A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Psoriasis is a chronic inflammatory skin disease featuring abnormal keratinocyte proliferation and differentiation. A genetic risk factor for psoriasis (PSORS4) is a deletion of LCE3B and LCE3C genes encoding structural proteins in differentiated keratinocytes. Because analogs of 1,25-dihydroxyvitamin D3 (1,25D) are used in psoriasis treatment, we hypothesized that 1,25D and other VDR ligands act via the vitamin D receptor (VDR) to upregulate expression of LCE 3A/3D/3E genes, potentially mitigating the absence of LCE3B/LCE3C gene products. This hypothesis was pursued using cultured keratinocytes, quantitative real time PCR (qPCR) to assess LCE gene expression, competition binding assays to assess direct ligand binding to VDR, and reporter gene assays to assess the ability of VDR ligands to activate transcription in a VDR- and VDR response element-dependent fashion. qPCR results in a human keratinocyte line, HaCaT, suggested that 1,25D and selected alternate or candidate VDR ligands might upregulate LCE transcripts. Further experiments in primary human keratinocytes confirmed that 1,25D and 10 µM delphinidin do indeed upregulate all five LCE3 genes (LCE3A–E), especially in calcium-differentiated cultures. Further, competition binding assays revealed that delphinidin does in fact bind VDR, but only weakly (IC50 approximately 1 mM). Finally, 20 µM delphinidin was shown to be capable of upregulating a luciferase reporter gene linked to a vitamin D responsive element found near the LCE3 gene cluster. Taken together, these results are consistent with delphinidin (or a metabolite) stimulating LCE3 transcription in a VDR/VDRE-dependent manner. We propose that upregulation of LCE genes may be part of the therapeutic effect of 1,25D to ameliorate psoriasis by providing sufficient LCE proteins, especially in individuals missing the LCE3B and 3C genes. Results with delphinidin further suggest that this compound or its metabolite(s) might offer an alternative to 1,25D in psoriasis therapy.

The vitamin D metabolite 1,25-dihydroxyvitamin D3 [1α,25(OH)2D3] plays an important role in the regulation of musculoskeletal growth and differentiation. 1α,25(OH)2D3 mediates its effects on cells, including chondrocytes and osteoblasts, through the classical nuclear 1α,25(OH)2D3 receptor. Additionally, recent evidence indicates that several cellular responses to 1α,25(OH)2D3 are mediated via a rapid, calcium-dependent membrane-mediated pathway. These actions of 1α,25(OH)2D3 can be blocked by antibodies to protein-disulfide isomerase family A, member 3 (Pdia3), indicating that it is part of the receptor complex; however, the pathway which is activated by this receptor is not fully understood. The overall goal of this thesis was to examine the roles of phospholipase A2 activating protein (PLAA) and calcium calmodulin-dependent kinase II (CaMKII) in 1α,25(OH)2D3 rapid membrane-mediated signaling. We further investigated the interaction between two pathways regulating growth plate cartilage and endochondral bone formation, 1α,25(OH)2D3 and Wnt5a, at the receptor complex level. Results indicated that PLAA was required for mediating 1α,25(OH)2D3 signal from Pdia3. Furthermore, CaM and CaMKII were identified as mediators of 1α,25(OH)2D3-stimulated PLAA-dependent activation of cPLA2 and PKCα, and downstream biological effects. Wnt5a and 1α,25(OH)2D3 are important regulators of endochondral bone formation. This study demonstrated that 1α,25(OH)2D3 and Wnt5a mediate their effects via similar receptor components in osteoblasts and chondrocytes suggesting that these pathways may interact.

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Vitamin D is a fat-soluble hormone primarily responsible in maintaining plasma calcium and phosphorus homeostasis in humans. Vitamin D insufficiency and deficiency is a global health issue. Very few foods naturally contain vitamin D; a major source is oily fish such as salmon. Several studies have analyzed vitamin D content in various fish, however studies concerning canned fish are lacking. In particular, this study was interested in evaluating the vitamin D content in canned sardines in not only the whole fish but also in the olive oil or water it was canned in. It was hypothesized that the vitamin D content in sardines canned in water would be greater than sardines canned in olive oil due to the fat-soluble nature of vitamin D to be more easily extracted into olive oil than water. Sardines (~100g) canned in olive oil had a slightly greater vitamin D content than the sardines in water (2,555.6±234.2 and 1,993.7±2,411.3 IUs (p<0.05) respectively). An evaluation of the vitamin D3 content in the olive oil and water used to can the sardines revealed 701.4±471.1 and 149.1±42.2 IUs in the total olive oil and water respectively recovered from the cans. It was determined that of the total vitamin D content in the can (sardines in olive oil or water) 20.9%±12.8% of vitamin D3 is found in the olive oil compared to only 14.2%±10.4% (p<0.05) vitamin D3 found in water. These results support the concept that sardines packed in olive oil may have less vitamin D3 than similar sardines packed in water. The analysis of the sardines revealed that they had more than 13 times the amount of vitamin D3 than that is reported in the USDA table of nutritional facts for canned sardines. This could be because the sardines were caught in the summer months when they are more likely to be consuming food containing vitamin D3 as a result of reduced synthesis of vitamin D3 in zooplankton and other lower life forms that the sardines consume. An alternative explanation for this increase in vitamin D3 content is the process of canning the sardines. Vital Choice, the supplier of the sardines, immediately ices the fish upon retrieval from the ocean (to ensure freshness) and then are canned in less than 5 hours after being caught. This process of freshness preservation could explain why the vitamin D content was so high; possibly an accurate representation of the original vitamin D content in the sardines.

