On the specificity of vitamin D compounds binding to chick pig intestinal 1,25-dihydroxyvitamin D3 receptor

The binding activity of four vitamin D metabolites and/or analogs for the intestinal 1,25-dihydroxyvitamin D3 receptor was evaluated after incubation at 25 degrees C for 1 h or at 0-4 degrees C for 18 h. The incubation conditions, which had no effect on the binding of 1,25-dihydroxyvitamin D3, had a dramatic effect on the binding of 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3 and a small but reproducible effect on 24,25-dihydroxyvitamin D3 binding to receptor. Affinities 10- to 20-fold higher were obtained for 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3, and affinities 3-fold higher were obtained for 24,25-dihydroxyvitamin D3 at the 0-4 degrees C/18-h incubation. A comparison of intestinal receptor from chick and pig with nine vitamin D compounds showed no major differences between the two species. The relative affinity of the vitamin D analogs to compete with tritiated 1,25-dihydroxyvitamin D3 for the receptor in pig nuclear extract, expressed as ratios of the molar concentration required for 50% binding of the tritiated 1,25-dihydroxyvitamin D3 compared to nonradioactive 1,25-dihydroxyvitamin D3, are as follows: 1,25-dihydroxyvitamin D3 (1) = 1,25-dihydroxyvitamin D2 = 24-homo-1,25-dihydroxyvitamin D3 greater than 1,24,25-trihydroxyvitamin D3 (4) greater than 25-hydroxyvitamin D3 (21) = 10-oxo-19-nor-25-hydroxyvitamin D3 = 1 alpha-hydroxyvitamin D3 (37) greater than 24,25-dihydroxyvitamin D2 (257) much much greater than vitamin D3 (greater than 10(6)).     

ZK 156718, a low calcemic, antiproliferative, and prodifferentiating vitamin D analog.

The physiologically active form of vitamin D, 1,25-dihydroxyvitamin D(3), plays an important role not only in the establishment and maintenance of calcium metabolism, but also in regulating cell growth and differentiation. Because the clinical usefulness of 1,25-dihydroxyvitamin D(3) is limited by its tendency to cause hypercalcemia, new analogs with a better therapeutic profile have been synthesized, including ZK 156718. We compared the effects of 1,25-dihydroxyvitamin D(3) and ZK 156718 on growth, differentiation, and on p21(Waf1/Cip1) and p27(Kip1) expression in human colon cancer cells (Caco-2). Whereas ZK 156718 at the concentration [10(-8) M] was as potent as 10(-6) M 1,25-dihydroxyvitamin D(3) in inducing differentiation and p21(Waf1/Cip1) expression, it was even more effective in inhibiting cell growth and stimulating p27(Kip1) expression than 1,25-dihydroxyvitamin D(3) itself. In summary, our study presents a new and potent vitamin D analog with a decreased metabolic stability, making it useful for the treatment of a diversity of clinical disorders.     

25-hydroxyvitamin D3-1alpha-hydroxylase expression in normal and malignant human colon.

1,25-dihydroxyvitamin D(3) 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 D(3)-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 D(3) could prevent intestinal tumor formation and progression.     

Regulation of the vitamin D receptor and cornifin beta expression in vaginal epithelium of the rats through vitamin D3

The aim of the present study was to determine the respective role of 1,25-dihydroxyvitamin D3 on vaginal epithelium and 1,25-dihydroxyvitamin D3 receptor expression in ovariectomized rats and vitamin D3 treated rats. Bilateral ovariectomies were performed in 20 mature, non-pregnant Wistar female rats. All the animals were divided into 2 groups consisting of 10 rats each. Group I served as control. In group II, animals were injected intramuscularly with vitamin D3 (50, 00 IU/kg). Two weeks after the injections, vaginas of animals in group I and group II were removed removed and processed for immunohistochemistry. Epithelial differentiation, 1,25-dihydroxyvitamin D3 receptor and cornifin beta expression were investigated in vaginal epithelium of control group (ovariectomized) and vitamin D3 treated rats. Vaginal epithelial cells from vitamin D3 treated animals changed into highly- stratified keratinizing layers. 1,25-dihydroxyvitamin D3 receptor and cornifin beta as a marker of squamous differentiation were present in ovariectomized rats treated with 1,25-dihydroxyvitamin D3. In contrast, cornifin beta and 1,25-dihydroxyvitamin D3 receptor were absent in all layers of vaginal epithelium in control group. We demonstrated for the first time that 1,25-dihydroxyvitamin D3 induced proliferation of vaginal epithelium consistent with the cornifin beta expression and 1,25-dihydroxyvitamin D3 up-regulated 1,25-dihydroxyvitamin D3 receptor expression in vaginal epithelium.     

