Vitamin D and Bone Health

1
Vitamin D Activity in BoneA (not so) innocent
bystander
Paul Anderson
Musculoskeletal Biology Research LabUniversity
of South Australia
2
Sunlight, Vitamin D, and Bone Health

• Rickets or Osteomalacia
• - bone-mineralising disease
• incidence increased as industrialisation
  increased in cities at the higher latitudes
• sunlight or vitamin D supplements is a cure

3
Vitamin D Synthesis and Activity
Vitamin D3
CYP2R1
Liver
25D
INACTIVE
Kidney
CYP24
CYP27B1
24,25(OH)2D3
1,25D
INACTIVE
ACTIVE
Ca homeostasis
Cellular differentiation
Immune system
4
Variation of Serum 25D and Daily Hours of
Sunlight in Adelaide (35ºS), 1982
Morris et al. Med J Aus 1984
5
Serum 1,25D levels and intestinal calcium
absorption in 319 patients with low vitamin D
status levels below 20 nmol/L reduce 1,25D and
calcium absorption
Need et al J Bone Miner Bone Res 2008 23
1859-1863
6
Vitamin D Deficiency in Rats
Severe vitamin D deficiency leads to
osteomalacia Less severe vitamin D depletion
leads to osteoporosis
?
Serum 25D
115 nmol/L
10 nmol/L
Ca
?
?
PTH
?
?
7
In terms of maintaining a healthy skeleton
What level of serum 25D is required to prevent
osteoporosis?
8
Modulation of Vitamin D Status by Diet Bone loss
occurs with 25Ds below 80nmol/L
30
25
20
BV/TV ()

15

10
5
0
20.2
46.2
83
92.8
115.2
Baseline
(
1.6
)
(4.4
)
(
3.0
)
(
8.0
)
(
9.4
)
(
)
)
(
)
(
(
Mean serum 25D, nmol/L (sem)
20.2
46.2
83
115.2
92.8
(1.6)
(4.4)
(3.0)
(9.4)
(8.0)
Mean serum 25D, nmol/L (sem)
Anderson et al, JBMR 2008
9
BV/TV is positively associated with serum
25D (not associated with 1,25D or PTH levels)
Anderson et al, JBMR 2008 23 1789-1797
10
In terms of maintaining a healthy skeleton
is vitamin D required simply to maintain blood
calcium levels or is their also a direct
requirement for vitamin D activity in the bone
cells?
11
RESORPTION
FORMATION
OSTEOCLASTS
OSTEOBLASTS
OSTEOCYTES
MINERALISED BONE
Time/Differentiation
12
Vitamin D Receptor Knock Out Mice
13
Vitamin D Receptor Knock Out Mice (VDRKO)
Wild-type
VDR KO
1 Ca diet
2 Ca diet
- failure of the osteoblast to form bone
14
Mature osteoblast-Specific VDR transgenic mouse
(OSVDR)
OSVDR transgene
Human VDR cDNA
Human osteocalcin promoter
SV40
poly-A tail
WT
OSVDR
0.75
16
25
85



20
80
0.5
14
m/day)

()
µ
Peak Load (N)
15
75
Tibial Cortical Area (nm2)
Oc.S
MAR (
0.25
12
10
70
0
5
10
65
Gardiner, E.M. et al,. Faseb J, 2000. 141908-16.
15
OSVDR animals maintain cortical bone during
vitamin D depletion when WTs do not
2


WT
OSVDR
Cortical bone volume (mm3)
1.6

1.2
0
26
29
150
157
Mean serum 25D levels, nmol/L
Plt0.05 vs WT, D-replete Plt0.01 vs WT
16
OSVDR animals accrue cortical bone during vitamin
D depletion when WT do not
Bone Formation
Bone Resorption

/day)
2
/um
Osteoclast number (/mm)
3
BFR (um
26
29
26
29
150
157
150
157
Mean serum 25D levels, nmol/L
Mean serum 25D levels, nmol/L
WT
OSVDR
17
Mechanical Loading of OSVDR mice increases
Newly Formed Woven Bone
35

