1. Vitamin D Fat soluble ‘vitamin’ Synthesised in skin, food sources include fish oils Physiological forms - vitamin D 3 (cholecalciferol) - vitamin D 2 (ergocalciferol) hormonal form synthesised in kidney Functions - intestine: calcium absorption - bone: promotes mineralization Deficiency syndromes - children: rickets - adults: osteomalacia Mechanism of action - receptor mediated

3. Synthesis of vitamin D 3

4. Sunlight as a source of vitamin D Adequate supplies of vitamin D 3 can be synthesized with sufficient exposure to solar ultraviolet B radiation Depends on latitude and season Summer sunlight in Cape Town = 2500 IU vitamin D 3 daily Melanin, clothing or sunscreens that absorb UVB will reduce cutaneous production of vitamin D 3

5. Metabolism of vitamin D

7. Actions of 1,25(OH) 2 D 3 on intestine Stimulates active calcium transport Induces expression of calbindin 9K (calcium binding protein)

8. Actions of 1,25(OH) 2 D 3 on bone Osteoblasts: –Simulates synthesis of collagen, osteocalcin and other bone matrix proteins Osteoclasts: –Stimulates osteoclastic bone resorption (indirect action) –Stimulates osteoclast recruitment

9. 1,25(OH) 2 D 3 and osteoclast formation

10. Abnormalities in vitamin D metabolism/action Vitamin D deficiency –Inadequate intake –Limited sunlight exposure Acquired or inherited disorders of vitamin D metabolism Inherited resistance to the actions of vitamin D

11. Calcium absorption and osteoporosis

12. Bone deformities in post-menopausal osteoporosis

13. 1,25-dihydroxyvitamin D 3 treatment of osteoporosis

14. The analogue 1  hydroxyvitamin D 3 is used to treat renal osteodystrophy Bone disease and renal failure

15. 1 alpha hydroxyvitamin D 3

16. VDR Enhanced mRNA synthesis From vitamin D responsive genes 1,25(OH) 2 D 3 cell cytoplasm nucleus DNA Mechanism of action of vitamin D

17. Homology between nuclear hormone receptors

19. Interaction of VDR with VDREs RXR VDR 3 5’-AGGTCAAGGTCA- 3’ Heterodimerization with RXR DR 3 VDRE in promotor region of target gene

20. Distribution of VDR in normal human tissues TissueImmunocytochemical staining Liver+/++ Kidney++/+++ Thyroid++/+++ Adrenal+/++ Stomach+/++ Duodenum++ Jejunum++ Colon+++ Skin++ Breast epithelium++ Skeletal muscle- (+) weak, (++) moderate, (+++) strong, (-) negative

21. Vitamin D regulated genes/gene products FunctionGene productTissue/cells Mineral homeostasiscalbindin 9Kintestinal mucosa osteocalcinosteoblast Peptide hormonesTSHrat pituitary cells PTHrat parathyroid Growth factors/TGF-  rat calvarial cells receptorsTNF-  leukaemia cells Oncogenesc-mycbreast cancer cells

22. Non-classical actions of vitamin D A wide variety of tissues and cells contain the VDR and respond to 1,25- dihydroxyvitamin D 3 Immunomodulatory actions and effects on cells growth and differentiation identified Development of analogues capable of modulating ‘non-classical’ actions of vitamin D

23. Immunomodulatory actions of 1,25-dihydroxyvitamin D 3 Activates monocytes and promotes differentiation of myeloid stem cells Suppresses lymphocyte proliferation, immunoglobulin production and cytokine synthesis

24. Differentiation of leukaemia cells by 1,25(OH) 2 D 3

25. Activity profile of calcitriol and its synthetic analogues Calcium mobilizing actions Anti-tumour/promotion of differentiation Immunosuppressive effects

26. Pathogenesis of psoriasis Chronic or chronically relapsing skin disease ? Susceptibility heritable Results form epidermal stem cell growth, initiated by lymphokines released from activated T cells

27. Vitamin D and psoriasis 1983 - 1,25-dihydroxyvitamin D 3 shown to promote keratinocyte differentiation 1985 - a patient with osteoporosis receiving 1  hydroxyvitamin D3 showed a dramatic improvement in her severe psoriasis Development of vitamin D analogues for topical treatment of psoriasis