INK4 cyclin-dependent kinase inhibitors as potential prognostic biomarkers and therapeutic targets in hepatocellular carcinoma

The INK4 family is an important family of cyclin-dependent kinase inhibitors (CDKIs) and consists of CDKN2A, CDKN2B, CDKN2, and CDKN2D. Abnormal expression of CDKN2A has been reported in hepatocellular carcinoma (HCC) and is associated with the prognosis of patients and infiltration of immune cells. However, there is a lack of systematic research on the roles of the other INK4 family members in the diagnosis, prognosis, and immune regulation of HCC. Using online public databases and clinical samples, we comprehensively analyzed the INK4 family in HCC. All four INK4 proteins were overexpressed in HCC and correlated with advanced cancer stage and poor prognosis. INK4 expression accurately distinguished tumor from normal tissue, particularly CDKN2A and CDKN2C. The INK4 family participated in cell-cycle regulation and the DNA damage repair pathway, which inhibited genotoxic-induced apoptosis in tumorigenesis. INK4 proteins were positively correlated with the infiltration of immune cells (B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells) and immune checkpoints (CTLA-4, PD1, and PD-L1). CDKN2D had the highest correlation (correlation coefficient >0.3) with all the above-mentioned infiltrating immune cells and immune checkpoints, indicating that it may be useful as an immunotherapy target. The INK4 family was valuable for diagnosis and predicting the prognosis of HCC and participated in the occurrence, progression, and immune regulation of HCC, demonstrating its potential as a diagnostic and prognostic biomarker and therapeutic target in HCC.     

Depletion of histone demethylase KDM2A inhibited cell proliferation of stem cells from apical papilla by de-repression of p15INK4B and p27Kip1

Mesenchymal stem cells (MSCs) are a reliable resource for tissue regeneration; although, the molecular mechanisms of their differentiation and proliferation are not clearly understood, which restricts the applications of MSCs. The histone demethylase, lysine (K)-specific demethylase 2A (KDM2A), and the mammalian paralog, lysine (K)-specific demethylase 2B (KDM2B), are evolutionarily conserved and ubiquitously expressed members of the JmjC-domain-containing histone demethylase family. A previous study determined that KDM2A and KDM2B can regulate the differentiation of MSCs, and KDM2B has been implicated in cell cycle regulation by de-repressing p15^INK4B (cyclin-dependent kinase inhibitor 2B). It is not known whether KDM2A is involved in the cell proliferation of MSCs. Here, we show that depletion of KDM2A by short hairpin RNAs can inhibit cell proliferation and arrest cell cycle progression at the G1/S-phase in human stem cells from apical papilla (SCAPs). The effect of KDM2A on cell proliferation was found to be mediated through de-repression of the cyclin-dependent kinase inhibitors, p15^INK4B and p27^Kip1 (cyclin-dependent kinase inhibitor 1B), in KDM2A knock-down SCAPs. Furthermore, chromatin immunoprecipitation assays demonstrated that silencing of KDM2A increased histone H3 Lysine 4 (H3K4) trimethylation at the p15^INK4B and p27^Kip1 loci and regulated its expression. Together, our results indicate that KDM2A is a H3K4 demethylase that regulates cell proliferation through p15^INK4B and p27^Kip1 in SCAPs.     

