Stimulation of D-glucose transport. A novel effect of vitamin D on intestinal membrane transport.

Vitamin D stimulates absorption of D-glucose in chick jejunum and ileum by a specific action on the maximal velocity of Na+-gradient driven D-glucose transport across the brush-border membrane of intestinal cells. Induction of D-glucose transport by either vitamin D-3 or 1,25-dihydroxyvitamin D-3 in embryonic intestine can be blocked by inhibitors of RNA and protein synthesis.

Vitamin D stimulates (Na+ + K+)-ATPase activity in chick small intestine.

Vitamin D3 and 1,25-dihydroxyvitamin D3 raise (Na+ + K+)-ATPase activity (ouabain-sensitive 86Rb+ uptake) in cultured embryonic and 4-week-old chick small intestine. Vitamin D stimulation of the sodium pump, which requires genomic action of the sterol, may lead to enhanced Ca2+ extrusion via a basolateral Na+/Ca2+ exchange mechanism, and, in addition, may provide a proliferative signal in undifferentiated enterocytes.

Growth regulation of human colon cancer cells by epidermal growth factor and 1,25-dihydroxyvitamin D3 is mediated by mutual modulation of receptor expression.

The human colon adenocarcinoma-derived cell line Caco-2 was used as a model system to study the interaction of epidermal growth factors (EGF) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in control of colorectal cancer cell growth. The mitogenic stimulus of EGF was rapidly transduced via apical and basal membrane receptors alike into elevation of c-myc expression, causing a shift of Caco-2 cells from the G0/G1 into the S phase of the cell cycle. The stimulatory effect of EGF on cell division was effectively counteracted by 1,25(OH)2D3: the presence of the steroid hormone prevents the negative effect of EGF on vitamin D receptor abundance and concurrently minimises ligand-occupied EGF receptor numbers on both sides of Caco-2 cell monolayers. Our data suggest that EGF and 1,25-(OH)2D3 actions on mutual receptor levels represent a specific feature of the potent antimitogenic effect of the steroid hormone on colon cancer cells.

Immunocytochemical localization of the extracellular calcium-sensing receptor in normal and malignant human large intestinal mucosa.

We identified the parathyroid type Ca(2+)-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.

Calcitriol-dependent, paracellular sodium transport in the embryonic chick intestine.

1,25-Dihydroxyvitamin D3 (calcitriol), or vitamin D3 itself, when added to cultures of 20-day-old embryonic chick small intestine, stimulated sodium (Na+) uptake from the mucosal surface. The calcitriol-mediated increase in Na+ uptake appeared to be related to increased tight-junctional or paracellular permeability. Support for this conclusion was, first, that the uptake of other ions, potassium (K+) and rubidium (Rb+), with tight-junctional permeabilities greater than Na+, was also stimulated by calcitriol, and second, perturbation of cellular Na+ and K+ fluxes by inhibition of Na+/K+-ATPase activity did not affect calcitriol-stimulated Na+, K+, or Rb+ transport. Calcitriol stimulation of Na+ fluxes across the brush border as an alternate possibility is unlikely for the following reason: the calcium ionophore A23187, while mimicking the stimulatory action of calcitriol on calcium (Ca2+) uptake, reduced epithelial Na+ uptake. It is therefore suggested that calcitriol, by virtue of its effect on Ca2+ transport, reduces rather than stimulates cellular Na+ uptake.

Vitamin D metabolism in human colon adenocarcinoma-derived Caco-2 cells: expression of 25-hydroxyvitamin D3-1alpha-hydroxylase activity and regulation of side-chain metabolism.

1Alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) and its synthetic analogues exhibit structure-related variations in their growth inhibitory actions in human colon adenocarcinoma-derived Caco-2 cells. Because this might be caused by differences in resistance against metabolic degradation, we used high performance liquid chromatography (HPLC) analysis to investigate pathways of vitamin D metabolism in two different Caco-2 cell clones. Importantly, when Caco-2 cells were incubated with tritium-labelled 25-hydroxyvitamin D3 (25(OH)D3) for up to 2 h they produced almost exclusively a metabolite, which was identified as 1alpha,25(OH)2D3 by co-chromatography with the synthetic standard in two different HPLC systems, and by a radioligand assay showing an identical binding affinity to the intestinal nuclear vitamin D receptor. Expression of the 25(OH)D3-1alpha-hydroxylase appears to be constitutive because almost identical enzyme activities are observed in any growth phase. 1Alpha,25(OH)2D3 can also activate side chain metabolism in Caco-2 cells: thereby, 1alpha,25(OH)2D3 or 25(OH)D3 are metabolized through the C-24 oxidative pathway into 1alpha,24(R),25(OH)3D3 and 24(R),25(OH)2D3, respectively, which undergo sequential metabolism into 1alpha,25(OH)2-24oxo-D3 and 24-oxo-25(OH)D3. Through C-23 oxidation these intermediary metabolites are further converted into 1alpha,23,25(OH)3-24-oxo-D3 and 23,25(OH)2-24-oxo-D3. Also direct C-23 oxidation of the substrates 1alpha,25(OH)2D3 and 25(OH)D3 generates 1alpha,23(S),25(OH)3D3 and 23(S),25(OH)2D3, respectively. In summary, our results demonstrated the presence of distinct pathways of vitamin D metabolism in Caco-2 cells: apart from metabolizing 1alpha,25(OH)2D3 along the C-24 and C-23 oxidative pathways, Caco-2 cells are able to synthesize 1alpha,25(OH)2D3 from 25(OH)D3 through constitutive expression of 25(OH)D3-1alpha-hydroxylase activity. The relevance of this finding for the intrinsic growth control of neoplastic colonocytes is discussed.

The pattern of prion-related protein expression in the gastrointestinal tract.

Prion diseases or transmissible spongiform encephalopathies have been shown to be communicated by oral ingestion of the infectious agent. However, the exact route of transmission is still unknown. In order to better understand the pathophysiology of these diseases, it is crucial to identify cell types of peripheral tissues in which the infectious agent may propagate. Since expression of cellular prion protein (PrPc) is a prerequisite for prion replication, we determined the expression of PrPc in the mucosa of the gastrointestinal tract using immunohistochemistry. Expression of PrPc was negative or weak in the neck region of the gastric mucosa and moderate to strong in crypts of both the small and the large bowel. PrPc was found to be upregulated in the mucosa of patients with Helicobacter pylori gastritis. In contrast, PrPc staining appeared to be downregulated in patients with inflammatory disorders of the large bowel and it remained moderate to strong in inflammatory disorders of the small bowel. Our results support the notion that epithelial cells of the gastrointestinal tract may represent a possible target for prion entry and replication.

In situ mRNA hybridization analysis and immunolocalization of the vitamin D receptor in normal and carcinomatous human colonic mucosa: relation to epidermal growth factor receptor expression.

There is evidence that vitamin D receptor (VDR)-mediated action of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) could limit colon cancer cell growth particularly when induced by activation of the epidermal growth factor receptor (EGFR). We therefore wanted to ascertain the relevance of this observation for human colon cancerogenesis. Utilizing in situ mRNA hybridization and immunocytochemical techniques, we analyzed cell-specific expression of VDR and EGFR in normal and malignant human colonic mucosa. In normal mucosa, VDR positivity is weak and observed only in a small number of luminal surface colonocytes. In contrast, EGFR expression at a relatively high level is also found in cells at the crypt base. The number of VDR-positive colonocytes increases remarkably during tumor progression. It reaches its maximum in low grade adenocarcinomas and returns to lower levels in highly malignant cancers. In both low- and high grade carcinomas, the great majority of tumor cells contain the EGFR message. The relative abundance of EGFR over VDR in normal mucosa and in high grade carcinomas would create a situation in which mitogenic effects from EGFR activation are only ineffectively counteracted by signaling from 1 alpha,25-(OH)2D3/VDR. In contrast, in well to moderately differentiated tumors, upregulation of VDR could retard further tumor progression.

