Identification of a vitamin D response element in the proximal promoter of the chicken carbonic anhydrase II gene

The carbonic anhydrase II gene, whose transcription is enhanced by 1,25-dihydroxyvitamin D-3 (1,25-(OH)(2)D-3), encodes an important enzyme in bone-resorbing cells derived from the fusion of monocytic progenitors. We analyzed the 1,25-(OH)(2)D-3-mediated activation of the avian gene by transient transfection assays with promoter/reporter constructs into HD11 chicken macrophages and by DNA mobility shift assays. Deletion and mobility shift analyses indicated that the -62/-29 region confers 1,25-(OH)(2)D-3 responsiveness and forms DNA-protein complexes. The addition of an anti-vitamin D receptor (VDR) antibody inhibited binding to this sequence, whereas anti-retinoid X receptor (RXR) antibody generated a lower mobility complex. Therefore, we concluded that this element binds a VDR RXR heterodimer, but the addition of extra 1,25-(OH)(2)D-3 had no effect on the formation of this complex. Moreover, the use of nuclear extracts from 1,25-(OH)(2)D-3-treated macrophages led to the formation of an additional high mobility complex also composed of VDR RXR heterodimer. Mutations provided evidence that the 1,25(OH)(2)D-3-mediated activation of the carbonic anhydrase II gene is mediated by VDR RXR heterodimers bound to a DR3-type vitamin D response element with sequence AGGGCAtggAGTTCG. This vitamin D response element is also functional in the ROS 17/2.8 osteoblasts.