osteoblasts - - ectsoc
TRANSCRIPT
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OSTEOBLASTSPierre J Marie, Ph.D.
Inserm Unit 606 & University Paris 7,Hopital Lariboisière, Paris, France
Osteoblasts
Bone marrow
Calcifiedbone
Unmineralizedmatrix
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Topics to be discussed
1. Bone formation
2. Methods used for analysis of osteoblasts
3. Main steps involved in osteoblastogenesis- Commitment- Differentiation- Function- Apoptosis
4. Main factors regulating osteoblastogenesis
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Bone
MesenchymalCells
Proliferation Zone(chondrocytes)
Calcification
Reserve Zone (chondroblasts)
Hypertrophic Zone
Osteoblasts
Membranous Bones (Cranial Vault)Long Bones (Humerus)
Bone Formation during Growth
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Lining cells
Osteoid
New bone
Osteocytes
Pre-osteoblasts
Osteoblasts
Inversionphase
Monocytes
Pre-osteoclasts
Osteoclast
Resorption
BoneBone RemodelingRemodeling in in AdultsAdultsFunctionFunction ofof osteoblastsosteoblasts: : rebuildrebuild thethe resorbedresorbed bonebone
Formation
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Topics to be discussed
1. Bone formation
2. Methods used for analysis of osteoblasts
3. Main steps involved in osteoblastogenesis- Commitment- Differentiation- Function- Apoptosis
4. Main factors regulating osteoblastogenesis
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Organ CultureAllows analysis of bone formation
(cranial & long bone)
Osteoblast proliferationMatrix formationLong bone
Cranial bone
Organ Culture
Prelabeling with3H-Thymidine/Proline
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Primary Osteoblast CulturesIsolated from cranial & long bone, or
bone marrow stroma
Osteoblast Cultures
Osteoblast proliferation, differentiation & apoptosis
Long bone
Cranial bone
Digestion/Migration
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Ex-vivo Cultures to Study Bone Formation
Allows comparison of theosteoblast phenotype(proliferation, differentiation, apoptosis) in vitro and in vivo
- in osteopenic rats or mice
- or in relation with a givendisease in humans(osteoporosis, …)
Marie PJ, JBMR, 1994
Bone Tissue:Histomorphometry
CulturedOsteoblasts
Bone Marrow
Bone Matrix
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STATINS
FGF1
BMP
CONTROL
Mundy et al., Science, 1999
In vivo Model: Local Injections
Ex: Statins Increase Bone Formation in Mice
Histology,Cell cultures
STATINS
CONTROL
STATINS
CONTROL
Cranial bone
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Principle: Use mouse genetics to determine the role of specific
genes in bone formation in relation to bone loss or bone gain
Methods:
- Inactivation of target gene in all tissues
- Tissue-specific deletion of a target gene in a tissue- or/and time-specific manner
- Overexpression of a target gene universally or in a tissue-specific manner
- Replace an endogenous gene with a modified variant (knock-inmodel)
Data :- Analyse the bone phenotype at all levels : DXA, µCT, histology, ex-vivo cultures…
Genetic models in mice
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Exemples of Gene Manipulation in Osteoblasts
- Transcription factors (Runx2, Osterix, PPARγ)
- Morphogens & regulators (BMP, noggin, gremlin, sHH…)
- Matrix bone proteins (biglycan, decorin, BSP, osteopontin…)
- Growth factors & receptors (IGF, IGF-BP, TGFβ, PTHrP-R, FGF)
- Mediators (LRP5, DKK1, Wnt…)
…More interesting models are those relevant to human pathology
Marie, Am J PharmacoGenomics, 2001
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Topics to be discussed
1. Bone formation
2. Methods used for analysis of osteoblasts
3. Main steps involved in osteoblastogenesis- Commitment- Differentiation- Function- Apoptosis
4. Main factors regulating osteoblastogenesis
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Osteoblasts : Origin and Plasticity
Pluripotent cell (Mesenchyme)
Chondrocyte Adipocyte
Fibroblast
Osteoblasts
Osteoprogenitor
?
