Claudia Zierold

Regulation of 25‐hydroxyvitamin D3‐24‐hydroxylase mRNA by 1,25‐dihydroxyvitamin D3 and parathyroid hormone

The 25‐hydroxyvitamin D3‐24‐hydroxylase mRNA is tightly and reciprocally regulated by 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) and parathyroid hormone (PTH). The upregulation of the 24‐hydroxylase by 1,25(OH)2D3 is well established and occurs at the transcriptional level through two vitamin D response elements in the promoter of the gene. However, this induction is blocked by the protein synthesis inhibitor cycloheximide (CHX) indicating a protein component in the regulation pathway. CHX treatment reduced total vitamin D receptor (VDR) protein levels in cells, but reintroduction of VDR and/or retinoid X receptor protein into cells by transfection did not reduce the inhibition by CHX. This indicates that production of another transcription factor or mRNA‐stabilizing protein synthesized in response to 1,25(OH)2D3 is required for optimal accumulation of 24‐hydroxylase mRNA. PTH downregulates the 24‐hydroxylase mRNA by affecting its stability. The half‐life of 24‐hydroxylase mRNA is reduced 4.2‐fold in AOK‐B50 cells by PTH. Untranslated regions of the 24‐hydroxylase mRNA in reporter gene assays did not confer PTH responsiveness. Further analysis of the coding region of the rat 24‐hydroxylase may reveal sites of action of PTH. J. Cell. Biochem. 88: 234–237, 2003.

Lithocholic acid can carry out in vivo functions of vitamin D.

The physiological ligand for the vitamin D receptor (VDR) is 1,25-dihydroxyvitamin D3. Lithocholic acid (LCA), a bile acid implicated in the progression of colon cancer, was recently shown to bind to VDR with low affinity and increase expression of the xenobiotic enzymes of the CYP3A family. Thus, LCA can induce its own catabolism through the VDR. We have now found that LCA can substitute for vitamin D in the elevation of serum calcium in vitamin D-deficient rats. Further, LCA in the diet will also replace vitamin D in the mobilization of calcium from bone. Further, LCA induces CYP24-hydroxylase mRNA gene expression in the kidney of vitamin D-deficient rats. It is clear, therefore, that LCA can be absorbed into the circulation to bind to the VDR at extra-intestinal sites. These findings lend support for the idea that the VDR may have evolved from an original role in detoxification.

Parathyroid hormone regulates 25-hydroxyvitamin D 3 -24-hydroxylase mRNA by altering its stability

The up-regulation of the 25-hydroxyvitamin D3-24-hydroxylase by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is well established and occurs at the transcriptional level through two vitamin D response elements in the promoter of the gene. However, the mechanism of down-regulation of the 24-hydroxylase by parathyroid hormone (PTH) has not yet been elucidated. To study the mechanism of PTH action, we used AOK-B50 cells, a porcine kidney-cell line with stably transfected opossum PTH receptor in which both the 24-hydroxylase mRNA and activity are down-regulated by PTH. Cells dosed with 1,25(OH)2D3 at 0 h, and subsequently at 0, 1, 2, or 4 h with 100 nM of PTH, showed levels of 24-hydroxylase mRNA equivalent to 72.6, 65.3, 57.2, and 37.1%, respectively, of the levels found in cells dosed with 1,25(OH)2D3 only. All cells were collected 7 h after the initial 1,25(OH)2D3 dose. This pattern of expression indicated that PTH does not act by repressing transcription but rather by making the mRNA for 24-hydroxylase susceptible to degradation. At least 1 h is required for PTH to act. Further RNA and protein syntheses are required for PTH to act. However, the sites and mechanism whereby PTH causes 24-hydroxylase mRNA degradation are unknown. Because the untranslated regions of genes can determine the stability of its transcripts, we studied the 5′ untranslated region and the 3′ untranslated region of the rat 24-hydroxylase gene by using reporter-gene strategy to identify possible PTH sites of action. None was found, suggesting that the destabilization site is elsewhere in the coding region.

Dysregulation of frizzled 6 is a critical component of B cell leukemogenesis in a mouse model of chronic lymphocytic leukemia

Wnt/Fzd signaling is known to play a key role in development, tissue-specific stem-cell maintenance, and tumorigenesis, particularly through the canonical pathway involving stabilization of beta-catenin. We have previously shown that Fzd9(-/-) mice have a deficiency in pre-B cells at a stage when self-renewing division is occurring in preference to further differentiation, before light chain immunoglobulin recombination. To determine whether pathologic usurpation of this pathway plays a role in B-cell leukemogenesis, we examined the expression of Wnt/Fzd pathway genes in the Emu-TCL1 mouse model of chronic lymphocytic leukemia. We find that, in the course of leukemogenesis, the expression of Wnt16, Wnt10alpha, Fzd1, and most dramatically, Fzd6, is progressively up-regulated in the transformed CD5(+) B cells of these mice, as are beta-catenin protein levels. Elimination of Fzd6 expression by crossing into Fzd6(-/-) mice significantly delays development of chronic lymphocytic leukemia in this model. Our findings suggest that the self-renewal signals mediated by Wnt/Fzd that are enlisted during B-cell development may be pathologically reactivated in the neoplastic transformation of mature B cells.

