Vitamin D receptor interaction with specific DNA. Association as a 1,25-dihydroxyvitamin D3-modulated heterodimer.

The vitamin D receptor (VDR) is a member of the steroid receptor gene family. In this report, we examine the nature of specific VDR DNA binding utilizing the vitamin D-responsive element derived from the human osteocalcin promoter. Association of the VDR with the human osteocalcin 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) responsive element (VDRE) in vitro was characterized on VDRE affinity columns by both weak and strong interactions. Weak interaction was a property of the VDR itself, monomeric in nature, and determined exclusively by the VDR's DNA-binding domain. Strong interaction, in contrast, was dependent upon an intact receptor molecule as well as a heterologous mammalian cell nuclear accessory factor (NAF). Heteromeric interaction between VDR and NAF was independent of the VDR DNA-binding domain, suggesting the presence of a functional dimerization domain separate from that for DNA binding. Direct association of NAF with immobilized VDR revealed that the interaction does not require the presence of DNA. Most importantly, while occupancy of the VDR by 1,25(OH)2D3 was not required for VDR interactions with either DNA or NAF, the presence of hormone increased the apparent relative affinity of the VDR for NAF approximately 10-fold. These studies suggest that high affinity association of the VDR with DNA requires both the DNA-binding domain as well as an additional independent structure located within the steroid-binding region. This protein subdomain interacts with NAF and is regulated by 1,25(OH)2D3.     

A 55-kilodalton accessory factor facilitates vitamin D receptor DNA binding.

The interaction of the vitamin D receptor with a vitamin D-responsive element (VDRE) derived from the human osteocalcin promoter in vitro has been shown to require a nuclear accessory factor (NAF) derived from monkey kidney cells. In this report we show that this factor is widely distributed in cells and tissues, including those that do not express the vitamin D receptor (VDR). NAF is required for VDR binding to a variety of known VDREs. VDR and NAF independently bind the VDRE weakly, as assessed by elution profiles generated during VDRE affinity chromatography. Together, however, both proteins coelute from this column with a profile that indicates a tighter strength of interaction. Analogous chromatography of the VDR derived from ROS 17/2.8 cells treated with 1,25-dihydroxyvitamin D3 in culture also reveals a dual profile of weak and strong binding, suggesting that in vivo modifications are unlikely to alter receptor DNA binding. NAF is a protein of 55 kDa, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and cross-linking experiments suggest that the VDR and NAF together form a heterodimer on a single VDRE with a mol wt of 103 kDa. These data demonstrate that NAF is required for VDR binding to specific DNA in vitro and suggest the possibility that NAF may be required for the transactivation capability of the VDR in vivo.     

Direct Repeat 3-Type Element Lacking the Ability to Bind to the Vitamin D Receptor Enhances the Function of a Vitamin D-responsive Element

In a previous study, we identified the element which allows the maximum response to 1,25(OH)₂D₃ in concert with two vitamin D-responsive elements (VDREs) in the rat 25-hydroxyvitamin D₃ 24-hydroxylase gene promoter, and designated it an accessory element [Ohyama, Y., Ozono, K., Uchida, M., Yoshimura, M., Shinki, T., Suda, T. and Yamamoto, O. Functional assessment of two vitamin D-responsive elements in the rat 25-hydroxyvitamin D₃ 24-hydroxylase gene. J. Biol. Chem., 1996, 271, 30381-30385]. The accessory element located adjacent to the proximal VDRE is not capable of binding to the vitamin D receptor (VDR), while its nucleotide sequence resembles the consensus sequence of VDREs, direct repeat 3 (DR3). To clarify the difference between the accessory element and VDREs, the function of the accessory element was compared with that of VDREs. The mutated accessory elements with a single nucleotide substitution showed the capability of binding to the VDR in vitro. However, these mutants still did not act as a VDRE when driven by the heterologous SV40 promoter. The accessory element did not enhance the function of a cAMP-responsive element. The corresponding site of the accessory element in the human 24-hydroxylase is a DR4-type element, and this element did not function as an accessory element. These results indicate that a critical nucleotide sequence is necessary for the binding to the VDR and for mediating the vitamin D effect, and suggest the different regulation between the rat and human 24-hydroxylase gene.     

