VDR-mediated Transactivation Research Articles

<p>Role Of Vitamin-D Supplementation In TB/HIV Co-Infected Patients</p>

ObjectiveThis review aimed to assess the role of vitamin D supplementation on the decrement of mortality and morbidity rate among tuberculosis (TB)/human immune deficiency virus (HIV) co-infected clients.Method: Pub Med, google scholar and google search were accessed to find out all document to describe this review article.ResultsNowadays TB/HIV co-infection has become a major global concern, particularly in low and middle-income countries. Mycobacterium tuberculosis and HIV infections are co-endemic and more susceptible to the progression of TB. Immunosuppression associated with HIV is a strong risk factor for the reactivation of latent TB to the active form. Immune cells like macrophages recognized Mycobacterium tuberculosis through TLR2/1, and it increases the expression of the vitamin D receptor (VDR) and CYP27B1. The synthesis of 1,25-dihydroxy vitamin D promotes VDR-mediated transactivation of the antimicrobial peptide cathelicidin and the killing of intracellular Mycobacterium tuberculosis. Cathelicidins have a direct antimicrobial effect through membrane disruption. Besides, it has also antiviral effects via inhibition of retrovirus (HIV) replication. In fact, as some studies showed, there was a lower induction of cathelicidin in monocytes who have low vitamin D levels.Conclusion: Therefore, vitamin D supplementation can be directly involved in the reduction of TB/HIV co-infection and its progression.

Modulation of Vitamin D Receptor Activity by the Corepressor Hairless: Differential Effects of Hairless Isoforms

The vitamin D receptor (VDR) and its corepressor Hairless (HR) are thought to regulate key steps in the hair cycle because mutations in VDR or HR cause alopecia in humans and mice. Many mammalian cells express two major HR isoforms due to alternative splicing of exon 17. HR isoform-a encodes an 1189-amino acid protein (full-length HR), and isoform-b encodes an 1134-amino acid protein (HRDelta1072-1126). We demonstrated that both HR isoforms are expressed in primary human keratinocytes and in the human keratinocyte cell line HaCaT. In transfected COS-7 cells, the full-length HR repressed VDR-mediated transactivation. In contrast, HRDelta1072-1126 failed to suppress and even stimulated VDR-mediated transactivation. In coimmunoprecipitation, both HR isoforms interacted with the VDR, but only the full-length HR interacted with histone deacetylase 1 (HDAC1). Alanine mutagenesis of two conserved glutamic acids residues (E1100A/E1101A) encoded by exon 17 completely eliminated HR corepressor activity and interactions with HDAC1. When the two HR isoforms were coexpressed in COS-7 cells, the corepressor activity of the full-length HR was not antagonized by the HRDelta1072-1126 isoform. When transfected into HaCaT cells, the full-length HR inhibited endogenous CYP24A1 basal gene expression as well as 1,25-dihydroxyvitamin D3-stimulated CYP24A1 expression. HRDelta1072-1126 failed to suppress basal or 1,25-dihydroxyvitamin D3-stimulated CYP24A1 gene expression. In conclusion, we have demonstrated that both HR isoforms are expressed in keratinocytes and that the HRDelta1072-1126 isoform lacks corepressor activity and is unable to bind HDACs. HRDelta1072-1126 may function as a coactivator in some settings by inhibiting HDAC recruitment to the VDR transcriptional complex.

Intestinal cell-specific vitamin D receptor (VDR)-mediated transcriptional regulation of CYP3A4 gene

CYP3A4 is the most important drug-metabolizing enzyme that is involved in biotransformation of more than 50% of drugs. Pregnane X receptor (PXR) dominantly controls CYP3A4 inducibility in the liver, whereas vitamin D receptor (VDR) transactivates CYP3A4 in the intestine by secondary bile acids. Four major functional PXR-binding response elements of CYP3A4 have been discovered and their cooperation was found to be crucial for maximal up-regulation of the gene in hepatocytes. VDR and PXR recognize similar response element motifs and share DR3(XREM) and proximal ER6 (prER6) response elements of the CYP3A4 gene.In this work, we tested whether the recently discovered PXR response elements DR4(eNR3A4) in the XREM module and the distal ER6 element in the CLEM4 module (CLEM4-ER6) bind VDR/RXRα heterodimer, whether the elements are involved in the intestinal transactivation, and whether their cooperation with other elements is essential for maximal intestinal expression of CYP3A4.Employing a series of gene reporter plasmids with various combinations of response element mutations transiently transfected into four intestinal cell lines, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP), we found that the CLEM4-ER6 motif interacts with VDR/RXRα heterodimer and partially cooperates with DR3(XREM) and prER6 in both basal and VDR-mediated inducible CYP3A4 regulation in intestinal cells. In contrast, eNR3A4 is involved only in the basal transactivation in intestinal cells and in the PXR-mediated rifampicin-induced transactivation of CYP3A4 in LS174T intestinal cells.We thus describe a specific ligand-induced VDR-mediated transactivation of the CYP3A4 gene in intestinal cells that differs from PXR-mediated CYP3A4 regulation in hepatocytes.