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Main Transcription FactorsInvolved in Postnatal Osteogenesis in vivo
Runx2 Osx
Runx2, ATF4AP-1 Family
Twist, Msx-2
Runx2
ALP, COLI…
COLI, BSP, ON, OP…
COLI, BSP, ON, OC…Apoptosis
Osteoblasts
Progenitor Cells
Pre-osteoblasts
Osteo-ChondroprogenitorCell
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Runx2: A Transcription FactorRequired for Osteogenesis
- Runx2: Runt family(DNA binding domain)
- Expressed in mesoderm and in osteoblastprecursor cells
- Invalidation in mice: no bone !
- Mutations (mice, humans): no binding to DNA: skeletal abnormalities (cleidocranialdysplasia)
DucyDucy et al., et al., CellCell, 1997, 1997KomoriKomori, , CellCell 19971997Komori, J Cell Biochem, 2002
Karsenty & Wagner, Dev Cell, 2002Lian & Stein, Curr Pharm Des, 2003
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Ducy et al , Genes & Dev, 1999
Runx2 is Also Involved in Postnatal Osteogenesis
3 weeks mice
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Ducy and Karsenty, 1997; Komori, 1997
Runx2 Controls Osteoblast Differentiation
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Pratap et al., Cancer Res. 2003Galindo et al., JBC 2005
Runx2 Controls Osteoblast Proliferation
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Runx2 is Highly Regulated by Multiple Proteins
Hoxa-2 (homeobox)Dlx3 (homeobox)Aj18 (ZNF protein, competes for DNA binding))Myeloid Elf-1 factor (MEF)Stat1Sox8, Sox9Nrf2
Msx2 (homeobox)Bapx1(homeobox)Dlx5 (homeobox)Rb (SATB2 (nuclear matrix protein)Hoxa-10 (homeobox)Taz (transcriptional activator)
Runx2Expression or function
Smurf1Schnurri-3Cyclin D1
CHIP(promotes proteasome degradation)
Marie, ABB, 2008
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Nakashima et al., Cell, 2002
E15.5 Newborn
Defective mineralization
Osterix (Osx) Controls Bone Formation
NFAT Nuclear factor of activated T cells)
Interacts with Osx
Koga et al., Nat Med, 2005Wang et al., J Cell Biol, 2006
Osx
p53(tumor suppressor)
Osx Regulation
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Osx Acts Downstream of Runx2
Col 1
Col 1BSPOsteonectin
Col 1BSPOsteonectinOsteocalcin
Pre-Osteoblast
Osteoblast
Ob progenitor
Pre-hypertrophicChondrocyte
Col 2Ihh
Col X
Runx2
Runx2Osx
Runx2Osx
Osteo-chondroprogenitor
Sox 5,6,9
HypertrophicChondrocyte
Nakashima et al., Cell, 2002
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PPARγ ControlsOsteoblast Plasticity
Osteoblast
Adipocyte
MesenchymalStem Cell PPARγ2
RUNX2
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Ex: Invalidation of PPARγ2 in Mice: Increased bone formation with age
PPARγ2
MesenchymalCell
Runx2
Akune et al, J Clin Invest 2004
IncreasedBone formation
DecreasedAdipogenesis
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Anabolic agents promote osteoblastogenesis and inhibitadipogenesis in the bone marrow stroma
Control of Osteoblast Plasticity
Osteoblast
Adipocyte
E2, Shh, leptin, BMP-2, TGFß+
-
Estrogens (Okasaki et coll. Endocrinology 2002)Sonic hedhehog (Spinella-Jaegle et al. J Cell Science 2001)Leptin (Thomas et coll. Endocrinology 1999) BMP-2 (Gimble et coll. JCB 1995)TGFß (Ahdjoudj et coll. JBMR 2002)
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• AP-1: c-fos, FosB, Fra-1, Fra-2, c-Jun, JunB,JunD
• Heterodimers Fos/Jun bind consensus sequences(TRE or CRE) in several target genes
• c-Fos: controls Runx2 in osteoblasts
• Fra2/JunD: activates Runx2 transcription
The AP-1 Family
Wagner, Ann rheum Dis, 2002
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• DeltaFosB (splice variant) overexpression : increasedbone formation, decreased adipogenesis, osteocondensation
• Fra1 overexpression: increased bone formation, osteocondensation
• Fra2 overexpression: increased bone formation, ostéocondensation
