Everything You Always Wanted to Know About Vitamin D but Were Afraid to Ask

Everything you always wanted to know about vitamin D but were afraid to ask

Hans van Leeuwen Department of Internal Medicine Erasmus MC Roerdam, The Netherlands Vitamin D

• Synthesis of Vitamin D – Skin, liver, kidney – Target ssue • Target ssue metabolism of vitamin D – Degradaon: CYP24 • Vitamin D binding protein (DBP) • Mechanism of acon – Vitamin D Receptor – Gene transcripon: inducon/repression • Biological Acon – Classical eﬀects: Calcium / phosphate homeostasis – Non‐classical eﬀects: Immune system / cancer / Vitamin D

skin

Cholecalciferol (vitamin D3) 7-dehydrocholesterol

liver Secondary source - diet

Vitamin D3 (fish, meat) Vitamin D2 (dietary supplements)

25-hydroxyvitamin D3

kidney 1,25-dihydroxyvitamin D3  Calcium/phosphate absorption from the diet in the intestine  Reabsorption of calcium and phosphate in the kidney  Bone mineralization Maintenance of the calcium/phosphate balance together with parathyroid hormone (FGF23, klotho) UV‐B (290‐300 nm)

Skin

7‐Dehydrocholesterol Pre‐vitamin D Vitamin D3 (Cholecalciferol) Liver

OH OH

Kidney

HO OH HO

1,25‐Dihydroxyvitamin D3 25‐Hydroxyvitamin D3 B

Ergosterol Vitamin D2 (Ergocalciferol) Impact of season, clothing and sunscreen on vitamin D producon

Boston Edmonton Bergen 25‐hydroxylase (CYP27A, CYP2D25, CYP3A4, CYP2R1)

• Lile if no eﬀect of vitamin D status (in case of suppleon in situaon of hypocalcemia; see ﬁgure) • Smulaon by dexamethasone 1α‐hydroxylase (CYP27B1), 12q13.1‐q13.3

Kidney • Smulaon by • parathyroid hormone, • low calcium, • low phosphate • calcitonin • Inhibion by • 1,25‐(OH)2D3 • FGF23 • Klotho • Anmycocs (ketoconazole, miconazole) Vitamin D

• Synthesis of Vitamin D – Skin, liver, kidney – Target ssue • Target ssue metabolism of vitamin D – Degradaon: CYP24 • Mechanism of acon – Vitamin D Receptor – Gene transcripon: inducon/repression • Biological Acon – Classical eﬀects: Calcium / phosphate homeostasis – Non‐classical eﬀects: Immune system / cancer / 1α‐hydroxylase is expressed in bone: in osteoblasts and osteoclasts

Osteoblasts

Also 25‐hydroxylase and DBP receptors are expressed in osteoblasts

25‐OHase

Van Driel et al., FASEB J Express 20:2417; 2006 (Van Driel et al., FASEB J. 2007) 1α‐hydroxylase is acve in human osteoblasts and acvity is blocked by ketoconazole

1 µM 25‐(OH)D3

48 hrs; 1 µM

48 hrs

 + ketoconazole

Van Driel et al., FASEB J Express 20:2417; 2006 (Van Driel et al., FASEB J. 2007) • 25‐(OH)D3 smulates CYP24 and osteocalcin expression in human osteoblasts • Smulaon is blocked by inhibion of 1α‐hydroxylase acvity

CYP24

Osteocalcin

(Van Driel et al., FASEB J. 2007) • Role of vitamin D in ssues that have a barrier NFkB funcon; immunological or physical.

• Hypothesis: vitamin D serves a similar role in osteoblasts: Regulang mineralizaon mediated by osteoblasts that cover the bone surface as a barrier.

• Current data may hold the clue to explain the

associaon studies between 25‐(OH)D3 and BMD, fractures.

