Cutting edge: 1,25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression.

The hormonal form of vitamin D(3), 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), is an immune system modulator and induces expression of the TLR coreceptor CD14. 1,25(OH)(2)D(3) signals through the vitamin D receptor, a ligand-stimulated transcription factor that recognizes specific DNA sequences called vitamin D response elements. In this study, we show that 1,25(OH)(2)D(3) is a direct regulator of antimicrobial innate immune responses. The promoters of the human cathelicidin antimicrobial peptide (camp) and defensin beta2 (defB2) genes contain consensus vitamin D response elements that mediate 1,25(OH)(2)D(3)-dependent gene expression. 1,25(OH)(2)D(3) induces antimicrobial peptide gene expression in isolated human keratinocytes, monocytes and neutrophils, and human cell lines, and 1,25(OH)(2)D(3) along with LPS synergistically induce camp expression in neutrophils. Moreover, 1,25(OH)(2)D(3) induces corresponding increases in antimicrobial proteins and secretion of antimicrobial activity against pathogens including Pseudomonas aeruginosa. 1,25(OH)(2)D(3) thus directly regulates antimicrobial peptide gene expression, revealing the potential of its analogues in treatment of opportunistic infections.

The pleiotropic actions of vitamin D.

General knowledge of the role of vitamin D3 in human physiology has been shaped by its discovery as a preventive agent of nutritional rickets, a defect in bone development due to inadequate uptake of dietary calcium. Studies on the function of the hormonal form of vitamin D3, 1alpha,25-dihydroxyvitamin D3, have been greatly accelerated by the molecular cloning and structural analysis of the vitamin D3 receptor, which is a ligand-activated regulator of gene transcription. Molecular genetic techniques including genomics have helped reveal that 1alpha,25-dihydroxyvitamin D3 can control more than calcium homeostasis. It has widespread effects on cellular differentiation and proliferation, and can modulate immune responsiveness, and central nervous system function. Moreover, accumulating epidemiological and molecular evidence suggests that 1alpha,25-dihydroxyvitamin D3 acts as a chemopreventive agent against several malignancies including cancers of the prostate and colon. Here, we survey the most-recent findings and discuss their implications for the potential therapeutic uses of vitamin D analogues.

Inhibition of F-Box protein p45(SKP2) expression and stabilization of cyclin-dependent kinase inhibitor p27(KIP1) in vitamin D analog-treated cancer cells.

Treatment of cancer cells with 1,25-dihydroxyvitamin D3 [1,25(OH)(2)D(3)] or its analogs induces growth arrest and expression of the cyclin-dependent kinase inhibitor p27(KIP1). Although 1,25(OH)(2)D(3) transiently enhances p27(kip1) gene transcription in some cells, its effects on p27(KIP1) protein levels are generally more gradual and sustained. This suggests that 1,25(OH)(2)D(3) treatment may be stabilizing p27(KIP1) protein, which is sensitive to modification by the SCF(SKP2) protein ubiquitin ligase and proteosomal degradation. Here, we show that treatment of AT-84 head and neck squamous carcinoma cells with the 1,25(OH)(2)D(3) analog EB1089 increases p27(KIP1) protein levels without significantly affecting expression of its mRNA. EB1089 treatment repressed expression of mRNAs encoding the F-box protein p45(SKP2), a marker of poor head and neck cancer prognosis, and the cyclin kinase subunit CKS1, which is essential for targeting p45(SKP2) to p27(KIP1). This coincided with a reduction of total p45(SKP2) protein, and p45(SKP2) associated with p27(KIP1). Consistent with these findings, turnover of p27(KIP1) protein was strongly inhibited in the presence of EB1089. A similar reduction in p45(SKP2) expression and stabilization of p27(KIP1) protein was observed in 1,25(OH)(2)D(3)-sensitive UF-1 promyelocytic leukemia cells, which also respond by transiently increasing p27(kip1) gene transcription. Our results reveal that 1,25(OH)(2)D(3) analogs increase levels of p27(KIP1) in different cell types by inhibiting expression of SCF(SKP2) subunits and reducing turnover of p27(KIP1) protein.

Ligand-dependent nuclear receptor corepressor LCoR functions by histone deacetylase-dependent and -independent mechanisms.

