The biological effects of 1,25-dihydroxyvitamin D (1,25D), the active metabolite of vitamin D, are assumed to be effected by interaction of the hormone with specific intracellular receptors and their subsequent interaction with cis-acting regulatory response elements within the promoter of the target gene. Lymphoid cells are unique targets of this 1,25D-effect, in that vitamin D receptors (VDR) are not constitutively expressed and prior """"""""activation events"""""""" are required for induction of receptor expression. The applicant has observed that in normal human B lymphocytes upregulation of VDR and productive ligand-receptor modulation of target genes are discrete events which may be differentially regulated, dependent upon whether cellular activation elicits """"""""competency-inducing"""""""" signals or """"""""progression-inducing"""""""" signals. For example, activation of B lymphocytes with anti-mu antibody or anti-CD40 antibody (competency-inducing activators) effectively upregulates VDR expression, yet B lymphocytes activated in this manner are still refractory to many (if not all) of the physiological effects of 1,25D; the hormone fails to modulate proliferation, to induce a more differentiated phenotype, to express VDR-containing nuclear protein complexes reactive with vitamin D response elements (VDREs), and to modulate transcription of vitamin D responsive genes. However, this apparent refractive response to the hormone can be overcome by activation of B lymphocytes with a combination of appropriate competency-inducing and progression-inducing signals, in conjunction with 1,25D. It is hypothesized by the applicant that induction of responsiveness to 1,25D may be determined by a threshold level of expression of VDR and the accessory nuclear receptors RXR (retinoid X receptor) and/or RAR (retinoic acid receptor). Therefore, the major objectives of this proposal are to examine the capacity of B lymphocyte polyclonal activators and B cell-tropic cytokines to upregulate responsiveness to 1,25D, and to correlate this event with cellular regulation of VDR, RXR and RAR nuclear receptor expression. B cell activation in the absence or presence of hormone will be analyzed for its capacity to render cells responsive to discrete stages of hormone-receptor interaction, including upregulation of VDR, RXR and RAR message and protein, formation of nuclear protein complexes interactive with the VDRE, induction of metabolic clearance of the hormone and establishment of biological effects. Biological effects to be examined include modulation of cellular RNA and DNA synthesis and phenotypic expression. Additionally, VDRE-reactive nuclear protein complexes will be characterized for the presence of VDR, and RAR and RXR isoforms. These studies will help to delineate mechanisms governing 1,25D-mediated gene regulation in normal human B lymphocytes, and may allow for further characterization of vitamin D as a mediator of the immune response.
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