Clonal differences in expression of 25-hydroxyvitamin D(3)-1alpha-hydroxylase,of 25-hydroxyvitamin D(3)-24-hydroxylase,and of the vitamin D receptor in human colon carcinoma cells: effects of epidermal growth factor and 1alpha,25-dihydroxyvitamin D(3)

Human colon carcinoma cells express 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1) and thus produce the vitamin D receptor (VDR) ligand 1alpha,25-dihydroxyvitamin D(3) (1,25-D3), which can be metabolized by 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24). Expression of VDR, CYP27B1, and CYP24 determines the efficacy of the antimitotic action of 1,25-D3 and is distinctly related to the degree of differentiation of cancerous lesions. In the present study we addressed the question of whether the effects of epidermal growth factor (EGF) and of 1,25-D3 on VDR, CYP27B1, and CYP24 gene expression in human colon carcinoma cell lines also depend on the degree of cellular differentiation. We were able to show that slowly dividing, highly differentiated Caco-2/15 cells responded in a dose-dependent manner to both EGF and 1,25-D3 by up-regulation of VDR and CYP27B1 expression, whereas in highly proliferative, less differentiated cell lines, such as Caco-2/AQ and COGA-1A and -1E, negative regulation was observed. CYP24 mRNA was inducible in all clones by 1,25-D3 but not by EGF. From the observed clonal differences in the regulatory effects of EGF and 1,25-D3 on VDR and CYP27B1 gene expression we suggest that VDR-mediated growth inhibition by 1,25-D3 would be efficient only in highly differentiated carcinomas even when under mitogenic stimulation by EGF.     

Monocyte-derived cells express CYP27A1 and convert vitamin D3 into its active metabolite

CYP27A1 catalyses hydroxylations in the biosynthesis of bile acids and the bioactivation of vitamin D3. We investigated the expression of CYP27A1 in human monocytes, monocyte-derived macrophages, and dendritic cells on mRNA and protein levels as well as its enzymatic activity in comparison with the expression of CYP27B1 and CYP24A1. Macrophages showed a strong expression of CYP27A1, whereas monocytes and dendritic cells expressed low levels of CYP27A1 mRNA. Immunohistochemistry revealed CYP27A1 and CYP27B1 protein expression in macrophages. Accordingly, macrophages converted vitamin D3 into the active metabolite 1,25(OH)2D3. Dendritic cells also metabolized vitamin D3 although to a lesser extent. This could be due to the high expression of CYP24A1, the enzyme that degrades 25(OH)D3 and 1,25(OH)2D3. Our results show that macrophages and dendritic cells are capable to perform both hydroxylation steps of the vitamin D3 metabolism suggesting a possible role of local 1,25(OH)2D3 synthesis by myeloid cells in the skin and gut.     

Expression,structure-function,and molecular modeling of vitamin D P450s

Although vitamin D(3) is a natural product of a sunlight-mediated process in the skin, the secosteroid's biological function is dependent upon specific cytochrome P450 enzymes that mediate the parent vitamin's bioactivation and inactivation. Cytochrome P450C1 (CYP27B1) is the regulatory rate-limiting enzyme that directs the bioactivation process through introduction of a C-1alpha hydroxyl group. The resultant 1,25-dihydroxyvitamin D(3) (1,25D) is the biologically active secosteroid hormone that directs the multitude of vitamin D-dependent actions involved with calcium homeostasis, cellular differentiation and growth, and the immune response. The circulating and cellular level of 1,25D is regulated through a coordinated process involving the hormone's synthesis and degradation. Central to the degradation and turnover of 1,25D is the regulatory multi-catalytic cytochrome P450C24 (CYP24) enzyme that directs the introduction of C-24R groups onto targeted 25-hydroxy substrates. Discussed in this article is the action of the rat CYP24 to catalyze the side-chain oxidation and cleavage of 25-hydroxylated vitamin D metabolites. Expression and characterization of purified recombinant rat CYP24 is discussed in light of mutations directed at the enzyme's active site.     