Growth inhibition of human colon adenocarcinoma-derived Caco-2 cells by 1,25-dihydroxyvitamin D3 and two synthetic analogs: relation to in vitro hypercalcemic potential.

The effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and of two synthetic analogs, 1,25S,26-tri-hydroxy-delta 22-vitamin D3 (1,25,26(OH)3-22ene-D3, Ro 23-4319) and 1,25-dihydroxy-delta 16-23yne-vitamin D3 (1,25(OH)2-16ene-23yne-D3, Ro 23-7553) on cell growth was evaluated by determination of [3H]thymidine incorporation into DNA of human colon adenocarcinoma-derived Caco-2 cells. The extent of growth inhibition by the vitamin D compounds varied between 20-40% (at 10(-8) M), depending on particular growth conditions of Caco-2 cells as well as on the molecular structure of the vitamin D sterols. In confluent, i.e., rather quiescent cells, all three vitamin D compounds were equipotent in suppressing growth. In rapidly dividing log phase cells, 1,25(OH)2-16ene-23yne-D3 or 1,25,26(OH)3-22ene-D3 were ten or five times, respectively, more efficient than 1,25(OH)2D3. A substantial effect on induction of the colonocyte differentiation marker alkaline phosphatase was only elicited by 1,25(OH)2-16ene-23yne-D3. The ability of the vitamin D compounds to raise intestinal calcium absorption was evaluated by determination of 45Ca2+ accumulation in embryonic chick duodenal explants. In this assay, both synthetic analogs were less effective than 1,25(OH)2D3 by a factor of 20. The intrinsic bone resorbing activities of the vitamin D analogs were compared in organ-cultured neonatal mouse calvariae. The most effective antiproliferative compound, 1,25(OH)2-16ene-23yene-D3, stimulated calcium release from cultured bones at concentrations less than 10(-11) M, and was thus ten times more potent than 1,25(OH)2D3 and hundred times more than 1,25,26(OH)3-22ene-D3.

Mechanism of antimitogenic action of vitamin D in human colon carcinoma cells: relevance for suppression of epidermal growth factor-stimulated cell growth.

Because the efficacy of 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3] in treatment of colon cancer might critically depend on its ability to specifically counteract epidermal growth factor (EGF)-stimulated tumor cell growth, we utilized human colon adenocarcinoma-derived cells in primary culture as well as the Caco-2 cell line to elucidate possible sites of interaction of 1alpha,25-(OH)2D3 with signaling from EGF receptor activation. In both types of colon cancer cells investigated, 10(-8) M 1alpha,25-(OH)2D3 reduced basal cell proliferation by about 50%, and prevented any rise in proliferation when colon cancer cells were treated with 25 ng/ml EGF: this can be explained by a marked inhibitory effect of 1alpha,25-(OH)2D3 on EGFR mRNA and protein expression. The steroid hormone also seemingly promotes EGF-induced internalization of apical and basolateral membrane EGFR. In addition, 1alpha,25-(OH)2D3 significantly reduced basal and EGF-stimulated expression of cyclin D1 at the mRNA and protein level in primary cultures as well as in the Caco-2 cell line. The ability of 1alpha,25-(OH)2D3 to interfere with a key event in cell cycle control and thereby to block mitogenic signaling from EGF could be seen as advantageous for the potential use of vitamin D compounds in colon cancer therapy.

25-Hydroxyvitamin D(3)-1alpha-hydroxylase and vitamin D receptor gene expression in human colonic mucosa is elevated during early cancerogenesis.