Effects of 1,25-dihydroxyvitamin D3 on proliferation and differentiation of porcine preadipocyte in vitro

1,25-Dihydroxyvitamin D3, the physiologically active form of vitamin D3, exerts its functions through a receptor-mediated mechanism and plays an important role in the cell differentiation. This study investigated the effects of 1,25-dihydroxyvitamin D3 on the proliferation and differentiation of porcine preadipocyte. Stromal-vascular cells containing preadipocytes were prepared from dorsal subcutaneous adipose tissue of approximately 3-day-old Chinese male crossbred pigs. After confluence, the differentiation was induced by transferrin, dexamethasone and insulin for 2 days, and then subsequently cultured for 6 days. The cells were treated with 1,25-dihydroxyvitamin D3 during the induction of differentiation (the early phase of differentiation) or throughout the differentiation period. The terminal differentiation markers, such as glycerol-3-phosphate dehydrogenase activity and lipid accumulation were measured during the process of cultures. The treatment with 1,25-dihydroxyvitamin D3 severely affected the induction of all differentiation markers throughout the differentiation period. 1,25-Dihydroxyvitamin D3 suppressed the expression of peroxisome proliferator-activated receptor gamma mRNA and interfered with the induction of retinoid X receptor alpha mRNA. The mRNAs of the adipogenesis-related genes, lipoprotein lipase, stearoyl-CoA desaturase, phosphoenolpyruvate carboxykinase, glycerol-3-phosphate dehydrogenase and glucose transporter 4 were reduced when 1,25-dihydroxyvitamin D3 was added into differentiation medium. Also, 1,25-dihydroxyvitamin D3 inhibited preadipocyte differentiation in dose-dependent manner. These results suggested that 1,25-dihydroxyvitamin D3 inhibited porcine preadipocyte differentiation through suppressing PPAR gamma and RXR alpha mRNA expressions and then down regulating the expression of adipogenesis-related genes.     

1,25-Dihydroxyvitamin D3-stimulated mRNAs in rat small intestine

The technique of differential hybridization has been employed to study gene expression associated with vitamin D action on the mammalian intestine. A cDNA library consisting of 10(6) independent recombinants was constructed from poly(A)+ RNA extracted from vitamin D-deficient rats given 1,25-dihydroxyvitamin D3. A survey of 20,000 clones resulted in identification of four distinct cDNAs whose corresponding mRNAs are significantly increased 12 h after an intrajugular dose of 1,25-dihydroxyvitamin D3 given to vitamin D-deficient rats. DNA sequence analysis identified these mRNAs as mitochondrial ATP synthetase, vitamin D-dependent calcium binding protein, cytochrome oxidase subunit I, and cytochrome oxidase subunit III. The time course of response of three of these mRNAs was similar, with maximum values at 12 h after dosing, while that of cytochrome oxidase subunit I showed two peaks at 6 and 18 h following a single dose of 1,25-dihydroxyvitamin D3. The levels of all four mRNAs were elevated in rats supplied with vitamin D when hypocalcemia was produced by dietary calcium restriction.     

Adenovirus-mediated overexpression of p15INK4B inhibits human glioma cell growth, induces replicative senescence, and inhibits telomerase activity similarly to p16INK4A.

