The vitamin D receptor (VDR) ligand, 1,25(OH)2D3, reduces proliferation and enhances differentiation and thus has been investigated for a role in preventing or treating cancer. Mice deficient for the VDR display a hyperproliferative response in the hair follicle and epidermis and decreased epidermal differentiation. Unlike their wild type littermates, when treated with 7,12 dimethylbenzanthracene (DMBA) or UVB, they develop skin tumors, including some characteristic of over-expression of the hedgehog (Hh) pathway. Both the epidermis and utricles of the VDR null animals over-express elements of the Hh pathway [Sonic Hedgehog (Shh, 2.02 fold), Patched1 1.58 fold, Smoothened 3.54 fold, Gli1 1.17 fold, and Gli2 1.66 fold]. This over-expression occurs at an age (11 weeks) where epidermal hyperproliferation is most visible and is spatially controlled in the epidermis. DMBA or UVB induced tumors in the VDR null mice also over-express elements of this pathway. Moreover, 1,25(OH)2D3 down-regulates the expression of some members of the Hh pathway in an epidermal explants culture system, suggesting a direct regulation by 1,25(OH)2D3. Our results suggest that increased expression of Shh in the keratinocytes of the VDR null animal activates the Hh pathway, predisposing the skin to the development of both malignant and benign epidermal neoplasms.

The objectives of this research were to study the effects of high intensity (0.5, 0.75, and 1.0 mW/cm (2)), dose (0.5, 1.0, and 1.5 J/cm (2)), and postharvest time (1 and 4 days) on the vitamin D 2 formation in Portabella mushrooms ( Agaricus bisporus) as a result of UV-B exposure, as well as the vitamin D 2 degradation in treated mushrooms during storage. Within each intensity application, dose had the largest effect where more exposure converted more vitamin D 2 from ergosterol. Similar dose across each intensity application resulted in similar vitamin D 2 concentration. Practical commercial production requires as short a treatment time as possible, and intensity was a major factor from this standpoint where the time it took to achieve a similar vitamin D 2 concentration for similar dose exposure was significantly reduced as intensity increased. By using an intensity of 1.0 mW/cm (2) at a dose of 0.5 J/cm (2), the concentration of vitamin D 2 produced was 3.83 microg/g dry solids of mushrooms in 8 min, whereas using an intensity of 0.5 mW/cm (2) at a dose of 0.5 J/cm (2), the concentration of vitamin D 2 produced was 3.75microg/g dry solids of mushrooms in 18 min. Also, postharvest time did not have a significant effect on vitamin D 2 formation in mushrooms that were treated 1 and 4 days after harvest. Vitamin D 2 degraded in treated mushrooms during storage by apparent first-order kinetics, where the degradation rate constant was 0.025 h (-1). The information provided in this study will help mushroom producers develop commercial-scale UV treatment processes to add value to their crop while improving consumer health.

The vitamin D receptor (VDR) regulates a diverse set of genes that control processes including bone mineral homeostasis, immune function and hair follicle cycling. Upon binding to its natural ligand, 1α,25(OH) 2 D 3 , the VDR undergoes a conformational change that allows the release of corepressor proteins and the binding of coactivator proteins necessary for gene transcription. We report the first comprehensive evaluation of the interaction of the VDR with a library of coregulator binding motifs in the presence of two ligands, the natural ligand 1α,25(OH) 2 D 3 and a synthetic, non-secosteroidal agonist LG190178. We show that the VDR has relatively high affinity for the second and third LxxLL motifs of SRC1, SRC2 and SRC3 and second LxxLL motif of DRIP205. This pattern is distinct in comparison to other nuclear receptors. The pattern of VDR-coregulator binding affinities was very similar for the two agonists investigated, suggesting that the biologic functions of LG190178 and 1α,25(OH) 2 D 3 are similar. Hairless binds the VDR in the presence of ligand through a LxxLL motif (Hr-1), repressing transcription in the presence and absence of ligand. The VDR binding patterns identified in this study may be used to predict functional differences among different tissues expressing different sets of coregulators, thus facilitating the goal of developing tissue and gene specific vitamin D response modulators.The vitamin D receptor (VDR), which binds 1α,25-dihydroxyvitamin D 3 (1α,25(OH) 2 D 3 ), contains several functional domains, including a ligand-binding domain (LBD), that mediates ligand-dependent gene regulation (1). A critical step in 1α,25(OH) 2 D 3 action is the induction of a LBD conformational change to form activation function 2 (AF-2)(2), a hydrophobic cleft formed by three helices and a short COOH-terminal amphipathic alpha helix (H12) (3), which serves as a binding surface for coactivators (4). Unliganded nuclear receptor (NR) heterodimers associate with corepressors such as the nuclear receptor corepressor (NCoR) and the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) (5,6) and associated histone deacetylases (7,8). These proteins function as adaptors to convey a repressive signal to the transcriptional apparatus by maintaining a closed chromatin structure with the histone Nterminal 'tails' in a charged state tightly associated with DNA (9). Ligand binding promotes the release of corepressors and the binding of coactivators, enhancing the transcription of ‡ These authors contributed equally to this work. * Corresponding author: Tel: 415-750-2089, Fax: 415-750-6929, e-mail: Daniel.bikle@ucsf NR corepressors NCoR and SMRT encode multiple, short receptor interaction domains composed of the sequence ΦxxΦΦ (Φ is leucine or isoleucine and x is any amino acid) (26,27). This motif is predicted to form an alpha helix, that is one turn longer than that formed by the LxxLL motif. In a manner analogous to the LxxLL-containing motifs, it has been suggested that this helix also binds the...

