Dissertations / Theses on the topic 'Vitamin D; bone cells'

While the major source of vitamin D is D₃ from ultraviolet exposure, some supplements supply D₂. The relative potency of vitamin D₂ versus vitamin D₃ remains controversial. The aims of the current study were, 1. To optimize the in vitro model, including use of cell lines, vitamin D concentrations, and outcome biomarkers. 2. To compare the potency of vitamin D₂ and D₃ metabolites on mouse and human bone cellular activity. 3. To explore the expression of VDR in osteoarthritic (OA) bone tissues as well as cellular responses to vitamin D₂ and D₃ metabolites ex-vivo. In mouse 2T3 osteoblasts, at physiological doses, both vitamin D₂ and D₃ metabolites increased ALP activity and mineralisation and up-regulated osteoblastic signature genes and proteins. At supra-physiological doses D₃ metabolites were more potent inhibitors of 2T3 function than D₂ metabolites. Although hBMS cell proliferation was inhibited by both 25(OH)D₂ and D₃, ALP activity was enhanced by both metabolites. However, 25(OH)D₃ was a more potent stimulator of ALP and mineralisation of hBMSCs. D₂ and D₃ equally stimulated expression of CX43 and PHEX markers in osteocytic cell lines. Immunohistochemistry of femoral heads showed much reduced VDR expression in OA osteocytes and osteoclasts, yet both 25(OH)D2 and D₃ increased OA-hBMSCs mineralisation more than non-OA-hBMSCs ex-vivo. While vitamin D₂ or D₃ increased mouse 2T3 osteoblastic activity at physiological doses, OA and non-OA hBMSCs differentiation was more responsive to 25(OH)D₃. Key bone cells such as osteocyte and osteoclasts expressed less VDR in OA. For the first time vitamin D₂ metabolites have been thoroughly examined and emerged as a potent stimulator of bone cell differentiation, at least in vitro. Vitamin D₃ in contrast is confirmed as highly potent in bone cells, but with toxicity at much lower doses than D₂.

Vitamin D is a precursor to a steroid hormone, 1,25 dihydroxyvitamin D (1,25(OH)2D). After its discovery and the characterization of its receptor, the vitamin D receptor (VDR), it was initially thought only to be involved in calcium homeostasis, but further research revealed an important role for vitamin D in the regulation of cell growth and differentiation of such cells as osteoblasts and bone marrow adipocytes. 1,25(OH)2D has also been shown to be a strong inhibitor and pro-differentiator of keratinocytes. The anti-proliferative and pro-differentiative properties of this hormone have led to studies where 1,25(OH)2D anticancer properties were assessed and initial findings that showed a requirement of other factors beyond VDR to induce 1,25(OH)2D signaling led to the identification of the retinoid X receptor, a common heterodimeric partner for several hormone receptors. The focus of thesis was to further elucidate the structure-function relationship of both the vitamin D receptor and the retinoid X receptor. Additionally, contributions to work directed towards further identifying the effects of vitamin D on osteoblast differentiation and survival. Interactions of 1,25(OH) 2D3 with its cognate receptor, identifying a key amino acid (Tryptophan 286) required for ligand contact and transcriptional activation, are described in Chapter 2. Mechanisms of vitamin D action on mesenchymal stem cell differentiation, promotion of osteoblast induction and maturation, and inhibition of adipocyte differentiation, are eluicidated in Chapter 3. Chapter 4 illustrates the effects of RAS/RAF/Mitogen-activated protein kinase mediated RXRalpha phosphorylation on the three-dimensional structure of the RXR/nuclear receptor partner heterodimers. Furthermore, this chapter reveals the inhibitory effect of the phosphorylation of a critical amino acid (serine 260) on the interaction of the AF-2 domain of the RXR with several coactivators, resulting in a decrease in the signaling potential of multiple steroid hormone receptors. The findings of this thesis further the knowledge of several areas of vitamin D biology, including both the canonical areas of bone formation, and the non-canonical area of vitamin D and cancer.

Most of the load-bearing demand placed on the human body is transduced by skeletal tissue, and the capacity of the skeleton to articulate in various opposing directions is essential for body movement and locomotion. Consequently, cartilage and bone defects due to trauma, disease, and developmental abnormalities result in disabling pain and immobility for millions of people worldwide. A novel way of promoting cartilage and bone regeneration is through the incorporation of either primary cells or multipotent progenitor cells in a three-dimensional (3D) biomaterial scaffold, and/or the addition of exogenous growth and differentiation factors. The first part of this study reports a protocol for using freshly isolated mature chondrocytes seeded in a 3D hydrogel biomaterial scaffold, developed to explore mechanotransduction of engineered cartilage constructs cultured in a designed bioreactor. The bioreactor was designed to allow the application of physiological mechanical forces (compression and fluid flow), as well as a non-invasive/non-destructive method for analyzing regenerating tissue in real time through ultrasound transducers and a computerized monitoring system. In the second part of this study, an engineered immortalized osteoprecursor cell line, designated OPC1 (osteoblastic precursor cell line 1), was used as a culture model system for exploring the effects of exogenous growth and differentiation factors, mainly vitamin D, on early bone development. OPC1 was previously designed to provide a consistent reproducible culture system for direct comparisons of engineered bone constructs, evaluating bone development and cell/biomaterial interactions, and for investigating putative bone differentiating factors. One of the objectives of this research effort was to explore tissue development and regeneration by culturing OPC1 in the presence of vitamin D metabolites vitaD3 and 1,25OH2D3, while assaying the concomitant biological response. Results indicate that OPC1 is capable of metabolizing the parental metabolite vitaD3, and thus 25OHD3, to the active vitamin D form 1,25OH2D3. The metabolism of vita3 resulted in an anti-proliferative and pro-differentiative influence on OPC-1. These results support the hypothesis that extra-endocrine synthesis of 1,25OH2D3 functions in a tissue specific manner to regulate growth and differentiation, in addition to the classic calcimic actions of the vitamin D endocrine pathway. Understanding the influence of vitamin D on bone development will have significant implications on healthy aging, including the susceptibility to skeletal disorders involved in development and aging, such as osteoarthritis (OA) and osteoporosis.

Une des causes d’échec les plus importantes dans la prise en charge des cancers est la rechute, reflet de la persistance de cellules souches cancéreuses. Les leucémies aiguës myéloïdes représentent la forme principale de leucémie aiguë chez l’adulte et sont caractérisées par une prolifération excessive de cellules immatures et un défaut d’apoptose. En haut de la hiérarchie clonale, les cellules souches leucémiques (CSL) au travers de leurs capacités fonctionnelles participent à l’initiation et la maintenance de la maladie. Ces cellules sont régulées à la fois de façon extrinsèque au travers du microenvironnement et de façon intrinsèque notamment les facteurs de transcription.Notre équipe travaille sur le récepteur à la vitamine D (VDR) et son ligand la vitamine D (VD) et a mis en évidence une synergie d’action entre chélation martiale et VD afin de lever le blocage de différenciation des LAM avec une toxicité réduite (Callens et al, JEM 2010). Une étude clinique rétrospective a été conduite mettant en évidence qu’un taux de vitamine D élevé chez les patients atteints de LAM avant tout traitement leur confère un meilleur pronostic (Paubelle, Zylbersztejn et al, Plos One 2013) . Nous poursuivons donc ce projet sur l’étude la voie VD/VDR dans la maintenance des cellules souches hématopoïétiques et sa dérégulation dans la LAM. Mon projet doctoral a pour objectif de déterminer l’implication du microenvironnement tumoral (voie des BMP et du VDR) dans le maintien des cellules souches leucémiques de LAM. Notre hypothèse de travail est que le récepteur à la vitamine D en plus de son rôle différenciant connu, aurait un impact sur le maintien des cellules souches hématopoïétiques normales et de LAM et que son mécanisme d’action passerait par la voie des Bone Morphogenetic Protein. Nous avons dans un premier temps démontré l’importance du VDR dans la régulation des CSH et pu tester l’intérêt de l’emploi de son ligand afin de cibler spécifiquement les cellules souches leucémiques dans des modèles pré-cliniques. Enfin nous avons pu confirmer la dérégulation de cette voie dans des cellules primaires de LAM et la régulation de ce récepteur par la voie des Bone Morphogenetic Protein. Ces travaux ouvrent de nouvelles perspectives dans la compréhension dans la biologie des CSH et de leur dérégulation dans la LAM
One of the most important causes of failure in the management of cancer is relapse, due to cancer stem cells persistence. Acute Myeloid Leukemias (AML) are the major form of acute leukemia in adults and are characterized by excessive proliferation of immature cells and apoptosis defect. At the top of the clonal hierarchy, leukemic stem cells (LSC) through their functional abilities participate in the initiation and maintenance of the disease. These cells are regulated both extrinsically mechanisms through the microenvironment and intrinsically by transcription factors.Our team is working on the Vitamin D Receptor (VDR) and its ligand Vitamin D (VD) and has demonstrated a synergistic action between iron chelation and VD in order to lift the differentiation blocking of AML with reduced toxicity (Callens et al, JEM 2010). A retrospective clinical study was conducted showing that a high vitamin D level in patients with AML before any treatment gives them a better prognosis (Paubelle, Zylbersztejn et al, Plos One 2013). We are continuing this project on the study of the VD/VDR pathway in the maintenance of hematopoietic stem cells (HSC) and its deregulation in AML. My project aims to determine the involvement of the tumor microenvironment (BMP and VDR pathway) in the maintenance of AML-LSC. Our working hypothesis is that the vitamin D receptor, in addition to its known differentiating role, would have an impact on the maintenance of normal HSC and AML and that its mechanism of action would be through the Bone Morphogenetic Protein pathway. We first demonstrated the importance of VDR in the regulation of HSCs and tested the interest of the use of its ligand to specifically target LSC in pre-clinical models. Finally we were able to confirm the deregulation of this pathway in primary AML cells and the regulation of this receptor by the Bone Morphogenetic Protein pathway. These works open up new perspectives in the understanding in CSH biology and their deregulation in AML

