Identification of the porcine intestinal accessory factor that enables DNA sequence recognition by vitamin D receptor.

Abstract

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]

Isolation of baculovirus‐expressed human vitamin D receptor: DNA responsive element interactions and phosphorylation of the purified receptor

“…Moreover, both complexes contained VDR as they were disrupted by the 4A5 antibody (lane 10). Thus, hVDR interacts with a RAF present in CV-1 cell nuclei to generate a protein:DNA complex that is distinct from the homodimeric complex formed by puri®ed VDR, and likely represents a heterodimer containing VDR and an RXR isoform, as has been demonstrated with nuclear extracts from HeLa cells [MacDonald et al, 1993] and pig intestine [Munder et al, 1995]. Recent experiments in our laboratory have shown the presence of RXRa in a very similar complex generated using nuclear extracts from COS-7 cells .…”

“…For example, enriched overexpressed VDR apparently does not bind to VDREs in DNA binding assays unless a mammalian nuclear extract is present [MacDonald et al, 1991;Sone et al, 1991;Ross et al, 1992]. A large body of data suggests that the major receptor auxiliary factors (RAFs) facilitating VDR DNA binding are the RXRs [Kliewer et al, 1992;MacDonald et al, 1993;Munder et al, 1995;Jin and Pike, 1996;Staal et al, 1996;Lemon et al, 1997].…”

DNA Affinity Chromatography of Transcription Factors

“…Nonidet P-40 (NP-40), a nonionic detergent, had been reported to be a necessary component in all the buffers used in some purification schemes (110 -112, 115, 118 -122), while it interferes and cannot be used in others (113,116,(123)(124)(125) or cannot be used with sequence-specific DNA affinity chromatography (126,127). Nonidet P-40 is no longer commercially available under that name.…”

Crk‐associated substrate (Cas) family member, NEDD9, is regulated in human neuroblastoma cells and in the embryonic hindbrain by all‐trans retinoic acid

“…Radiolabeled oligonucleotide probe was separated from free nucleotides using a quick spin Sephadex G-50 (Roche). Human RAR␥1 (ϳ50 fmol; Repa et al, 1997) and a mouse RXR␥ (ϳ60 fmol; Munder et al, 1995) were incubated for 15 min on ice in the presence of 83 g/ml poly dI-dC, 6 mM Tris, pH 7.4; 6.7% glycerol, 17 mM NaCl, 0.5% dithiothreitol, and 3.3 mg/ml bovine serum albumin. Radiolabeled probe (150,000 cpm) was added and incubated for an additional 10 min at room temperature, followed by separation on a 4% nondenaturing polyacrylamide gel.…”