Irx4 forms an inhibitory complex with the vitamin D and retinoic X receptors to regulate cardiac chamber-specific slow MyHC3 expression

The slow myosin heavy chain 3 gene (slow MyHC3) is restricted in its expression to the atrial chambers of the heart. Understanding its regulation provides a basis for determination of the mechanisms controlling chamber-specific gene expression in heart development. The observed chamber distribution results from repression of slow MyHC3 gene expression in the ventricles. A binding site, the vitamin D response element (VDRE), for a heterodimer of vitamin D receptor (VDR) and retinoic X receptor alpha (RXR alpha) within the slow MyHC3 promoter mediates chamber-specific expression of the gene. Irx4, an Iroquois family homeobox gene whose expression is restricted to the ventricular chambers at all stages of development, inhibits AMHC1, the chick homolog of quail slow MyHC3, gene expression within developing ventricles. Repression of the slow MyHC3 gene in ventricular cardiomyocytes by Irx4 requires the VDRE. Unlike VDR and RXR alpha, Irx4 does not bind directly to the VDRE. Instead two-hybrid and co-immunoprecipitation assays show that Irx4 interacts with the RXR alpha component of the VDR/RXR alpha heterodimer and that the amino terminus of the Irx4 protein is required for its inhibitory action. These observations indicate that the mechanism of atrial chamber-specific expression requires the formation of an inhibitory protein complex composed of VDR, RXR alpha, and Irx4 that binds at the VDRE inhibiting slow MyHC3 expression in the ventricles.     

Competition between c-fos and 1,25(OH)2 vitamin D3 in the transcriptional control of type I collagen synthesis in MC3T3-E1 osteoblastic cells

Interaction between c-fos and 1,25(OH)₂ vitamin D₃ (VD) on the type I collagen synthesis was studied. VD inhibited collagen synthesis and type I collagen mRNA expression in MC3T3-E1 osteoblastic cells. In contrast, VD reversed the inhibition of collagen synthesis and mRNA expression of the c-fos transfectants that overexpressed c-fos gene to a comparable level as those of the control transfectants. The gel shift assay showed that vitamin D receptor (VDR) complex binding to vitamin D responsive element (VDRE) was inhibited under constitutively expressed c-fos gene, suggesting that c-fos gene product, c-Fos, may inhibit the binding of VDR complex to VDRE by making a c-Fos-VDR complex. The result suggests the existence of a fine tuning between c-fos and VD in the bone metabolism which may be relevant to the pathogenesis of rheumatoid bone lesion. © 1995 Wiley-Liss, Inc.     

The vitamin D-responsive element in the human osteocalcin gene. Association with a nuclear proto-oncogene enhancer

A vitamin D-responsive element (VDRE) locus within the 5' region of the human osteocalcin gene promoter contains a steroid response-like half-site immediately proximal to a consensus site for the AP-1 nuclear oncogene family. In the studies described here, internal mutagenesis of the osteocalcin promoter coupled to functional assays reveal that the interaction of the vitamin D receptor is limited to the proximal region of the VDRE locus. Mutations within the distal AP-1 consensus site reduce the basal activity of the promoter but have little effect on vitamin D inducibility. The absolute level of promoter activity induced by hormone, however, is dramatically reduced in the absence of an intact AP-1 site suggesting a functional synergism between the receptor and AP-1-related proteins. In vitro receptor-DNA binding studies confirm the lack of requirement for the distal component in receptor binding. These results suggest that the osteocalcin VDRE is limited to 15 nucleotides closely juxtaposed to a distal functional AP-1 site. The close association of the two sites may lead to proto-oncogene and steroid receptor interactions that result in interesting functional consequences.     

The trascriptional activation of the human copper/zinc superoxide dismutase gene by 2,3,7,8-tetrachlorodibenzo-p-dioxin through two different regulator sites,the anitoxidant responsive element and xenobiotic responsive element

Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced during biological oxidations and environmental stress. The most toxic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), induces SOD1 in human liver cells. Deletion analyses showed that the promoter region between -400 and -239 was responsible for the induction, in which two different characteristic regulatory elements, the antioxidant responsive element (ARE) and xenobiotic responsive element (XRE), are located. When the cells transfected with the plasmid containing those two cis-elements, the transactivation of SOD1 promoter was about 4-fold by TCDD, whereas mutation either on the ARE or XRE elevated the promoter activity by about 2-fold. Functional analyses of these two elements by deletion, mutation in the natural context, heterologous promoter assay, and gel mobility shift assay supported the notion that the activation of the SOD1 promoter was induced by TCDD through these two regulatory elements ARE and XRE. These results alongside our previous data indicate that the induction of SOD1 in response to TCDD is mediated by either Nrf2 protein or Ah receptor protein through ARE and XRE, respectively. These results also imply that the SOD1 can be induced by dioxin either in combination with or independently of these two regulatory elements to effectively defend cells from oxidative stress.