• General perspecve: these data contribute to the emerging concept of steroid hormone producon in target ssues (glucocorcoids, estradiol)

• This implicates the transion from a hormone acng at a distant site of synthesis to a local

factor acng in an auto/paracrine manner Van Driel et al., FASEB J Express 20:2417; 2006 Vitamin D

Vitamin D3

vitamin D3 25-hydroxylase; CYP27A, CYP2D25 Liver O2

24-hydroxylase; CYP24, all vitamin D target cells

25-Hydroxyvitamin D3 24,25-Dihydroxyvitamin D3

O2 1α-hydroxylase; CYP27B1, Kidney O2 O2

1,25-Dihydroxyvitamin D3 1,24,25-Trihydroxyvitamin D3

O2 24‐hydroxylase (CYP24), 20q13

. Stimulation by 1,25-(OH)2D3 . Inhibition by parathyroid hormone (in hypocalcemia) Vitamin D C-24 Oxidation degradation pathway to Calcitroic acid 24-hydroxylase activity and biological action

higher 1,25-(OH)2D3 level in Inhibition of 24-hydroxylase activity gives prostate cancer cells and…….

+ liarozole

- liarozole Inhibition of 24-hydroxylase activity, increased growth inhibition

Ly et al. Endocrinology 140:2071; 1999

Quantitative mapping of amplicon structure by array Comparative Genomic Hybridization (CGH) identifies CYP24 as a candidate oncogene. Albertson et al., Nature Genetics 25(2):144; 2000. Relation VDR level and 24-hydroxylase activity

Increase in VDR level …… Increase in induction of 24-hydroxylase 24-hydroxylation is the rate-limiting step of the degradation pathway but it does not

directly abolishes the action of 1,25-(OH)2D3

1α-hydroxylation is very important for the eventual activity of the vitamin D metabolites, which is conceivable considering the significance of the C-1α

position for VDR binding

1,25-(OH)2D3

1,24,25-(OH)3D3

24,25-(OH)2D3 Target tissue metabolism

. Conversion of active compound into inactive compound

1,25-Dihydroxyvitamin D3  1,24,25-trihydroxyvitamin D3  calcitroic acid

. Vitamin D induces its own degradation to limit its activity

. Intervention with degradation pathway in target cells enhances biological effect

. Demonstrates the significance of hormone degradation pathways Vitamin D

. Synthesis of Vitamin D . Skin, liver, kidney . Target tissue . Target tissue metabolism of vitamin D . Degradation: CYP24 . Vitamin D binding protein (DBP) . Mechanism of action . Vitamin D Receptor . Gene transcription: induction/repression . Biological Action . Classical effects: Calcium / phosphate homeostasis . Non-classical effects: Immune system / cancer / Vitamin D-binding protein (DBP); 4q11-q13

. Polymorphic, monomeric serum α-globulin of about 58 kDA (dependent on glycosylation); hDBP 458 aa. long

. Located closely together near centromere: DBP-albumin-α-fetoprotein, and α-albumin.

. Patterns of high structural similarity with proteins and genes of albumin, α-fetoprotein, and α-albumin.

. Identified in 1959; then function not known. . Present in all major vertebrate phyla

. Binding, solubilization, and serum transport of vitamin D sterols.

. It may have an additional spectrum of biological activities, including roles in inflammation and immune system (DBP-MAF)

. The DBP gene is widely used in population genetics and forensic medicine Vitamin D-binding protein (DBP)

. Circulates in micromolar range; primarily produced in the liver

. Most studies failed to link vitamin D status of disturbances in mineral homeostasis with alternations in DBP concentration.

. DBP increases during pregnancy and under influence of female sex hormones.

. Albeit having weak, non-specific associations with lipoproteins and albumin, the vitamin D sterols in the circulation form a specific, high-capacity, high-affinity association with DBP. Vitamin D-binding protein (DBP)

DBP-Macrophage activating factor (DBP-MAF)

. DBP can be converted into a DBP-MAF by the deglycosylation of DBP at the non-sterol binding domain.

. DBP-MAF plays a role in osteoclast differentiation and mediates bone resorption by directly activating osteoclasts.

. DBP-MAF treated osteopetrosis rats have increased number and activity of osteoclasts, and decreased bone mass.

