Identification and Characterization of a Vitamin D₃Response Element of Chicken Carbonic Anhydrase-II

Abstract

1,25-Dihydroxyvitamin D3 (VD) controls multiple aspects of homeostasis, cell growth, and differentiation by the action of its nuclear receptor (VDR), which binds to, and activates transcription from, response elements in the promoter region of its target genes. Carbonic anhydrase-II (CA-II), an enzyme important to osteoclast function, has been shown to be regulated by VD. We screened the promoter of chicken CA-II for VDR binding sites and identified a functional VDRE, between positions -1,203 and -1,187. Like the majority of the VDREs described to date, this response element consists of two directly repeated hexameric core binding motifs spaced by three nucleotides and is bound by a heterodimer formed by the VDR and the retinoid X receptor (RXR). We show that the polarity of the binding of this heterodimer is 5'-VDR-RXR-3' in the CA-II VDRE, whereas on a "classical" DR3-type VDRE, such as that of the mouse osteopontin gene, this polarity is reversed to 5'-RXR-VDR-3'. We also show that the polarity of the heterodimeric complex in relation to the basic transcriptional machinery influences the sensitivity of the transcriptional activity to VD. This suggests that the orientation of a hormone response element in its natural promoter context constitutes an additional level of gene regulation.

Competition for a Unique Response Element Mediates Retinoic Acid Inhibition of Vitamin D3-stimulated Transcription

“…Whereas the order on two direct repeats separated by three bases was (5Ј to 3Ј) TR-VDR, this was reversed on the VDRE separated by four bases. The same authors also find that orientation of VDR-RXR is reversed when comparing the DNA sequences in the mouse osteopontin and avian carbonic anhydrase genes, both of which comprise direct repeats separated by three bases (48). Finally, novel VDRE sequences comprising inverted palindromes separated by nine nucleotides have been identified in the human calbindin D 9k and rat osteocalcin genes (33).…”

“…To study the effect of heterodimer polarity on transcriptional activity, constructs containing TR-RXR or VDR-RXR response elements placed in both orientations relative to a basal promoter were transfected into target cells, which were assayed for subsequent responsiveness to steroid hormone treatment (23,33,48). In each instance orientation of the response element significantly influenced the level of reporter activity.…”

Vitamin D and Osteogenic Differentiation in the Artery Wall

“…It is interesting that OPG also reduces hyperlipidemia-induced calcification, independent of vitamin D levels (50). Osteoclast-like cells have been found in the artery wall, and their resorptive activity is driven by carbonic anhydrase, which has a vitamin D response element in its gene promoter (51). One positive modifier of vitamin D-induced vascular calcification is magnesium deficiency.…”

Identification of a Vitamin D Response Element in the Proximal Promoter of the Chicken Carbonic Anhydrase II Gene

“…The mouse osteopontin VDRE has been shown to bind VDR homodimers with low affinity (3,6,12,13) but VDR⅐RXR heterodimers with high affinity (3,9,14,15). DR3-type elements have also been found in numerous promoters such as the rat osteocalcin gene (16,17), the rat calbindin D-9k (18), the avian integrin ␤3 subunit gene (19), the rat 24-hydroxylase gene (20 -22), the avian carbonic anhydrase II (CAII) gene (23), and mouse p21 (24). However, the binding sites of the VDREs characterized so far vary considerably in their sequences, preventing definition of a real VDRE consensus sequence (7,(25)(26)(27).…”

“…CAII gene expression is also activated transcriptionally by thyroid hormone in normal erythrocytic cells (46), and several domains in the avian CAII promoter have been shown to control the thyroid hormone regulation of transcription (47,48). In previous work, we identified a VDRE between positions Ϫ1203 and Ϫ1187 of the CAII promoter which mediates 1,25-(OH) 2 D 3 responsiveness to the herpes simplex virus thymidine kinase (tk) minimal promoter in the Drosophila SL3 cell line and in human MCF-7 cells (23). This VDRE, bound by a VDR⅐RXR heterodimer, is however not functional in an avian macrophage cell line.…”