• c-Fos overexpression : Osteosarcoma (mice, humans), Paget’s disease, fibrous dysplasia (McCune Albright)
• Mice lacking Jun B show defective bone formation andbone loss
The AP-1 Family is Important for Bone Formation
Wagner, Ann rheum Dis, 2002
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•• bHLHbHLH formsforms homodimershomodimers or or heterodimersheterodimers withwith otherothermoleculesmolecules bHLHbHLH (E12/E47)(E12/E47)
•• Basic Basic domaindomain bindsbinds a a sequencesequence CANNTG CANNTG (E box) in (E box) in promoterpromoter ofof targettarget genesgenes
• Twist mutations induce degraded/truncated Twist protein and craniosynostosis
El Ghouzzi et al 1997Bourgeois et al 1998
Twist: A Transcription FactorControlling Osteoblasts
NH2 COOHDNA
BindingHelix I Loop Helix II
bHLH
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Mesenchymalcells
OsteoprogenitorsOsteoblasts
Proliferation Differentiation
Twist interacts with Runx2 during skeletal development in mice
Bialek et al., Dev Cell 2004
Twist +Runx2 -OC -
Twist +/-Runx2 +OC +/-
Twist -Runx2 +OC +
Runx2Bindingto OSE2
OC
Twist(Twist Box)
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Differentiation
ALPCol1A1
Runx2OPBSPOCFGFR2
Twist Regulates Runx2 during Postnatal Osteoblast Differentiation (Human)
Twist haploinsufficiency
(bHLH domain)
Yousfi et al., J Clin Invest, 2001Yousfi et al., BBRC, 2002Guenou et al., Hum Mol Gen, 2005
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-ATF4 accumulation (low degradation) activates OSE1 in theosteocalcin promoter and induces osteocalcin expression
- Enhances collagen synthesis postranscriptionally
ATF4 regulates osteoblasts
Yang et al., Cell, 2004; JBC, 2004
•Coffin-Lowry syndrome: mutation in RSK2 (growthfactor related kinase): inactivation of RSK2 and boneloss
RSK2 phosphorylation
ATF4 (cAMP-response element-binding protein)
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Krishnan et al. J. Clin. Invest. 2006
Wnt/β-catenin signaling regulates osteogenesis
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Topics to be discussed
1. Bone formation
2. Methods used for analysis of osteoblasts
3. Main steps involved in osteoblastogenesis- Commitment- Differentiation- Function- Apoptosis
4. Main factors regulating osteoblastogenesis
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Function: Bone Formation and Mineralization
• Extracellular matrixType 1 collagen
Matrix proteins: OsteopontinBone sialoproteinOsteonectinThrombospondinPhosphoproteinsProteoglycans
• MineralCalcium, PhosphateImmature calcium-phosphate crystalsHydroxyapatite (mature)
Osteoblasts synthesize and export type I collagen fibers
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Osteoblasts contribute to bone matrixmineralization
Young, OI 2003
Collagen fibers Mineral associated with collagen
Role of calcium and phosphate:
Calcium or phosphate deficiency: osteomalacia
Role of alkaline phosphatase:
Cleaves orthophosphateHypophosphatasia: osteomalacia
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Topics to be discussed
1. Bone formation
2. Methods used for analysis of osteoblasts
3. Main steps involved in osteoblastogenesis- Commitment- Differentiation- Function- Apoptosis
4. Main factors regulating osteoblastogenesis
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Osteoblast Apoptosis
Dying OsteoblastDyingOsteoblast
x 250 x 1000
5-10 % osteoblasts and osteocytes die by apoptosis
Tunel staining, rat bone
Dying Osteoblast
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Importance of Osteoblast Apoptosis in the Control ofBone Formation
• End of matrix formation: Osteoblast detachment resultsin cell death
• Increased osteoblast apoptosis in glucocortioid-inducedosteoporosis and immobilization