O objetivo deste estudo foi analisar o metabolismo ósseo de pacientes com proteinúria glomerular sem uso prévio de drogas que afetassem esse metabolismo. Dezessete pacientes foram estudados com biópsia óssea para análise histomorfométrica e fragmentos ósseos foram obtidos para cultura de célula (n=13) na qual nós avaliamos proliferação de osteoblasto. A comparação dos achados histomorfométricos a controles de literatura demonstrou uma diminuição da remodelação óssea e comprometimento de sua microarquitetura. Corroborando com esse resultado houve diminuição da proliferação dos osteoblastos dos pacientes quando comparados a controles (n=5) doadores de órgãos. Análise bioquímica revelou correlação negativa da 25(OH)D3 com a proteinúria e positiva com a proliferação dos osteoblastos em cultura
The objective of this study was to analyze bone metabolism in proteinuria glomerular patients not having previously used drugs affecting bone metabolism. Seventeen patients were studied with histomorphometric analysis of bone biopsies and bone fragments were obtained for cell culture (n = 13), in which we evaluated osteoblastic proliferation. Comparing patients to controls of literature indicate reduced bone remodeling and altered bone microarchitecture. In corroboration, mean osteoblast proliferation was lower in patient samples when compared with those for normal osteoblasts obtained from age-matched, gender-matched donor organs (n = 5). Concentrations of 25-hydroxyvitamin-D3 correlated negatively with proteinuria and positively with osteoblast proliferation in culture

In addition to its established role in bone health, vitamin D (2S(OH)D) may also have a role in modulating immune function and early life development. Despite recent advances, there is a lack of consensus with regards to the optimal vitamin D cut-offs for multiple health outcomes and this uncertainty is further compounded by the wide measurement variability for the vitamin. Consequently, the work described in this thesis aimed to explore these areas of controversy. Using data from ongoing studies at the University of Ulster, a comparison study (n 131), of vitamin D status in the two most widely used methods (liquid chromatography mass spectrometry (LC-MS/MS) and enzyme immunoassay (ELISA)) of measurement was undertaken. Significant variation in definition of status was observed, with overestimation of vitamin D concentrations by ELISA >2S% compared to LC-MS/MS. In a second study, using LC-MS IMS, the vitamin D status and markers of bone health of a sample of older Irish adults (n 1936) form the Trinity, Ulster Department of Agriculture (TUDA) study was assessed. A total of 16% were vitamin D deficient «2Snmolll) and 42% were deemed to be insufficient (2S-S0nmolll). These levels of nonoptimal vitamin D concentrations were coupled with high rates of impaired bone health (31 % classified as osteopenic and 18% osteoporotic from BMD measures). A higher prevalence of impaired bone health and vitamin D inadequacy was observed in females compared to males while individuals who were vitamin D deficient or insufficient were significantly more likely to be osteoporotic than those who were sufficient (>SO nmol/l). These data provide additional evidence to support the recent 10M recommendation of a 2S(OH)D concentration of 50 nmol/l for optimal bone health. In a third study, the association between vitamin D status, immune markers of inflammation and the ratio of pro: anti-cytokines was investigated in a sub-sample of TUDA participants (n 998). Vitamin D was significantly correlated with pro-inflammatory markers and a 25(OH)D concentration >75nmol was associated with an improved inflammatory (profile as determined by the pro:anti cytokine ratio) compared to individuals with a 25(OH)D status <25 or 25-75nmol/l. In a fourth study, vitamin D status was assessed within a sample (n 260) of pregnant women from a sunny equatorial country (5degS) (Seychelles). Maternal vitamin D status was observed to be >75nmol/l through all sample periods of pregnancy and was significantly associated with higher birth weight and length with no apparent upper limit of effect. These results demonstrate the importance of optimal vitamin D status during pregnancy and the need for adequate dietary recommendations in order to achieve this level within far latitude populations that are exposed to low UVB sun light. In conclusion, the results within the current thesis suggest concentrations of vitamin D greater than recently recommended cut-offs for bone health (50nmol/l) are associated with extra-skeletal health benefits. Furthermore, consideration needs to be given to the current vitamin D dietary recommendations within the UK and Ireland in order to address the high level of deficiency observed in the older adult population and to achieve the optimal vitamin D concentration in terms of benefits for bone health, immune function and neonatal health outcomes for the whole population

Since vitamin D deficiency is common at birth, the objective of this study was to test if postnatal vitamin D supplementation would normalize bone mineralization. Forty guinea pigs were randomized to receive a diet with or without vitamin D3 during pregnancy. Newborn pups were randomized to receive 10 IU of vitamin D3 or a placebo daily until d28. Measurements at birth and d28 included whole body and regional bone mass, osteocalcin and deoxypyridinoline, plus biomechanical testing of excised tibias and femurs. Offspring from deficient sows had lower body weight, whole body and tibia bone mineral content (BMC) and lower osteocalcin and biomechanical integrity. By d28 this group had lower whole body bone density and femur BMC, unless supplemented. Interactions with gender showed males continued to have low 25(OH)D despite supplementation. Therefore, neonates born to sows with dietary vitamin D deficiency require supplemental vitamin D to support normal bone mineral accretion.

Background: Between 1961-1971 vitamin D deficiency was recognized as a public health issue in the UK, because of the lack of effective sunlight and the population mix [1, 2]. In recent years, health care professionals have cited evidence suggesting a re-emergence of the vitamin D deficiency linked to a number of health consequences as a concern [3-6]. Evidence from observational studies has linked low vitamin D status with impairment in glucose homeostasis and immune dysfunction [7-9]. However, interventional studies, particularly those focused on paediatric populations, have been limited and inconsistent. There is a need for detailed studies, to clarify the therapeutic benefits of vitamin D in these important clinical areas. Objective: The aims of this PhD thesis were two-fold. Firstly, to perform preliminary work assessing the association between vitamin D deficiency and bone status, glucose homeostasis and immune function, and to explore any changes in these parameters following short term vitamin D3 replacement therapy. Secondly, to assess the effectiveness of an electronic surveillance system (ScotPSU) as a tool to determine the current incidence of hospital-based presentation of childhood vitamin D deficiency in Scotland. Methods: Active surveillance was performed for a period of two years as a part of an electronic web-based surveillance programme performed by the Scottish Paediatric Surveillance Unit (ScotPSU). The validity of the system was assessed by identifying cases with profound vitamin D deficiency (in Glasgow and Edinburgh) from the regional laboratory. All clinical details were checked against those identified using the surveillance system. Thirty-seven children aged 3 months to 10 years, who had been diagnosed with vitamin D deficiency, were recruited for the bone, glucose and immunity studies over a period of 24 months. Twenty-five samples were analysed for the glucose and bone studies; of these, 18 samples were further analysed for immune study. Treatment consisted of six weeks taking 5000 IU units cholecalciferol orally once a day. At baseline and after completion of treatment, 25 hydroxyvitamin D (25(OH)D), parathyroid hormone (PTH), alkaline phosphatase (ALP), collagen type 1 cross-linked C-telopeptide (CTX), osteocalcin (OCN), calcium, phosphate, insulin, glucose, homeostasis model assessment index, estimated insulin resistance (HOMA IR), glycated hemoglobin (HbA1c), sex hormone binding globulin (SHBG), lipids profiles, T helper 1 (Th1) cytokines (interleukin-2 ( IL-2), tumor necrosis factors-alpha (TNF-α), interferon-gamma (INF-γ)), T helper 2 (Th2) cytokines (interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6)), T helper 17 (Th17) cytokine (interleukin-17 (IL-17)), Regulatory T (Treg) cytokine (interleukin-10 (IL-10)) and chemokines/cytokines, linked with Th1/Th2 subset balance and/or differentiation (interleukin-8 (IL-8), interleukin-12 (IL-12), eosinophil chemotactic protein ( EOTAXIN), macrophage inflammatory proteins-1beta (MIP-1β), interferon-gamma-induced protein-10 (IP-10), regulated on activation, normal T cell expressed and secreted (RANTES), monocyte chemoattractant protein-1(MCP-1)) were measured. Leukoocyte subset analysis was performed for T cells, B cells and T regulatory cells and a luminex assay was used to measure the cytokiens. Results: Between September 2009 and August 2011, 163 cases of vitamin D deficiency were brought to the attention of the ScotPSU, and the majority of cases (n = 82) were reported in Glasgow. The cross-validation checking in Glasgow and Edinburgh over a one-year period revealed only 3 (11%) cases of clearly symptomatic vitamin D deficiency, which had been missed by the ScotPSU survey in Glasgow. While 16 (67%) symptomatic cases had failed to be reported through the ScotPSU survey in Edinburgh. For the 23 children who are included in bone and glucose studies, 22 (96%) children had basal serum 25(OH)D in the deficiency range (< 50 nmol/l) and one (4%) child had serum 25(OH)D in the insufficiency range (51-75 nmol/l). Following vitamin D3 treatment, 2 (9%) children had final serum 25(OH)D lower than 50 nmol/l, 6 (26%) children had final serum 25(OH)D between >50-75 nmol/l, 12 (52%) children reached a final serum 25(OH)D >75-150 nmol/l and finally 3 (13%) exceeded the normal reference range with a final 25(OH)D >150 nmol/l. Markers for remodelling ALP and PTH had significantly decreased (p = 0.001 and <0.0001 for ALP and PTH respectively). In 17 patients for whom insulin and HOMA IR data were available and enrolled in glucose study, significant improvements in insulin resistance (p = 0.04) with a trend toward a reduction in serum insulin (p = 0.05) was observed. Of those 14 children who had their cytokines profile data analysed and enrolled in the immunity study, insulin and HOMA IR data were missed in one child. A significant increase in the main Th2 secreted cytokine IL-4 (p = 0.001) and a tendency for significant increases in other Th2 secreted cytokines IL-5 (p = 0.05) and IL-6 (p = 0.05) was observed following vitamin D3 supplementation. Conclusion: An electronic surveillance system can provide data for studying the epidemiology of vitamin D deficiency. However, it may underestimate the number of positive cases. Improving vitamin D status in vitamin D deficient otherwise healthy children significantly improved their vitamin D deficient status, and was associated with an improvement in bone profile, improvements in insulin resistance and an alteration in main Th2 secreting cytokines.