Biologically active 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) binds the vitamin D receptor (VDR) to exert its effect on target cells. VDR expression is found in a number of immune cells including professional antigen-presenting cells such as dendritic cells. It has been found that the actions of 1,25-(OH)2D3 on the immune system are mainly immunosuppressive. The cross-presentation pathway allows for exogenously derived antigens to be presented by pAPCs on MHC-I molecules to CD8+ T cells. CD8+ T cell activation results in the expansion of epitope-specific T cell populations that confer host protection. These epitopes can be organized into an immunodominance hierarchy. Previous work demonstrated that introducing LCMV-NP via the cross-priming pathway significantly alters the immunodominance hierarchy of a subsequent LCMV infection. Building upon these observations, our study assessed the effects of LCMV-NP cross priming in the presence of a single dose of 1,25-(OH)2D3. Treatment with 1,25-(OH)2D3 was found to have biological effects in our model system. In vitro pAPCs were demonstrated to up-regulate IL-10 and CYP24A1 mRNA, in addition to the transactivation of cellular VDR, as demonstrated by a relocalization to the nuclear region. Mice treated with 1,25-(OH)2D3 were found to produce up-regulated IL-10 and CYP24A1 transcripts. Expression of VDR was increased at both the transcript and protein level. Our results demonstrate that a single dose of 1,25-(OH)2D3 does not affect the cross-priming pathway in this system. Treatment with 1,25-(OH)2D3 did not influence the ability of differentiated pAPCs to phagocytose or cross-present exogenous antigen to epitope-specific CD8+ T cells. Furthermore, 1,25-(OH)2D3 did not alter cross-priming or the establishment of the LCMV immunodominance hierarchy in vivo. By confirming that 1,25-(OH)2D3 does not suppress cross-priming in our model, our study helps to expand the understanding of the immunomodulatory role of exogenous 1,25-(OH)2D3 on the outcome of virus infection. Collectively, our data supports the observation that the role of 1,25-(OH)2D3 in the immune system is not always associated with suppressive effects. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2011-08-29 14:53:18.766

Epidemiological evidence from season of birth and migrant studies has led to the proposal that developmental vitamin D3 (DVD) deficiency may be a risk factor for schizophrenia. Concurrently, there has been recent intense interest in vitamin D3 as a modifying factor in the development of immune responses. However, the effects of DVD deficiency on immune function remains unknown. Thus, a model of DVD deficiency has been developed in Sprague-Dawley rats. Briefly, female rats were maintained on a vitamin D3 deficient diet prior to and during gestation. At birth dams were returned to a vitamin D3 replete diet. Post-weaning their offspring, the DVD-deficient rats, were maintained on a vitamin D3 replete diet. Therefore, this model represents a transient, prenatal vitamin D3 deficiency. A number of structural and cellular changes have been described in the DVD-deficient rat brain, including ventriculomegaly, decreased growth factor expression, and increased rates of mitosis. These changes are correlated with altered adult behaviour in the DVD-deficient rat, including a locomotor sensitivity to novelty. This thesis will focus on extending these findings and characterising immune function in the DVD-deficient rat. Vitamin D3 can affect cellular differentiation and is anti-proliferative and pro-apoptotic in many tissues including the brain. The effect of DVD deficiency on brain development has been restricted to neuronal cells in vivo and in vitro. However, the effect of this exposure on glial cell maturation and phenotype was unknown. The experiments reported in this thesis demonstrated that primary cortical glial cell cultures from DVD-deficient rat neonates displayed a similar phenotype and maturational status compared with cells from control rats. Learning and memory was examined in this model by exploring the phenomenon of latent inhibition. DVD-deficient rats displayed normal latent inhibition, however, they exhibited a subtle performance acquisition deficit during the early stages of the conditioned avoidance learning task. Extended handling and pre-exposure were able to ameliorate this deficit, though with this treatment normal latent inhibition was also abolished in both control and DVD-deficient rats. Ultrasonic vocalization and nociceptive threshold testing confirmed that alterations in peripheral sensation could not explain this performance acquisition deficit. The results suggested that anxiety or attentional mechanisms may have contributed to this rate of learning deficit. Finally, as vitamin D3 is a powerful immunoregulator, there is the potential for a transient DVD deficiency to induce a persistent alteration in the development and function of the immune system. This hypothesis was supported by findings that showed DVD deficiency resulted in a primed immune system, as indicated by an enlarged spleen, thymus and peripheral lymph tissue as well as increased pro-inflammatory cytokine production in response to an in vitro stimulation. However, these findings did not lead to an alteration in cell mediated immune response in vivo. The results from the research reported in this thesis indicate that a transient, prenatal vitamin D3 deficiency had a subtle yet significant effect on the immune system and behaviour of the adult rat. These findings add further weight to the body of evidence that link prenatal vitamin D3 status to various adverse health outcomes. The DVD-deficient rat model is an integral step in understanding prenatal vitamin D3 deficiency as a potential environment risk factor in the development of immune and psychiatric disorders.

