Vitamin D• Fat soluble ‘vitamin’

• Synthesised in skin, food sources include fish oils

• Physiological forms - vitamin D3 (cholecalciferol)

- vitamin D2 (ergocalciferol)

hormonal form synthesised in kidney

• Functions - intestine: calcium absorption

- bone: promotes mineralization

• Deficiency syndromes - children: rickets

- adults: osteomalacia

• Mechanism of action - receptor mediated

Synthesis of vitamin D3

Sunlight as a source of vitamin D

• Adequate supplies of vitamin D3 can be synthesized with sufficient exposure to solar ultraviolet B radiation

• Depends on latitude and season

• Summer sunlight in Cape Town = 2500 IU vitamin D3 daily

• Melanin, clothing or sunscreens that absorb UVB will reduce cutaneous production of vitamin D3

Metabolism of vitamin D

Actions of 1,25(OH)2D3 on intestine

• Stimulates active calcium transport

• Induces expression of calbindin 9K (calcium binding protein)

Actions of 1,25(OH)2D3 on bone

• Osteoblasts:

– Simulates synthesis of collagen, osteocalcin and other bone matrix proteins

• Osteoclasts:

– Stimulates osteoclastic bone resorption (indirect action)

– Stimulates osteoclast recruitment

1,25(OH)2D3 and osteoclast formation

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

Calcium absorption and osteoporosis

Bone deformities in post-menopausal osteoporosis

1,25-dihydroxyvitamin D3 treatment of osteoporosis

The analogue 1 hydroxyvitamin D3 is used to treat renal osteodystrophy

Bone disease and renal failure

OH OH

H

H

1 alpha hydroxyvitamin D3

VDR

Enhanced mRNA synthesisFrom vitamin D responsivegenes

1,25(OH)2D3

cellcytoplasm nucleus

DNA

Mechanism of action of vitamin D

Homology between nuclear hormone receptors

Interaction of VDR with VDREs

RXR VDR

35’-AGGTCA AGGTCA- 3’

Heterodimerization with RXR

DR 3 VDREin promotor region oftarget gene

Distribution of VDR in normal human tissues

Tissue Immunocytochemical stainingLiver +/++Kidney ++/+++Thyroid ++/+++Adrenal +/++Stomach +/++Duodenum ++Jejunum ++Colon +++Skin ++Breast epithelium ++Skeletal muscle - (+) weak, (++) moderate, (+++) strong, (-) negative

Vitamin D regulated genes/gene products

Function Gene product Tissue/cells

Mineral homeostasis calbindin 9K intestinal mucosaosteocalcin osteoblast

Peptide hormones TSH rat pituitary cellsPTH rat parathyroid

Growth factors/ TGF- rat calvarial cellsreceptors TNF- leukaemia cells

Oncogenes c-myc breast cancer cells

Non-classical actions of vitamin D

• A wide variety of tissues and cells contain the VDR and respond to 1,25-dihydroxyvitamin D3

• Immunomodulatory actions and effects on cells growth and differentiation identified

• Development of analogues capable of modulating ‘non-classical’ actions of vitamin D

Immunomodulatory actions of 1,25-dihydroxyvitamin D3

• Activates monocytes and promotes differentiation of myeloid stem cells

• Suppresses lymphocyte proliferation, immunoglobulin production and cytokine synthesis

Differentiation of leukaemia cells by 1,25(OH)2D3

Activity profile of calcitriol and its synthetic analogues

• Calcium mobilizing actions

• Anti-tumour/promotion of differentiation

• Immunosuppressive effects

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

Vitamin D and psoriasis

• 1983 - 1,25-dihydroxyvitamin D3 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

Treatment of psoriasis with vitamin D

OHHO

OH

1,25--vit. D3

OHHO

OH

EB 1089CB 1093

OHHO

OH

O

OHHO

OH

MC 903

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

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

Topical calcipotriol treatment in advanced breast cancer

chest wall of patient treated with calcipotriol ointment

at start of treatment after 5 weeks

Vitamin D analogues: profile of activity

analogue inhibition of calcaemic activity cell growth

__________________________________________________________________

1,25(OH)2D3 1 1

MC 903 2 0.05

CB 966 6 0.2

EB 1089 60 0.5

CB 1093 160 0.27

KH 1060 1000 1.3

Effects of vitamin D analogues on progression NMU-induced rat

mammary tumours

5432100

100

200

300

EB 1089 1ug/kg

control

week

% o

f in

itia

l tum

or v

olum

e

******

2.54+/-0.04mmol/l

3.06+/-0.07mmol/l

5432100

50

100

150

200

CB 1093 1ug/kgcontrol

week

% o

f in

itia

l tum

or v

olum

e

**

****

2.67+/-0.02mmol/l

2.98+/-0.07mmol/l

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

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

Ia Ib Ic II III IV V VI VII VIII IX

FokI start codon polymorphismF = 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 disequilibriumb = a = T = L

exon

Polymorphisms in the VDR Gene

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

VDR polymorphisms and breast cancer

Vitamin D appears to be protective against breast and prostatecancer. It can inhibit growth and induce apoptosis in in vitromodels of both.