Oncogenic nucleoporin CAN/Nup214 interacts with vitamin D receptor and modulates its function

Vitamin D receptor (VDR) is a ligand-dependent transcription factor and should be located in nucleus to transactivate target genes. To explore the molecules that interact with VDR and facilitate its nuclear localization, we screened a human kidney cDNA library using the yeast two-hybrid approach, and found that VDR binds to the carboxy-terminal region of an oncogenic nucleoporin, CAN/Nup214. CAN/Nup214 was originally identified through its involvement in a certain type of acute myeloid leukemia, and is a component of nuclear pore complex (NPC). Co-immunoprecipitation experiments confirmed the interaction between VDR and the carboxy-terminus of CAN/Nup214 containing a cluster of the phenylalanine-glycine (FG) repeat in mammalian cells. The exogenously expressed full-length CAN/Nup214 was localized predominantly at the nuclear envelope, suggesting its integration in the NPCs. We then examined the effects of exogenous expression of full-length CAN/Nup214 and its carboxy-terminal fragment on the VDR-mediated transactivation. The overexpression of full-length CAN/Nup214 facilitated the VDR-mediated transactivation, while the expression of the carboxy-terminal fragment suppressed it. The DNA-binding domain of VDR was required for the facilitation of the VDR-dependent transactivation by CAN/Nup214. Although the subcellular distribution of VDR was not obviously altered by the overexpression of full-length CAN/Nup214 or the carboxy-terminal fragment, the expression of the carboxy-terminal fragment inhibited the interaction between full-length CAN/Nup214 and VDR. These results indicate that CAN/Nup214 interacts with VDR and modulates its function as a transcription factor.

Steroid receptor coactivator 3 maintains circulating insulin-like growth factor I (IGF-I) by controlling IGF-binding protein 3 expression.

Steroid receptor coactivator 3 (SRC-3/AIB1/ACTR/NCoA-3) is a transcriptional coactivator for nuclear receptors including vitamin D receptor (VDR). Growth hormone (GH) regulates insulin-like growth factor I (IGF-I) expression, and IGF-I forms complexes with acid-labile subunit (ALS) and IGF-binding protein 3 (IGFBP-3) to maintain its circulating concentration and endocrine function. This study demonstrated that the circulating IGF-I was significantly reduced in SRC-3(-/-) mice with the C57BL/6J background. However, SRC-3 deficiency affected neither GH nor ALS expression. The low IGF-I level in SRC-3(-/-) mice was not due to the failure of IGF-I mRNA and protein synthesis but was a consequence of rapid degradation. The rapid IGF-I degradation was associated with drastically reduced IGFBP-3 levels. Because IGF-I and IGFBP-3 stabilize each other, SRC-3(-/-) mice were crossbred with the liver-specific transthyretin (TTR)-IGF-I transgenic mice to assess the relationship between reduced IGF-I and IGFBP-3. In SRC-3(-/-)/TTR-IGF-I mice, the IGF-I level was significantly increased over that in SRC-3(-/-) mice, but the IGFBP-3 level failed to increase proportionally, indicating that the low IGFBP-3 level is a responsible factor that limits the IGF-I level in SRC-3(-/-) mice. Furthermore, IGFBP-3 mRNA was reduced in SRC-3(-/-) mice. The IGFBP-3 promoter activity induced by vitamin D, through VDR, was diminished in SRC-3(-/-) cells, suggesting an important role of SRC-3 in VDR-mediated transactivation of the IGFBP-3 gene. In agreement with the role of SRC-3 in VDR function, the expression of several VDR target genes was also reduced in SRC-3(-/-) mice. Therefore, SRC-3 maintains IGF-I in the circulation through enhancing VDR-regulated IGFBP-3 expression.

