T he dendritic cell (DC) plays a central role in orchestrating cellular immune responses to self and foreign antigens. A process of ''maturation'' that occurs after the acquisition of antigen results in up-regulation of MHC͞peptide and costimulatory ligands on the surface of DCs and in the secretion of immunomodulatory cytokines. In the mature state the DC is primed to activate T cells in an antigen-specific fashion. DC maturation is not, however, a consequence of antigen acquisition per se. Exposure to substances within the tissue microenvironment regulate DC maturational events. Among the factors that induce DC maturation are components of pathogenic microorganisms, products secreted by parenchymal cells and macrophages, and contact with activated T cells (1-6). Whether the preservation of DCs in an immature state results from the absence of maturational stimuli or is also actively maintained is not known.We

The centrosome plays an important role in maintenance of cell polarity and in progression through the cell cycle by determining the number, polarity, and organization of interphase and mitotic microtubules. By examining a set of 35 high grade human breast tumors, we show that centrosomes of adenocarcinoma cells generally display abnormal structure, aberrant protein phosphorylation, and increased microtubule nucleating capacity in comparison to centrosomes of normal breast epithelial and stromal tissues. These structural and functional centrosome defects have important implications for understanding the mechanisms by which genomic instability and loss of cell polarity develop in solid tumors.

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Once during each cell cycle, mitotic spindle poles arise by separation of newly duplicated centrosomes. We report here the involvement of phosphorylation of the centrosomal protein centrin in this process. We show that centrin is phosphorylated at serine residue 170 during the G 2 /M phase of the cell cycle. Indirect immunofluorescence staining of HeLa cells using a phosphocentrin-specific antibody reveals intense labeling of mitotic spindle poles during prophase and metaphase of the cell division cycle, with diminished staining of anaphase and no staining of telophase and interphase centrosomes. Cultured cells undergo a dramatic increase in centrin phosphorylation following the experimental elevation of PKA activity, suggesting that this kinase can phosphorylate centrin in vivo. Surprisingly, elevated PKA activity also resulted intense phosphocentrin antibody labeling of interphase centrosomes and in the concurrent movement of individual centrioles apart from one another. Taken together, these results suggest that centrin phosphorylation signals the separation of centrosomes at prophase and implicates centrin phosphorylation in centriole separation that normally precedes centrosome duplication.

immune responses through their ability to mingle with and activate naïve T cells (1, 2). The differentiation of bone marrowderived precursors toward DC lineages and the functional plasticity necessary for an effective spectrum of DC͞T cell interactions is tightly regulated. Identification of individual gene products that are essential to normal DC differentiation and function has provided critical insights for understanding DC biology and for therapeutic modulation of DCs. The clearest example of such a gene product is the NF-B component RelB. Expression of RelB appears late in embryogenesis and is concentrated in the thymus and secondary lymphoid organs: specifically, in interdigitating DCs (iDCs) (3). Deletion of relB resulted in the virtual absence of iDCs with accompanying deficits in cognate immunity (4,5). Functional analysis of the remaining DCs or ex vivo derivation of relB Ϫ/Ϫ DCs revealed failure to up-regulate MHC and costimulatory proteins and deficiency in the capacity to stimulate naïve T cells, cross-present MHC I-restricted peptides, and facilitate Ig isotype switching (6). Significantly, RelB-deficient bone marrow-derived DCs (BMDCs) or BMDCs in which RelB activity is inhibited have the potential to induce antigen-specific immune tolerance in vivo (7).RelB intracellular levels and signaling are enhanced during DC maturation and participate in up-regulation of immunostimulatory proteins (8, 9). Transcriptional regulation of relB has not been extensively studied, although it is known to be inducible by means of NF-B response elements (10). We observed reduced intracellular levels of RelB in BMDCs generated in the presence of the active form of vitamin D 3 , 1␣,25-dihydroxyvitamin D 3 (1␣,25(OH) 2 D 3 ), and related analogs (D 3 analogs) (11,12). This effect depended on expression of the vitamin D receptor (VDR), which is a member of the nuclear receptor (NR) family of DNA binding proteins, and was mediated through binding of VDR͞retinoic acid X receptor ␣ (RXR␣) to vitamin D response elements (VDREs) in the promoter (12). These findings added to a growing body of evidence linking 1␣,25(OH) 2 D 3 ͞VDR to negative regulation of Th1-type immune responses and to inhibition of DC maturation (13-16). The implications of this literature are best exemplified by animal models (allograft rejection, autoimmune diabetes mellitus, and experimental allergic encephalomyelitis) that are ameliorated by 1␣,25(OH) 2 D 3 or D 3 analogs (13-17), by evidence that D 3 analog-conditioned DCs mediate immune tolerance in vivo (13)(14)(15)(16)18), and by epidemiologic studies that identify VDR genotype and vitamin D status as risk factors for autoimmunity (19,20).The interaction of VDR with chromosomal DNA is necessary for positive transcriptional responses, but NR association with additional proteins (coactivators, corepressors, and histone-modifying enzymes) is also crucial (21,22). Although transcriptional suppression by 1␣25(OH) 2 D 3 ͞VDR may be mediated by competitive displacement of positive regulatory factors, activ...

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Transforming growth factor-beta (TGF-beta) is one of the most abundant growth factors secreted by bone cells, and regulation of TGF-beta expression is crucial for bone development and growth. Previous studies from our laboratory demonstrated that 1 alpha, 25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) inhibits human osteoblast and keratinocyte growth by increasing TGF-beta2 secretion and synthesis. To examine the mechanism by which 1 alpha,25(OH)2D3 regulates TGF-beta 2 transcription in osteoblasts, we ligated segments of the human TGF-beta 2 promoter 5' of a growth hormone reporter gene in a growth hormone reporter plasmid and examined the effects of 1 alpha,25(OH)2D3 administration on the expression of growth hormone in cells transfected with such chimeric promoter-reporter plasmids. The promoter region extending from -973 to +68 bp of the transcription start site elicited a 7-fold increase in reporter gene activity in transiently transfected osteoblasts after 1 alpha,25(OH)2D3 treatment, whereas the region from -553 to +68 bp of the transcription start site showed no response following 1 alpha,25(OH)2D3 treatment. Transfection of osteoblasts with reporter plasmids containing TGF-beta 2 promoter DNA from -869 to -658 bp revealed a 3.8-fold increase in reporter gene activity. DNA fragments from this region (-743 to -676 bp and -786 to -728 bp) ligated into reporter plasmids also showed increases in reporter activity. Gel retardation assay experiments showed that DNA fragments from -774 to -735 bp and -714 to -675 bp both formed a DNA-protein complex with bacterially expressed human retinoic acid X receptor alpha (RXR alpha) and vitamin D receptor (VDR) and with nuclear extracts from human bone cells. Addition of a vitamin D receptor antibody to reactions containing the aforesaid DNA fragments and bone cell nuclear extract resulted in further retardation of the mobility of the DNA-protein complex (super-shifting). Removal of two putative direct repeat DNA fragments in this region abolished VDR-RXR alpha-vitamin D response element complex formation. The TGF-beta 2 promoter contains two imperfect direct repeat DNA sequences: TGTAGAACAAGTAGA and AATGAAGTTGGTGGA that mediate the effect of 1 alpha,25(OH)2D3.

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