LCoR (ligand-dependent corepressor) is a transcriptional corepressor widely expressed in fetal and adult tissues that is recruited to agonist-bound nuclear receptors through a single LXXLL motif. LCoR binding to estrogen receptor alpha depends in part on residues in the coactivator binding pocket distinct from those bound by TIF-2. Repression by LCoR is abolished by histone deacetylase inhibitor trichostatin A in a receptor-dependent fashion, indicating HDAC-dependent and -independent modes of action. LCoR binds directly to specific HDACs in vitro and in vivo. Moreover, LCoR functions by recruiting C-terminal binding protein corepressors through two consensus binding motifs and colocalizes with CtBPs in the nucleus. LCoR represents a class of corepressor that attenuates agonist-activated nuclear receptor signaling by multiple mechanisms.

1Alpha,25-dihydroxyvitamin D3 promotes vascularization of the chondro-osseous junction by stimulating expression of vascular endothelial growth factor and matrix metalloproteinase 9.

Vitamin D deficiency results in defects in endochondral bone development characteristic of rickets, which include elongation of the cartilaginous growth plates and disorganization of the primary spongiosa. These defects are caused in part by impaired cartilage mineralization and vascularization of the chondro-osseous junction. Blood vessel invasion of mineralized cartilage is an essential step in endochondral ossification, providing access for cells that degrade cartilage as well as those that form bone. Vascular endothelial growth factor (VEGF) was shown to be a key regulator of this process when infusion of a dominant negative VEGF receptor effectively blocked vascular invasion and endochondral ossification in the growth plates of juvenile mice. Here, we show that the active metabolite of vitamin D 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] directly stimulates transcription of mRNAs encoding VEGF121 and -165 isoforms in the CFK2 chondrogenic cell line. Enhanced VEGF expression also was evident in growth plate chondrocytes and osteoblasts in the tibia of juvenile mice treated systemically with 1alpha,25(OH)2D3. This was seen in conjunction with enhanced expression of matrix metalloproteinase (MMP) 9, which activates VEGF stored in the cartilage matrix, in osteoclastic cells adjacent to the chondro-osseous junction. The alterations in VEGF and MMP-9 expression were accompanied by enhanced vascular invasion of mineralized cartilage, as assessed by CD31 immunoreactivity. These results provide evidence that 1alpha,25(OH)2D3 signaling stimulates VEGF and MMP-9 gene expression and promotes neovascularization of the epiphyseal growth plate in vivo through increased availability of active growth factor.

Expression profiling in squamous carcinoma cells reveals pleiotropic effects of vitamin D3 analog EB1089 signaling on cell proliferation, differentiation, and immune system regulation.

The active form of vitamin D3, 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is key mediator of calcium homeostasis and is a component of the complex homeostatic system of the skin. 1,25-(OH)2D3 regulates cellular differentiation and proliferation and has broad potential as an anticancer agent. Oligonucleotide microarrays were used to assess profiles of target gene regulation at several points over a 48 h period by the low calcemic 1,25-(OH)2D3 analog EB1089 in human SCC25 head and neck squamous carcinoma cells. One hundred fifty-two targets were identified, composed of 89 up- and 63 down-regulated genes distributed in multiple profiles of regulation. Results are consistent with EB1089 driving SCC25 cells toward a less malignant phenotype, similar to that of basal keratinocytes. Targets identified control inter- and intra-cellular signaling, G protein-coupled receptor function, intracellular redox balance, cell adhesion, and extracellular matrix composition, cell cycle progression, steroid metabolism, and more than 20 genes modulating immune system function. The data indicate that EB1089 performs three key functions of a cancer chemoprevention agent; it is antiproliferative, it induces cellular differentiation, and has potential genoprotective effects. While no evidence was found for gene-specific differences in efficacy of 1,25-(OH)2D3 and EB1089, gene regulation by 1,25-(OH)2D3 was generally more transient. Treatment of cells with 1,25-(OH)2D3 and the cytochrome P450 inhibitor ketoconazole produced profiles of regulation essentially identical to those observed with EB1089 alone, indicating that the more sustained regulation by EB1089 was due to its resistance to inactivation by induced 24-hydroxylase activity. This suggests that differences in action of the two compounds arise more from their relative sensitivities to metabolism than from differing effects on VDR function.