BONE STRUCTURE
and FUNCTION and
BONE REMODELING
Dolores Shoback, MD
Professor of Medicine, UCSF
San Francisco VA Medical Center
July 15, 2010
”BONE BASICS”
• BONE CELLS - function
• Bone remodeling
– Resorption - RANK-L, RANK, OPG
– Formation - Wnt/LRP5
– Regulators of Wnt signaling
“Imbalances” in bone remodeling
& formation - osteoporosis targets for therapy
Disclosures: Speaker - Novartis; consultant - Amgen
BONE CELLS
OSTEOBLASTS
• “Bone-forming” cells
– Synthesize matrix proteins:
• OSTEOBLASTS
• OSTEOCYTES
• OSTEOCLASTS
• Type 1 collagen, alkaline phosphatase, osteocalcin,
osteopontin, fibronectin, collagenase…
– Mineralize bone “osteoid” (matrix)
– Receptors for large # of hormones & cytokines
(eg, PTH, vitamin D, BMPs, TGF etc.)
– Express receptors for Wnt & proteins downstream
in signaling pathway
“Executive cells”
• Direct bone remodeling
• Express RANK-ligand on cell - surface
1
OSTEOBLASTS
LIFE CYCLE of OSTEOBLAST
*NO
commitment
turning
back
Lian JB and Stein GS, Osteoporosis, 2001
OSTEOCYTES
• Comprise > 90% of cells in bone
• Least understood bone cell -- “buried” in
bone -- difficult to isolate/study
– “Mechano-sensing”
– Communicate physical forces to & from
bone surface to bone interior
• Characteristics -• Long projections from cell body through
matrix to surface osteoblasts &
osteocytes
• Encased in lacunae
OSTEOCYTES
• Synthesize some matrix proteins & can be
involved in responses to hormones - PTH
• MAIN source for SCLEROSTIN
– Important protein in Wnt pathway
– Negative regulator of osteoblast activity
– New “target” being explored to treat osteoporosis
2
OSTEOCYTE
OSTEOCYTES - Bone
Mechanical forces, strain, loading
OSTEOCYTES - Bone Section
How Osteocytes Mediate Mechanotransduction
Bonewald and Johnson, Bone 2008
3
Osteoclast Development
BONE CELLS
• OSTEOCLASTS
– Multinucleated cells
– Derived from monocyte - macrophage
lineage (BONE MARROW)
monocyte
RANK-
NFAT
Promonocyte
RANK+
Horowitz et al. Immunol
Rev 2005; 208: 141
OSTEOCLASTS
NF-κ
κB, c-Fos
Pu.1
M-CSF
Multipotent
progenitor
macrophage
RANKL
mononuclear
osteoclast
precursor
RANK = receptor
activator for NF
kappa B
mature
osteoclast
M-CSF = macrophage
colony stimulating
factor
OSTEOCLASTS (blue arrows)
• Primary job - bone resorption
• Highly specialized cytoskeletal structures – “Ruffled border”
– “Sealing zone”
– Attach to & dissolve bone matrix
• Produce TRAP, lysosomal enzymes,
cathepsin K, integrins
• Express calcitonin receptors & RANK
MAJOR drug target - osteoporosis
4
Bone Remodeling Cycle: Normal Bone
OCs digest bone
within a sealed
resorption vacuole
Resorption
Resting
Reversal
Apoptotic
OCs
Critical check-point in control
of bone remodeling
– Regulation of osteoclast
formation, number, activity &
lifespan
Preosteoblasts
RANK--L, RANK, OPG system
RANK
Mature osteoblasts
building osteoid
tissue
Formation
Mineralization
Mundy. Calcium Homeostasis: Hypercalcemia and Hypocalcemia. 1989.
Factors Stimulate OB Expression of RANK-L:
RANK+RANK-L Mediate OC Survival & Fct
CFU-M
Glucocorticoids
PTH
RANKL
Pre-fusion Osteoclast
PGE2
Osteoprotegerin (OPG) Prevents RANK-L/RANK
Binding & Inhibits OC Activity
CFU-M
RANK
Pre-fusion
Osteoclast
RANKL
RANK
Vitamin D
IL-11
IL-6
+mCSF
IL-1
PTHrP
TNF-α
α
Activated
Osteoclast
Osteoblasts &
BM Stromal Cells
CFU-M = colony-forming unit-macrophage
Boyle WJ et al. Nature 2003;423:337; Hofbauer LC,
Schoppet M. JAMA 2004;292:490
.