The primary function of vitamin D in humans is to maintain sufficient circulating calcium concentrations. Low vitamin D levels could result in excessive calcium resorption from bone. Vitamin deficiency may therefore decrease bone mineral density (BMD), resulting in an increased risk of fracture. This thesis sought to determine the association between vitamin D intake and bone health and to estimate circulating levels of vitamin D optimal for bone health without increasing the risk for non-bone disease. Furthermore, the thesis assessed the difference in performance between common serum vitamin D assays and the genetic influence of vitamin D status. In prospective population-based cohorts, blood concentrations <40 nmol/L (lowest 5%) increased the risk of fracture in elderly men. Low levels were further associated with a slight decrease in lumbar spine BMD. Both high (>98 nmol/L) and low (<46 nmol/L) vitamin D levels were associated with higher cancer and overall mortality. In another cohort, also of older men and women, no association was found between vitamin D levels and fracture. Low vitamin D levels were weakly associated with decreased total body BMD in men but not in women. Dietary intake of vitamin D over a 20-year period in more than 60,000 Swedish women was not associated with osteoporosis or fracture, regardless of calcium intake. During summer, dietary vitamin D intake and other life style habits are of minor importance for the variation in vitamin D levels relative to sun exposure and genes. In summer time, genes explain about half  of the variation in vitamin D levels, but none of the variance in winter time. The variability between vitamin D assays was substantial. Three assays classified 8, 22 and 43% of the same study population as vitamin D insufficient if <50 nmol/L was set as the insufficiency level. Based on the results in this thesis, low 25(OH)D levels and low dietary vitamin D intake are not a major cause of fractures in community-dwelling elderly Swedish women and men. Differences in assay performance and potential negative health outcomes of high 25(OH)D levels need to be considered.

Includes bibliographical references (leaves 226-273.) Investigates the regulation of the expression of CYP27B1, CYP24 and vitamin D receptor (VDR) mRNA, both in the bone and in the kidney, with the aim to determine whether the regulation of the vitamin D metabolism in the bone is independent from that in the kidney. The effects of age, dietary calcium and vitamin D status on the expression of these genes in both the kidney and the bone, as well as on a number of biochemical factors known to regulate the renal metabolism of 1,25D, such as PTH, calcium and 1,25D itself, were examined. CYP27B1 mRNA expression was also studied in histological sections of rat femoral bone.

Bone formation and remodeling require continuous generation of osteoprogenitors from bone marrow stromal cells (MSC), which are regulated by local growth factors and hormones with putative roles in mesenchymal proliferation and differentiation. Hepatocyte growth factor (HGF) and its receptor c-Met are widely expressed in MSC and are thought to play a key role in the interactions between cells. 1,25-dihydroxyvitamin D (1,25OHD) is the most active metabolite of vitamin D. 1,25OHD binds to its nuclear/membrane vitamin D receptor (VDR) and generates appropriate biological responses. The purpose of this study was to investigate the regulation of proliferation and differentiation by HGF in human bone marrow-derived stromal cells (hMSC). We examined the impact of HGF on hMSC cell-cycle regulation and the combination effects of HGF and 1,25OHD on hMSC osteogenic differentiation to enhance our knowledge of hMSC regulation. hMSC isolated from bone marrow were plated and grown in DMEM supplemented with 3% FBS incubated at 37C with 5% CO2 in air. HGF treatment of hMSCs reduced the rate of cell proliferation and this result was not due to apoptosis or cell senescence. Real-time RT-PCR and Western blot analysis showed increased gene and protein expression of the cell-cycle inhibitors p53, p21, and p27 after HGF treatment. These results appear to be specific because HGF did not significantly alter the gene expression level of other cell-cycle mediators such as RB, cyclin D1, CDK2, CDK4, or CDK6. Transfection of siRNA specific for cMet, the HGF receptor, eliminated the HGF anti-proliferation effect. cMet siRNA also eliminated the increase in p53, p21, and p27, further supporting a role for these cell-cycle inhibitors in HGF¡¯s regulation of hMSC. These results suggest that treatment of hMSC with HGF slows cell proliferation by increasing the expression of p53, p21, and p27. The reduced rate of cell proliferation did not appear to be due to cell differentiation, because treatment of hMSC with HGF alone did not induce cell differentiation. However, HGF in combination with a known osteogenic differentiation activator, 1,25OHD, significantly increased cell maturation/differentiation compared to 1,25D alone, as indicated by an increase in osteocalcin mRNA (a marker for osteogenic differentiation). Whereas HGF had no effect on 1,25OHD synthesis per se, HGF did induce 1,26OHD receptor (VDR) gene expression. HGF up-regulated the expression of the p63 gene, a member of the p53 family. Knocking down the p63 gene reduced the HGF effect on VDR expression and eliminated the HGF-induced up-regulation of the osteogenic differentiation markers osteopontin (OPN) and bone sialoprotein (BSP). Moreover, the ChIP assay shows that p63 was able to bind to the VDR promoter, possibly explaining the mechanism of p63-mediated VDR up-regulation. These results indicate that HGF can also induce hMSC osteogenic differentiation when combined with 1,25OHD by up-regulating 1,25OHD receptor VDR expression.

The CYP27B1 enzyme that synthesizes 1alpha,25-(OH) 2D, is expressed in chondrocytes, suggesting that local production of 1alpha,25-(OH)2D could play an autocrine or paracrine role in the differentiation of these cells. To test this hypothesis, we have engineered mutant mice that do not express the Cyp27b1 gene in chondrocytes. This led to increased width of the hypertrophic zone of the growth plate at E15.5, increased bone mass in neonatal long bones, and increased expression of the chondrocytic differentiation markers Indian Hedgehog and PTH/PTHrP receptor. VEGF mRNA levels were decreased, accompanied by decreased PECAM-1 immunostaining, suggesting a delay in vascularization. We have also engineered mice overexpressing a Cyp27b1 transgene in chondrocytes. The transgenic mice showed a partial mirror image phenotype compared to the tissue-specific inactivation model. These results support an autocrine/paracrine role of 1alpha,25-(OH) 2D in endochondral ossification and chondrocyte development in vivo.
The CYP24A1 enzyme is involved in the catabolic breakdown of 1alpha,25-(OH)2D but also synthesizes the 24R,25-(OH) 2D metabolite. Studies in chicken suggest a role for 24R,25-(OH) 2D in fracture repair. We induced stabilized transverse mid-diaphysial fractures of the tibia in four-month-old wild-type and Cyp24a1-deficient mice. Examination of the callus sections showed delayed hard callus formation in the homozygous mutant animals compared to wild-type littermates. RT-qPCR showed perturbed levels of type X collagen transcripts in mutant mice at 14 and 21 days post-fracture, reflecting the delayed healing. Rescue of the impaired healing by subcutaneous injection of 24R,25-(OH)2D3 normalized the histological appearance of the callus, static histomorphometric index and type X collagen mRNA expression, while 1alpha,25-(OH)2D 3 did not. These results show that Cyp24a1 deficiency delays fracture repair and strongly suggest that vitamin D metabolites hydroxylated at position 24, such as 24R,25-(OH)2D, play an important role in the mechanisms leading to normal fracture healing.