Human colorectal cancer cells not only express the nuclear vitamin D receptor (VDR) but are also endowed with 25-hydroxy-vitamin D(3)-1alpha-hydroxylase activity and therefore are able to produce the specific ligand for the VDR, the hormonally active steroid 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)). In the present study we show by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) as well as by Western blotting and immunohistochemical methods, that in human large intestinal carcinomas expression of the genes encoding the 25-(OH)D(3)-1alpha-hydroxylase as well as the VDR increases in parallel with ongoing dedifferentiation in the early phase of cancerogenesis, whereas in poorly differentiated late stage carcinomas only low levels of the respective mRNAs can be detected. This indicates that, through up-regulation of this intrinsic 1alpha,25(OH)(2)D(3)/VDR system which mediates the anti-mitotic effects of the steroid hormone, colorectal cancer cells are apparently able to increase their potential for an autocrine counter-regulatory response to neoplastic cell growth, particularly in the early stages of malignancy.

Induction of epidermal growth factor receptor expression and mitogenesis by alcohol in human colon adenocarcinoma-derived Caco-2 cells.

Epidemiologic studies suggest that alcohol may be an inducing factor in human colon tumorigenesis. As colon cells are frequently under autocrine control by growth factors, involvement of the EGFR pathway in alcohol-related colon tumor progression was investigated in the human colon adenocarcinoma-derived cell line Caco-2 which shows EGFR distribution mainly in basolateral cell membranes. EGF treatment results in almost complete downregulation of the basolateral receptor. Low concentrations of ethanol (0.22 mM, 0.1%) however, lead to significantly increased EGFR mRNA and protein expression and a raised mitotic rate mainly in basolaterally treated cells. Alcohol-induced overexpression of EGFR is paralleled by increased cyclin D1 expression. This suggests a possible mechanism for low blood levels of alcohol to stimulate in vivo proliferation of colonocytes by elevating transcription of a growth factor receptor as well as by modifying expression of a cell cycle regulator.

Vitamin D receptor and cytokeratin expression may be progression indicators in human colon cancer.

Epidemiological data suggest the protective role of vitamin D against the development of colorectal carcinoma in man. This could be due to the anti-mitogenic effect of the steroid hormone on human colon carcinoma cells which is mediated by a specific nuclear vitamin D receptor (VDR). Western blot analysis showed that VDR expression increases during the transition from normal mucosa to polyps and later to pT3 tumors. In later stages, however, VDR is dramatically reduced. Cytokeratin 20, which was monitored as a differentiation marker, decreases in parallel with advancing proliferation and disappears from "normal" mucosa adjacent to later stage carcinoma. Interestingly, VDR density was conspicuously higher in all tumors tested when compared to adjacent "normal" tissue. This suggest that, up to a certain degree of dedifferentiation, malignant colonocytes can upregulate the VDR, probably as a counteractive measure in response to tumor cell growth, but that this ability is finally lost in highly undifferentiated carcinoma cells.

Vitamin D receptor activity and prevention of colonic hyperproliferation and oxidative stress.

Unimpaired vitamin D action has been implicated in human cancer prevention. We have previously demonstrated the effectiveness of 1 alpha-dihydroxyvitamin D3 (1,25-D3) to reduce proliferation and increase differentiation in human colon cancer cells. The aim of this study was to investigate, on the one hand, expression of the vitamin D receptor (VDR) and of 25-hydroxyvitamin D(3)-1 alpha-hydroxylase (1 alpha-hydroxylase) in human normal and malignant colonic tissue and, on the other hand, to determine consequences of reduced or lacking VDR action in a VDR knockout mouse model. In low-grade malignancies of the human colon we found increased VDR and 1 alpha-hydroxylase mRNA expression. However, in late-stage high-grade tumors the vitamin D system is severely compromised. In the mouse colon we found an inverse relationship between VDR levels and proliferation in colon descendens, a tissue known to be specifically affected by nutrients during carcinogenesis. Expression of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, was significantly augmented with complete loss of VDR. These data suggest that genomic 1,25-D(3) action is necessary to protect against nutrition-linked hyperproliferation and oxidative DNA damage.

Interspecies comparative pathology of colorectal neoplasms: relevance for treatment.

