J. Wesley Pike

Jun-Fos and receptors for vitamins A and D recognize a common response element in the human osteocalcin gene

We present evidence that the vitamin D response element in the human osteocalcin gene confers responsiveness to the vitamin A metabolite, retinoic acid. Retinoic acid receptor (RAR) expressed in E. coli binds to this sequence in vitro. Transfection of RAR expression vectors in cultured cells activates heterologous promoters containing this sequence in vivo. This response element contains a consensus AP-1 site TGACTCA and in vitro is bound by the Jun-Fos complex. Unexpectedly, cotransfection of Jun and Fos expression vectors suppresses basal level transcription of the osteocalcin gene and suppresses induction by both retinoic acid and vitamin D3. Additional studies delimit an 11 nucleotide segment as a minimal hormone response element containing the AP-1 site as its core. These results indicate that two distinct classes of transcription factors can recognize common regulatory sequences, a phenomenon we refer to as cross-coupling.

The caudal-related homeodomain protein Cdx-2 regulates vitamin D receptor gene expression in the small intestine

The actions of 1,25‐dihydroxyvitamin D3 (1,25(OH)2 D3) are mediated through the nuclear vitamin D receptor (VDR). The regulation of VDR abundance plays an important role in determining the magnitude of the target cell response to 1,25(OH)2D3. The major physiological activity of 1,25(OH)2D3 is the regulation of calcium absorption in the small intestine, and the level of VDR is an important factor in this regulation. However, the characterization of VDR gene expression in the small intestine remains unknown. In the present study, we investigated the regulation of the human VDR (hVDR) gene expression in the small intestine. The 4.0 kb of the 5′‐flanking region of the hVDR gene promoter was cloned and characterized by the measurement of luciferase activity and an electrophoretic mobility‐shift assay (EMSA). With the EMSA, we found that Cdx‐2 (a homeodomain protein‐related caudal) binds to the sequence 5′‐ATAAAAACTTAT‐3′ at −3731 to −3720 bp (hVD‐SIF1) relative to the transcription start site of the hVDR promoter. This sequence is very similar to the human sucrase‐isomaltase footprint 1 (SIF1) element. With a competition analysis and specific antibodies for Cdx‐2, we demonstrated that Cdx‐2 is able to activate VDR gene transcription by binding to this element. The mutation of the hVD‐SIF1 sequence in the hVDR gene promoter markedly suppressed the transactivation of the reporter gene in Caco‐2 cells. In addition, the DNA fragment (−3996 to −3286) containing the hVD‐SIF1 binding site increased transcription when placed upstream of the herpes simplex virus thymidine kinase promoter. These findings suggest that Cdx‐2 plays an important role in the intestine‐specific transcription of the hVDR gene.

Biochemical evidence for 1,25-dihydroxyvitamin D receptor macromolecules in parathyroid, pancreatic, pituitary, and placental tissues.

DNA-cellulose chromatography has been recently employed in our laboratory as an extremely effective and sensitive technique with which to identify macromolecules which specifically bind 1,25-dihydroxyvitamin D (1,25-(OH)2D). Chromatography of cytosols prepared from rachitic chick intestine, parathyroid gland, pancreas, pituitary gland, and normal rat placenta all demonstrate binding components for 1,25-(OH)2D which interact with the DNA affinity ligand under low salt conditions (< 0.15M), and can be eluted as a single macromolecular peak during a linear KCl gradient between 0.25–0.30M. Further, sucrose gradient analysis of these DNA-cellulose purified components under high salt conditions (0.3M KCl) indicates a common sedimentation coefficient of 3.3S. Since the receptor properties of this macromolecule in rachitic chick intestine have been previously characterized, it seems likely that these components in the parathyroid, pituitary, pancreas, and placenta represent typical receptors for 1,25-(OH)2D.

IL-4 inhibits osteoclast formation through a direct action on osteoclast precursors via peroxisome proliferator-activated receptor γ1

IL-4 is a pleiotropic immune cytokine secreted by activated TH2 cells that inhibits bone resorption both in vitro and in vivo. The cellular targets of IL-4 action as well as its intracellular mechanism of action remain to be determined. We show here that IL-4 inhibits receptor activator of NF-κB ligand-induced osteoclast differentiation through an action on osteoclast precursors that is independent of stromal cells. Interestingly, this inhibitory effect can be mimicked by both natural as well as synthetic peroxisome proliferator-activated receptor γ1 (PPARγ1) ligands and can be blocked by the irreversible PPARγ antagonist GW 9662. These findings suggest that the actions of IL-4 on osteoclast differentiation are mediated by PPARγ1, an interpretation strengthened by the observation that IL-4 can activate a PPARγ1-sensitive luciferase reporter gene in RAW264.7 cells. We also show that inhibitors of enzymes such as 12/15-lipoxygenase and the cyclooxygenases that produce known PPARγ1 ligands do not abrogate the IL-4 effect. These findings, together with the observation that bone marrow cells from 12/15-lipoxygenase-deficient mice retain sensitivity to IL-4, suggest that the cytokine may induce novel PPARγ1 ligands. Our results reveal that PPARγ1 plays an important role in the suppression of osteoclast formation by IL-4 and may explain the beneficial effects of the thiazolidinedione class of PPARγ1 ligands on bone loss in diabetic patients.