Cross sectional and observational data suggest that HIV-positive individuals and those receiving antiretroviral (ARV) therapy are prone to higher rates of osteoporosis and osteopaenia than HIV-negative individuals. Likewise, HIV-positive individuals often have low vitamin D status. Evidence is emerging more generally of a strong association between HIV infection and poor bone health. There is also evidence that treatment with ARV therapy (ART) and suboptimal vitamin D status may exacerbate this problem (Brown et al, 2006a, 2010). But, to date, causal relationships have not been fully established. This thesis explores the interactions between these separate factors and provides novel data about the effects of HIV infection and its treatment, on bone health in a particular group of black, South African women. Bone loss and poor vitamin D status in the context of HIV infection are important global health issues because these conditions may affect millions of individuals. If HIVassociated bone loss is causally associated with an increased risk of bone fracture then it is possible that there will be an epidemic of HIV-associated fractures in coming decades, particularly in the developing world, including Africa. Study data have so far often been limited by several factors, including cross-sectional design, absence of control groups, a preponderance of attention to bone outcomes in males and in Caucasians, and a lack of good quality data in Africans living in Africa. Th is study aimed to -assess the magnitude of HIV- and ART-associated changes in areal bone mineral density (aBMD), size-adjusted bone mineral content (SA-BMC) and vitamin D status in adult, premenopausal women living in Johannesburg, South Africa. Ninetyeight HIV-negative (Negative reference: Nref) and 149 HIV-positive women were enrolled to allow for comparison between groups. The HIV-positive women were recruited into those eligible to start ART (Positive low CD4 : Plow, n=75) and those unlikely to require ART (Positive preserved CD4 : Ppres, n=74) during a 12-month followup period. The design was longitudinal with visits at 0, 6 and 12 months for measurement of body composition, bone measures and dietary assessment. Blood and urine samples were collected for the evaluation of relevant musculoskeletal analytes, including 25(0H)D at each time point. Most women ( > 80%) who received ART during the course of the study received South African standard first-line therapy consisting of lamivudine, tenofovir and efavirenz. A post hoe analysis of possible effects of ART was performed by retrospectively dividing HIV-positive women into ART-unexposed (n=66) and ART-exposed (n=74). At baseline there was a high prevalence of overweight with 65%, 65% and 44% with BMI > 25 kg/m2 in Nref, Ppres and Plow respectively. Plow had lower weight, BMI, fat mass, lean mass, waist and hip circumferences than the other groups. Nref and Ppres were not different from each other. There were no differences in aBMD or SA-BMC 1 between groups at baseline and no significant differences in vitamin D status between the groups. The mean ±SD serum 25(0H)D concentrations were 59.7 ±16.5, 59.2 ±16.5 and 61.6 ±22.3 nmol/1 in Nref, Ppres and Plow respectively. Plow had significantly lower serum albumin concentration (p < 0.0001) and higher serum phosphate concentration (p < 0.0001). The magnitude of differences in serum phosphate was: Ppres-Nref = 12. 7 ±2.9%; Plow-Nref = 20.3 ±2.9% and Plow-Ppres = 7.6 ±3.1% (p < 0.001). Tubular maximum Reabsorption of Phosphate/Glomerular Filtration Rate (TmP/GFR) was 11.2 ±3.2% and 27.4 ±3.2% respectively greater in Ppres and Plow than Nref (p < 0.0001), and higher in the Plow compared to Ppres 16.2 ±3.4%, (p=0.0002). Serum alkaline phosphatase and urine phosphate to creatinine ratio were not significantly different (p > 0.05). At the 12-month follow-up, Plow subjects remained lighter than their Nref and Ppres counterparts. However, there was a 3.9 ±0.9% increase in mean weight in the Plow group over 12 months (p < 0.001), which represented 10.2 ±0.8% (p < 0.001) increase in fat, rather than lean, mass accumulation. There were significant mean decreases in aBMD and SA-BMC in Plow subjects, and those exposed to ART of the order of 2-3% at total hip, femoral neck and lumbar spine. There were no significant differences in mean vitamin D status between the groups and no significant changes, the mean 25(0H)D concentrations were 63.3 ±17.7, 66.0 ±18.4 and 61.1 ±20.1 nmol/1 in Nref, Ppres and Plow respectively. Serum albumin concentrations had risen by a mean of 9.1 ±1.1% in the Plow group to reach comparable concentrations with the other groups. Alkaline phosphatase activity had significantly risen in the Plow group compared with the other groups (p < 0.001). Serum phosphate concentration remained higher in Plow than the other groups, though the mean value had not increased. Serum phosphate had significantly increased in Nref from baseline to 12 months 7.0 ±2.3% (p=0.05) and non-significantly in Ppres 5.2 ±2.4%. TmP/GFR had declined from baseline by 11.2 ±3.6% in Plow and non-significantly increased in Nref and Ppres (6.4 ±3.3% and 3.8 ±3.5% respectively). These data suggest that HIV infection in South African women is associated with differences in body composition but not with differences in bone measures or vitamin D status. However, being in the Plow group, and ART exposure, was associated with a significant decrease in mean aBMD and SA-BMC, of the order of 2-3%, over 12 months of observation at the hip, femoral neck and lumbar spine. These decreases, in young women, exceed those seen in early menopause, which is of the order of 1-2% annual decrease. The decreases were evident despite the fact that HIV-positive women exposed to ART had increases in fat mass, weight and serum albumin and alkaline phosphatase over time. In this group serum phosphate concentration and TmP/GFR decreased after the introduction of ART, suggesting an effect of ART on renal phosphate handling. ART exposure was not associated with change in vitamin D status. In the post hoe analysis the biochemical results in ART-unexposed compared to ARTexposed was very similar to that in Ppres compared with Plow. Further studies to assess skeletal effects over a longer time in HIV-positive, ARTexposed and na"ive women are warranted. Studies are also required in post-menopausal women, children and men. Given the high prevalence of overweight and obesity recorded in the study population, there may also be a need for interventions to reduce cardiometabolic disease risk in this population.

The objective of this study was to investigate the extent to which vitamin D and K are associated with bone health in pediatric cystic fibrosis (CF) patients. We hypothesized that: (1) the prevalence of vitamin D and K deficiencies would be high despite routine vitamin therapy, (2) bone health would be reduced and (3) vitamin K and D status would be associated with bone health.
Our results showed poor bone mineral mass in these CF children despite mild disease and good nutritional status. Neither vitamin K nor D was a predictor of bone health but weight and height Z-scores, fat-free mass, physical activity and lung function were all consistent predictors.
These results indicate that nutritional status as well as physical activity are key determinants of bone health in CF children and offer a unique opportunity in the prevention of CF-related bone disease. Further vitamin intervention research needs to be done in this population.

In addition to the more traditional roles associated with Vitamin D, such as the formation and maintenance of healthy bones, regulation of calcium-phosphate homeostasis and parathyroid hormone regulation, Vitamin D is involved in many other processes in the body including regulation of immune function, neurological function and control of cellular proliferation. Many studies have shown the chemoprotective and therapeutic effects of Vitamin D in the treatment of many ailments including cancer, with many studies investigating as a potential treatment option. Vitamin D therapy has limited success in prostate cancer with tumours becoming resistant to treatment and developing a more aggressive phenotype. Intracrine signalling has been put forward as a theory as to how tumours continue to grow in patients who have undergone androgen ablation therapy; this hypothesis suggests that androgens are synthesized de novo within the tumour microenvironment from adrenal precursors. Cytochrome P450 enzymes are involved in the metabolism of endogenous and exogenous compounds, including steroid hormines, to prevent toxic accumulation. Previous studies in this laboratory have shown that the major hepatic CYP, CYP3A4, can be regulated by the Vitamin D Receptor (VDR) in epithelial cells of the colon when it is liganded to toxic bile acids which in turn facilitates their excretion. This thesis demonstrates a novel finding whereby the VDR directly modulates expression of CYP3A4 in the well known prostate cancer cell line LNCaP. CYP3A4 expression is found to be up-regulated in the presence of the most biologically active form of the vitamin (1,25D) in a LNCaP cells, with a subsequent increase in enzymatic activity and metabolism of testosterone as evidenced by HPLC MSIMS analysis. Interestingly mutation analysis uncovered the finding that the more novel vitamin D response element, ER-6, has important roles to play in the VDR mediated transcriptional response. Additionally VDR was also found to interact with the receptor interacting protein of 140kDa (RIP140) in a ligand dependent manner resulting in dose dependent repression of VDR transcriptional activation while gene silencing of RIP140 resulted in a significant increase in CYP3A4 expression. Considering these results together, CYP3A4 and RIP140 may provide new therapeutic targets in the treatment of prostate cancer.

The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), is a key calcium (Ca++) regulatory hormone. It is also associated with functions unrelated to Ca++ homeostasis. Here, special attention is paid towards the anticancer properties of 1,25D. 1,25D strongly inhibits the growth of well-differentiated head and neck squamous cell carcinoma (HNSCC) derived cell lines. However, advanced, less differentiated, HNSCC cell lines (e.g. SCC4) are partially resistant to 1,25D. Resistance to nuclear receptor (NR) agonists is a common event that occurs in other NR-related treatments. For example, some leukemias develop resistance to the usually effective retinoic acid (RA) treatment. However, treating RA-resistant cells with HDAC inhibitors (HDACi) sensitizes them to RA. Thus, this study aims to investigate how treatment with TSA, an HDACi, would affect the response of SCC4 cell lines to 1,25D. We found that TSA had a variety of effects on 1,25D-regulated gene expression. Combined treatment with 1,25D and TSA increased the expression of cell-cycle regulating proteins, but also enhanced the downregulation of key target genes. Given the potential of the 1,25D/HDACi combination in combating cancers, two chimeric compounds, each containing parts of 1,25D and an HDACi, were synthesized in collaboration with Dr. James Gleason (Dept. of Chemistry, McGill). These 1,25D analogs have the HDACi-like structure replacing the 1,25D side chain. Both compounds proved to be agonists of the vitamin D receptor. Moreover, the TSA-substituted compound, called triciferol, effectively induced a-tubulin as well as histones acetylation. This study underlines the potential of combining 1,25D and TSA in cancer treatment, and reveals that bi-functional 1,25D analogs can be produced with potentially enhanced therapeutic activity.

Osteoporosis is a disease characterized by low bone mineral density, deteriorated bone microstructure and increased fracture risk. About 50% of all women and 25% of all men will have an osteoporotic fracture. Given that there is no effective cure in established osteoporosis, prevention is of high importance. Bone mineral density (BMD) is accumulated during childhood and adolescence with a peak at about 20 years of age. Peak BMD has been suggested to explain at least half of the variation in BMD up to old age. Thus, to increase peak BMD could decrease the risk of later fractures. The purpose of the present thesis was to investigate the influence of physical activity, vitamins A and D, and fatty acids on peak bone mass in men. The influence of physical activity on bone accrual was studied in two cohorts. In the first cohort 46 ice hockey players, 18 badminton players and 27 controls, all 17 years of age at baseline, were followed for four years. During the follow up the badminton players gained more bone mass at the hip compared to both the ice hockey players and controls. In the second cohort the associations between physical activity and BMD were investigated in 62 female and 62 male young medical students. The estimated high impact activity per week was associated with bone mass at all sites in the male medical students (r=0.27-0.53, p<0.05). In the female cohort different estimates of physical activity were not related to bone mass at any site. In both males and females correlations between bone mass and body constitution parameters were observed. Levels of vitamin D3, vitamin D2, retinol, retinol-binding-protein-4 (RBP-4) and fatty acids were measured in 78 young men with a mean age of 22.6 years. BMD at various sites were measured using Dual-Energy X-ray absorptiometry. Levels of vitamin D3 showed a significant positive association with all BMD sites and also lean body mass (r=0.23-0.35, p<0.05). Levels of vitamin D2, however, showed a significant negative correlation with BMD of the total body (r=-0.28, p=0.01) and spine (r=-0.27, p=0.02). There was also a significant negative relationship between levels of vitamin D3 and D2 (r=-0.31, p=0.006). Concentrations of n-3 (omega-3) fatty acids showed a positive association with BMD at the total body (r=0.27, p=0.02) and spine BMD (r=0.25, p=0.02). There was also a positive association between levels of n-3 fatty acids and changes in BMD of the spine between 16 and 22 years of age (r=0.26, p=0.02). The significant associations found seemed to be related mostly to the concentration of the n-3 fatty acid docosahexaenoic acid. Levels of retinol and RBP-4 were not related to BMD but to levels of osteocalcin, which is a marker of bone formation. This association disappeared when adjusting for the influence of abdominal fat mass. In summary, the present thesis suggests that many modifiable factors may influence the accumulation of peak bone mass in males, such as physical activity, vitamins, and fatty acids. Further studies are needed to investigate whether optimizing these factors in youth may decrease the risk of osteoporosis later in life.