Role of distal upstream sequence in vitamin D-induced expression of human CYP24 gene

The level of CYP24 mRNA in cultured human fibroblasts increases up to 20,000-fold in response to 1,25-dihydroxyvitamin D(3). Two vitamin D-responsive elements (VDREs) located immediately upstream of the CYP24 gene are primarily responsible for the induction. We studied roles of other regions in the 5'-flanking sequence of this gene. A series of deletion constructs between nucleotides -1918 and +209 of the gene were examined for their promoter activities employing the luciferase reporter assay. We found that the VDREs were not sufficient to account for the extent of induction. The sequence between nucleotides -548 and -294, which is located immediately upstream of the VDREs and includes three potential Sp1 sites, acted synergistically with the VDREs for the induction. Further upstream sequence and the 5'-untranslated region did not appear to play a major role in the vitamin D response.     

Bioengineering anabolic vitamin D-25-hydroxylase activity into the human vitamin D catabolic enzyme, cytochrome P450 CYP24A1, by a V391L mutation

CYP24A1 is a mitochondrial cytochrome P450 (CYP) that catabolizes 1α,25-dihydroxyvitamin D(3) (1α,25-(OH)(2)D(3)) to different products: calcitroic acid or 1α,25-(OH)(2)D(3)-26,23-lactone via multistep pathways commencing with C24 and C23 hydroxylation, respectively. Despite the ability of CYP24A1 to catabolize a wide range of 25-hydroxylated analogs including 25-hydroxyvitamin D(3), the enzyme is unable to metabolize the synthetic prodrug, 1α-hydroxyvitamin D(3) (1α-OH-D(3)), presumably because it lacks a C25-hydroxyl. In the current study we show that a single V391L amino acid substitution in the β3a-strand of human CYP24A1 converts this enzyme from a catabolic 1α,25-(OH)(2)D(3)-24-hydroxylase into an anabolic 1α-OH-D(3)-25-hydroxylase, thereby forming the hormone, 1α,25-(OH)(2)D(3). Furthermore, because the mutant enzyme retains its basal ability to catabolize 1α,25-(OH)(2)D(3) via C24 hydroxylation, it can also make calcitroic acid. Previous work has shown that an A326G mutation is responsible for the regioselectivity differences observed between human (primarily C24-hydroxylating) and opossum (C23-hydroxylating) CYP24A1. When the V391L and A326G mutations were combined (V391L/A326G), the mutant enzyme continued to form 1α,25-(OH)(2)D(3) from 1α-OH-D(3), but this initial product was diverted via the C23 hydroxylation pathway into the 26,23-lactone. The relative position of Val-391 in the β3a-strand of a homology model and the crystal structure of rat CYP24A1 is consistent with hydrophobic contact of Val-391 and the substrate side chain near C21. We interpret that the substrate specificity of V391L-modified human CYP24A1 toward 1α-OH-D(3) is enabled by an altered contact with the substrate side chain that optimally positions C25 of the 1α-OH-D(3) above the heme for hydroxylation.     

Cloning of a functional 25‐hydroxyvitamin D‐1α‐hydroxylase in zebrafish (Danio rerio)

Activation of precursor 25‐hydroxyvitamin D₃ (25D) to hormonal 1,25‐dihydroxyvitamin D₃ (1,25D) is a pivotal step in vitamin D physiology, catalysed by the enzyme 25‐hydroxyvitamin D‐1α‐hydroxylase (1α‐hydroxylase). To establish new models for assessing the physiological importance of the 1α‐hydroxylase‐25D‐axis, we used Danio rerio (zebrafish) to characterize expression and biological activity of the gene for 1α‐hydroxylase (cyp27b1). Treatment of day 5 zebrafish larvae with inactive 25D (5–150 nM) or active 1,25D (0.1–10 nM) induced dose responsive expression (15–95‐fold) of the vitamin D‐target gene cyp24a1 relative to larvae treated with vehicle, suggesting the presence of Cyp27b1 activity. A full‐length zebrafish cyp27b1 cDNA was then generated using RACE and RT‐PCR methods. Sequencing of the resulting clone revealed an open reading frame encoding a protein of 505 amino acids with 54% identity to human CYP27B1. Transfection of a cyp27b1 expression vector into HKC‐8, a human kidney proximal tubular epithelial cell line, enhanced intracrine metabolism of 25D to 1,25D resulting in greater than twofold induction of CYP24A1 mRNA expression and a 25‐fold increase in 1,25D production compared to empty vector. These data indicate that we have cloned a functional zebrafish CYP27B1, representing a phylogenetically distant branch from mammals of this key enzyme in vitamin D metabolism. Further analysis of cyp27b1 expression and activity in zebrafish may provide new perspectives on the biological importance of 25D metabolism. Copyright © 2014 John Wiley & Sons, Ltd.     