X
Multinucleated
Osteoclast
Bone
Resorption
OPG
Multinucleated
Osteoclast
Hormones
Growth
Factors
Cytokines
X
Activated
Osteoclast
Osteoblasts
Bone Formation
Boyle WJ et al. Nature 2003;423:337
X
Bone Resorption
5
Why ? - BONE REMODELING
MAb (Denosumab) Binds RANK-L
RANKL
Pre-fusion
Osteoclast
CFU-M
Allows skeleton to --
RANK
OPG
MAb to RANKRANKligand
Hormones
Growth Factors
Cytokines
Osteoclast Formation,
Function,
& Survival Inhibited
• Respond to mechanical loading
• Repair microdamage (“wear and tear”) & prevent
accumulation
• Maintains “quality control”
• Release minerals (Ca and phosphorus) & growth
factors stored in matrix into circulation
Osteoblasts
Bone Formation
Resorption Inhibited
All bone cells participate in
remodeling
~Pathway dead~
Wnt - LRP5 Pathway
Dkk
WIF
sFRP
Wnt
Sclerostin
Unliganded
state
Liganded
state
Frizzled
Frizzled
axin
Anabolic Pathway to Bone
Formation
APC
axin
Multiple Targets
Dsh
Frat-1
APC
GSK3
P
β -Catenin
GSK3
LRP
(No
new
nucleus
bone
β -Catenin
made)
TCF/LEF
OS TE OB LAS T
β -Catenin
β -Catenin
β -Catenin
nuclear
localization
SMRT/
NCoR
p300/CBP
altered
transcription
of genes
BONE
FORMATION
6
~Pathway dead~
Dkk
WIF
sFRP
Wnt
Sclerostin
Unliganded
state
Liganded
state
Frizzled
Frizzled
axin
axin
APC
GSK3
P
β -Catenin
GSK3
LRP
β -Catenin
Proteosomal
degradation
(No
new
nucleus
bone
β -Catenin
made)
TCF/LEF
OS TE OB LAS T
β -Catenin
β -Catenin
nuclear
localization
SMRT/
NCoR
p300/CBP
altered
transcription
of genes
• Genetic disorders & mouse models -– Loss of function mutations in LRP5*
(severe osteoporosis & fractures)
– Gain of function mutations in LRP5
(high bone mass “trait”)
Dsh
Frat-1
APC
Wnt/LRP 5 Signaling - Bone
Formation
BONE
FORMATION
Wnt/LRP 5 Pathway - Bone
Formation
• Wnt/LRP5 pathway is essential – OB* proliferation, differentiation, & survival
– Activity (work done) during a phase of
bone formation
LRP5* = LDL receptor related protein 5
Sclerostin Secreted by Osteocytes
Negatively Regulates Bone Formation
Sclerostin*
Natural inhibitors - working locally
– Soluble decoy receptors, proteins - WIF,
sFRP
– SOST gene product - sclerostin
– Dkk (dickkopf)
Mature
Osteoblasts
X
Pre-osteoblast
lining cells
Mesenchymal
stem cells
X
New bone
Osteocyte
Ott SM. JCEM 2005; 90: 6741-6743
Semenov MV, et al. JBC 2006; 281: 38276
Bone
Semenov M, et al. JBC 2005; 280: 26770
Li X, et al. JBC 2005; 280: 19883
7
Sclerostin Binds LRP5 on
Osteoblasts & Inhibits Wnt Signaling
• Human genetic disorders
Less bone formation
FZD
LRP5
Wnt
Sclerostin: Key Mediator of Bone
Formation ~ Evidence
Scl
• Mouse models
• Clinical studies
Scl = sclerostin
FZD = frizzled receptor
LRP5 = LDL receptor-related protein 5
SCLEROSTEOSIS - both SOST genes
are mutated (AR)
• Loss of function mutation
BMD’s of Homozygotes with
Sclerosteosis & Heterozygous
Family Members
A: Child with sclerosteosis
B: Adult with sclerosteosis
C: Heterozygous carrier of mutation
• BMD -in pts w/sclerosteosis: Z scores
+7.7 to +14.4
• Heterozygotes:
• Sclerosteosis -• Tall stature
• Nerve root & cranial nerve
entrapment, foramen magnum
compression (sudden death), large
jaw
Z scores from + 0.1 to + 5.2
• NO bony complications with 1 defective Sost
gene
• Goal - therapies to titrate down levels of
sclerostin - but NOT to ZERO
Gardner JC et al, JCEM, 2005
Gardner JC et al, JCEM, 2005