The “Bald Mill Hill” Mutation in the Mouse Is Associated with an Abnormal, Mislocalized HR bmh Protein

We have previously identified a mutation in the mouse hairless locus-hairless rhino bald Mill Hill (Hr(rhbmh)). The genetic alteration in these mice consists in a large 296 bp deletion at the 3' part of the hairless gene (ID:MGI:3039558; J:89321). Here, we show that this deletion removes the stop codon and creates a new reading frame at the C terminus of the hairless protein, generating a larger mutant protein harboring an additional sequence of 117 amino acids. The mutant hairless gene mRNA is expressed during the embryonic and post-natal development of the hair follicle. The mutant protein is identified in bmh mouse skin at different stages of development by a specific antibody. We demonstrate that the HR bmh protein is able to interact with the vitamin D receptor (VDR), but is not able to repress VDR-mediated transactivation. Immunofluorescence analysis reveals that HR bmh protein displays an abnormal cellular localization in transfected cell lines, as well as in the epidermis and hair follicle of bmh mutant mice. We discuss the relevance of the hairless protein mis localization in cell signalling pathways and with respect to the specific skin phenotype of mouse hairless mutants.

Culture serum-induced conversion from agonist to antagonist of a Vitamin D analog, TEI-9647

The nuclear receptor for Vitamin D (VDR) mediates many of the effects of Vitamin D in target tissues by regulating gene expression. The transactivation function of ligand-bound VDR in target tissues is thought to depend on the tissue-type and the cellular-environment, but the molecular basis for these differences has not been fully understood. In this study, during characterization of TEI-9647 as a synthetic ligand for the VDR, we found that depletion of serum from the culture medium converted TEI-9647 from an antagonist to an agonist of VDR-mediated transactivation, whereas it retained antagonistic activity in the presence of serum. Consistent with these results, using a mammalian two-hybrid system, we found that TEI-9647 recruited different coactivators to the VDR in the presence and absence of serum. These findings suggest that an unknown serum factor modulates the transactivation function of the VDR.

Physical and Functional Interaction between the Vitamin D Receptor and Hairless Corepressor, Two Proteins Required for Hair Cycling

Both the vitamin D receptor (VDR) and hairless (hr) genes play a role in the mammalian hair cycle, as inactivating mutations in either result in total alopecia. VDR is a nuclear receptor that functions as a ligand-activated transcription factor, whereas the hairless gene product (Hr) acts as a corepressor of both the thyroid hormone receptor (TR) and the orphan nuclear receptor, RORalpha. In the present study, we show that VDR-mediated transactivation is strikingly inhibited by coexpression of rat Hr. The repressive effect of Hr is observed on both synthetic and naturally occurring VDR-responsive promoters and also when VDR-mediated transactivation is augmented by overexpression of its heterodimeric partner, retinoid X receptor. Utilizing in vitro pull down methods, we find that Hr binds directly to VDR but insignificantly to nuclear receptors that are not functionally repressed by Hr. Coimmunoprecipitation data demonstrate that Hr and VDR associate in a cellular milieu, suggesting in vivo interaction. The Hr contact site in human VDR is localized to the central portion of the ligand binding domain, a known corepressor docking region in other nuclear receptors separate from the activation function-2 domain. Coimmunoprecipitation and functional studies of Hr deletants reveal that VDR contacts a C-terminal region of Hr that includes motifs required for TR and RORalpha binding. Finally, in situ hybridization analysis of hr and VDR mRNAs in mouse skin demonstrates colocalization in cells of the hair follicle, consistent with a hypothesized intracellular interaction between these proteins to repress VDR target gene expression, in vivo.

Retinoid X receptor is a nonsilent major contributor to vitamin D receptor-mediated transcriptional activation.