. Hence, the contribution of the DBP to bone metabolism is not only through assisting the vitamin D endocrine system, but may also directly influences bone resorption. Vitamin D

. Member of the nuclear receptor family / ligand-induced transcription factors Vitamin D Receptor (VDR); 12q13.11 (1α-hydroxylase: 12q13.1-q13.3) Homologous and heterologous regulation of VDR expression

Various hormones, growth factors etc. have been shown to regulate VDR • Variable results: i.e. either up or down or no effect. • Regulation seems to be tissue specificity

• PTH, phosphorus: up- and down regulation Homologous regulation • Normocalcemic: up-regulation • Hypocalcemic: no effect on up-regulation

Why down-regulation in a state of high calcium need? Mechanism of action of VDR Liganded VDR binds to specific DNA recognition sites (VDREs) as heterodimers with Retinoic X receptorr (RXR)

RXR-RAR

VDR prefers RGTTCA motifs over RXR- VDR RGGTCA motifs (R = A or G).

VDR binds efficiently to DR3-, DR4-and RXR- PXR ER9-type REs.

1-5 rule Umesono, Cell 1991

Coacvator and corepressor complexes are required for nuclear receptor‐mediated transcriponal regulaon Gene expression Up regulaon CYP 24 Human/rat osteocalcin Mouse osteoponn NPT2 (Phosphate transporter) Carbonic anhydrase Integrin β3

Down regulaon ………………. you name one Mouse osteocalcin Rat Cbfa1 (in preosteoblasts) Rat bone sialoprotein Rat PTHrP in tumors Interleukin 2 No vitamin D response element VDR‐RXR heterodimer blocks NFATp/AP‐1 complex formaon and then stably associates with the NF‐AT‐1 element. This direct inhibion by a nuclear hormone receptor of transcriponal acvators of the IL‐2 gene may provide a mechanisc explanaon of how vitamin derivaves can act as potent immunosuppressive agents. Vitamin D

Calcium / phosphate homeostasis Vitamin D

Linear trend surface map of ultraviolet radiation in 3,073 Linear trend surface map of age-adjusted prostate cancer counties of the contiguous U.S. Figure redrawn from Cancer, among white men, 1970–1979 in 3,073 counties of the 70, No. 12, 1992, p. 2865. Copyright 1992, American Cancer contiguous U.S. Figure redrawn from Cancer, 70, No. 12, Society. Reprinted by permission of Wiley-Liss, Inc. A 1992, p. 2865. Copyright 1992, American Cancer Society. subsidiary of John Wiley & Sons, Inc. Reprinted by permission of Wiley-Liss, Inc. A subsidiary of John Wiley & Sons, Inc. Non‐classical eﬀects (i.e. not primarily related to calcium homeostasis)

In general the non-classical effects are related to

Inhibition of cell growth - stimulation of cell differentiation

• Inhibition of growth of wide variety of tumor cells • Prostate • Colon • Breast • Hematological malignancy

• Immune suppressive role (inhibition of T-cell proliferation)

• Hyperproliferative disorder such as psoriasis Other tissues / processes and vitamin D action

• Vitamin D Metabolism in Pregnancy and Lactation • Vitamin D and Reproductive Organs • Vitamin D Receptor as a Sensor for Toxic Bile Acids • Vitamin D and the Renin-Angiotensin System • Vitamin D and Muscle • Vitamin D and Cardiovascular Medicine What can hamper the applicaon of vitamin D in the treatment of these deseases?

Development of hypercalcemia

Development of vitamin D analogs Retain growth inhibitory effects while have no or reduced calcemic activity SUMMARY

• 1,25-(OH)2D3 is biologically most active vitamin D compound • produced via multistep process in various tissues: skin, liver, kidney

• Target tissues also can produce biologically active 1,25-(OH)2D3

• 1α,25-(OH)2D3 is an endocrine regulator but can also act in an auto/ paracrine manner

• Signiﬁcance of target tissue catabolism/inactivation of 1,25-(OH)2D3

• 1,25-(OH)2D3 is the most potent inducer of its own degradation • Transported in serum bound to DBP SUMMARY

• Transported in serum bound to DBP

• Acts intracellular via the VDR, a member of the nuclear receptor family

• VDR is widely distributed and present in almost all tissues

• Directly binds to speciﬁc sequences in the DNA

• Both induction and repression of gene expression

• Central regulator of calcium and phosphate homeostasis

• Non-classical effects on growth and differentiation of a wide variety of cells not primarily involved in calcium homeostasis