30

25
WT
OSVDR
OSVDR
20
Volume (mm2)
WT
WT
15
10

OSVDR
OSVDR
OSVDR
5
ND
ND
WT
WT
0
900
1800
1200
1500
Baldock, Thomas, Forwood, unpublished
Equilibrated Microstrain
18
SUMMARY (1)
Osteoblast are a target for direct activity of
vitamin D via the VDR The presence of VDR is
necessary to regulate bone formation Enhanced VDR
in osteoblasts and/or osteocytes increases bone
formation and markedly increase the level of
mechanically loaded new bone formation
19
RESORPTION
FORMATION
OSTEOCLASTS
OSTEOCLASTS
MINERALISED BONE
Time/Differentiation
20
Skin
UV
Vitamin D3
7-dehydrocholesterol
Liver
25(OH)D3
Kidney
CYP27B1
1,25(OH)2D3
21
CYP27B1 Knock Out Mice (CYP27B1KO)
22
CYP27B1 Activity in Many Tissues
CYP27B1 promoter Luciferase reporter gene
Anderson et al, MCE 2008
23
Vitamin D Synthesis and Bone Mineralisation
CYP27B1 promoter expression associated with
growth region and trabecular structures
Anderson et al, MCE 2008
Anderson et al, BONE 2005
24
siRNA knock-down of CYP27B1 mRNA in HOS
osteoblast abrogates 1,25D synthesis and
Osteocalcin expression
1,25D
CYP27B1
7
300
6
250
5
200
1,25D (pmoll/L)
CYP27B1b-Actin mRNA (10-4)
4
150
3
100
2
50
1
0
0
0
400
0
400
25D (nM)
25D (nM)
control siRNA
CYP27B1 siRNA
Atkins et al, BONE 2007
Anderson et al, JSBMB 2007
25
Osteoblast-Specific CYP27B1 Transgenic Mouse
human CYP27B1 cDNA
human osteocalcin promoter
SV40
poly-A tail
26
OSC mice bones are partially protectedfrom bone
loss as they age
L1 Vertebrae
11 Plt0.05
10 Plt0.05
15 Plt0.01
20
20
18
18
BV/TV ()
BV/TV ()
16
16
14
14
12
12
WT
OSC
WT
OSC
WT
OSC
WT
OSC
7 wks old
20 wks old
7 wks old
20 wks old
Micro-CT analyses of male and female OSC mice at
7 and 20 weeks of age in the L1 lumbar veterbra.
ngt10.
27
No change in osteoclastic recruitment, but a
strong trend towards increased bone formation in
20w OSC mice
TRAP ive osteoclasts per bone perimeter and bone
formation rate in 20 wk old female mice. While
osteoclast surface was unchanged, BFR was highly
correlated with BV/TV in OSC mice (R20.67,
Plt0.01, data not shown).
28
SUMMARY (2)
Osteoblast are a target for direct activity of
vitamin D via the local synthesis of active
1,25D The presence of CYP27B1 is necessary to
regulate bone formation Enhanced CYP27B1 in
osteoblasts and/or osteocytes increases bone
formation.
29
RESORPTION
FORMATION
OSTEOCLASTS
OSTEOCLASTS
MINERALISED BONE
Time/Differentiation
30
Vitamin D Synthesis and Osteoclast
31
Vitamin D Synthesis and Osteoclast
Osteoclast Formation reduced by CYP27B1 knock-down
25D treatment reduced osteoclastic resorption
32
SUMMARY (3)
Osteoclast are a target for direct activity of
vitamin D via VDR and the local synthesis of
active 1,25D The presence of CYP27B1 appears to
play a role in stimulating osteoclast formation
and yet attenuating the resorptive activity of
existing osteoclasts.
33
RESORPTION
FORMATION
OSTEOCLASTS
OSTEOCLASTS
MINERALISED BONE
Time/Differentiation
34
UniSA / SA Pathology Prof Howard Morris Dr
Andrew Turner Rebecca Sawyer Helen
Tsangari Yolandi Starczak PhD Students Dongqing
Yang Jessica Laurence Rahma Triliana Jackson
Ryan Daniel Reinke Alison Leviton
SA Assoc Prof Gerald Atkins, Adel Uni Assoc
Prof Nico Voelcker, UniSA
National Collaborators
Dr Rachel Davey (Uni of Melb) Dr Paul Baldock
(Gavan Institute) Dr Gethin Thomas (UQ) Prof Mark
Forwood (Griffiths Uni)
International Collaborators
Prof Rene St-Arnaud (Montreal) Prof David
Goltzman (Montreal) Prof Shigeaki Kato
(Tokyo) Prof Lynda Bonewald (Kansas)
NHMRC 453310, 565192, 565372, 1003433, 1029926,
1029756
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36
DUAL ROLES FOR VITAMIN D MAY DEPENDENT ON SOURCE
OF 1,25D AND THE LEVELS OF 1,25D
25D
1,25D
25D
1,25D
1,25D
VDR
VDR
1
1
VDRE
VDRE
ANABOLIC
CATABOLIC ANTI-ANABOLIC