Osteoporosis is a common highly prevalence public health problem affecting both gender in all age stages in worldwide. Little information is known about the bone health and lifestyle characteristics of women living in Middle Eastern countries in general and Saudi Arabia in specific. The complete information and statistical figures of osteoporosis prevalent and vitamin D deficiency among population it has not yet been identified. The strong correlation between dietary intake and bone health has been explored in Western populations but no data are available in Middle Eastern countries. The aims of this study were four fold: (i) to examine the extent of poor bone health in the Saudi population of postmenopausal and premenopausal women; (ii) to investigate the effect of lifestyle factors including physical activity levels and sun-time exposure on bone integrity; (iii) to determine the extent of vitamin D deficiency in the population and the effect of this status on bone mass and calcium/bone metabolism; (iv) to evaluate the dietary quality and quantity in Saudi women and investigate fully the effect of diet on bone health indices. As part of our investigation, a total of 212 Saudi Arabian apparently healthy women were voluntarily participated in this study. A total of 112 postmenopausal and 100 premenopausal women. They were aged 45-60 years and 20-30 years respectively. Bone mineral density (BMD) was determined at the lumbar spine (L2-L4) and femoral neck using dual x-ray absorptiometry (DXA). Calcaneal bone mass was measured by broadband ultrasound attenuation (BUA). All subjects were interviewed concerning their habitual dietary intake, physical activity levels and general lifestyle. Information on dietary intake of each individual was obtained using 3-day estimated food diaries. The amount of food consumed (in grams) for the five food groups was calculated for each subject. Intakes were converted to frequency of consumption (time/d) by dividing food groups by average portion sizes. Using the only existing Food Composition Table for the Middle East, the nutrient values for energy, protein, fat, fibre, calcium, phosphorus, iron, vitamin C, vitamin D and potassium were chosen for the five food groups identified and calculated per 100g. Bone resorption was assessed by measurement of pyridinium crosslinks (PYD) and (DPD) using a second morning urine sample. Bone formation was assessed by bone specific alkaline phosphatase (BSAP) and osteocalcin (OC). Serum 25(OH)D, 1,25(OH)D, PTH, calcium, and phosphorus were measured. Bone health indices indicated a high prevalence of low bone mass in these groups. According to WHO criteria (WHO, 1994), a total of 52% of postmenopausal and 37% of premenopausal women were osteopenic at the lumbar spine. Osteoporotic prevalence was 13% and 2% respectively. Similar results were found for the femoral neck. Physical activity levels were low and exposure to sunlight was low. A significant correlation was found between period of sunlight exposure (min/d) and axial BMD and calcaneal bone mass. The 'quality' of food consumption by Saudi Arabian women does not follow the recommended food guidelines, and the intakes of energy, fibre and potassium in women are lower than those recommended in western population but intakes of phosphorus are somewhat higher. Calcium, vitamin C and iron are around recommended levels. Vitamin D deficiency is highly prevalent in Saudi women, with 78% of women being below the classical threshold of 12ng/ml. A low milk consumption was associated with higher bone resorption in both postmenopausal and premenopausal women which remained significant after adjustment for the key confounding factors of age, weight, height and menopausal status. A low intake of fruit and vegetables and nutrients associated with high fruit and vegetable intake including vitamin C, potassium and estimates of net endogenous acid production were found to be related to poorer indices of bone health. These results are a cause for concern. It indicates that bone health is poor and dietary and lifestyle factors are not favourable to skeletal integrity in Saudi Arabian women.

Low serum 25-hydroxyvitamin D (25(OH)D) concentrations and calcium intakes and a high risk of fracture have been separately reported in Inuit women. We examined vitamin D status, nutrient intake, and forearm bone mineral density (fBMD) in Inuit women (n=419, aged 40-90 y) from Nunavut. Fasting serum was assayed for 25(OH)D, osteocalcin (OC) and parathyroid hormone (PTH). Vitamin D status was below optimal (≤ 75 nmol/L) in 69.4% of pre- and 37.6% of postmenopausal women. Vitamin D and calcium intakes (per 1000 kcal) tended to increase with age. Low fBMD was observed in 33% of post- (T-score < -1.5) and 2% of premenopausal women (Z-score < -2). Predictors of fBMD included body mass index and OC, and age in the post- and PTH in the premenopausal group. Lower vitamin D status and dietary nutrient density among premenopausal women suggests a greater risk for osteoporosis with aging compared to the previous generation.
Des bas niveaux sériques de vitamine D (25(OH)D), un apport alimentaire insuffisant en calcium, ainsi qu'un risque élevé de fracture ont été rapportés séparément chez les femmes Inuit. Nous avons évalué le statut de vitamine D, l'apport alimentaire de nutriments, et la densité minérale osseuse à l'avant-bras (aDMO) chez les femmes Inuit (n=419, âgées de 40 à 90 ans) du Nunavut participant dans l'Enquête de Santé Inuit 2007/2008. Les taux sériques de 25(OH)D, d'ostéocalcine (OC) et de parathormone (PTH) ont été mesurés. Le statut de vitamine D était sous-optimal (≤ 75 nmol/L) dans chez 69.4% des femmes pré-ménopausées et 37.6% des femmes ménopausées. Les apports alimentaires de vitamine D et de calcium (par 1000 kcal) avaient tendance à augmenter avec l'âge. La aDMO était basse chez 33% des femmes ménopausées (T-score < -1.5) et 2% des femmes pré-ménopausées (Z-score < -2). Les prédicteurs de la aDMO comprenaient l'indice de masse corporelle et l'OC, ainsi que l'âge dans les femmes ménopausées et la PTH dans les femmes pré ménopausées. L'alimentation et le statut de vitamine D des femmes pré ménopausées suggèrent que, avec l'âge, leur risque pour l'ostéoporose sera plus élevé que la génération précédente.

There is growing evidence that females in the Middle East are not obtaining sufficient amounts of vitamin D, the perceived reason being their unhealthy lifestyle. This situation is a cause for concern particularly for adolescent females, because adequate vitamin D status has been shown to promote bone mineral accrual in adolescence. Achieving a high peak bone mass (PBM) may help decrease the risk of postmenopausal osteoporosis and fragility fractures. The aim of this study was to assess the vitamin D status among Kuwaiti adolescent girls and to assess its impact and those of lifestyle, on their bone mass.

Background: Emerging epidemiological evidence implicates Vitamin D insufficiency in the risk of Inflammatory Bowel Disease (IBD). Dendritic cells (DC) are thought to play a pivotal role in the dysregulated intestinal mucosal T-cell mediated immune responses which characterise IBD. In vitro human blood studies and in vivo animal models of IBD, suggest that vitamin D is immunomodulatory inducing a tolerogenic DC phenotype and influencing leucocyte homing properties with potential therapeutic applications. Aims: 1) Determine Vitamin D status in Crohn’s Disease (CD) patients and identify associations with patient and disease phenotype; 2) Determine homing molecule expression by circulating DC and T-cells, and the phenotypic properties of gamma-delta (γδ) T-cell populations in IBD patients and healthy controls; 3) Determine in vitro immunomodulatory effects of 1,25-dihydroxyvitamin D3 (1,25-OH2D3) on co-stimulatory and functional phenotype of monocyte-derived DC and low density cells (LDC) enriched for DC isolated from peripheral blood and the terminal ileum. Results: Vitamin D insufficiency was common in CD. Serum 25-hydroxyvitamin D negatively correlated with the Harvey Bradshaw Index and Physicians’ Rating of Disease Activity, but was not associated with disease phenotype including distribution and use of immunosuppression. There were no significant differences in the expression of gut and skin homing markers on circulating DC or T-cells in patients with CD compared with healthy controls. The circulating γδ T-cell population was reduced in active IBD compared with controls. Aberrant expression of skin-homing molecules on γδ T-cells, conventional T-cells and DC was demonstrated in a CD patient with an extra-intestinal cutaneous manifestation of IBD, abrogated upon clinical resolution in response to treatment with corticosteroids. 1,25OH2D3 promoted a functionally immature tolerogenic phenotype and monocyte-like state in LDC and monocyte-derived DC in vitro. Initial investigations suggest that vitamin D impairs the ability of DC from the terminal ileum to stimulate T-cell proliferation. Conclusion: Vitamin D insufficiency is common in IBD. Vitamin D status correlated with clinical disease activity in Crohn’s Disease. Vitamin D exerts immunomodulatory effects in vitro on MoDC and LDC, supporting the potential application of Vitamin D for therapeutic use in IBD.

