Interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα) are proinflammatory cytokines that play a critical role in inflammatory bowel disease, as well as in colorectal tumorigenesis. We hypothesize that these cytokines modulate the expression and thus activity of the vitamin D system in colonic epithelial cells. We treated the colon cancer cell line COGA-1A for 6, 12, and 24 h with 1,25-dihydroxyvitamin D3 (1,25-D3), IL-6, TNFα, and with combinations of these compounds. Using quantitative RT-PCR, we analyzed mRNA expression of genes activating and catabolizing 1,25-D3 (1α-hydroxylase (CYP27B1), 24-hydroxylase (CYP24A1)), expression of several vitamin D target genes, as well as expression of cyclooxygenase 2 (COX-2) and 15-hydroxyprostaglandin dehydrogenase. As expected, treatment with 1,25-D3 resulted in an upregulation of CYP24A1, whereas expression of CYP27B1 was not affected. Treatment with TNFα and IL-6 led to decreased expression of the vitamin D activating enzyme CYP27B1. The strong inflammatory property of TNFα was mirrored by its activation of COX-2 and inhibition of prostaglandin E2 (PGE2) catabolism. Interestingly, expression of the calcium ion channel TRPV6 was markedly decreased by TNFα.We conclude from these results that the presence of proinflammatory cytokines might impair activation of 1,25-D3, limiting its anti-inflammatory action.This article is part of a Special Issue entitled ‘16th Vitamin D Workshop’.

BackgroundThe calcium sensing receptor (CaSR), a calcium-binding G protein-coupled receptor is expressed also in tissues not directly involved in calcium homeostasis like the colon. We have previously reported that CaSR expression is down-regulated in colorectal cancer (CRC) and that loss of CaSR provides growth advantage to transformed cells. However, detailed mechanisms underlying these processes are largely unknown.Methods and resultsIn a cohort of 111 CRC patients, we found significant inverse correlation between CaSR expression and markers of epithelial-to-mesenchymal transition (EMT), a process involved in tumor development in CRC. The colon of CaSR/PTH double-knockout, as well as the intestine-specific CaSR knockout mice showed significantly increased expression of markers involved in the EMT process. In vitro, stable expression of the CaSR (HT29CaSR) gave a more epithelial-like morphology to HT29 colon cancer cells with increased levels of E-Cadherin compared with control cells (HT29EMP). The HT29CaSR cells had reduced invasive potential, which was attributed to the inhibition of the Wnt/β-catenin pathway as measured by a decrease in nuclear translocation of β-catenin and transcriptional regulation of genes like GSK-3β and Cyclin D1. Expression of a spectrum of different mesenchymal markers was significantly down-regulated in HT29CaSR cells. The CaSR was able to block upregulation of mesenchymal markers even in an EMT-inducing environment. Moreover, overexpression of the CaSR led to down-regulation of stem cell-like phenotype.ConclusionsThe results from this study demonstrate that the CaSR inhibits epithelial-to-mesenchymal transition and the acquisition of a stem cell-like phenotype in the colon of mice lacking the CaSR as well as colorectal cancer cells, identifying the CaSR as a key molecule in preventing tumor progression. Our results support the rationale to develop new strategies either preventing CaSR loss or reversing its silencing.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0330-4) contains supplementary material, which is available to authorized users.

Vitamin D (VD) is essential for the human body and involved in a wide variety of critical physiological processes including bone, muscle, and cardiovascular health, as well as innate immunity and antimicrobial responses. Here, we elucidated the significance of the VD system in cytomegalovirus (CMV) infection, which is one of the most common opportunistic infections in immunocompromised or -suppressed patients. We found that expression of vitamin D receptor (VDR) was downregulated in CMV-infected cells within 12 hours [hrs] post infection [p.i.] to 12 % relative to VDR expression in mock-infected fibroblasts and did not recover during the CMV replication cycle of 96 hrs. None of the biologically active metabolites of VD, cholecalciferol, calcidiol, or calcitriol, inhibit CMV replication significantly in human fibroblasts. In a feedback loop, expression of CYP24A1 dropped to 3 % by 12 hrs p.i. and expression of CYP27B1 increased gradually during the replication cycle of CMV to 970 % probably as a consequence of VDR inhibition. VDR expression was not downregulated during influenza virus or adenovirus replication. The potent synthetic vitamin D analog EB-1089 was not able to inhibit CMV replication or antagonize its effect on VDR expression. Only CMV replication, and none of the other viral pathogens evaluated, inhibited the vitamin D system in vitro. In view of the pleiotropism of VDR, CMV-mediated downregulation may have far-reaching virological, immunological, and clinical implications and thus warrant further evaluations in vitro and in vivo.

Studies have shown that the calcium‐sensing receptor (CaSR) mediates the antitumorigenic effects of calcium against colorectal cancer (CRC). Expression of the CaSR in colorectal tumors is often reduced. We have reported previously that silencing of CaSR in CRC is caused in part by methylation of CaSR promoter 2 and loss of histone acetylation. We investigated the impact of aberrant microRNA expression on loss of CaSR expression. A microarray study in two Caco‐2 subclones (Caco2/AQ and Caco2/15) that have similar genetic background, but different CaSR expression levels (Caco2/AQ expressing more CaSR than Caco2/15), identified 22 differentially expressed microRNAs that potentially target the CaSR. We validated these results by performing gain‐ and loss‐of‐function studies with the top candidates: miR‐9, miR‐27a, miR‐135b, and miR‐146b. Modulation of miR‐135b or miR‐146b expression by mimicking or inhibiting their expression regulated CaSR protein levels in two different colon cancer cell lines: Caco2/AQ (moderate endogenous CaSR expression) and HT29 (low endogenous CaSR levels). Inhibition of miR‐135b and miR‐146b expression led to high CaSR levels and significantly reduced proliferation. In samples of colorectal tumors we observed overexpression of miR‐135b and miR‐146b, and this correlated inversely with CaSR expression (miR‐135b: r = −0.684, p < 0.001 and miR‐146b: r = −0.448, p < 0.001), supporting our in vitro findings. We demonstrate that miR‐135b and miR‐146b target the CaSR and reduce its expression in colorectal tumors, reducing the antiproliferative and prodifferentiating actions of calcium. This provides a new approach for finding means to prevent CaSR loss, developing better treatment strategies for CRC.

No abstract