19nor-1,25-dihydroxyvitamin D2 specifically induces CYP3A9 in rat intestine more strongly than 1,25-dihydroxyvitamin D3 in vivo and in vitro

In the intestine, the vitamin D receptor is activated by 1α, 25-dihydroxyvitamin D3 [1,25(OH)2D3] to perform its function in calcium homeostasis, or it is activated by lithocholic acid when its levels are elevated after a meal. Both ligands transcriptionally up-regulate the mRNA of enzymes belonging to the CYP3A subfamily, increasing the metabolism of a variety of carcinogens, drugs, and hormones. Of the cytochrome P450 enzymes, the CYP3A subfamily is the most abundant in liver and intestine and has the widest range of substrate specificity. In addition to being a ligand for the vitamin D receptor, lithocholic acid is also a substrate for CYP3A enzymes. Lithocholic acid causes colon cancer; thus, decreasing lithocholic acid levels in the intestine by up-regulating CYP3A enzymes with 1,25(OH)2 D3 analogs may have therapeutic value in the prevention of colon cancer. We investigated the induction of CYP3A9 by 1,25(OH)2D3 and 19nor-1α,25-dihydroxyvitamin D2[19nor-1,25(OH)2 D2]. We observed the that latter analog, currently used to treat renal osteodystrophy, is more efficacious than 1,25(OH)2 D3 in inducing CYP3A9 in rat intestines. CYP3A9 mRNA was maximally elevated 5 to 7 h after a single dose of 1,25(OH)2 D3 to rats and then gradually returned to baseline. We performed promoter deletion analysis of the rat CYP3A9 promoter and identified one proximal vitamin D response element located at -119 to -133 from the transcriptional start site, which is responsible for a large part of the 1,25(OH)2D3 response, and two other vitamin D response elements located at -726 to -744 and at -754 to -776, which together are responsible for the increased sensitivity of CYP3A9 to 19nor-1,25(OH)2D2.

Protein synthesis is required for optimal induction of 25-hydroxyvitamin D3-24-hydroxylase, osteocalcin, and osteopontin mRNA by 1,25-dihydroxyvitamin D3

The regulation of the 25-hydroxyvitamin D(3)-24-hydroxylase gene by 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) has been extensively studied. It is well established that two vitamin D response elements in the promoter are responsible for the 1,25(OH)(2)D(3) induction of transcription. Surprisingly, this induction is blocked by the protein synthesis inhibitor, cycloheximide (CHX). In AOK-B50 cells, 1,25(OH)(2)D(3) caused a large induction of 24-hydroxylase mRNA by 7h; however, the addition of CHX simultaneously or 2h after 1,25(OH)(2)D(3) addition caused 76.4+/-13.0 and 37.1+/-18.8% reductions in the mRNA, respectively. Addition of CHX 4h after 1,25(OH)(2)D(3) had the opposite effect, and 21.7+/-17.2% more mRNA was observed after 7h. Similar patterns of mRNA expression were observed in other cell lines. CHX also decreased the induction by 1,25(OH)(2)D(3) of osteocalcin and osteopontin mRNA in ROS17/2.8 cells when added together with 1,25(OH)(2)D(3). The effect of CHX on the expression of a stably transfected luciferase construct under the control of 1400bp of 24-hydroxylase promoter indicates that a 1,25(OH)(2)D(3)-inducible transcription factor(s) that acts in the promoter region may at least in part be responsible for the effect of CHX on mRNA production of target genes.

Isolation and characterization of the chicken vitamin D receptor gene and its promoter.

The sequences from several independent cDNA clones encoding the chicken vitamin D receptor as well as primer extension assay have clearly delineated the 5′ terminus and the transcriptional start site. Screening a chicken genomic library produced genomic clones containing vitamin D receptor (VDR) gene fragments. Restriction map of clone 8 showed that the 18.6‐kb chicken VDR fragment has exons 1 and 2, intron 1, part of intron 2, and 7‐kb 5′ flanking region. Exons 1, 2 , and 3 found in the chicken VDR gene shares low homology with its mammalian counterparts (i.e., E1A, E1B, and E1C in human). By contrast, the fourth exon and following exons for the coding region of VDR gene are highly conserved between avian and mammalian species. While the fourth exon bears the ATG sites for translation initiation in mammals, the third exon in birds has two extra ATG sites for leaky translation as determined previously. Thus, the avian VDR has more N‐terminal sequence than the mammalian VDR and is found in two distinct forms. The 5′ flanking region from genomic clone 8 shares considerable homology in several regions with the human and mouse VDR promoters. Moreover, the 5′ flanking region of chicken VDR gene possesses promoter activity, as shown by its ability to drive the luciferase reporter gene in cell transfection assays. Like other steroid receptor promoters, the chicken VDR promoter contains no TATA box but possesses several GC boxes or SP1 sites. A series of deletional promoter constructs established that the proximal GC boxes are the major drivers of gene transcription, while the more upstream sequences have repressive elements. J. Cell. Biochem. 77:92–102, 2000.