Hormone-dependent phosphorylation of the 1,25-dihydroxyvitamin D3 receptor in mouse fibroblasts.

Experimental results, employing several immunologic techniques, suggest that the mouse receptor for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) undergoes hormone-dependent phosphorylation in intact cells. Treatment of monolayer cultures of mouse 3T6 fibroblasts with 1,25(OH)2D3 reveals that the occupied 1,25(OH)2D3 receptor displays a minor reduction in electrophoretic mobility as compared to its unoccupied 54,500 dalton counterpart, a change consistent with covalent modification. Similar results were obtained by immunoprecipitation of metabolically-labeled receptors after incubation of 3T6 cells with [35S]methionine. This technique also provided greater insight into the precursor-product relationship between the two receptor forms. [32P]Orthophosphate-labeling of 3T6 cells, followed by immunoprecipitation indicated that only the form exhibiting covalent modification was phosphorylated. The temporal correspondence between the binding of 1,25(OH)2D3 to its cellular receptor and its phosphorylation suggests that the biochemical role of 1,25(OH)2D3 may be to induce a conformational change susceptible to phosphorylation and possibly functional activation.

Analysis of osteocalcin expression in transgenic mice reveals a species difference in vitamin D regulation of mouse and human osteocalcin genes

A line of transgenic mice expressing a human osteocalcin genomic fragment (hOClocus) and a murine MC3T3‐E1 cell line containing a stably integrated human osteocalcin promoter construct have been developed to characterize the osteogenic and hormonal regulation of human osteocalcin in vivo and in vitro. In this study, we used these models to demonstrate a species difference in the regulation of the mouse and human osteocalcin genes by vitamin D. Repeated administration of 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) to mice carrying the hOClocus transgene resulted in striking increases in serum human osteocalcin, whereas serum mouse osteocalcin levels were unchanged after 24 h and only modestly increased 48 h after the second dose of hormone. 1,25(OH)2D3 increased human calvarial mRNA expression by 1.8‐fold and slightly decreased mouse osteocalcin mRNA levels by approximately 1.2‐fold. Furthermore, treatment of primary calvarial osteoblasts from these mice with 1,25(OH)2D3 increased human osteocalcin production but inhibited mouse osteocalcin protein accumulation. To investigate further the mechanism for the apparent species difference in vitamin D3 induction of mouse and human osteocalcin, we examined the effect of 1,25(OH)2D3 in an MC3T3‐E1 cell line (MC4) containing a stably integrated 3900 bp osteocalcin promoter‐luciferase construct. Treatment of MC4 cells with ascorbic acid resulted in parallel increases of the endogenous mouse osteocalcin protein and luciferase reporter activity over a 12‐day period. Continuous exposure of MC4 cells to 1,25(OH)2D3 resulted in time‐and dose‐dependent increases in the activity of the phOC3900 luciferase construct. By contrast, the hormone had no effect on mouse osteocalcin protein concentrations and inhibited its induction by ascorbic acid. However, when cells were treated acutely with 1,25(OH)2D3 at later times during growth in ascorbic acid, the induction of mouse osteocalcin protein was only partially inhibited. In conclusion, our results indicate that common osteogenic signals regulate both mouse and human osteocalcin gene expression, but the mouse gene is resistant to induction by vitamin D. This species difference in vitamin D regulation of osteocalcin appears to result from the failure of 1,25(OH)2D3 to transcriptionally activate the mouse osteocalcin gene.

An improved radioreceptor assay for 1,25-dihydroxyvitamin D in human plasma

Abstract We describe a modified assay technique for quantitating 1,25-dihydroxyvitamin D in plasma. The method involves a rapid extraction of the hormone using minicolumn (made of granular diatomaceous earth) chromatography followed by single-step purification on high-performance liquid chromatography. Quantitation of plasma 1,25-dihydroxyvitamin D is achieved by a radioligand receptor assay employing lyophilized cytosolic receptor protein from chick intestine and high-specific-activity 1,25-dihydroxy[3H]vitamin D3 (166 Ci/mmol). A new incubation medium including an ethanol extract of vitamin D-deficient chick serum yields high specific binding and improves the precision of the radioassay. Bound and free hormone are separated with dextran-coated charcoal of equivalent particle size. The method is sensitive to 0.5 pg/tube with a practical detection range of 1–20 pg/tube, permitting duplicate assay of endogenous 1,25-dihydroxyvitamin D in plasma volumes as small as 0.5 ml. The intra- and interassay coefficient of variation are 5 and 9%, respectively, and the method is valid over a wide-range sample dilution. This assay technique was applied to the measurement of plasma 1,25-dihydroxyvitamin D hormone concentration in normal young adults (55.2 ± 13.6 pg/ml; n = 20) and in patients with chronic renal failure (13.5 ± 5.2 pg/ml; n = 9) and primary hyperparathyroidism (83.3 ± 18 pg/ml; n = 10).

