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.

Prostate cancer is the most occurring form of cancer in men in Sweden and new candidates for treatment towards advanced phases of prostate cancer needs to be investigated. One suggested treatment is vitamin D which will mediate effect via VDR and PDIA3 and cause cell cycle arrest.  Another treatment is digitoxin which will cause accumulation of intracellular Ca2+ leading to apoptosis. The aim of the project of was to investigate potential synergistic effects of 25-hydroxyvitamin D3 and digitoxin in prostate cancer cell lines and find out effects mediated by PDIA3. DU145 and LNCaP cells were seeded and treated with 25-hydroxyvitamin D3 (10-10, 10-9, 10-7 M), digitoxin (25 ng/ml and 50 ng/ml) and four combinations of 25-hydroxyvitamin D3 and digitoxin. Cell viability assay was performed for determining the number of viable cells. Treatment with 25-hydroxyvitamin D3 10-7M + digitoxin 50ng/ml (68%), 25-hydroxyvitamin D3 10-9M + digitoxin 25ng/ml (39%), 25-hydroxyvitamin D3 10-9M + digitoxin 50ng/ml (69%), digitoxin 25ng/ml (26%) and digitoxin 50 ng/ml (44%) was statistically significant with increased cell viability compared to untreated control in DU145 after 48h of treatment. Treatment with 25-hydroxyvitamin D3 10-7M (12%) and 25-hydroxyvitamin D3 10-9M (12%) was statistically significant with increased cell viability compared to untreated control in LNCaP after 24h of treatment. The conclusion based on results from this study is that a combination of digitoxin and 25-hydroxyvitamin D3 does not inhibit cell viability in DU145 or LNCaP cancer cell lines.

25-Hydroxyvitamin D3[25(OH)D3] is a metabolite of vitamin D3 that has long been considered to be an inactive precursor of the hormonally active metabolite 1,25- dihydroxyvitamin D3[l,25(OH)2D3]; consequently very few studies have addressed the potential biological activity of 25(OH)2D3. However, it is known that 100 nM 25(OH)2D3 increases calcium transport in the perfused duodenal loop of the chicken to 200% of controls within 20 minutes. The hypothesis of the current study is that 25(OH)2D3 may be a hormonally active metabolite and its effects can be studied in isolated chick enterocytes. To begin testing this postulate, time course studies of 45Ca uptake were undertaken in isolated intestinal cells (from 7 wk chicks). After establishing the basal uptake of 45Ca for 5 minutes, cells were treated with vehicle(< 0.01% v/v ethanol, final concentration) or 25 nM, 50 nM, 100 nM, or 300 nM 25(OH)2D3 and samples were taken at T = 1, 3, 5, 7, and 10 min. With increasing concentrations of steroid, the enterocyte 45Ca decreased. The optimal concentration of 100 nM 25(OH)2D3 induced the most rapid response: within 1 min 45Ca decreased to 54% of controls (P < 0.001) and 70% of the controls at T= 3, 5, and 7 min (P < 0.01 to < 0.05, relative to controls). Comparison of the 7- min time points for 25 nM, 50 nM, 100 nM, and 300 nM 25(OH)2D3 appeared to yield a biphasic dose response curve with values of 45Ca observed at 99% (NS, not significant), 75% (P < 0.05), 70%(P < 0.01%), and 80% (NS) of corresponding controls, respectively. Physiological levels of 24,25(OH)2D3 (6.5 nM) inhibited the action of 100 nM 25(OH)2D3 in isolated chick enterocytes. Time course studies with isolated enterocytes from 14 wk and 28 wk chickens treated with 100 nM 25(OH)2D3 also showed decreased responsiveness: at T= 1 min 45Ca levels in 7 wk, 14 wk, and 28 wk were 54% (P < 0.01), 83% (NS), and 80% (NS) of corresponding controls, respectively. Experiments with the calcium channel activator BAY K8644 (2 μM) and protein kinase A (PKA) activator forskolin (20 μM) revealed enhanced levels of 45Ca at T= 10 min that were 132% and 140% of corresponding controls, respectively (each, P < 0.05). Phorbol ester treatment of the cells resulted in significant increases in the levels of 45Ca between the treated cells and corresponding controls at T=7 and 10 min. Cells treated with 100 nM 25(OH)2D3 revealed 89.8% and 78.4% increases above controls in PKA activity at T =1 min (P < 0.05) and T=3 min, relative to corresponding controls. However, there was no evidence for the activation of PKC by 25(OH)2D3 during the time period studied.