28. Treatment of psoriasis with vitamin D

30. Vitamin D and breast cancer Risk of breast cancer inversely related to intensity of local sunlight and 1,25-D levels Low serum 1,25-D levels correlated with disease progression and development of bone metastases >80% breast tumour specimens VDR positive and presence of receptor is associated with increased disease free survival 1,25-D and its analogues inhibit growth and promote apoptosis in vitro and in vivo

31. Trial of topical calcipotriol (MC903) therapy in advanced breast cancer 19 patients with locally advance or metastatic breast cancer and evaluable cutaneous deposits were treated daily with one gram calcipotriol (MC903) ointment All patients were normocalcaemic at entry 14 patients completed 6 weeks of treatment. 3 showed a partial and one a minimal response

32. Topical calcipotriol treatment in advanced breast cancer chest wall of patient treated with calcipotriol ointment at start of treatmentafter 5 weeks

33. Vitamin D analogues: profile of activity analogue inhibition of calcaemic activity cell growth __________________________________________________________________ 1,25(OH) 2 D 3 11 MC 90320.05 CB 96660.2 EB 1089600.5 CB 10931600.27 KH 106010001.3

34. Effects of vitamin D analogues on progression NMU-induced rat mammary tumours

35. Processes involved in the tumour suppressive activity of vitamin D analogues Inhibition of cell proliferation Induction of apoptosis Promotion of cell differentiation Inhibition of angiogenesis Altered elaboration or response to growth factors Inhibition of metastasis

36. Polymorphisms in the VDR gene Vitamin D receptor gene polymorphisms have been identified These include single base change mutations in the 3’ UTR region These SNPs have been reported to be associated with altered risk for certain diseases It is not yet known in what way these differences in the gene may affect the activity of the translated receptor protein

37. Ia Ib Ic II III IV V VI VII VIII IX FokI start codon polymorphism F = VDR (424 amino acids) f = VDR (427 amino acids) BsmI (B/b) ApaI (A/a) TaqI (T/t) Long/Short (L/S) poly (A) microsatellite Linkage disequilibrium b = a = T = L exon Polymorphisms in the VDR Gene

38. Association between VDR polymorphisms and disease Bone mineral density Early postnatal growth Diabetes mellitus Psoriasis TB and hepatitis B virus infection Primary hyperparathyroidism Prostatic and breast cancer

39. VDR polymorphisms and breast cancer

40. 20.7 - 21.020.1 -20.4 19.8 - 20.120.4 - 20.7 Prostate cancer mortality amongst white men per 100 000 >33.431.4 - 33.4 <29.429.4 - 31.4 Calculated UV radiation From Hanchette CL et al. Cancer 1992; 70(12); 2861-9 Geography of Prostate Cancer in USA

41. Studies of association between VDR genotype and prostate cancer

42. To determine VDR polymorphism frequencies and their association with breast cancer risk* *Bretherton-Watt et al 2001. Br J Cancer vol 85, 171-175, Project Aim

43. Volunteers

44. Identification of Bsm I SNP The polymorphism constitutes a single nucleotide change (G to T) which deletes a Bsm I endonuclease restriction site CGCATTC CTCATTC Restriction site present (b) Restriction site absent (B)

45. Genotyping SNPs: the traditional way }}} bbBbBB Presence/absence of restriction site is indicated by b/B Therefore: bb – homozygous, restriction site BB – homozygous, no restriction site Bb – heterozygote

46. Results

47. VDR polymorphisms in control and breast cancer patients p< 0.01 patients 43.1% 46.4% 10.5% bb Bb BB controls 28.6% 55.2% 16.2%  2 analysis p> 0.2 15.5% 44.8% 39.8% ff Ff FF 16.2% 48.1% 35.7% BsmI  2 analysis FokI

48. VDR More/less VDR produced? Different/less stable VDR produced? Altered ability to switch on/regulate genes? Altered sensitivity to hormones and environmental factors? 1,25(OH) 2 D 3 bb/LL BB/SS high risk low risk

49. Summary Recent research has identified the possibility of developing analogues capable of modulating non-classical actions of vitamin D Polymorphisms in the VDR gene may contribute to susceptibility to certain diseases