Chronic obstructive pulmonary disease (COPD) is a multifactorial and systemic disease driven by inflammation and oxidative stress, predominantly caused by smoking, but exacerbated by infection and pollution. Current therapies aim to treat specific aspects of the disease and/or symptoms rather than the disease as a whole. Vitamin D, more commonly known for its importance in calcium and phosphorus homeostasis, has now been established as an important immunomodulatory agent. Recent studies have shown vitamin D deficiency to be associated with reduced lung function and COPD disease severity. Equally vitamin D has been shown to modulate a number of inflammatory mediators which are important in COPD pathology. Together, these suggest that vitamin D may be beneficial in COPD. Airway epithelial cells play a central role in COPD pathogenesis and have been shown to constitutively activate vitamin D. This study investigated the effects of vitamin D on inflammation and oxidative stress in human airway epithelial cells, crucially on mediators important in COPD pathology and also investigated the potential mechanisms involved in the action of vitamin D. Three different human airway epithelial cell lines A549, NCI-H292, 16HBE14o- and primary human small airway epithelial cells were shown to express similar inflammatory mediators, with the 16HBE14o- cells shown to be a useful model of primary cells. Vitamin D significantly modulated IL-6 mRNA and protein expression in the primary cells and reduced MUC5AC mRNA and protein expression in the mucous producing NCI-H292 cells. Interestingly, vitamin D treatment gave a biphasic effect on induction of inflammatory mediators by TNFα in the NCI-H292 cells. Vitamin D also modulated the induction of oxidative stress, antioxidant gene expression and Nrf2 protein expression in the NCI-H292 cells. This data emphasises the complexity of vitamin D modulation of inflammatory mediators and oxidative stress in airway epithelial cells and provides new novel avenues of future study.

Thesis (M.S.)--University of Cincinnati, 2008.
Committee/Advisors: Shanil Juma PhD (Committee Chair), Hageman Gilbert PhD (Committee Member). Title from electronic thesis title page (viewed Aug.29, 2008). Includes abstract. Keywords: calcium and vitamin D; osteoporosis. Includes bibliographical references.

Background: Studies have not examined the relationships between serum vitamin D (SVitD), serum cortisol (SCort), bone mineral density (BMD), and body fat percent (BF%) in elite figure skaters. However, studies of non-athletes have found that BMD is inversely related to SCort and directly related to SVitD, and BF% is inversely related to SVitD and directly related to SCort. It was, therefore, the purpose of this study to assess the relationships between SCort, SVitD, BMD, and BF% in elite figure skaters. Methods: U.S. national team figure skaters were assessed at a national training camp during the summer, 2012. BMD and body composition were measured by dual energy x-ray absorptiometry (DEXA). Blood chemistry values for SVitD and SCort were obtained via venous puncture after an overnight fast, the same morning as the DEXA measurement. Georgia State University Institutional Review Board approval was obtained for the assessment of data collected at this training camp. Results: 24 out of 39 training camp attendees (61.5%) volunteered to be assessed as part of this study. Subjects ranged from 17 to 34 years and included males (n=11) and females (n=12). In all skaters statistically significant negative correlations (2-tailed Spearman) were found between SCort and BMD of the spine (r=-0.458, p=0.032), pelvis (r=-0.532, p=0.011), ribs (r=-0.517, p=0.014), and trunk (r=-0.538, p=0.010). In females, SCort was negatively correlated with BMD of the pelvis (r=-0.664, p=0.026) and trunk (r=-0.609, p=0.047), and was positively correlated with total BF% (r=0.657, p=0.020) and trunk fat % (r=0.708, p=0.010). In males, SCort was significantly correlated with BMD of the ribs (r=-0.627, p=0.039). The 3 skaters (all female) with SCort > 28 mcg/dL had significantly lower mean BMD of the total body, left femoral neck, legs, trunk, and pelvis, and significantly greater BF% of the total body and trunk when compared to the 20 skaters with SCort 7-28 mcg/dL. No significant correlations between SVitD and BMD or BF% were found. A Mann-Whitney U test found no significant differences in BMD and BF% between the 8 skaters with SVitD ≥ 30 ng/mL compared to the 15 skaters with SVitD < 30 ng/mL (p>0.05). Females with SVitD ≥ 30 ng/mL had significantly higher BMD (p=0.041) of the right femoral neck when compared to those with lower SVitD. Conclusions: Correlations consistently found negative associations between SCort cortisol and BMD in multiple assessment areas, particularly those composed of trabecular bone. Higher SCort was also associated with higher BF% in female skaters. Despite spending a great deal of time in indoor facilities, limiting vitamin D creation through sunlight exposure, no significant correlation between SVitD and BMD was found. Female athletes in ‘appearance’ sports, may be predisposed to restrained eating behaviors, which may be associated with elevated SCort. These findings suggest a need for further study of the interaction between SCort, BMD, and BF% in these athletes. The lack of a statistically significant relationship between SVitD and BMD suggests the need to investigate additional factors associated with bone injury risk in athletes.

Introduction: Young male jockeys compromise bone health by engaging in caloric restriction and high volumes of physical activity during periods of musculoskeletal growth and development. Failure to attain peak bone mass (PBM) during growth can have adverse short and long term musculoskeletal effects, with numerous studies demonstrating inferior bone health in jockey populations. However, no study to date has addressed counteracting the deleterious effects that participation in this sport has on bone health. The purpose of this six-month double-blind randomised placebo controlled trial was to examine the efficacy of 800mg calcium and 400 IU vitamin D daily supplementation on improving bone mineral properties at the tibia (weight-bearing) and radius (non-weight-bearing) using peripheral Quantitative Computed Tomography (pQCT) and blood-borne markers of bone turnover. Three inter-related studies were designed following the findings of a systematic review and meta-analysis examining the effects of calcium and vitamin D supplementation on bone mineral density in healthy males. Study one examined the effect that the protocol had on markers of bone metabolism and bone properties of the radius. Study two was designed to investigate the impact of six months supplementation on weight-bearing bone while study three further explored alterations to radial and polar cortical bone properties at the tibial mid-shaft following the clinical trial. Methods: Twenty-nine male jockeys (age=20.18 ± 3.23yrs) were originally recruited to the study with 17 completing the intervention. Bone properties at the ultra-distal (4%) and proximal (66%) radius and tibia using pQCT and serum vitamin D, Procollagen type 1 N propeptide (P1NP) and C-terminal telopeptide of type I collagen (CTx) were assessed at baseline and six months. Bone properties at the 66% tibial site were further analysed using BoneJ pQCT distribution plug-in. Polar and radial volumetric bone mineral density (vBMD) was measured in 36, ten degree cortical sectors (polar) and three concentric cortical divisions (radial). Polar distribution was further consolidated into four, 90 degree quadrants aligned to anatomical planes. Cortical mineral mass, endocortical and pericortical radii were also analysed. Results: After co-varying for height, body mass and baseline bone measurements, the analysis of co-variance (ANCOVA) results of these combined studies demonstrate that six months calcium and vitamin D supplementation stimulated a reduction in bone resorption together with significant improvements to bone material properties at the proximal tibia in the supplemented group. ANCOVA serum analysis indicated significantly higher vitamin D levels (18.1%, p=0.014) and lower CTx (ng/L) (-24.8%, p=0.011) in the supplemented group with P1NP unchanged. The supplemented group displayed greater post-intervention bone properties at the 66% proximal site with cortical content (mg·mm) 6.6% greater (p<0.001), cortical area (mm2) 5.9% larger (p<0.001), cortical density (mg·cm3) 1.3% greater (p=0.001), and total area (mm2) 4% larger (p=0.003). No alterations were observed to bone material properties at the radius, nor the ultra-distal tibia or bone strength indices. When cortical bone of the proximal tibia was examined in greater detail the supplemented group demonstrated greater endocortical vBMD in the posterior region of bone (1140.5 ± 6.3 vs 1116.2 ± 5.9; p=0.018) with a trend suggesting supplementation improved mineral mass and stimulated bone apposition in the posterior and lateral regions of the tibia. Conclusion: This is the first randomised controlled trial to examine the efficacy of calcium and vitamin D supplementation in improving bone properties in a highly vulnerable, young athletic, weight-restricted population. Results indicate beneficial effects of supplementation on bone properties in as little as six months. Although the study size is small, this intervention appears promising as a strategy for improving bone health in young athletes in weight-restricted sports.

Vitamin D is very well known for its classical role in the maintenance of calcium and phosphorus homeostasis as well as in the prevention of rickets. More recent findings of its ability to inhibit cell proliferation, induce apoptosis, induce differentiation, inhibit angiogenesis, and modulate the immune system have made it a current topic of intense research, particularly in the field of cancer research. We used a murine model of breast cancer metastasis to bone to investigate the effect of vitamin D deficiency on the growth of breast cancer tumour cells within bone. We also established that these breast cancer tumour cells express the enzyme CYP27B1 (1α-hydroxylase) which is able to convert the inactive vitamin D precursor 25-hydroxyvitamin D (25(OH)D) to the active metabolite 1,25-dihydroxyvitamin D (1,25(OH)2D). We next examined the effect of the local activation of vitamin D by tumoral CYP27B1 on the growth of these tumour cells within bone. Although we did not see a significant difference in the growth of breast cancer tumour cells in the bones of vitamin D deficient mice as compared to vitamin D sufficient mice, we have demonstrated that breast cancer tumour cells that do not express CYP27B1 grow much more aggressively within bone than breast cancer tumour cells which express CYP27B1. This suggests a very important role for the local activation of vitamin D by extra-renal CYP27B1 on the growth of breast cancer tumour cells within the bone microenvironment. These findings suggest a potential use for 25(OH)D as a treatment for breast cancer metastasis to bone either alone or in combination.
La vitamine D est bien connue pour son rôle dans le maintien des concentrations de calcium et du phosphore dans la circulation ainsi que dans la prévention du rachitisme. La découverte plus récente de sa capacité d'inhiber la prolifération cellulaire, induire leur différentiation ainsi que l'apoptose cellulaire, inhiber l'angiogenèse, et moduler le système immunitaire rend son étude un sujet de recherche très intéressant surtout dans le domaine de la recherche sur le cancer. Nous avons étudié l'effet de la carence en vitamine D sur la croissance tumorale dans un modèle murin de métastases osseuses du cancer du sein. Nous avons aussi établi que ces cellules expriment l'enzyme CYP27B1 (1α-hydroxylase) et sont donc capables d'activer la vitamine D en son métabolite actif la 1,25-dihydroxyvitamine D (1,25(OH)2D) à partir du métabolite inactif, la 25-hydroxyvitamine D (25(OH)D). Nous avons ensuite examiné l'effet de l'activation locale de la vitamine D par les cellules tumorales dérivées du sein sur la croissance de ces cellules dans le microenvironnement osseux. Nous n'avons constaté aucune différence significative entre la croissance des cellules tumorales du cancer du sein dans l'os chez les souris carencées en vitamine D en comparaison aux souris non carencées en vitamine D. Cependant, nous avons démontré que les cellules tumorales du cancer du sein qui expriment le CYP27B1 croissent beaucoup moins vite dans l'os que les cellules tumorales qui n'expriment pas le CYP27B1. Ces résultats suggèrent un rôle très important de l'activation extra-rénale de la vitamine D par les cellules tumorales du cancer du sein pour inhiber la croissance de ces cellules dans l'os. En conclusion, ces travaux indiquent que le précurseur inactif 25(OH)D pourrait être utilisé seul ou en combinaison pour le traitement des métastases osseuses du cancer du sein.