The vitamin D receptor (VDR) belongs to the thyroid hormone/retinoid receptor subfamily of nuclear receptors and functions as a heterodimer with retinoid X receptor (RXR). The RXR-VDR heterodimer, in contrast to other members of the class II nuclear receptor subfamily, is nonpermissive where RXR does not bind its cognate ligand, and therefore its role in VDR-mediated transactivation by liganded RXR-VDR has not been fully characterized. Here, we show a unique facet of the intermolecular RXR-VDR interaction, in which RXR actively participates in vitamin D3-dependent gene transcription. Using helix 3 and helix 12 mutants of VDR and RXR, we provide functional evidence that liganded VDR allosterically modifies RXR from an apo (unliganded)- to a holo (liganded)-receptor conformation, in the absence of RXR ligand. As a result of the proposed allosteric modification of RXR by liganded VDR, the heterodimerized RXR shows the "phantom ligand effect" and thus acquires the capability to recruit coactivators steroid receptor coactivator 1, transcriptional intermediary factor 2, and amplified in breast cancer-1. Finally, using a biochemical approach with purified proteins, we show that RXR augments the 1,25-dihydroxyvitamin D3-dependent recruitment of transcriptional intermediary factor 2 in the context of RXR-VDR heterodimer. These results confirm and extend the previous observations suggesting that RXR is a significant contributor to VDR-mediated gene expression and provide a mechanism by which RXR acts as a major contributor to vitamin D3-dependent transcription.

BAG1L Enhances Trans-activation Function of the Vitamin D Receptor

The vitamin D receptor (VDR) is a member of the steroid/retinoid receptor superfamily of nuclear receptors that has potential tumor-suppressive functions. We show here that VDR interacts with and is regulated by BAG1L, a nuclear protein that binds heat shock 70-kDa (Hsp70) family molecular chaperones. Endogenous BAG1L can be co-immunoprecipitated with VDR from prostate cancer cells (ALVA31; LNCaP) in a ligand-dependent manner. BAG1L, but not shorter non-nuclear isoforms of this protein (BAG1; BAG1M/Rap46), markedly enhanced, in a ligand-dependent manner, the ability of VDR to trans-activate reporter gene plasmids containing a vitamin D response element in transient transfection assays. Mutant BAG1L lacking the C-terminal Hsc70-binding domain suppressed (in a concentration-dependent fashion) VDR-mediated trans-activation of vitamin D response element-containing reporter gene plasmids, without altering levels of VDR or endogenous BAG1L protein, suggesting that it operates as a trans-dominant inhibitor of BAG1L. Gene transfer-mediated elevations in BAG1L protein levels in a prostate cancer cell line (PC3), which is moderately responsive to VDR ligands, increased the ability of natural (1alpha,25(OH)(2) vitamin D(3)) and synthetic (1alpha, 25-dihydroxy-19-nor-22(E)-vitamin D(3)) VDR ligands to induce expression of the VDR target gene, p21(Waf1), and suppress DNA synthesis. Thus, BAG1L is a direct regulator of VDR, which enhances its trans-activation function and improves tumor cell responses to growth-suppressive VDR ligands.

Characterization of the activation function-2 domain of the human 1,25-dihydroxyvitamin D 3 receptor

In this study, we determined the ligand-dependent activation function domain 2 (AF-2) of the human vitamin D receptor (hVDR) and characterized it using site-directed mutagenesis. A single mutation at glutamic acid-420 (E420Q) and an additional mutation at leucine-417 (L417A-E420Q) eliminated ligand-dependent transcriptional activation. In addition, lysine-264 was also demonstrated to be vital for ligand-induced transactivation. However, bacterial-overexpressed transcriptional factor IIB (TFIIB) was able to bind to both AF-2 and lysine-264 mutant hVDRs in vitro. The ligand-dependent transactivation via wild type hVDR was interfered with weakly only when a 10-fold molar excess of L417A-E420Q plasmid was co-transfected. This suppressive effect was diminished by introducing an additional mutation at a cysteine residue in the DNA binding domain. Thus, we conclude that the AF-2 domain of the hVDR located between amino acids 417 and 420, as well as lysine-264, are essential for ligand-dependent transactivation, and that TFIIB was not necessary for the function of these two regions of the hVDR. Our finding that AF-2 mutant hVDRs exhibit only very weak suppressive effect may indicate a difference in the molecular mechanism of the VDR-mediated transactivation from other nuclear receptors.

Evidence for ligand-dependent intramolecular folding of the AF-2 domain in vitamin D receptor-activated transcription and coactivator interaction.

