Recombinant 1,25-dihydroxyvitamin D3 receptor from a baculovirus expression system requires a mammalian-derived nuclear accessory protein for binding to a vitamin D response element (DRE). This was established by electrophoretic mobility shift analyses using radiolabeled DNA probes consisting of DREs from two vitamin D-responsive genes. Mammalian nuclear extract was also required for the binding of wild-type porcine vitamin D receptor to a DRE. Surprisingly, the accessory factor-dependent formation of receptor-DRE complex was independent of exogenous 1,25-dihydroxyvitamin D3. A 59-to 64-kDa accessory protein from porcine intestinal nuclear extract was identified by sizeexclusion chromatography. Nuclear extracts from rat liver and kidney contained accessory factor, whereas smaller amounts were detected in heart muscle. Spleen and skeletal muscle contained no detectable accessory factor.An understanding of the biological actions of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] requires a comprehensive study of its intracellular receptor (1, 2). It is through this receptor that 1,25-(OH)2D3 exhibits hormonal activities that include stimulation of calcium absorption in the intestine, calcium mobilization from bone, and calcium reabsorption in kidney (1). A probable mechanism for these activities involves an interaction between hormonally altered receptor protein and chromatin, influencing the expression of genes whose products are involved in those functions (2).The molecular cloning of cDNAs encoding human (3) and rat (4) vitamin D receptors has permitted a structural analysis based on their deduced amino acid sequences. Similarities in structural domains of these receptor proteins include a cysteine-rich region at the amino terminus, characteristic of a DNA binding motif, and a hydrophobic pocket near the carboxyl terminus that is thought to form the site of ligand binding. The domains were more clearly defined within the human vitamin D receptor protein using deletion analysis and expression studies of the cDNA (5). These structural determinations have resulted in the inclusion of the 1,25-(OH)2D3 receptor as a member ofthe steroid, thyroid, and retinoic acid superfamily of receptor proteins (6,7).Several recent advances have provided tools by which the interaction between the vitamin D receptor and nucleic acid could be examined. For example, the realization of cis-acting nucleotide sequence response elements that confer vitamin D responsiveness within the transcriptional control regions of the mouse osteopontin gene (8) and the human (9, 10) and rat (11) osteocalcin genes has provided target sequences for in vitro studies of 1,25-(OH)2D3 receptor binding. Studies using these vitamin D response elements (DREs) have defined a transcriptional effect of 1,25-(OH)2D3 on homologous and heterologous promoter activities (10). Other progress has resulted from the production of elevated levels of recombinant vitamin D receptor from a yeast expression system using the human cDNA (12) and from a baculovirus expression sy...
Extracellular matrix (ECM) turnover is regulated by matrix metalloproteinases (MMPs) and plays an important role in cardiac remodeling. Previous studies from our lab demonstrated an increase in gelatinolytic‐MMP‐2 and ‐9 activities in endocardial tissue from ischemic cardiomyopathic (ICM) and idiopathic dilated cardiomyopathic (DCM) hearts. The signaling mechanism responsible for the left ventricular (LV) remodeling, however, is unclear. Administration of cardiac specific inhibitor of metalloproteinase (CIMP) prevented the activation of MMP‐2 and ‐9 in ailing to failing myocardium. Activation of MMP‐2 and ‐9 leads to induction of proteinase activated receptor‐1 (PAR‐1). We hypothesize that the early induction of MMP‐9 is a key regulator for modulating intracellular signaling through activation of PAR and various downstream events which are implicated in development of cardiac fibrosis in an extracellular receptor mediated kinase‐1 (ERK‐1) and focal adhesion kinase (FAK) dependent manner. To test this hypothesis, explanted human heart tissues from ICM and DCM patients were obtained at the time of orthotopic cardiac transplants. Quantitative analysis of MMP‐2 and ‐9 gelatinolytic activities was made by real‐time quantitative zymography. Gel phosphorylation staining for PAR‐1 showed a significant increase in ICM hearts. Western blot and RT‐PCR analysis and in‐situ labeling, showed significant increased expression of PAR‐1, ERK‐1and FAK in ICM and DCM. These observations suggest that the enhanced expression and potentially increased activity of LV myocardial MMP‐9 triggers the signal cascade instigating cardiac remodeling. This early mechanism for the initiation of LV remodeling appears to have a role in end‐stage human heart failure.
The nuclear accessory protein in porcine intestinal nuclear extracts that activates the binding of the vitamin D receptor to its vitamin D response elements has been highly purified. It contains a protein that binds 9-cis- [H] retinoic acid, was detected on immunoblots with an anti-retinoid X receptor (RXR) peptide antibody, and supports the binding of retinoic acid receptor y to the retinoic acid receptor ,B gene response element. Most important, the two specific complexes formed by porcine nuclear extract with the vitamin D response elements from either the osteocalcin gene or the rat 24-hydroxylase gene are shifted to a larger complex by both an anti-vitamin D receptor antibody and an anti-RXR antibody, leaving no doubt that in vivo the nuclear accessory factor for the vitamin D receptor in the intestine is an RXR protein.Vitamin D regulates calcium homeostasis by the mobilization of calcium from bone, reabsorption of calcium in the kidney, and absorption of calcium from the intestine. The physiologically active metabolite of vitamin D 1,25-dihydroxyvitamin D3 [1,2D3] is known to exert these functions by binding to a transcription factor, the vitamin D receptor (VDR) (1). The VDR is a member of the steroid/thyroid receptor superfamily, which also includes nonsteroid binding receptors such as the retinoic acid receptors (RARs) and retinoid X receptors (RXRs) (2). VDR and RAR are members of a subset of this family that can bind to response elements as heterodimers and recognize response elements consisting of direct repeats (3).Several reports have demonstrated that RXRs can function in vitro as accessory factor(s) (e.g., heterodimerize) with VDR and other receptors. Heterodimerization of RXRs with VDRs, thyroid receptors, and RARs increases the affinity of these receptors for their response elements. When RXR cDNAs are cotransfected with these receptors, the transcriptional response to the respective ligand is increased (4-9). These studies provide evidence for a possible role of RXRs as a link between several ligand-induced pathways.A mammalian-derived nuclear accessory factor (NAF) required for tight binding of VDR to vitamin D response elements (DREs) has been described (10-12). The determination of the molecular weight of NAF from pig intestinal nuclear extract (PINE), a major vitamin D target tissue, revealed a molecular mass of 59-64 kDa (12). MacDonald et al. (13) MATERIALS AND METHODS Chemicals. 1,25-(OH)2D3 was purchased from Tetrionics (Madison, WI). 9-cis-Retinoic acid (RA) was synthesized by silver oxide oxidation of 9-cisretinaldehyde (Aldrich). Radioactive 9-cis-RA was synthesized from all-trans-[11,12-3H]RA (48.7 Ci/mmol; 1 Ci = 37 GBq; DuPont/NEN) by photochemical isomerization (14) and was purified by HPLC using a C18 Zorbax-ODS column as described (14). The purity of 9-cis- [11,12-3H]
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.