Additional protein factors play a role in the formation of VDR/RXR complexes on vitamin D response elements

The vitamin D receptor (VDR) elicits a transcriptional response to 1,25‐dihydroxyvitamin D3 by binding to specific response elements (VDRE) in the promoter of target genes. Retinoic X receptor (RXR) is required for formation of the VDR‐VDRE complex when VDR is supplied at physiologic concentrations. When porcine intestinal nuclear extract is used as a source of VDR, two distinct complexes are always observed with native gel electrophoresis. Both complexes contain VDR and RXR. We now show that the faster‐migrating complex requires another heretofore unknown nuclear factor for its formation. In addition, we provide evidence that the formation of the slower‐migrating complex is enhanced by transcription factor IIB (TFIIB). Using ligand binding assays, we determined that both complexes contain the same ratio of VDR to VDRE. Using RXR subtype‐specific antibodies in gel shift assays, we show that the complexes contain more than one RXR subtype. Therefore, the present results demonstrate VDR‐RXR‐VDRE complexes formed with pig intestinal nuclear extracts contain other proteins and that the complexes formed between VDR and VDRE are not simply heterodimers of VDR and RXR. J. Cell. Biochem. 71:515–523, 1998.

Vitamin D Metabolism in Normal and Chronic Kidney Disease States

Vitamin D is a prohormone synthesized in the skin from the precursor molecule 7-dehydrocholesterol by the action of sunlight. It is found in low amounts in food, with fortified dairy and fish oils being the most abundant source. Vitamin D undergoes an important 2-step bio-activation process required to produce the active metabolite 1,25-dihydroxyvitamin D (1,25(OH)2D). The bio-activation process comprises the synthesis of 25-hydroxyvitamin D in the liver by 25-hydroxylation, followed by the conversion to 1,25(OH)2D by the 1α-hydroxylase in kidney under very tightly regulated physiological conditions. 1,25(OH)2D is responsible for maintaining adequate levels of calcium and phosphorus in the blood. Calcium is essential for muscles and nervous system functions, and through the actions of 1,25(OH)2D on intestine, kidney, and bone, the body prevents imbalances of both calcium and phosphate via an intricate system. In addition, 1,25(OH)2D plays an important role in many biological non-calcemic functions throughout the body. 1,25(OH)2D must bind to the vitamin D receptor to carry out its functions. The highly active and lipid soluble 1,25(OH)2D is inactivated by the 24-hydroxylase, which is the enzyme responsible for the major catabolic pathway that ultimately results in the water soluble calcitroic acid for excretion in the urine. Regulation of key players in vitamin D metabolism is reciprocal and very tight. The activating enzyme 1α-hydroxylase, and the catabolic enzyme 24-hydroxylase are reciprocally regulated by PTH, 1,25(OH)2D, and FGF23. Chronic kidney disease is associated with abnormalities of phosphorus homeostasis and altered vitamin D metabolism, and if left untreated, result in significant morbidity and mortality.

General Principles of Vitamin D Action and Mechanism-Based Search for Analogs with Specific Actions

There is no doubt that vitamin D which is normally formed in skin or obtained in the diet must be altered before it can carry out its functions (DeLuca 1974, 1988). First, 25-hydroxylation is carried out in the microsomes and mitochondria of liver to produce the circulating form of vitamin D 25-hydroxyvitamin D3 (25-OH-D3). Secondly, 1α-hydroxylation produces the final hormonal or active form 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). In normal humans and animals, this occurs exclusively in the proximal convoluted tubule cells of the kidney, with the notable exception of the placenta (DeLuca 1974, 1988). There is abundant evidence to support the idea that 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) is the metabolically active form of vitamin D (DeLuca 1974, 1988). It is believed to carry out both the classical functions of the vitamin and some of the more recently found functions in differentiation and development and in suppression of the parathyroid glands (Darwish and DeLuca 1993; DeLuca 1988, 1992). Furthermore, new and unknown functions of vitamin D will likely be discovered, as, for example, in the female reproductive system (Halloran and DeLuca 1980; Kwiecinski et al. 1989), in the islet cells of the pancreas (Chertow et al. 1983), and in the keratinocytes of skin (Smith et al. 1986). This list will probably become longer and will include abnormal sites such as in malignant tissue that contains significant amounts of vitamin D receptor (VDR) (Eisman 1984).