A ligand-dependent transcriptional activation domain (AF-2) exists in region E of the nuclear receptors. This highly conserved domain may contact several coactivators that are putatively involved in nuclear receptor-mediated transcription. In this study, a panel of vitamin D receptor (VDR) AF-2 mutants was created to examine the importance of several conserved residues in VDR-activated transcription. Two AF-2 mutants (L417S and E420Q) exhibited normal ligand binding, heterodimerization with retinoid X receptor, and vitamin D-responsive element interaction, but they were transcriptionally inactive in a VDR-responsive reporter gene assay. All AF-2 mutations that abolished VDR-mediated transactivation also eliminated interactions between VDR and several putative coactivator proteins including suppressor of gal1 (SUG1), steroid hormone receptor coactivator-1 (SRC-1), or receptor interacting protein (RIP140), suggesting that coactivator interaction is important for AF-2-mediated transcription. In support of this concept, the minimal AF-2 domain [VDR(408-427)] fused to the gal4 DNA binding domain was sufficient to mediate transactivation as well as interaction with putative coactivators. Introducing the L417S and E420Q mutations into the minimal AF-2 domain abolished this autonomous transactivation and coactivator interactions. Finally, we demonstrate that the minimal AF-2 domain interacted with an AF-2 deletion mutant of the VDR in a 1,25-(OH)2D3-dependent manner, suggesting a ligand-induced intramolecular folding of the VDR AF-2 domain. The L417S mutant of this domain disrupted the interaction with VDR ligand-binding domain, while the E420Q mutant did not affect this interaction. These studies suggest that the conserved AF-2 motif may mediate transactivation through ligand-dependent intermolecular interaction with coactivators and through ligand-induced intramolecular contacts with the VDR ligand-binding domain itself.

Retinoid X receptor:vitamin D3 receptor heterodimers promote stable preinitiation complex formation and direct 1,25-dihydroxyvitamin D3-dependent cell-free transcription.

The numerous members of the steroid/nuclear hormone receptor superfamily act as direct transducers of circulating signals, such as steroids, thyroid hormone, and vitamin or lipid metabolites, and modulate the transcription of specific target genes, primarily as dimeric complexes. The receptors for 9-cis retinoic acid and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], RXR and VDR, respectively, as members of this superfamily, form a heterodimeric complex and bind cooperatively to vitamin D responsive elements (VDREs) to activate or repress the transcription of a multitude of genes which regulate a variety of physiological functions. To directly investigate RXR- and VDR-mediated transactivation, we developed a cell-free transcription system for 1,25(OH)2D3 signaling by utilizing crude nuclear extracts and a G-free cassette-based assay. Transcriptional enhancement in vitro was dependent on purified, exogenous RXR and VDR and was responsive to physiological concentrations of 1,25(OH)2D3. We found that RXR and VDR transactivated selectively from VDRE-linked templates exclusively as a heterodimeric complex, since neither receptor alone enhanced transcription in vitro. By the addition of low concentrations of the anionic detergent Sarkosyl to limit cell-free transcription to a single round and the use of agarose gel mobility shift experiments to assay factor complex assembly, we observed that 1,25(OH)2D3 enhanced RXR:VDR-mediated stabilization or assembly of preinitiation complexes to effect transcriptional enhancement from VDRE-linked promoter-containing DNA.

Vitamin D-influenced gene expression via a ligand-independent, receptor-DNA complex intermediate.

A lingering question regarding the regulation of target gene expression by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] has been the delineation of vitamin D receptor (VDR)-DNA binding and transactivation. This report confirms that initial VDR-DNA interaction occurs in a ligand-independent fashion. An electrophoretic mobility-shift analysis demonstrated that VDR, derived from extracts of the small intestines of vitamin D-deficient rats, is capable of binding a vitamin D response element (DRE). Additional mobility-shift studies using either porcine-derived VDR or recombinant rat VDR from insect cells revealed DRE-binding capability in the absence of 1,25-(OH)2D3. The reactions were performed in various salt environments, with the maximum of porcine VDR-DRE and rat VDR-DRE binding detected at 100 mM and 150 mM KCl, respectively. The addition of 1,25-(OH)2D3 to an identical set of reaction mixtures resulted in increased DRE binding with greater affinities exhibited by both VDR types. These two phenomena were confirmed upon examination of an elution profile of VDR bound to DRE-linked Sepharose. When a linear KCl gradient was used for elution without the addition of 1,25-(OH)2D3, the peak of VDR was 205 mM KCl; the presence of exogenous hormone shifted the maximum VDR elution to a position corresponding to 265 mM KCl. Based on these data and previous reports on VDR-mediated transactivation, we propose a model for 1,25-(OH)2D3-influenced target gene expression.