La vitamina D ha estat durant molt de temps coneguda pel seu important paper en la
regulacio dels nivells de calci i fosfor, i en la mineralitzacio de l'os. Els efectes biologics de la
forma hormonalment activa de vitamina D (1,25(OH)2D3, o calcitriol) estan mediats per la
unio al receptor de vitamina D (VDR), que alhora actua sobre els elements de resposta
especifics (VDRE) en el promotor dels gens diana de vitamina D, modificant-ne l'expressio.
En les ultimes decades, s'ha demostrat que el VDR no solament es present en les dianes
classiques de la vitamina D com els ossos, els ronyons i l'intesti, sino tambe en molts altres
teixits, com els musculs esqueletics, llisos, i cardiacs, i tambe en el cervell, la pell i el fetge.
A mes del control de l'homeostasi mineral, la vitamina D exerceix accions pleiotropiques en
diferents tipus cel.lulars, influint en processos fisiologics importants com la proliferacio, la
diferenciacio cel.lular, la resposta immune, aixi com tambe en les diferents condicions
patologiques: el cancer, les malalties cardiovasculars, metaboliques i autoimmunes. A mes,
juntament amb els seus efectes endocrins, la vitamina D te funcions importants
autocrines/paracrines. En les cel.lules de muscul llis vascular (CMLV), la vitamina D regula la
proliferacio i la calcificacio, aquests dos processos, juntament amb la senescencia de les
CMLV es produeixen durant el proces aterosclerotic. Per poder-se dividir, les CMLV
necessiten l'energia de l'ATP produida per les mitocondries. Les alteracions de la funcio
mitocondrial estan relacionades amb una major produccio de radicals lliures. A la paret
arterial, l'angiotensina II (Ang II) actua com un potent mediador d'estres oxidatiu, provocant
senescencia prematura induida per l'estres (SIPS). Els nivells d'Ang II son controlats per la
renina, l'expressio de la qual es regulada per la vitamina D. Per tant, un defecte en la
senyalitzacio de la vitamina D pot conduir a un augment en els nivells d'Ang II i produir els
seus efectes adversos en la paret de l'arteria.
Quan es van cultivar les CMLV, obtingudes de ratolins knockout per VDR (VDRKO) es
va trobar un augment en la produccio d'Ang II en el medi de cultiu d'aquestes cel.lules en
comparacio amb el tipus salvatge (WT), que estava d'acord amb la major activacio del
sistema renina-angiotensina a nivell sistemic, que es va trobar en els ratolins mutants de
VDR. D'una banda, una disminucio en les taxes de proliferacio de les CMLV VDRKO es va
trobar in vitro i in vivo. Aquest descens va ser en paral.lel a un augment en el volum
cel.lular. En estat de repos, les cel.lules VDRKO presentaven un augment significatiu de
l'expressio de p57Kip2 juntament amb els nivells elevats de p19Arf, p21Cip1 i p27Kip1, i nivells
inferiors de PRB fosforilada, ciclines D i E, i axi es preve la seva proliferacio. A mes, les
cel.lules VDRKO van mostrar un augment en l'expressio de la catepsina D, un enzim amb
activitat com renina, i del receptor d'Ang II tipus 1. A mes, la produccio d'anio superoxid va
ser major en les cel.lules mutants, i va ser inhibida tant amb losartan com amb DPI. D'altra
banda, les cel.lules VDRKO presenten una disminucio significativa en l'expressio de l'enzim
mitocondrial antioxidant, la superoxid dismutasa de manganes (SOD2) i l'oxid nitric sintetasa
induible que produeix la molecula vasodilatadora NO,. Per respirometria d'alta resolucio es va
determinar que les mitocondries de VDRKO CMLV van ser menys eficients que podria explicar
el menor contingut d'ATP en aquestes cel.lules. Tots aquests factors es van relacionar amb el
fenotip senescent, que presentaven al voltant del 20% de les CMLV sense VDR en cultiu.
En conclusio, l'absencia de senyalitzacio per VDR en CMLV porta a SIPS causada per
l'augment en la produccio local d'Ang II, amb el conseguent augment dels radicals lliures,
produits per l'activacio de la NADPH oxidasa, el que suggereix un possible paper del sistema
de la vitamina D en malalties vasculars, que mostren una hiperproliferacio de les CMLV.

La vitamina D ha sido durante mucho tiempo conocida por su importante papel en la
regulacion de los niveles de calcio y fosforo, y en la mineralizacion del hueso. Los efectos
biologicos de la forma hormonalmente activa de la vitamina D (1,25(OH)2D3, o calcitriol)
estan mediados por la union al receptor de la vitamina D (VDR), que a su vez actua sobre los
elementos de respuesta a la vitamina D (VDRE) en el promotor de los genes diana,
modificando su expresion. En las ultimas decadas, se ha demostrado que el VDR esta
presente no solo en las dianas clasicas de la vitamina D, como los huesos, los rinones y el
intestino, sino tambien en muchos otros tejidos, como los musculos esqueleticos, lisos, y
cardiacos, y en el cerebro, la piel y el higado. Ademas del control de la homeostasis mineral,
la vitamina D ejerce acciones pleiotropicos en diferentes tipos celulares, influyendo en
procesos fisiologicos importantes tales como la proliferacion y diferenciacion celular, la
respuesta inmune, asi como diferentes condiciones patologicas como el cancer, las
enfermedades cardiovasculares, metabolicas y autoinmunes. Ademas, junto con sus efectos
endocrinos, la vitamina D tiene papeles autocrinas/paracrinas importantes. En las celulas de
musculo liso vascular (CMLV), la vitamina D regula la proliferacion y la calcificacion. Ambos
procesos, junto con la senescencia de las CMLV surgen durante el proceso aterosclerotico.
Para poder dividirse, las CMLV necesitan la energia del ATP producido por las mitocondrias.
Las alteraciones en la funcion mitocondrial estan relacionadas con una mayor produccion de
radicales libres. En la pared arterial, la angiotensina II (Ang II) actua como un potente
mediador de estres oxidativo, provocando senescencia prematura inducida por estres (SIPS).
Los niveles de Ang II son controlados por la renina, cuya expresion genica se regula por la
vitamina D. Por lo tanto, un defecto en la senalizacion de la vitamina D puede conducir a un
aumento en los niveles de Ang II y sus efectos adversos en la pared arterial.
Cuando se cultivaron CMLV, obtenidas de ratones knockout para VDR (VDRKO) se
encontro un aumento en la produccion de Ang II en el medio de cultivo de estas celulas,
comparando con el tipo salvaje (WT), lo que estaba de acuerdo con la mayor activacion de la
sistema renina-angiotensina a nivel sistemico, que fue encontrada en los ratones mutantes
de VDR. Por otra parte, una disminucion en la tasa de proliferacion de las VDRKO CMLV fue
encontrada in vitro e in vivo. Este descenso era en paralelo a un aumento en el volumen
celular. En estado de reposo, las celulas VDRKO presentaron un aumento significativo en la
expresion de p57Kip2 junto con niveles elevados de p19Arf, p21Cip1 y p27Kip1, y menores
niveles de pRb fosforilada y ciclinas D y E, asi previniendo su proliferacion. Ademas, las
celulas VDRKO mostraron un aumento en la expresion de la catepsina D, una enzima con
actividad parecida a la del renina, y del receptor de Ang II tipo 1. Ademas, la produccion del
anion superoxido fue mayor en las celulas mutantes, y fue inhibida tanto con losartan como
con DPI. Por otra parte, las celulas VDRKO presentaron una disminucion significativa en la
expresion de la enzima antioxidante mitocondrial superoxido dismutasa de manganeso
(SOD2) y la oxido nitrico sintetasa inducible, que produce la molecula vasodilatadora NO. Por
respirometria de alta resolucion fue determinado que las mitocondrias de las CMLV VDRKO
fueron menos eficientes que podria explicar el menor contenido de ATP en estas celulas.
Todos estos factores se relacionaron con el fenotipo senescente, presentado por alrededor de
20% de las CMLV que carecian VDR en cultivo.
En conclusion, la ausencia de senalizacion por VDR en CMLV lleva a SIPS debido al
aumento en la produccion local de Ang II, con el consiguiente aumento de los radicales
libres, producidos por la activacion de la NADPH oxidasa, lo que sugiere un posible papel del
sistema de la vitamina D en enfermedades vasculares, que muestran una hiperproliferacion
de las CMLV.