Comparative pathology may serve as a practical tool for therapy by comparison of normal and abnormal structures of the digestive tract in animals and men. A better understanding of colon cancer as the most common solid neoplasm after lung cancer in the industrialized world is sought. In the so-called developed nations and in animals colon cancer is less frequent. The pathogenesis of colon cancer involves environmental and genetic factors. Several types of colorectal cancer can be discerned and the species distribution ranges from invertebrates to man. Colorectal neoplastic progression is species-specific. An intraspecies-specific comparison of large bowel cancer is also valuable. Alteration of signal transduction pathways and somatic mutations of oncogenes are described, as well as the occurrence, research and current treatment. Metastasis of neoplasms of the colon and of the rectum can be studied by intraspecies-specific comparison. Sections of this review deal with vitamin D and cancer and close with present therapies for colorectal cancer.

Vitamin D and calcium insufficiency-related chronic diseases: molecular and cellular pathophysiology.

A compromised vitamin D status, characterized by low 25-hydroxyvitamin D (25-(OH)D) serum levels, and a nutritional calcium deficit are widely encountered in European and North American countries, independent of age or gender. Both conditions are linked to the pathogenesis of many degenerative, malignant, inflammatory and metabolic diseases. Studies on tissue-specific expression and activity of vitamin D metabolizing enzymes, 25-(OH)D-1 alpha-hydroxylase and 25-(OH)D-24-hydroxylase, and of the extracellular calcium-sensing receptor (CaR) have led to the understanding of how, in non-renal tissues and cellular systems, locally produced 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) and extracellular Ca(2+) act jointly as key regulators of cellular proliferation, differentiation and function. Impairment of cooperative signalling from the 1,25-(OH)(2)D(3)-activated vitamin D receptor (VDR) and from the CaR in vitamin D and calcium insufficiency causes cellular dysfunction in many organs and biological systems, and, therefore, increases the risk of diseases, particularly of osteoporosis, colorectal and breast cancer, inflammatory bowel disease, insulin-dependent diabetes mellitus type I, metabolic syndrome, diabetes mellitus type II, hypertension and cardiovascular disease. Understanding the underlying molecular and cellular processes provides a rationale for advocating adequate intake of vitamin D and calcium in all populations, thereby preventing many chronic diseases worldwide.

Vitamin D and calcium deficits predispose for multiple chronic diseases.

There is evidence from both observational studies and clinical trials that calcium malnutrition and hypovitaminosis D are predisposing conditions for various common chronic diseases. In addition to skeletal disorders, calcium and vitamin D deficits increase the risk of malignancies, particularly of colon, breast and prostate gland, of chronic inflammatory and autoimmune diseases (e.g. insulin-dependent diabetes mellitus, inflammatory bowel disease, multiple sclerosis), as well as of metabolic disorders (metabolic syndrome, hypertension). The aim of the present review was to provide improved understanding of the molecular and cellular processes by which deficits in calcium and vitamin D cause specific changes in cell and organ functions and thereby increase the risk for chronic diseases of different aetiology. 1,25-Dihydroxyvitamin D(3) and extracellular Ca(++) are both key regulators of proliferation, differentiation and function at the cellular level. However, the efficiency of vitamin D receptor-mediated intracellular signalling is limited by the negative effects of hypovitaminosis D on extrarenal 25-hydroxyvitamin D-1alpha-hydroxylase activity and thus on the production of 1,25-dihydroxyvitamin D(3). Calcium malnutrition eventually causes a decrease in calcium concentration in extracellular fluid compartments, resulting in organ-specific modulation of calcium-sensing receptor activity. Hence, attenuation of signal transduction from the ligand-activated vitamin D receptor and calcium-sensing receptor seems to be the prime mechanism by which calcium and vitamin D insufficiencies cause perturbation of cellular functions in bone, kidney, intestine, mammary and prostate glands, endocrine pancreas, vascular endothelium, and, importantly, in the immune system. The wide range of diseases associated with deficits in calcium and vitamin D in combination with the high prevalence of these conditions represents a special challenge for preventive medicine.