8
Preclinical Studies: Sclerostin - 1
Preclinical Studies: Sclerostin - 2
• Transgenic overexpression - SOST -- low bone
mass (Loots et al, Gen Res, 2005)
•
Sclerostin MAb treatment of OVX rat
(@ 6 mos) (Li et al, JBMR, 2009)
– Complete reversal of one-year of
estrogen deficiency-induced bone loss
– Bone mass (& strength) increased to
levels above non-OVX controls
– Reversible
•
Knockout mouse (SOST -/-) (Li et al, JBMR, 2008)
– >50% increase in BMD (LS, femur)
– Micro-CT: increased BV/TV in trab & cortical
bone
– Histomorph: >9-fold increase in OB surface
(formation), NO change in OC’s
(uncoupling)
– Mech testing -- strength signif increased
Lumbar spine BMD
Sclerostin & PTH - 1
OVX + Sclerostin Ab
Control
OVX X 1 year
Femur-tibia BMD
•
PTH administration (anabolic doses) -– REDUCES sclerostin levels (via PTH1-R & cyclic AMP pathway)
– DECREASES # osteocytes staining
for sclerostin
Control
Li et al, JBMR, 2009
Bellido et al, Endo, 2005; Keller and Kneissel, Bone, 2005; Kremer et al, TEM, 2010
9
Sclerostin & PTH - 2
Sclerostin - Human Studies
PTH administered intermittently to Sost
overexpressing (Tg) and Sost-deficient (KO)
mice - – WT: PTH induced substantial gains in BMD,
BMC, cort thickness, increased BFR
• Serum sclerostin levels
Postmenopausal women 1.16 +/- 0.38 ng/ml
Premenopausal women 0.48 +/- 0.15 ng/ml**
(**p < 0.001)
– PTH-induced gains - BLUNTED - in Sost
overexpressing & Sost deficient mice at all
doses & all sites
• Negative correlations between sclerostin and
free estrogen indices in postmenopausal women
(p< 0.002)
Sclerostin (osteocytes) - critical role in
PTH anabolism
Kremer et al, JBMR, 2010
Sclerostin MAb in Humans
Mizra et al, JCEM, 2010
Sclerostin MAb in Humans
• Randomized, DB, PC, phase I
trial, ~85 days
• SC & IV single doses of antisclerostin Ab to PM women &
men (N=72)
• BTM, BMD, safety
• Responses to SC doses
Uncoupling
Padhi et al, JBMR, 2010
BMD responses to SC single doses of anti-Scl
MAb over time (LS + 5.3%, hip + 2.8%) (~ 6
Padhi
et al, et
JBMR,
2010 2010
mos TPTD)
Padhi
al, JBMR,
10
DKK - Natural Inhibitor of Wnt Pathway
Dkk Functions - Bone - 1
Dkk required for head development & limb
formation (KO mice)
•
• LRP5 gain of function mutations (causing high
BMD) - work by disrupting (inhibitory) interaction
between Dkk and LRP5 (+++Wnt signaling)
• Dkk1 overexpression in osteoblasts - osteopenia
• Dkk1 increases RANK-L & decreases OPG
production (by Ob’s)
Pinzone et al,Blood, 2009
Summary & Conclusions
Dkk Functions - Bone - 2
•
•
Dkk1 inhibits fracture repair
• Dkk1 in OBs - UPREGULATED - in glucocorticoidinduced osteoporosis & E2 deficiency
• Osteolytic lesions in myeloma, breast & prostate
cancers -- Dkk1 implicated
– Blood Dkk1 levels are higher in breast ca pts with bone
mets (vs breast ca w/out mets or w/non-bone mets)
– Myeloma cells release Dkk1
– Serum & marrow levels of Dkk1 are high in myeloma
– Blocks key steps in OB differentiation - in myeloma
BONE
–
–
–
–
–
Diverse cells with specialized functions
Multiple pathways/ongoing cycles
RANK-L/RANK/OPG
Wnt/LRP5
Regulatory molecules - control bone formation help treat osteoporosis & bone metastases
Understanding basic mechanisms of
bone biology & remodeling – give better
understanding of disease & treatment
strategies
Dkk1 involved in bad things in bone !