Clonal Differences in Expression of 25-Hydroxyvitamin D3-1α-hydroxylase, of 25-Hydroxyvitamin D3-24-hydroxylase, and of the Vitamin D Receptor in Human Colon Carcinoma Cells: Effects of Epidermal Growth Factor and 1α,25-Dihydroxyvitamin D3

Human colon carcinoma cells express 25-hydroxyvitamin D₃-1α-hydroxylase (CYP27B1) and thus produce the vitamin D receptor (VDR) ligand 1α,25-dihydroxyvitamin D₃ (1,25-D3), which can be metabolized by 25-hydroxyvitamin D₃-24-hydroxylase (CYP24). Expression of VDR, CYP27B1, and CYP24 determines the efficacy of the antimitotic action of 1,25-D3 and is distinctly related to the degree of differentiation of cancerous lesions. In the present study we addressed the question of whether the effects of epidermal growth factor (EGF) and of 1,25-D3 on VDR, CYP27B1, and CYP24 gene expression in human colon carcinoma cell lines also depend on the degree of cellular differentiation. We were able to show that slowly dividing, highly differentiated Caco-2/15 cells responded in a dose-dependent manner to both EGF and 1,25-D3 by up-regulation of VDR and CYP27B1 expression, whereas in highly proliferative, less differentiated cell lines, such as Caco-2/AQ and COGA-1A and -1E, negative regulation was observed. CYP24 mRNA was inducible in all clones by 1,25-D3 but not by EGF. From the observed clonal differences in the regulatory effects of EGF and 1,25-D3 on VDR and CYP27B1 gene expression we suggest that VDR-mediated growth inhibition by 1,25-D3 would be efficient only in highly differentiated carcinomas even when under mitogenic stimulation by EGF.     

Constitutive and inducible expression of cytochromes P4501A (CYP1A1 and CYP1A2) in normal prostate and prostate cancer cells

Constitutive and benzo[a]pyrene (B[a]P) inducible expression of CYP1A1 and CYP1A2 in prostate cancer and normal prostate epithelial cells were examined by immunoblotting. Androgen independent prostate cancer cell lines DU145 and PC3 have constitutive expression of CYP1A and CYP1A1 and CYP1A2, respectively. Four micromolar B[a]P did not appear to induce CYP1A1 or CYP1A2 expression in DU145 or PC3 cells. The androgen dependent prostate cancer cell line, LnCap, also has constitutive expression of CYP1A1 and CYP1A2. However, both CYP1A1 and CYP1A2 are induced by treatment of LnCap cells with 4 microM B[a]P. Untreated normal prostate and primary prostate tumor cells have no detectable CYP1A1 expression. Treatment with 4 microM B[a]P induced CYP1A1 expression in both normal and primary tumor prostate cells. Constitutive CYP1A2 expression was detected in normal prostate cells with little or no induction by exposure to 4 microM B[a]P. Primary prostate tumor cells did not show constitutive expression of CYP1A2. However, CYP1A2 was induced by 4 microM B[a]P in primary prostate tumor cells. These observations indicate that hormonal and cancer specific factors affect the expression and induction of the phase I metabolic enzymes, CYP1A1 and CYP1A2 in prostate cells. These observations may be related to the potential smoking-linked higher risk of prostate cancer development and morbidity of prostate cancer patients who smoke.     

Regulation of extrarenal synthesis of 1,25-dihydroxyvitamin D3--relevance for colonic cancer prevention and therapy

Epidemiological studies have demonstrated a high incidence of colonic tumors in populations living in areas of low sunlight exposure. This suggests 1,25-dihydroxyvitamin D3, an antimitotic prodifferentiating steroid hormone, as a potentially preventive factor since levels of the precursor 25-hydroxyvitamin D3 in serum are, to a major part, dependent upon sun exposure. Conversion into the active metabolite from the precursor is effected by CYP27B1, and degradation by CYP24. Both p450 hydroxylases are known to be located in the kidney. However, we were able to demonstrate presence, and activity of both enzymes also in the colon. We have shown also that during early tumor progression expression of CYP27B1 and of the vitamin D receptor is upregulated. Therefore the vitamin D system may function as a potent physiological defense against further tumor progression in cancer patients. We suggest that estrogenic substances, and also phytoestrogens present in soy food could, by increasing tumor tissue-located CYP27B1 activity and decreasing degradative CYP24 activity, augment tumor-localized 1,25-dihydroxyvitamin D3 levels and activity.     