The vitamin d receptor: A primitive steroid receptor related to thyroid hormone receptor

We have previously reported the cloning and sequencing of both the chicken and human vitamin D3 receptor cDNAs. A comparison of their deduced amino acid sequence with that of the other classic steroid hormone receptors and the receptor for thyroid hormone indicates that there are two regions of conservation between these molecules. The first is a 70 amino acid, cysteine-rich sequence (C1), the second region (C2) is a 62 amino acid region located towards the carboxyl terminus of the proteins. In other systems the former has been identified as a region responsible for DNA binding activity, whereas the latter represents the NH2-terminal boundary of the hormone binding domain. We present here evidence utilizing eucaryotic expression of cDNA encoding the hVDR C1 domain, followed by a DNA cellulose chromatography assay, which confirms that the DNA binding activity resides in this region of the receptor for vitamin D3. Additionally, the vitamin D3 receptor contains a 60 amino acid portion at its carboxyl terminus (C3) which exhibits homology with the receptor for thyroid hormone. Conservation in this region of the molecule is found only between homologous or closely related receptors. This indicates a relationship between the vitamin D3 receptor and the receptor for thyroid hormone and may suggest that they evolved from a single primordial gene.

Evidence for a reactive sulfhydryl in the DNA binding domain of the 1,25-dihydroxyvitamin D3 receptor.

Abstract Evidence is presented here that organomercurial binding to a reactive sulfhydryl group is capable of altering the DNA-binding characteristics of the 1,25-dihydroxyvitamin D receptor (D-receptor). Accordingly, hormone-free receptor (R o ) binding to DNA-cellulose is inhibited in a concentration-dependent fashion with both HgCl 2 and p-chloromercuribenzene sulfonate (pCMBS) with complete inhibition evident at 1.0 mM. Further, low concentrations (0.5 mM) of mercurials are also capable of dissociating preformed DNA-receptor complexes, a process reversible with excess thiol reagent such as monothioglycerol. These findings are in contrast to alkylating reagents such as iodoacetamide, which is capable of only partially inhibiting the formation of the receptor-DNA duplex (37% at 25 mM). Once created, however, the duplex is completely insensitive to dissociation (even at 25 mM). These results imply that in addition to the association of a cysteine(s) moiety in or near the sterol binding site, modification of a similarly reactive group(s) can also alter the D-receptors DNA-binding domain.

1,25-Dihydroxyvitamin D regulates expression of the tryptophan hydroxylase 2 and leptin genes: implication for behavioral influences of vitamin D

To investigate vitamin D‐related control of brain‐expressed genes, candidate vitamin D responsive elements (VDREs) at ‐7/‐10 kb in human tryptophan hydroxylase (TPH)2 were probed. Both VDREs bound the vitamin D receptor (VDR)‐retinoid X receptor (RXR) complex and drove reporter gene transcription in response to 1,25‐dihydroxyvitamin D3 (1,25D). Brain TPH2 mRNA, encoding the rate‐limiting enzyme in serotonin synthesis, was induced 2.2‐fold by 10 nM 1,25D in human U87 glioblastoma cells and 47.8‐fold in rat serotonergic RN46A‐B14 cells. 1,25D regulation of leptin (Lep), encoding a serotoninlike satiety factor, was also examined. In mouse adipocytes, 1,25D repressed leptin mRNA levels by at least 84%, whereas 1,25D induced leptin mRNA 15.1‐fold in human glioblastoma cells. Chromatin immunoprecipitation sequencing analysis of the mouse Lep gene in response to 1,25D revealed a cluster of regulatory sites (cis‐regulatory module; CRM) at ‐28 kb that 1,25D‐dependendy docked VDR, RXR, C/EBPp, and RUNX2. This CRM harbored 3 VDREs and single C/EBPβ and RUNX2 sites. Therefore, the expression of human TPH2 and mouse Lep are governed by 1,25D, potentially via respective VDREs located at ‐7/‐10 kb and ‐28 kb. These results imply that vitamin D affects brain serotonin concentrations, which may be relevant to psychiatric disorders, such as autism, and may control leptin levels and affect eating behavior.—Kaneko, I., Sabir, M. S., Dussik, C. M., Whitfield, G. K., Karrys, A., Hseih, J.‐C., Haussler, M. R., Meyer, M. B., Pike, J. W., Jurutka, P. W. 1,25‐dihydroxyvitamin D regulates expression of the tryptophan hydroxylase 2 and leptin genes: implication for behavioral influences of vitamin D. FASEB J. 29, 4023‐4035 (2015). www.fasebj.org