Mice null for the Vitamin D receptor (VdrKO) have a disrupted first hair follicle cycle and aborted subsequent hair follicle cycling. We examined the expression of different markers and mediators of hair follicle cycling in the hair follicle of the VdrKO mouse during days 13–22 when the hair follicle normally initiates and completes the first catagen. We compared the expression of those genes in mice with a nonsense mutation in hairless (Rhino), which have a similar alopecia phenotype, and to Cyp27b1 null mice which are deficient in the production of 1,25(OH)2D3, the Vdr ligand, but display normal hair follicle cycling. Our results demonstrate the down regulation of hair follicle markers and the alteration of expression of hedgehog (Hh), Wnt, Fgf, and Tgfβ pathways in VdrKO and Rhino mice, but not in Cyp27b1KO mice. Treatment of VdrKO mice with an agonist to the Hh pathway partially restored hair follicle cycling, suggesting a role of this pathway in the regulation of hair follicle cycling by VDR. These results suggest that Vdr regulates directly or indirectly the expression of genes required for hair follicle cycling, including Hh signaling, independent of 1,25(OH)2D3.

Vitamin D sufficiency is associated with protection against malignancy in a number of tissues clinically, and a strong body of evidence from animal and cell culture studies supports this protective role. Cancers in the skin differ, however, in that higher serum levels of 25OHD are associated with increased basal cell carcinomas (BCC), the most common form of epidermal malignancy. This result may be interpreted as indicating the role of UVR (spectrum 280–320) in producing vitamin D in the skin as well as causing those DNA mutations and proliferative changes that lead to epidermal malignancies. Recent animal studies have shown that mice lacking the vitamin D receptor (VDR) are predisposed to developing skin tumors either from chemical carcinogens such as 7,12 dimethylbenzanthracene (DMBA) or chronic UVR exposure. Such studies suggest that vitamin D production and subsequent signaling through the VDR in the skin may have evolved in part as a protective mechanism against UVR induced epidermal cancer formation. In this manuscript we provide evidence indicating that vitamin D signaling protects the skin from cancer formation by controlling keratinocyte proliferation and differentiation, facilitating DNA repair, and suppressing activation of the hedgehog (Hh) pathway following UVR exposure.

Ultra violet (UV) irradiation, in particular UVB, is the single most important carcinogen for skin tumor formation. UVB induces genetic mutations and immune suppression, which lead to abnormal cell proliferation and eventually tumor formation. Previously studies from our group and others demonstrated that both global and epidermal specific VDR knock out mice are predisposed to either chemical (DMBA)-or long-term UVB-induced skin tumor formation, paralleled by an increase in β-catenin signaling. Using primary cultured human keratinocytes, we further demonstrated that 1,25(OH)2-dihydroxyvitamin D3 (1,25(OH)2D3) suppresses cyclin D1 and Gli1 which are regulated by β-catenin/TCF signaling and have a critical role in epidermal carcinogenesis. Blockage of VDR by siRNA resulted in hyperproliferation of keratinocytes, and increased expression of cyclin D1 and Gli1. In addition, we also showed that 1,25(OH)2D3/VDR directly regulates transcriptional activity of β-catenin/TCF signaling using the –catenin reporter TopGlow. Using K14 driven tamoxifen-induced cre recombinase to delete both VDR and β-catenin in keratinocytes of mice following the first hair follicle cycle, we found that ablation of epidermal specific β-catenin cannot rescue VDR null mice from UVB-induced skin tumor formation. Further study using VDR or β-catenin single null mice is necessary to compare with the data from double null mice.

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The transcriptional activity of the vitamin D receptor (VDR) is regulated by a number of coactivator and corepressor complexes, which bind to the VDR in a ligand (1,25(OH) 2 D 3 ) dependent (coactivators) or inhibited (corepressors) process. In the keratinocyte the major coactivator complexes include the vitamin D interacting protein (DRIP) complex and the steroid receptor coactivator (SRC) complexes. These coactivator complexes are not interchangeable in their regulation of keratinocyte proliferation and differentiation. We found that the DRIP complex is the main complex binding to VDR in the proliferating keratinocyte, whereas SRC2 and 3 and their associated proteins are the major coactivators binding to VDR in the differentiated keratinocyte. Moreover, we have found a specific role for DRIP205 in the regulation of β-catenin pathways regulating keratinocyte proliferation, whereas SRC3 uniquely regulates the ability of 1,25(OH) 2 D 3 to induce more differentiated functions such as lipid synthesis and processing required for permeability barrier formation and the innate immune response triggered by disruption of the barrier. These findings provide a basis by which we can understand how one receptor (VDR) and one ligand (1,25(OH) 2 D 3 ) can regulate a large number of genes in a sequential and differentiation specific fashion. KeywordsVitamin D receptor; coactivators; keratinocytes; differentiation; proliferation; β-catenin; innate immunity; permeability barrier INTRODUCTIONThe epidermis is composed of four layers of keratinocytes at different stages of differentiation (Fig. 1). The basal layer (stratum basale, SB) rests on the basal lamina separating the dermis and epidermis. Within this layer are the stem cells. These cells proliferate, providing the cells for the upper differentiating layers. They contain an extensive keratin network comprised of keratins K5 and K14. The layer above the basal cells is the © 2010 Elsevier Ltd. All rights reserved. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. spinous layer (stratum spinosum, SS). These cells initiate the production of the keratins K1 and K10, which are the keratins characteristic of the more differentiated layers of the epidermis. Cornified envelope precursors such as involucrin also appear in the spinous layer as does the enzyme transglutaminase, responsible for the ε-(γ-glutamyl) lysine cross-linking of these substrates into the insoluble cornified envelope. The granular layer (stratum granulosum,SG), lying above the spinous layer, is characterized by electron-dense keratohyalin granules containing profilaggr...