Micro RNA‐550a interferes with vitamin D metabolism in peripheral B cells of patients with diabetes

The pathogenesis of diabetes is to be further investigated. Vitamin D3 (VitD3) can improve diabetes. Micro RNAs (miR) are involved in regulating cell activities. This study tests a hypothesis that miR‐550a interferes with the metabolism of VitD3 in peripheral B cells. In this study, blood samples were collected from patients with diabetes and healthy persons. The B cells were isolated from the blood samples to be treated with tumor necrosis factor (TNF)‐α. The B cells were then collected and analyzed for the expression of miR‐550a and cyp27b1. The results showed that B cells from healthy subjects were capable of converting VitD metabolite calcidiol to calcitriol, which was impaired in B cells collected from diabetic patients. The diabetic patients showed lower bone mineral density than that in healthy subject. The miR‐550a was negatively correlated with bone mineral density and the Levels of cyp27b1 in peripheral B cells of patients with diabetes. In vitro study showed that TNF‐α increased miR‐550a expression and inhibited the expression of cyp27b1 in B cells. miR‐550a mediated the effects of TNF‐α on inducing chromatin remodeling at the cyp27b1 gene locus. In conclusion, miR‐550a mediates the TNF‐α‐induced suppression of cyp27b1 expression in peripheral B cells of patients with diabetes, which can be blocked by inhibition of miR‐550a.     

Vitamin D3 modulated gene expression patterns in human primary normal and cancer prostate cells

The vitamin D receptor (VDR) is a member of the steroid/retinoid receptor superfamily of nuclear receptors and has potential tumor-suppressive functions in prostate and other cancer types. Vitamin D3 (VD3) exerts its biological actions by binding within cells to VDR. The VDR then interacts with specific regions of the DNA in cells, and triggers changes in the activity of genes involved in cell division, cell survival, and cellular function. Using human primary cultures and the prostate cancer (PCa) cell line, ALVA-31, we examined the effects of VD3 under different culture conditions. Complete G0/G1 arrest of ALVA-31 cells and approximately 50% inhibition of tumor stromal cell growth was observed. To determine changes in gene expression patterns related to VD3 activity, microarray analysis was performed. More than approximately 20,000 genes were evaluated for twofold relative increases and decreases in expression levels. A number of the gene targets that were up- and down-regulated are related to potential mechanisms of prostatic growth regulation. These include estrogen receptor (ER), heat shock proteins: 70 and 90, Apaf1, Her-2/neu, and paxillin. Utilizing antibodies generated against these targets, we were able to confirm the changes at the protein level. These newly reported gene expression patterns provide novel information not only potential markers, but also on the genes involved in VD3 induced apoptosis in PCa.     

Caffeine decreases vitamin D receptor protein expression and 1,25(OH)2D3 stimulated alkaline phosphatase activity in human osteoblast cells

Of the various risk factors contributing to osteoporosis, dietary/lifestyle factors are important. In a clinical study we reported that women with caffeine intakes >300 mg/day had higher bone loss and women with vitamin D receptor (VDR) variant, tt were at a greater risk for this deleterious effect of caffeine. However, the mechanism of how caffeine effects bone metabolism is not clear. 1,25-Dihydroxy vitamin D₃ (1,25(OH)₂D₃) plays a critical role in regulating bone metabolism. The receptor for 1,25(OH)₂D₃, VDR has been demonstrated in osteoblast cells and it belongs to the superfamily of nuclear hormone receptors. To understand the molecular mechanism of the role of caffeine in relation to bone, we tested the effect of caffeine on VDR expression and 1,25(OH)₂D₃ mediated actions in bone. We therefore examined the effect of different doses of caffeine (0.2, 0.5, 1.0 and 10 mM) on 1,25(OH)₂D₃ induced VDR protein expression in human osteoblast cells. We also tested the effect of different doses of caffeine on 1,25(OH)₂D₃ induced alkaline phosphatase (ALP) activity, a widely used marker of osteoblastic activity. Caffeine dose dependently decreased the 1,25(OH)₂D₃ induced VDR expression and at concentrations of 1 and 10 mM, VDR expression was decreased by about 50–70%, respectively. In addition, the 1,25(OH)₂D₃ induced alkaline phosphatase activity was also reduced at similar doses thus affecting the osteoblastic function. The basal ALP activity was not affected with increasing doses of caffeine. Overall, our results suggest that caffeine affects 1,25(OH)₂D₃ stimulated VDR protein expression and 1,25(OH)₂D₃ mediated actions in human osteoblast cells.     