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates the uptake of phosphate in isolated chick intestinal cells , while the steroid 24,25- dihydroxyvitamin D3 [24,25(OH)2D3] inhibits the rapid stimulation by l,25(OH)2D3. Earlier work in this laboratory has indicated that a cellular binding protein for the 24,25(OH)2D3 is the enzyme catalase. Since binding resulted in decreased catalase activity and increased H2O2 production, studies were undertaken to determine if pro-oxidant conditions mimicked the inhibitory actions of 24,25(OH)2D3, and anti-oxidant conditions prevented the inhibitory actions of 24,25(OH)2D3. An antibody against a putative 24,25(OH)2D3 binding protein was found to neutralize the inhibitory effect of the steroid on 1,25(OH)2D3-mediated 32P uptake (P2D3, each in Cells exposed to hormone alone again showed an increased accumulation of 32P from T=5-10 min, while cells treated with catalase inhibitor and hormone had uptake levels that were indistinguishable from controls. We tested whether inactivation of protein kinase C (PKC), the signaling pathway for 32P uptake, occurred. Incubation of cells with 100 nM phorbol-13-myristate (PMA) increased 32P uptake to 143% of controls, while cells pretreated with 50 μM H2O2 prior to PMA did not exhibit increased uptake. Likewise, PMA significantly increased PKC activity at T=1-3 min (P2O2 prior to PMA did not. It is concluded that catalase has a central role in mediating rapid responses to steroid hormones.

Abstrakt BakgrundProstatacancer är ett mycket vanligt tillstånd som påverkar miljoner människor varje år över världen. Faktum är att cancer är en grupp med cirka 200 olika sjukdomar som orsakas av okontrollerad celltillväxt och dessa celler delar sig okontrollerat tills en tumör uppkommer vilken kan sprider sig över hela kroppen. Cancer beror på fel som uppstår i DNA, så kallade mutationer. Prostatacancer är en av de vanligaste orsakerna till cancerdödlighet hos män, det vill säga att chanser att bota sjukdomen är större om diagnosen sker tidigt i förloppet.SyftetSyftet med detta arbete är att undersöka om vitamin D kan påverka utvecklingen av prostatacancer. Metoden och MaterialI denna arbetet gjordes på litteraturstudien genom sökord i PubMed. Efter de artikelsökningen gjordes om mitt arbete är baserat på sex artiklar och att dessa identifierade med hjälp av databasen PubMed och specifika sökord i kombination med inkludering-och exkluderingskriterier.Resultat Studien har visat att låga nivåer av vitamin D kan också kopplats till en ökad risk för prostatacancer. Det har visat att patienter med prostatacancer medelhöga eller höga vitamin D nivåer i blodet kan vara kopplade till bättre resultat än lägre nivåer det vill säga att högre nivåer av vitamin D är associerat med förbättrad överlevnad. Resultatet redovisade av de artiklarna att vitamin D påverkar utvecklingen av prostatacancer genom att bromsa utvecklingen av cancer.slutsatsI denna litteraturstudie har visat att det finns ett samband mellan 25-hydroxivitamin D och 1,25-dihroxivitamin D är skydda mot patienter som lever med prostatacancer. Det har majoriteten visat på att män med förhöjda av 1,25-dihroxivitamin D, vilket ger bättre överlevnad.

