The vitamin D receptor knockout (VDR-KO) mouse presents with a skeletal phenotype typical for complete lack of genomic 1,25-dihydroxycholecalciferol effects. Our previous data from human colorectal tissue suggest that the steroid hormone and its receptor may have protective function against tumour progression. In order to investigate the relevance of the vitamin D system for pre-malignant site-directed changes in the colon, we characterized the amount and site-specific distribution of the VDR along the large intestine in wild-type (WT), heterozygote (HT) and KO mice. We also evaluated expression of proliferating cell nuclear antigen (PCNA), of cyclin D1 and the levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a marker of oxidative stress. In colon ascendens, proliferative cells were dispersed all along the crypt and expression levels of all three markers were high in WT mice. A decrease of VDR expression did not affect expression significantly. In colon descendens, however, fewer proliferative cells were solely located in the lower third of the crypt, and an inverse relationship between VDR reduction, PCNA positivity and cyclin D1 expression was found in HT and KO mice. In parallel to enhanced proliferation a highly significant increase of 8-OHdG positivity occurred. Therefore, the sigmoid colon of VDR-KO mice, fed on an appropriate lactose/calcium-enriched diet to alleviate impaired calcium homeostasis-related phenotypic changes, is an excellent model for investigating induction and prevention of pre-malignant changes in one of the hotspots for human colorectal cancer incidence.

The main autocrine/paracrine role of the active metabolite of vitamin D₃, 1α,25-dihydroxyvitamin D₃ (1,25-D₃), is inhibition of cell growth and induction of cell differentiation and/or apoptosis. Synthesis and degradation of the secosteroid occurs not only in the kidney but also in normal tissue or malignant extrarenal tissues such as the colon. Because 25-hydroxyvitamin D₃ 24-hydroxylase (CYP24A1) is considered to be the main enzyme determining the biological half-life of 1,25-D₃, we have examined expression of the CYP24A1 mRNA (by real-time RT-PCR) and protein (by immunohistochemistry) in normal human colon mucosa, colorectal adenomas, and adenocarcinomas in 111 patients. Although 76% of the normal and benign colonic tissue was either completely devoid of or expressed very low levels of CYP24A1, in the majority of the adenocarcinomas (69%), the enzyme was present at high concentrations. A parallel increased expression of the proliferation marker Ki-67 in the same samples suggests that overexpression of CYP24A1 reduced local 1,25-D₃ availability, decreasing its antiproliferative effect.

1,25-dihydroxyvitamin D₃ has anti-mitotic, pro-differentiating, and pro-apoptotic activity in tumor cells. We demonstrated that the secosteroid can be synthesized and degraded not only in the kidney but also extrarenally in intestinal cells. Evaluation of 1,25-dihydroxyvitamin D₃-synthesizing CYP27B1 hydroxylase mRNA (real-time PCR) and protein (immunoblotting, immunofluorescence) showed enhanced expression in high- to medium-differentiated human colon tumors compared with tumor-adjacent normal mucosa or with colon mucosa from non-cancer patients. In high-grade undifferentiated tumor areas expression was lost. Many cells co-expressed CYP27B1 and the vitamin D receptor. We suggest that autocrine/paracrine antimitotic activity of 1,25-dihydroxyvitamin D₃ could prevent intestinal tumor formation and progression. (J Histochem Cytochem 52:985–989, 2004)

SUMMARY We identified the parathyroid type Ca²⁺-sensing receptor (CaR) in normal human colon mucosa and in cancerous lesions at the mRNA and protein level. Polymerase chain reaction produced an amplification product from reverse-transcribed large intestinal RNA which corresponded in size and length to a 537-bp sequence from exon 7 of the CaR gene. With a specific antiserum against its extracellular domain, the CaR could be detected by immunostaining in normal human colon mucosa in cells preferentially located at the crypt base. The CaR protein was also expressed in tumors of the large bowel in all 20 patients examined. However, the great majority of CaR-positive cells in the adenocarcinomas inspected were confined to more differentiated areas exhibiting glandular-tubular structures. Poorly or undifferentiated regions were either devoid of specific immunoreactivity or contained only isolated CaR-positive cells. In the normal mucosa and in glandular-tubular structures of cancerous lesions, the CaR was exclusively expressed in chromogranin A-positive enteroendocrine cells and in only a small fraction of PCNA-positive cells.