Analyses in human urothelial cells identify methylation of miR-152, miR-200b and miR-10a genes as candidate bladder cancer biomarkers

Urinary miRNAs are discussed as potential biomarkers for bladder cancer. The majority of miRNAs, however, are downregulated, making it difficult to utilize reduced miRNA signals as reliable diagnostic tools. Because the downregulation of miRNAs is frequently associated with hypermethylation of the respective regulative sequences, we studied whether DNA hypermethylation might serve as an improved diagnostic tool compared to measuring downregulated miRNAs. miRNA expression arrays and individual qPCR were used to identify and confirm miRNAs that were downregulated in malignant urothelial cells (RT4, 5637 and J82) when compared to primary, non-malignant urothelial cells (HUEPC). DNA methylation was determined by customized PCR-arrays subsequent to methylation-sensitive DNA-restriction and by mass spectrometry. miRNA expression and DNA methylation were determined in untreated cells and in cultures treated with the demethylating agent 5-Aza-2′-deoxycytidine. miR-200b, miR-152 and miR-10a displayed differential expression and methylation among untreated cancer cell lines. In addition, reduced miRNA expression of miR-200b, miR-152, and miR-10a was associated with increased DNA methylation in malignant cells versus HUEPC. Finally, the demethylation approach revealed a causal relationship between both parameters for miR-152 in 5637 and also suggests a causal connection of both parameters for miR-200b in J82 and miR-10a in 5637. In conclusion, our studies in multiple bladder cancer cell lines and primary non-malignant urothelial cells suggest that hypermethylation of miR-152, miR-10a and miR-200b regulative DNA sequences might serve as epigenetic bladder cancer biomarkers.     

The interplay between epigenetic silencing,oncogenic KRas and HIF-1 regulatory pathways in control of BNIP3 expression in human colorectal cancer cells

Bcl-2/adenovirus E1B-19 kDa-interacting protein 3 (BNIP3) is an important mediator of cell survival and a member of the Bcl-2 family of proteins that regulate programmed cell death and autophagy. We have previously established a link between the expression of oncogenic HRas and up-regulation of BNIP3 and the control of autophagy in cancer cells. However, in view of varied expression of BNIP3 in different tumor types and emerging uncertainties as to the role of epigenetic silencing, oncogenic regulation and the role of BNIP3 in cancer are still poorly understood.     

Effects of 1,25-dihydroxyvitamin D3 on the distribution of androgen and vitamin D receptors in human prostate neonatal epithelial cells

Although many studies have examined the mechanisms of 1,25-dihydroxyvitamin D(3) (calcitriol or 1,25 D) action in different prostate cancer cell lines, little is known regarding the influence of this steroid on the normal prostate. The presence of both VDR and AR in normal prostatic tissues raises the distinct possibility of an important role for this hormone in the normal gland. In order to ascertain the possible role of 1,25 D on both AR and VDR in the normal prostate, the effects of calcitriol and dihydrotestosterone (DHT) on the normal human neonatal prostatic epithelial cell line, 267B-1, were examined. These studies were approached by focusing on how 1,25 D in the presence or absence of DHT affects the distribution of AR and VDR in the cytoplasmic and nuclear compartments of the cells in terms of their protein levels and DNA binding activities. Immunoblot analyses show that 1,25 D increases the AR protein level in both the cytoplasmic and nuclear fractions but not the VDR protein level. On the other hand, the gel shift assays demonstrate that 1,25 D increases both the AR- and VDR-DNA binding activities in the nuclear fraction, whereas there is no increase in DNA binding activities in the cytoplasmic fraction. Addition of DHT along with 1,25 D does not affect the DNA binding activities of both AR and VDR. Overall, these studies suggest that 1,25 D actions on the normal prostate cells may be mediated independently through AR and VDR, respectively.