• Mechanical strain reduces osteoblast survival
Jilka, Bone 1998
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Control rat
Unloaded rat
Reduced mechanical strain increasesosteoblast apoptosis in vivo
Integrinα5β1
Strain
APOPTOSIS
PI3Kp85 p110
SURVIVAL
BaxBcl-2
Bcl-xLBad
Bad-P
Akt
Dufour et al., 2008
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Regulation of osteoblast apoptosis
Apoptosis Survival
TNFalpha, IL1BMP-2Glucocorticoids
-+
PTHEstrogens
Wnt/β-cateninFGF
IGF, TGFβStrain
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Mesenchymal Osteo- Immature Mature cell progenitor cell osteoblast osteoblast
Runx2 Runx2 Runx2
OsxATF4
Fra1∆FosBc-FosJunD
Twist Msx2β-Catenin
ALP, Collagen type I, osteopontin
Marie PJ. Arch Biochem Biophys, 2008
Cell death
Osteocyte
BSP DNAOsteocalcin Fragmentation
Summary: Crucial steps in bone formation
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Topics to be discussed
1. Bone formation
2. Methods used for analysis of osteoblasts
3. Main steps involved in osteoblastogenesis- Commitment- Differentiation- Function- Apoptosis
4. Main factors regulating osteoblastogenesis
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Hormonal Regulation
Hormones(PTH, GC, Estrogens,
GH, Vit.D…) Bone Growth
Bone Formation
Bone Mass
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Main Hormones Regulating Bone Formation
Osteoblast Number Osteoblast Activity
1,25 (OH)2 Vitamin D −
Estrogens
Growth Hormone
Glucocorticoids
Parathyroid hormone (PTH)
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Continuous PTH
↑ RANKL↓ OPG
↑ Osteoclastogenesis
↑ Bone Resorption
Bone Loss
PTH: A Bipotent Molecule Active on Bone Cells
Lanske et coll. JCI 1999
Intermittent PTH
↓Osteoblast/osteocyteapoptosis
↑Number of osteoblasts
↑ Trabecular Bone Formation
↑ Bone Gain
↑ Pre-osteoblast
number
↓ BMC adipogenesis
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cAMP
PLCPKC
PTH Receptor
PTH Signaling in OsteoblastsPTH
ERK1/2
Partidge, Gene 2002; Rey et al., Bone 2007; JBC 2006; Yamamoto et al., Bone 2007; Yang et al., Bone 2006; Ferrari et al., Endo. 2005; Chen et al., JBC 2004
PKA
Osteoblast Proliferation Differentiation Survival
Src
PI3K/Akt
β−Catenin
Adenylyl cyclase
Gi Gs Gq/G11
β-arrestin2
ERK1/2 β−Cateninp38
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Target Genes induced by PTH Signaling in Osteoblasts
Jilka, Bone 2007
c-foscyclin D1
Osteoblastproliferation
Runx2Runx2-PRunx2 degradationOsterix
Decreased PPARγ
Osteoblastcommitment
IGF-IFGF2TGFβSclerostin
Osteoblastfunction
Bad-Pβ-catenin
Osteoblastsurvival
PTH Signaling+
+ ++
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Estrogens and Bone Formation
- Important role in the acquisition of peakbone mass during skeletal growth
- Act on osteoblasts through genomic andnongenomic effects
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Estrogen Receptor Pathways
E2
Osteoblastsurvival
ERK1/2Smad
PI3K
Akt
Aquirre JBC 2007; Kousteni MCB 2007
Membrane ER
Osteoblastdifferentiation
Wnt
Genomic (simplified) Non-Genomic
ERα
ERβ
E2
E2
ERE
Src
CoAct
Genes(ALP, COL1A1,
Cytokines…)
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Estrogens Promote Bone Formation ThroughGrowth Factor Expression
IGF-ITGFß
COLIA1
E+
+
-
Cell proliferation
Collagen synthesis
Apoptosis
E
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OPG
Differentiation
RANKLRANKLs
Estrogens Control RANKL/OPG Expression by Osteoblasts
RANK
OsteoclastPrecursor
Osteoblast / Stromal Cells
Mature Ostoclast
Bone
17 β-Estradiol
-
+ 17 β-Estradiol
Bone
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Growth factors
Local regulation of Osteogenesis
Bone Growth
Bone Formation
Bone Mass
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• Soluble factors, produced locally by osteoblasts