Vitamin D exerts its cellular actions by binding to a specificintracellular receptor, the VDR. The ligand/receptor complex thenacts as a TF leading to increased or decreased transcription ofsensitive genes.

The human VDR gene contains a number of SNPs that may alter theactivity or function of the VDR

SNPs in the VDR gene have previously been associated withdisorders of bone metabolism eg. osteoporosis and altered bonemineral density

We investigated whether SNPs in the VDR gene were associatedwith risk of breast cancer

20.7 - 21.020.1 -20.4

19.8 - 20.1 20.4 - 20.7

Prostate cancer mortality amongst white men per 100 000

>33.431.4 - 33.4

<29.4 29.4 - 31.4

Calculated UV radiation

From Hanchette CL et al. Cancer 1992; 70(12); 2861-9

Geography of Prostate Cancer in USA

STUDY PATIENTS POLY OR Ingles et al 57 non hispanic whites Poly A 4.61 Taylor et al 108 men - radical prostatectomy Taq I 0.32 p<0.01 (Black and White) Ma J et al 327 (questionairre identified) Bsm Not significant Taq I Not significant Correa-Cerro 132 Poly A Not significant Taq I Not significant Kibel et al 41 men - metastatic disease Poly A Not significant Taq I Not significant

Studies of association between VDR genotype and prostate cancer

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

Controls (n=241, median age 55.2 years,range 51-79 years).Breast cancer patients (n=181, median age61.2 years, range 29-91, median time sincediagnosis 4.3 years, range 0.4-27.5 years).

Inclusion criteria to the study were:1) A recent mammogram and breast history;2) Caucasian (due to ethnic variations in

polymorphism distribution).Written informed consent was obtainedprior to collection of a blood sample.

Volunteers

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)

Step TimeGenomic DNA extracted from whole blood 40 minsPCR amplification of the polymorphismperformed using VDR specific primers.

2.5 hours

PCR product is digested 65oC with BsmIrestriction enzyme

1 hour

Digest is analysed by agarose gelelectrophoresis.

2 hours

Genotyping SNPs: the traditional way

} } }

bb Bb BB

Presence/absence of restriction site isindicated by b/BTherefore:bb – homozygous, restriction site BB – homozygous, no restriction site

Bb – heterozygote

TThheerree wwaass aa ssiiggnniiffiiccaanntt ddiiffffeerreennccee bbeettwweeeennBBssmmII//PPoollyy AA ggeennoottyyppee ffrreeqquueenncciieess iinn ccaanncceerrppaattiieennttss ccoommppaarreedd ttoo hheeaalltthhyy ccoonnttrroollss..TThhiiss ddiiffffeerreennccee wwaass ssuucchh tthhaatt tthhee ooddddss ooff bbrreeaassttccaanncceerr ffoorr aa wwoommaann ooff ggeennoottyyppee bbbb//LLLL aarree ttwwiicceetthhoossee ffoorr aa wwoommaann ooff ggeennoottyyppee BBBB//SSSS..

TThheerree wwaass nnoo aassssoocciiaattiioonn wwiitthh tthhee FFookkIIppoollyymmoorrpphhiissmm aanndd bbrreeaasstt ccaanncceerr.. HHoowweevveerr,, FFookkIIppoollyymmoorrpphhiissmm aappppeeaarreedd ttoo mmoodduullaattee tthhee rriisskkaassssoocciiaatteedd wwiitthh BBssmmII//PPoollyy ((AA)) ggeennoottyyppee..PPoosssseessssiioonn ooff FFFF ggeennoottyyppee rreedduucceedd tthhee rriisskkaassssoocciiaatteedd wwiitthh ggeennoottyyppee bbbb//LLLL..

Results

VDR polymorphisms in control and breast cancer patients

p< 0.01

patients

43.1%46.4%

10.5%bbBbBB

controls

28.6%

55.2%

16.2%2 analysis

p> 0.2

15.5%

44.8%

39.8%ffFfFF

16.2%

48.1%

35.7%

BsmI

2 analysis

FokI

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)2D3

bb/LL

BB/SS

high risk

low risk

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