It is now accepted that 1,25(OH)2D3 mediates its rapid actions on the control of phosphate and calcium homeostasis through its membrane receptor termed the 1,25D3-MARRS (membrane associated rapid response steroid binding) protein. I determined the various PKC isotypes involved in the rapid regulation of phosphate uptake and calcium extrusion in chick intestinal cells. 1,25(OH)2D3-mediated phosphate uptake was stimulated within 1 min after addition of the hormone. Western blot analyses on isolated intestinal cells treated with steroid hormone resulted in dose-dependent increases in PKC alpha and PKC beta in postnuclear centrifugation fractions, but not in the low speed centrifugation fractions. The highest immunoreactivity of PKC alpha was found after treatment of the cells with 300 pM 1,25(OH)2D3 and declined at 650 pM hormone, relative to corresponding controls, while the highest immunoreactivity of PKC beta was found in cells treated with either 300 pM or 650 pM 1,25(OH)2D3. Therefore, PKC alpha and PKC beta redistribution are likely to relate to the dose-response curve for both phosphate uptake and calcium efflux, respectively. Using transfection of primary cultures of intestinal cells with siRNA for these two isotypes, I found decreased 32P uptake in cells transfected with siRNA to either PKC alpha or PKC beta in both controls (relative to untransfected controls), and hormone-treated cells. Further study of the effect of chemical blockers for PKC alpha or PKC beta on phosphate uptake was conducted in suspensions of isolated intestinal cells. The results from these experiments also confirmed the findings from the siRNA experiments and demonstrated decreased 32P uptake in cells treated with 1,25(OH)2D3 plus blockers in comparison with cells treated with 1,25(OH)2D3 alone. The effects of PKC alpha and PKC beta in steroid-mediated calcium extrusion were further investigated using siRNA for PKC alpha or PKC beta. We found the siRNA to PKC beta alone caused decreased calcium extrusion. We also found that the inhibitors of PKC beta, but not PKC alpha caused significantly enhanced calcium uptake by decreasing calcium efflux from the cells. This result suggested that PKC beta might be involved in the rapid response of 1,25(OH)2D3-stimulated calcium extrusion. I used confocal microscopy to study the redistribution of PKC alpha and PKC beta in cells exposed to steroid hormone for 30 sec. PKC alpha was found to increase significantly in the apical membrane after a 30 sec exposure of cells to 300- or 650 pM 1,25(OH)2D3. By comparison, anti-PKC beta immunofluorescence was found to increase significantly in the basal region of cells, relative to controls, following exposure of cells to 300 pM seco-steroid. These combined results, lead me to conclude the involvement of both PKC alpha and PKC beta in the signal transduction mechanism of 1,25(OH)2D3-mediated phosphate uptake while PKC beta is involved in the mechanism of 1,25(OH)2D3-mediated calcium efflux in chick intestinal epithelial cells.

Over 200,000 cases of breast cancer are diagnosed every year. Nearly 20% of these patients supplement their diets with some form of vitamin D. This high frequency of vitamin D supplement use may be due in part to research suggesting that cancer patients with higher serum vitamin D3 levels have better prognoses than patients with low serum vitamin D3. However, double-blind clinical trials on the efficacy of vitamin D3 supplementation in breast cancer have been inconclusive. A recent meta-analysis showed evidence of reduced cancer recurrence in patients taking vitamin D3 supplements who had ‘estrogen receptor positive’ (ERα66+) breast cancer, but not those who had estrogen receptor negative’ (ERα66-) breast cancer. Once ingested, vitamin D3 is metabolized in the liver into the circulating pre-hormone 25(OH)D3, which is then further metabolized into 1a,25(OH)2D3 and 24R,25(OH)2D3. 24R,25(OH)2D3 has been shown to activate a number of membrane signaling pathways, some of which overlap with 17b-estradiol (E2) signaling through ERα36, a membrane isoform of ERα66. The central hypothesis of this thesis was that 24R,25(OH)2D3 is tumorigenic in certain cancers and that this tumorigenicity is mediated in part by ERa isoforms. E2 signaling through ERa36 has been described in the ERa66-, ERa36+ breast cancer cell line HCC381. Specific aim 1 determined whether E2 signaling through ERa36 was tumorigenic other cancers with different ERa profiles. Specific aim 2 determined how 24R,25(OH)2D3 affected tumorigenicity in breast cancer using the common breast cancer cell line MCF7 (ERa66+, ERa36+) as a model. Specific aim 3 investigated the role of ERa isoforms in 24R,25(OH)2D3 signaling in breast cancer cell lines by comparing the tumorigenic effects of 24R,25(OH)2D3 in MCF7 cells (ERa66+, ERa36+) and HCC38 cells (ERa66-, ERa36+). To determine whether ERa66 regulates the effects of 24R,25(OH)2D3, ERa66 was expressed in two ERα66- cell lines. The effect of 24R,25(OH)2D3 on apoptosis was assessed in wild-type and ERa-expressing cell lines.