The genes encoding the cyclin-dependent kinase inhibitors p16INK4A (CDKN2A) and p15INK4B (CDKN2B) are frequently homozygously deleted in a variety of tumor cell lines and primary tumors, including glioblastomas in which 40-50% of primary tumors display homozygous deletions of these two loci. Although the role of p16 as a tumor suppressor has been well documented, it has remained less well studied whether p15 plays a similar growth-suppressing role. Here, we have used replication-defective recombinant adenoviruses to compare the effects of expressing wild-type p16 and p15 in glioma cell lines. After infection, high levels of p16 and p15 were observed in two human glioma cell lines (U251 MG and U373 MG). Both inhibitors were found in complex with CDK4 and CDK6. Expression of p16 and p15 had indistinguishable effects on U251 MG, which has homozygous deletion of CDKN2A and CDKN2B, but a wild-type retinoblastoma (RB) gene. Cells were growth-arrested, showed no increased apoptosis, and displayed a markedly altered cellular morphology and repression of telomerase activity. Transduced cells became enlarged and flattened and expressed senescence-associated beta-galactosidase, thus fulfilling criteria for replicative senescence. In contrast, the growth and morphology of U373 MG, which expresses p16 and p15 endogenously, but undetectable levels of RB protein, were not affected by exogenous overexpression of either inhibitor. Thus, we conclude that overexpression of p15 has a similar ability to inhibit cell proliferation, to cause replicative senescence, and to inhibit telomerase activity as p16 in glioma cells with an intact RB protein pathway.     

Differential effects of Vitamin D analogs on calcium transport

It is well known that the efficiency of intestinal active calcium transport is regulated by the Vitamin D receptor pathway and Vitamin D analogs seem to exhibit differential effects on intestinal active calcium transport. To investigate the molecular basis for the difference among Vitamin D analogs, we tested three Vitamin D analogs: 1,25-dihydroxyvitamin D(3), 19-nor-1,25-dihydroxyvitamin D(2), and 1alpha-hydroxyvitamin D(2) ex vivo and in vitro. In 5/6 nephrectomized rat intestinal active calcium transport, 19-nor-1,25-dihydroxyvitamin D(2) did not show a significant effects on intestinal active calcium transport at all the concentrations tested, while 1alpha-hydroxyvitamin D(2) at 0.33 and 0.67 microg/kg and 1,25-dihydroxyvitamin D(3) at 1microg/kg significantly stimulated calcium transport. In Caco-2 cells, 19-nor-1,25-dihydroxyvitamin D(2) did not show a significant effect on calcium transport, while 1,25-dihydroxyvitamin D(3) and 1,25-dihydroxyvitamin D(2) (the active form of 1alpha-hydroxyvitamin D(2)) stimulated calcium transport by 934 and 501% at 0.1microM, respectively. 1,25-Dihydroxyvitamin D(2) potently induced the expression of CALB3 and TRPV6 mRNA with an EC(50) of 0.3 and 1.0nM, whereas 19-nor-1,25-dihydroxyvitamin D(2) was 10-fold less potent than 1,25-dihydroxyvitamin D(2) in inducing CALB3 and TRPV6 mRNA. The three Vitamin D analogs had no significant effect on the expression of PMCA1 mRNA. These Vitamin D analogs did not change the expression of Vitamin D receptor (VDR) up to 10nM, but stimulated CYP24A1 expression in a dose-dependent manner with the potency in the order of 1,25-dihydroxyvitamin D(3)>1,25-dihydroxyvitamin D(2)=19-nor-1,25-dihydroxyvitamin D(2). These results suggest that the differential effect of Vitamin D analogs on stimulating intestinal and Caco-2 calcium transport may be in part due to its different effect on stimulating CALB3 and TRPV6 mRNA expression.     

Isolation and identification of 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3, a new metabolite of 1,25-dihydroxyvitamin D3 produced in rat kidney