No description available.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 83-100).

Les patients atteints de maladies inflammatoires de l'intestin (MII) ont un risque accru de développer un cancer colorectal dû aux lésions épithéliales secondaires à l’inflammation chronique. La vitamine D (vD) régule NOD2, gène impliqué dans la réponse inflammatoire et dans la susceptibilité aux MII, et induit son expression dans les monocytes et dans l’épithélium intestinal. Dans ce projet, nous avons d’abord induit le cancer colorectal associé à la colite ulcéreuse (CAC) en administrant un traitement combiné d’azoxyméthane (AOM) et de dextran de sulfate de sodium (DSS) aux souris C57BL/6J. Par la suite, nous avons étudié l'effet d’une carence en vD3 sur le développement du CAC et évalué la capacité préventive d’une supplémentation en vD3 sur la tumorigenèse, et vérifié si cet effet est médié par NOD2, en utilisant les souris Nod2-/-. Les C57BL/6J et les Nod2-/-, ayant reçu une diète déficiente en vD3, étaient moins résistantes au CAC par rapport aux souris supplémentées. Le pourcentage de perte de poids, l’indice d’activation de la maladie (DAI), le taux de mortalité et le poids relatif du côlon (mg/cm) chez les souris déficientes en vD3 étaient plus élevés en comparaison avec celles supplémentées en vD3. Une augmentation du score d'inflammation et de la multiplicité tumorale corrélait avec une expression accentuée de l’Il6 dans les colonocytes des souris déficientes en vD3. La vD3 régulait l’expression génétique de Cyp24, Vdr et de gènes pro-inflammatoires chez les C57BL/6, comme chez les Nod2-/-. En conclusion, la supplémentation en vD3 peut prévenir le développement du CAC indépendamment de NOD2. / Patients with inflammatory bowel disease (IBD) have an increased risk of developing colorectal cancer due to continuing epithelial cell injury from the chronic inflammatory process. Vitamin D (vD) regulates NOD2, a gene involved in the inflammatory response and in IBD susceptibility, and induces its expression in monocytes and intestinal epithelial cells. In this project, we first established an azoxymethane (AOM)/dextran sodium sulfate (DSS) murine model of colitis-associated colorectal cancer (CAC) using C57Bl/6J. We then investigated the effect of vD3 deficiency on CAC development, and evaluated the ability of vD3 supplementation to prevent tumorigenesis. Lastly, we assessed whether the preventive benefits of vD3 on colon carcinogenesis are mediated via NOD2 using Nod2 knockout mice (Nod2-/-). vD3 deficient C57Bl/6J and Nod2-/- mice displayed increased severity of AOM/DSS-induced CAC compared to mice given vD3 supplemented diets. In vD3 deficient mice, body weight loss, Disease Activity Index (DAI), mortality rate and the colon weight/length ratio were higher compared to vD3-supplemented mice. An increased inflammation score was observed in the mucosa of vD3 deficient mice along with augmentation in the expression level of IL-6. Higher tumour multiplicity was also observed in vD3 deficient groups compared to vD3-supplemented groups. In both C57Bl/6J and Nod2-/- mice, vD3 regulated Cyp24, Vdr and pro-inflammatory genes. In conclusion, vD3 supplementation can prevent CAC independently of NOD2.