathway activation play a critical role in inflammation-cancer progression,and our research group has found that IL-6 and TNF-a might stimulate Wnt signaling pathway though NF-κB pathway activation in colon cancer cells.This research is conducted to explore the role of CYP24A1 in activation of Wnt signaling pathway induced by proinflammatory cytokines,and provide the possibility of strategies to specifically counteract CYP24A1 activity,restore tissue responsiveness to vitamin D and prevent ulcerative colitis related colorectal cancer.MedthodsIn colon cancer cell lines HCT-116 and Caco-2,we analyzed the impression of IL-6 and TNF-a on CYP24A1 expression using Western Blot and on activation level of NF-κB pathway using EMSA.Specific inhibitor PDTC of NF-κB pathway wasgiven and then impression of IL-6 and TNF-a on CYP24A1 expression was measured again by Western Blot.CYP24A1 was knocked down by siRNA,and then activation of Wnt signaling pathway induced by IL-6 and TNF-a was measured by dual luciferase reporter gene assay and nuclear β-catenin levels were measured by Western Blot.ResultsIL-6 and TNF-a treatment up-regulated CYP24A1 expression dose-and time-dependently.IL-6 and TNF-a treatment(IL-6 100ng/ml 6h and TNF-a 50ng/ml 6h on Caco-2,IL-6 50ng/ml 6h and TNF-a 100ng/ml 6h on HCT-116)activated NF-κB pathway and PDTC treatment partly inhibited the up-regulation of CYP24A1 induced by these cytokines.SiRNA-induced knockdown of CYP24A1 expression partly antagonized the increase in β-catenin nuclear distribution and Wnt signaling pathway activation induced by IL-6 and TNF-α(relative luciferase activity,Caco-2:IL-63.297±0.1610 vs 2.527±0.03323,p=0.0221;TNF-α 4.130±0.4289 vs 2.780±0.3092,p=0.0249;HCT116:IL-6 1.357±0.03422 vs 1.086±0.07545,p=0.0048;TNF-α2.374±0.1955 vs 1.802±0.06163,p=0.0313).ConclusionsIL-6 and TNF-α treatment may up-regulate CYP24A1 expression through NF-κB pathway activation,and then activate Wnt signaling pathway by promoting the nuclear import of β-catenin.Part 2:The Effect of Proinflammatory Cytokines on CYP24A1 Expression in a Mice Model of Inflammation-Associated Colorectal CancerPurposeTo explore the effect of TNF-α and NF-κB pathway on CYP24A1 expression in a inflammation-associated colorectal cancer model induced by azoxymethane(AOM)dextran sulfate sodium(DSS).MedthodsC57BL/6 mice were randomly divided into 4 groups.Group 1 served as normal control without AOM/DSS.Group 2-4 served intervention model,which were treated with AOM 12.5 mg/kg by intraperitoneal injection and a 5-days DSS treatment(2.5%in drinking water)one week later.Group 2 served as model control.Group 3 was given NF-κB antisense oligonucleotide by enema every 2 weeks.Group 4 was given TNF-a monocolonal antibody by intraperitoneal injection every 2 weeks.All groups were sacrificed at the end of 12th week.Macroscopic evaluation of tumor load(TL)as well as pathologic evaluation was carried out to assess carcinogenesis severity.The expression level of CYP24A1 was determined by inmmunohistochemistry.ResultsCYP24A1 expression was significantly higher in model control(n=8,2.500 ±0.3273)than in normal control(n=6,1 · 167 ± 0.1667).NF-κB antisense oligonucleotide group had a lower tumor load(n=5,1.130 ± 0.1678 cm)and a inhibited NF-κB activity than model control group(n=8,2.294 ±0.3638 cm)(p=0.0354).CYP24A1 expression was lower in NF-κB antisense oligonucleotide group than in model control(1.400 ± 0.2449 vs 2.500 ± 0.3273,p=0.0362).Tumor load of TNF-a monocolonal antibody group(n=5,1.780 ± 0.5142)was lower than that of model control(n=8,2.294 ± 0.3638),but the differences had no significance(p=0.4195).TNF-a monocolonal antibody group had a inhibited NF-κB activity and a lower CYP24A1 expression than model control(1.167 ± 0.1667 vs 2.500 ± 0.3273,p=0.0144).ConclusionsCYP24A1 expression may be unregulated by TNF-α and activation of NF-κB pathway.