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

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-[3H]retinoic acid, was detected on immunoblots with an anti-retinoid X receptor (RXR) peptide antibody, and supports the binding of retinoic acid receptor gamma to the retinoic acid receptor beta 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-influenced gene expression via a ligand-independent, receptor-DNA complex intermediate.

A lingering question regarding the regulation of target gene expression by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] has been the delineation of vitamin D receptor (VDR)-DNA binding and transactivation. This report confirms that initial VDR-DNA interaction occurs in a ligand-independent fashion. An electrophoretic mobility-shift analysis demonstrated that VDR, derived from extracts of the small intestines of vitamin D-deficient rats, is capable of binding a vitamin D response element (DRE). Additional mobility-shift studies using either porcine-derived VDR or recombinant rat VDR from insect cells revealed DRE-binding capability in the absence of 1,25-(OH)2D3. The reactions were performed in various salt environments, with the maximum of porcine VDR-DRE and rat VDR-DRE binding detected at 100 mM and 150 mM KCl, respectively. The addition of 1,25-(OH)2D3 to an identical set of reaction mixtures resulted in increased DRE binding with greater affinities exhibited by both VDR types. These two phenomena were confirmed upon examination of an elution profile of VDR bound to DRE-linked Sepharose. When a linear KCl gradient was used for elution without the addition of 1,25-(OH)2D3, the peak of VDR was 205 mM KCl; the presence of exogenous hormone shifted the maximum VDR elution to a position corresponding to 265 mM KCl. Based on these data and previous reports on VDR-mediated transactivation, we propose a model for 1,25-(OH)2D3-influenced target gene expression.

Phosphorylation is involved in transcriptional activation by the 1,25-dihydroxyvitamin D3 receptor.

The 1,25-dihydroxyvitamin D3 receptor becomes phosphorylated upon treatment with 1,25-dihydroxyvitamin D3. We have investigated the role of phosphorylation in the transcriptional activity induced by 1,25-dihydroxyvitamin D3 through its receptor. An active 1,25-dihydroxyvitamin D3-dependent transcription system was reconstituted in CV-1 cells by co-transfection of plasmids containing the rat 1,25-(OH)2D3 receptor DNA and a functional vitamin D response element (DRE) in a reporter gene construct. Treatment of these transiently transfected CV-1 cells with modulators of protein kinase A (8-Br-cAMP, PKIA and H-9) and phosphatases (Okadaic acid) resulted in mimicking or abolishing the transcriptional activity of 1,25-dihydroxyvitamin D3 in a receptor-dependent fashion. These modulators directly altered 1,25-dihydroxyvitamin D3 receptor phosphorylation. Therefore, the present results strongly suggest that phosphorylation plays a central role in the transcriptional activity of the 1,25-dihydroxyvitamin D3 receptor.

A nuclear protein essential for binding of rat 1,25-dihydroxyvitamin D3 receptor to its response elements.

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 size-exclusion 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 unusual variant of T-CLL: evidence for the existence of a hitherto unrecognized T cell subset.

A case of T-cell chronic lymphocytic leukaemia (T-CLL) with an unusual mature membrane phenotype: E+, CD3+, CD4+, CD8-, M1+, Leu-15+, Fc gamma+, is described. The cells were large granular lymphocytes with slight immature features. Functionally these cells lacked helper, suppressor and NK activity but possessed normal levels of K activity. These findings demonstrate several features not previously described in T-CLL: the coexpression of the antigens detected by T4, M1 and Leu-15 the presence of Fc gamma receptors on CD4+ lymphocytes and the lack of NK activity in M1+, Fc gamma+ cells. This study broadens the known heterogeneity of T-CLL and suggests the existence of a hitherto unrecognized normal T-lymphocyte subset with the same functional and phenotypic characteristics as in the case described here.