Vitamin D has long been known for its important role in regulating the levels
of calcium and phosphorus, and in mineralization of bone. The biological effects of
the hormonally active form of vitamin D (1,25(OH)2D3, or calcitriol) are mediated
by the binding to the vitamin D receptor (VDR) which in turn acts over specific
responsive elements (VDRE) in the promoter region of the vitamin D - target genes,
modifying their expression. In the last decades, it has been shown that VDR is
present not only in the classical vitamin D targets such as bone, kidney, and
intestine, but also in many other tissues, like skeletal, smooth, and heart muscles,
and in brain, skin, and liver. In addition to the control of the mineral homeostasis,
vitamin D exerts pleiotropic actions in a variety of cell types, influencing important
physiological processes such as cellular proliferation and differentiation, the
immune response as well as different pathological conditions like cancer,
cardiovascular, metabolic and autoimmune diseases. Moreover, together with its
endocrine effects, vitamin D has important autocrine/paracrine roles. In vascular
smooth muscle cells (VSMC), vitamin D regulates proliferation and calcification.
Both processes, together with VSMC senescence occur during the atherosclerotic
process. To be able to divide, VSMC need the energy of ATP produced by
mitochondria. Alterations in the mitochondrial function are related to increased
production of free radicals. In the artery wall, angiotensin II (Ang II) serves as a
potent mediator of oxidative stress, provoking stress-induced premature
senescence (SIPS). Ang II levels are controlled by renin, which gene expression is
regulated by vitamin D. Thus, a defect of vitamin D signalling could lead to an
increase in Ang II levels and its adverse effects in the artery wall.
When we cultured VSMC, obtained from VDR knockout (VDRKO) mice we
found an increment in the production of Ang II in the culture medium of these cells
in comparison with the wild type (WT), which was in accordance with the increased
activation of the renin-angiotensin system at systemic level, found in the VDR
mutant mice. Moreover, a decrease in the proliferation rates of VDRKO VSMC was
found in vitro and in vivo. This decrease was in parallel to an increase in the cell
volume. In quiescent state, VDRKO cells presented significantly increased
expression of p57Kip2 together with elevated levels of p19Arf, p21Cip1 and p27Kip1, and
lower levels of phosphorilated pRb and cyclins D and E, thus preventing their
proliferation. Furthermore, VDRKO cells showed an increase in the expression of
cathepsin D, an enzyme with renin-like activity, and of the Ang II receptor type 1.
Also, superoxide anion production was higher in the mutant cells, and was inhibited
with both, losartan and DPI. Moreover, the VDR ablated cells presented a significant
decrease in the expression of the mitochondrial antioxidant enzyme manganese
superoxide dismutase (SOD2) and the inducible nitric oxide synthase, which
produces the vasodilatator molecule NO. By high-resolution respirometry we found
that the mitochondria of VDRKO VSMC were less efficient which could explain the
lower content of ATP in these cells. All of these findings were associated with the
senescent phenotype, presented by around 20% of the VSMC lacking VDR in
culture.
In conclusion, the absence of VDR signalling in VSMC leads to SIPS due to the
increased local production of Ang II, with a subsequent increase in free radicals,
produced by the activation of the NADPH oxidase, which suggests a possible role of
the vitamin D system in vascular conditions which show hyperproliferation of VSMC.

Vitamin D (VitD) deficiency has been implicated in the pathogenesis of multiple diseases including chronic kidney disease (CKD). VitD has direct effects on most cells in the innate and adaptive immune system including; CD4+ T cells and dendritic cells (DCs) both of which express the vitamin D receptor (VDR). This thesis, divided into two distinct parts, dissects the effects of: a) in vivo repletion of VitD in a placebo controlled, double blinded clinical trial in CKD patients. Here we hypothesised that repletion with cholecalciferol in VitD insufficient/deficient adult patients would ameliorate systemic inflammation. And the effects of b) VitD treatment of CD4+ T cells in vitro using multiple techniques to delineate the mechanisms influencing cytokine regulation. Here we hypothesised that VitD treatment of CD4+ T cells would, through binding of liganded VDR, lead to epigenetic modifications affecting genes involved in the regulation of cytokine production. In vivo we show that VitD repletion in VitD-deficient and insufficient patients with early stage CKD has immunoregulatory effects on circulating myeloid DCs by reducing expression of HLA-DR (a marker of mature DC phenotype). In vitro we identify a novel signaling pathway in CD4+ T cells, induced by VitD. The hallmark effect of VitD on CD4+ T cells was the induction of an immunoregulatory phenotype characterized by inhibited Th1 and Th17 cytokines and induction of the antiinflammatory cytokine IL-10, a process we show to be regulated by induction of IL-6 and subsequent STAT3 signalling. We further show that these processes are genetically regulated by histone modifications driven by liganded VDR, both at putative enhancer and promoter sites. These findings, most notably, have implications for dysregulated immunoregulation in the setting of inflammatory skin diseases and the development of novel therapeutics for the treatment of these conditions.

Involvement of Cellular Retinoic Acid (CRABP) or Retinol (CRBP) Binding Proteins and 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) receptors in the response of cultured cells to retinoic acid and 1,25(OH)₂D₃ was examined. A new method for saturation analysis of CRABP and CRBP was applied to human tumors, human neuroblastoma cells, which retinoic acid causes to differentiate, and a bioselected subline resistant to retinoic acid. These data suggest that CRABP may not mediate cell differentiation by retinoic acid. In other studies, 1,25(OH)₂D₃ receptors and bioresponses were characterized in cultured primate cells. Rhesus monkey kidney cells (LLC-MK₂) were resistant to 1,25(OH)₂D₃-dependent induction of 25(OH)D-24-hydroxylase enzyme. The ED₅₀ in LLC-MK₂ cells was 10-100 fold higher than in other cultured cells. This resistance resulted from a low affinity receptor. Since the LLC-MK₂ variant receptor did not differ in size from the wild type rhesus 1,25(OH)₂D₃ receptor, (Mᵣ = 52 kDa) a subtle alteration in the receptor likely caused the decreased ligand affinity. Also of interest was the possible cellular resistance to 1,25(OH)₂D₃, in the owl monkey (Aotus trivurgatus), which generally occurs in new world primates. Owl monkey kidney (OMK) cells had the same content of receptors for 1,25(OH)₂D₃ and sensitivity to this hormone as cells from the rhesus monkey (old world primate). The ED₅₀ for induction of 24hydroxylase was 2-3 nM in both the OMK cells and the rhesus monkey fibroblasts. Both cells contained 2300 high affinity receptor molecules per cell, which bound DNA and were characterized by immunoblot as 52 kDa proteins. 1,25(OH)₂D₃ treatment increased the content of 1,25(OH)₂D₃ receptors in OMK cells, by increasing the synthesis of receptor mRNA. These data indicate the owl monkey is not resistant to 1,25(OH)₂D₃, unlike other new world primates. This finding was confirmed independently by demonstration that the owl monkey maintained mean serum 1,25(OH)₂D₃ levels (29 pg/ml) in the range of old world primates (33 pg/ml) and humans, in contrast to the elevated 1,25(OH)₂D₃ in other new world primates (97-129 pg/ml). This result suggests the alteration of 1,25(OH)₂D₃-endocrine dynamics in new world primates occurred subsequent to the evolutionary divergence of the owl monkey.

The standard of care for unresectable lung cancer is chemoradiation. However, therapeutic options are limited and patients are rarely cured. While Radiation therapy is effective at killing tumor cells or inhibiting their growth initially, development of resistance to treatments and recurrence of tumors are major issues. One of the major goals of Dr. Gewirtz’s laboratory has been to develop strategies to overcome the resistance and attenuate disease recurrence. One of these attempts involve employing vitamin D and its analogs in combination with radiation therapy. Our proposed studies were based on a previous finding where vitamin D and vitamin D analogs such as EB 1089, were shown to enhance the response to radiation in breast cancer through the promotion of autophagy. We extended these studies to non-small cell lung cancer (NSCLC) and were able to validate that 1,25-D3 (the hormonally active form of vitamin D) and EB 1089 does in fact sensitize A549 and H460 cells and prolonged the growth arrest induced by radiation alone and suppressed proliferative recovery, which translated to a significant reduction in clonogenic survival. In H838 or H358 NSCLC cells, which lack the vitamin D receptor or functional p53, respectively, 1,25-D3 failed to modify the extent of radiation-induced growth arrest or suppress proliferative recovery post irradiation. Sensitization to radiation in H1299 NSCLC cells was evident only when p53 was induced in otherwise p53 null H1299 NSCLC cells. Sensitization by 1,25-D3/ EB 1089 was not associated with increased DNA damage, decreased DNA repair or an increase in apoptosis, necrosis or senescence. Instead sensitization appeared to be a consequence of the conversion of the cytoprotective autophagy induced by radiation alone to a novel cytostatic form of autophagy by the combination of 1,25-D3 or EB 1089 with radiation. While both pharmacological and genetic suppression of autophagy or inhibition of AMPK phosphorylation sensitized the NSCLC cells to radiation alone, inhibition of the cytostatic autophagy induced by the combination treatment reversed sensitization. Evidence for selectivity was provided by lack of radiosensitization in normal human bronchial cells and cardiomyocytes. Taken together, these studies have identified a unique cytostatic function of autophagy that appears to be mediated by the vitamin D receptor, p53 and possibly AMPK in the promotion of an enhanced response to radiation by 1,25-D3 and EB 1089 in NSCLC.

Thesis (Ph.D)--Materials Science and Engineering, Georgia Institute of Technology, 2010.
Committee Chair: Barbara Boyan; Committee Member: Andres Garcia; Committee Member: Anthony Norman; Committee Member: Nael McCarty; Committee Member: Zvi Schwartz. Part of the SMARTech Electronic Thesis and Dissertation Collection.