The Vitamin D Response Element-binding Protein

Abstract

Vitamin D resistance in certain primate genera is associated with the constitutive overexpression of a nonvitamin D receptor (VDR)-related, vitamin D response element-binding protein (VDRE-BP) and squelching of vitamin D-directed transactivation. We used DNA affinity chromatography to purify proteins associated with non-VDR-VDRE binding activity from vitamin D-resistant New World primate cells. In electrophoretic mobility shift assays, these proteins bound specifically to either single-strand or double-strand oligonucleotides harboring the VDRE. Amino acid sequencing of tryptic peptides from a 34-kDa (VDRE-BP1) and 38-kDa species (VDRE-BP-2) possessed sequence homology with human heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and hnRNPA2, respectively. cDNAs bearing the open reading frame for both VDRE-BPs were cloned and used to transfect wild-type, hormone-responsive primate cells. Transient and stable overexpression of the VDRE-BP2 cDNA, but not the VDRE-BP1 cDNA, in wild-type cells with a VDRE-luciferase reporter resulted in significant reduction in reporter activity. These data suggest that the hnRNPA2-related VDRE-BP2 is a dominantnegative regulator of vitamin D action. Dominant-negative control of steroid hormone-regulated gene transcription has been traditionally attributed to: 1) expression of transcriptionally silent receptor molecules, which compete with transactivating receptor dimer pairs for binding to enhancer elements (1, 2); and 2) expression of non-DNAbinding co-repressor molecules, which interact with other constituents of the transcriptional complex to halt or slow transcription (3). More recently, another family of dominantnegative-acting proteins which squelch transcription have been identified. These proteins are in the heterogeneous nuclear ribonuclear protein (hnRNP) 1 superfamily (4 -8). hnRNPs were initially recognized for their ability to bind to singlestrand pre-mRNA (9, 10) and control the modification and movement of mRNA in the cell. The fact that hnRNPs could function as dominant-negative regulators of transcription was discovered in New World primates, a nonhuman primate suborder that is characterized phenotypically by relative targettissue resistance to adrenal, gonadal, and vitamin D sterol/ steroid hormones (11)(12)(13)(14)(15)(16)(17)(18)(19)(20).We recently cloned and characterized the first member of the hnRNP family capable of modifying steroid hormone-directed transactivation (21,22), the estrogen response element-binding protein (ERE-BP). The 42-kDa ERE-BP is highly homologous to proteins in the hnRNPC subfamily (23). It acts to squelch estrogen receptor (ER)-estrogen response element (ERE)-directed transactivation by competing with the ER homodimer for binding to the ERE. Although discovered because of its overexpression in estrogen-resistant New World primate species, the ERE-BP is also expressed in Old World primate species including man (21). The vitamin D response elementbinding protein or VDRE-BP described here is in the second set of non-receptor sterol/stero...

Functional Characterization of Heterogeneous Nuclear Ribonuclear Protein C1/C2 in Vitamin D Resistance

“…The hnRNPs, first recognized for their ability to bind single strand ribopolynucleotides (16,17), are a family of more than 20 proteins that contribute to the complex associated with nascent pre-mRNA and are thus able to modulate RNA processing, including the stabilization of pre-mRNAs for nuclear export and translation (18 -20). More recently, hnRNPs have been shown to be capable of binding DNA in both single-(16, 17) and double-stranded formats (14,15). Acting in their capacity as double-stranded DNA-binding proteins, we have shown previously that individual hnRNPs may function as dominant-negative modulators of steroid hormone-mediated transcription by competing with the VDR-RXR (14, 15) and the estrogen receptor (21-23) for VDRE and estrogen response element, respectively.…”

An Hsp27-related, Dominant-negative-acting Intracellular Estradiol-binding Protein

“…In the first class of epigenetic factors are dominantnegative-acting inhibitors of 1,25(OH) 2 D 3 -and E 2 -directed transactivation. These proteins are in the hnRNP family of cis-acting proteins (16,18). They squelch steroid hormonedirected transactivation by competing in trans with activated receptor proteins for the cognate hormone response elements of the receptor in the promoter of hormone-regulated genes.…”

“…Western Blot Analysis-Denatured cell extracts were subjected to electrophoresis using 4 -12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes as described previously (16). The membranes were blocked with 5% nonfat dry milk for 1 h and then incubated with monoclonal anti-human Hsp27 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 2 h and with horseradish peroxidaseconjugated secondary antibody for another 1 h prior to detection of antibody-reactive proteins with chemiluminescence reagent (ECL, Amersham Biosciences).…”

“…For example, studies of glucocorticoid resistance in NWP cells have shown increased expression of the heat shock protein (Hsp)90-associated FK506-binding immunophilin FKBP51, which inhibits ligand binding to glucocorticoid receptors (GR) (15). By contrast, vitamin D resistance in NWPs appears to be caused by aberrant expression of a dominantnegative-acting vitamin D response element-binding protein (VDRE-BP), the protein being homologous to human heterogeneous nuclear ribonucleoprotein A (hnRNPA) (16). Estrogen resistance in NWP is associated with the overexpression of two proteins, a nonreceptor-related estrogen response element (ERE)-binding protein (ERE-BP) (17,18) and an intracellular estradiol-binding protein (IEBP) (19,20).…”

“…Estrogen resistance in NWP is associated with the overexpression of two proteins, a nonreceptor-related estrogen response element (ERE)-binding protein (ERE-BP) (17,18) and an intracellular estradiol-binding protein (IEBP) (19,20). ERE-BP is a member of hnRNPC-like family and acts to squelch estrogen receptor (ER)-ERE transactivation by competing with the ER for binding to ERE (16,17). IEBP is a member of the Hsp27 family and is overexpressed in female and male estrogen-resistant NWP cells and tissue (19).…”