A new metabolite of vitamin D3 was produced in vitro by perfusing rat kidneys with 1,25-dihydroxyvitamin D3 (4 X 10(-6) M). It was isolated and purified from the lipid extract of the kidney perfusate by high-performance liquid chromatography. By means of ultraviolet absorption spectrophotometry, mass spectrometry, chemical derivatization, and chemical synthesis, the new metabolite was identified as 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3. Along with the new metabolite, three other previously identified metabolites, namely, 1,24,25-trihydroxyvitamin D3, 1,25-dihydroxy-24-oxovitamin D3, and 1,23,25-trihydroxy-24-oxovitamin D3, were also isolated. The new metabolite was also formed when 1,23,25-trihydroxy-24-oxovitamin D3 was used as the substrate. Thus, the new metabolite fits into the following metabolic pathway: 1,25-dihydroxyvitamin D3----1,24(R),25-trihydroxyvitamin D3----1,25-dihydroxy-24-oxovitamin D3----1,23,25-trihydroxy-24-oxovitamin D3----1,23-dihydroxy-24,25,26,27-tetranorvitamin D3. Further, we used 1 alpha,25-dihydroxy[1 beta-3H]vitamin D3 in the kidney perfusion system and demonstrated 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 as the major further metabolite of 1,25-dihydroxyvitamin D3, circulating in the final perfusate when kidneys were perfused with 1,25-dihydroxyvitamin D3 (6 X 10(-10) M) for 4 h. The biological activity of 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 (C-3 alcohol) and its metabolic relationship to 1-hydroxy-23-carboxy-24,25,26,27-tetranorvitamin D3 (calcitroic acid or C-23 acid), the other previously identified side-chain cleavage metabolite of 1,25-dihydroxyvitamin D3, are unknown and are presently undergoing investigation.     

Convergence of vitamin D and retinoic acid signalling at a common hormone response element

Although 1,25-dihydroxyvitamin D3 (1,25D3) and retinoic acid (RA) have distinct developmental and physiological roles, both regulate the cell cycle. We provide molecular and genomic evidence that their cognate nuclear receptors regulate common genes through everted repeat TGA(C/T)TPyN8PuG(G/T)TCA (ER8) response elements. ER8 motifs were found in the promoters of several target genes of 1,25D3 and/or RA. Notably, an element was characterized in the cyclin-dependent kinase (CDK) inhibitor p19ink4d gene, and 1,25D3- or RA-induced p19INK4D) expression. P19ink4d knockdown together with depletion of p27kip1, another CDK inhibitor regulated by 1,25D3 and RA, rendered cells resistant to ligand-induced growth arrest. Remarkably, p19INK4D-deficient cells showed increased autophagic cell death, which was markedly enhanced by 1,25D3, but not RA, and attenuated by loss of p27KIP1. These results show a limited crosstalk between 1,25D3 and RA signalling by means of overlapping nuclear receptor DNA binding specificities, and uncover a role for p19INK4D in control of cell survival.     

End product inhibition of hepatic 25-hydroxyvitamin D production in the rat: specificity and kinetics

The role of vitamin D metabolites in the regulation of hepatic 25-hydroxyvitamin D production was investigated by examining the effects of 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, and 24,25-dihydroxyvitamin D on the synthesis of [25-3H]hydroxyvitamin D by rachitic rat liver homogenates. Production of [25-3H]hydroxyvitamin D was inhibited by 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, but not by 24,25-dihydroxyvitamin D. 25-Hydroxyvitamin D increased the Km of the vitamin D-25-hydroxylase enzyme(s), while 1,25-dihydroxyvitamin D decreased the Vmax with a Ki of 88.7 ng/ml. Inhibition of hepatic 25-hydroxyvitamin D production by 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D may be another control mechanism to regulate circulating vitamin D levels.     

Up-regulation of the intestinal 1,25-dihydroxyvitamin D receptor during hypervitaminosis D: a comparison between vitamin D2 and vitamin D3

Concentrations of intestinal 1,25-dihydroxyvitamin D receptor were measured in rats receiving pharmacological amounts (25,000 IU/rat daily for 6 days) of either vitamin D2 or vitamin D3. The data showed that both hypervitaminosis D2 and hypervitaminosis D3 resulted in significant up-regulation of intestinal 1,25-dihydroxyvitamin D receptor (fmol/mg protein) relative to controls (409 +/- 24, vitamin D2-treated; 525 +/- 41, vitamin D3-treated; and 249 +/- 19, control). The 1,25-dihydroxyvitamin D receptor enhancement also was accompanied by elevated plasma 25-hydroxyvitamin D and hypercalcemia. These data suggest that increased target-tissue 1,25-dihydroxyvitamin D receptor may play a role in enhancing target-tissue responsiveness and, thus, have a significant role in mediating the toxic effects of hypervitaminosis D.