11
Micro-CT Images
(rat)
HOW DOES BONE
REMODELING via RANKLigand and Wnt/LRP5
PATHWAY
HELP to UNDERSTAND
OSTEOPOROSIS?
sham
Ovariectomy
Li et al, JBMR, 2009
OVX + Sclerostin Ab
…& mechanically strong
*
12
OSTEOPOROSIS
Imbalance in RANKRANK-L/OPG & Anabolic Signals
via LRP5/Wnt -- Bone Loss
• Reduction in bone mass
• Disruption of bone micro-architecture
CHANGES in BIOMECHANICAL
STRENGTH →→→ FRACTURES
• Osteoporosis ~ “IMBALANCE” in bone
remodeling cycle
anabolic
signals
OPG
OSTEOBLAST
RANKL
Medications/
interventions
- shift the
remodeling
imbalance in
favorable
direction
OSTEOCLAST
Hofbauer and Schoppet. JAMA. 2004;292:490.
Many Factors Regulate
Bone Remodeling
Wnt, LRP5, Sclerostin Pathway
Resorption
Formation
GM-CSF
IL-1
IL-6
RANKL
PGE2
TNF-α
α
Reversal
Resting
Resorption
Formation
OPG
TGF-β
β
Estrogen
Mundy. Calcium Homeostasis: Hypercalcemia and Hypocalcemia. 1989.
Osteoblastic
gene
expression
Krishnan et al, JCI, 2006
13
Wnt/LRP 5/Sclerostin Pathway Definitions
•
•
•
•
•
•
•
•
•
LRP5 = LDL receptor related protein 5
Wnt = wingless
Frizzled = 7 TM G-protein coupled receptor
APC = adenomatous polyposis coli
SOST = sclerostin
DKK = Dickkopf
GSK 3 = glycogen synthetase kinase 3
Dsh = Dishevelled
Beta catenin = transcription factor
• When sclerostin (Sost gene) is
mutated (AR) -- SCLEROSTEOSIS
A: Child with sclerosteosis
B: Adult with sclerosteosis
C: Heterozyg carrier of mutation
• BMD -in pts w/sclerosteosis at all
sites: Z scores ranging from +7.7 to
+14.4 !!!!
• Heterozygotes:
from +0.1 to +5.2
Z scores range
• Heterozygotes have no other bony
complications of having 1 defective Sost
gene
• Therapies that titrate levels of
sclerostin - with MAb’s
Gardner JC et al, JCEM, 2005
Unopposed RANK-L/RANK Activity&
Inadequate Bone Formation:
Senile & Steroid-induced Osteoporosis
• Processes that produce unfavorably alter ratio between
formation & resorption
Molecules
- formation
OPG
RANKL
Medications/
interventions
- shift the
remodeling
imbalance in
favorable
direction
RANK--ligand Pathway - Clinical
RANK
MAbs block RANK-L & RANK interaction
-- suppress xs bone remodeling &
resorption & OC formation
Exploited as therapeutic strategy to treat
bone loss - cancer, arthritis, menopause
Osteoclastic Activity
Hofbauer and Schoppet. JAMA. 2004;292:490.
14
2. RANK-Ligand Expression - Mediates
OC Formation, Function, & Survival
CFU-M
Pre-fusion
Osteoclast
Sclerostin Binds LRP5 on
Osteoblasts & Inhibits Wnt Signaling
RANKL
RANK
Multinucleated
Osteoclast
Wnt signaling in bone
Inhibition of Wnt signaling by Scl
More bone formation
Less bone formation
LRP5
FZD
Hormones
Growth
Factors
Cytokines
Wnt
Scl
Activated
Osteoclast
Osteoblasts
Scl = sclerostin
FZD = frizzled receptor
LRP5 = LDL receptor-related protein 5
Bone Formation
Boyle WJ et al. Nature 2003;423:337
LRP5
FZD
Wnt
Bone Resorption
Ellies D, et al. JBMR 2006;21:1738
Nusse R. Nature 2001;411:255
Semenov M, He X. JBC 2006;281:38276
Final steps - OSTEOCLAST Formation
Multinucleated cell
PTH
Sost
Li X, et al. JBC 2005;280:19883
Semenov M, et al. JBC 2005;280:26770
Kinetics of Bone Remodeling
•
•
•
•
Lifespan of the BMU*
Osteoclast lifespan
Active lifespan - osteoblast
Rate of turnover of skeleton
“Sealing zone”
≈ 6-9 months
≈ 2 weeks
≈ 3 months
≈ 10%/year
- Treatment & Monitoring - considerations
“Ruffled border”
Dissolved matrix
“Resorption pit”
Ross FP and Teitelbaum SL, Osteoporosis, 2001
* BMU = basic multicellular unit
Parfitt et al, J Cell Biochem, 1994
15