• Some are included in the bone matrix
• Act locally on bone cells
• Are regulated by themselves, hormones and mechanicalforces
• Regulate cell proliferation, function and survival
Growth Factors in Bone
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Main growth factorsregulating bone formation
Number ofosteoblasts
Osteoblastactivity
PDGF
EGF
IGF
FGF
TGFβ
BMP
-
-
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Bone Morphogenetic Proteins (BMPs)
- Members of TGF-β superfamily
- Extracted initially from demineralized bone matrix(Urist, 1965)
- Induce cartilage and bone formation in nonskeletal sites
- BMP-2 , 3, 7 and 8 are osteogenic
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BMP Signaling is Important in Post-Natal BoneFormation
Invalidation of BMP-RIB: Decreased bone formation and bone loss
Zhao et al., JCB 2002
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Canonical Pathway of Bone Morphogenetic Proteins
Cytoplasm
BMPs
PP
BMPR-IIBMPR-I
Smad-1/5 Smad-1/5
Smad-4
Smad-6
Smad-7
+
P
Smad-1/5Smad-4
P
Runx2
Nucleus
Osx
Bone Formation
Ten Dijke, et al., 2003
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IIBMP-2
P
I PKC
Bax/Bcl-2
Caspases
Apoptosis
MitochondriaP
ERK1/2
Runx2Osx, Msx2, Dlx5
Osteoblast Genes
Guicheux et al., JBMR 2003 Lemonnier et al., JBC 2004
Rawadi et al., JBMR 2003Hay et al. JBC 2001
p38 MAPK
Wnts
Wnt signaling
JNK
PKD
BMP Non-Canonical Pathways
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Cytoplasm
BMPs
PP
BMPR-IIBMPR-I
Smad-1/5
Smad-4
P No Transcription
Noggin, Gremlin, …
Nucleus
+
Canalis, End Rev, 2003Chen et al., Growth factors, 2004
Negative Regulation of BMP Signaling by Antagonists
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Transforming Growth factor beta (TGF-ß): a positive regulator of bone formtion
• Expressed by osteoblasts
• Promotes osteoblast proliferation
• Promotes osteoblast function: - collagen synthesis- proteoglycans
• Increases osteoblast survival
• Promotes osteogenesis in vivo
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Insulin Growth Factors I & II (IGF-I, IGF-II)
- Expressed by osteoblasts
- Binds to IGF binding proteins that regulate itsbinding to receptors
- Promotes osteoblast proliferation-Promotes osteoblast function:
-collagen synthesis-proteoglycans
- Inhibits osteoblast apoptosis- Promotes osteogenesis in vivo
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Zhang, M. et al. J. Biol. Chem. 2002
Disruption of the Igf1 receptor reduces bone formation rate
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Fibroblast Growth Factors (FGFs)
-FGF-2,-9,-18:Expressed by osteoblasts
Complex effects:
- FGF2 promotes osteoblast proliferation
- Modulates osteoblast apoptosis
- Promotes osteogenesis in vivo
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Montero et al., J Clin Invest, 2000FGF2-/-
FGF2+/+8 months mice: bone loss
FGF2 -/-FGF2 +/+
FGF2 Invalidation Reduces Bone Formation
Defective bone formation
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Mesenchyme
Pre-Osteoblasts
Bone Bone
Mesenchyme
Pre-Osteoblasts
Activation of FGF Receptor-2 Increases Bone Formation
Normal Suture Apert Suture (Activating FGFR2 Mutation)
Lomri & Marie, J Clin Invest, 1998; Ornitz & Marie; Genes and Dev, 2002
Human Coronal Suture
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PPARγC/EBPα
Immature Matureprogenitor osteoblast
GC
WntsPTH; E2LoadingGC
β-Catenin/LEF-1
Adipocyte
Mesenchymalcell
Osx
PTHBMP2, IGF1
PTHBMP2TGFβLoading
PTH, 1,25vit.DGHBMP2; TGFβ, WntsFGF2/FGFR2 PGE2; IhhLoading
Runx2
Cellsurvival
Celldeath
Summary: Hormonal and LocalRegulation of Osteoblastogenesis
Marie PJ. Arch Biochem Biophys, 2008
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Hypothalamus(leptin, …)
Bone Formation
Bone mass
Central Regulation of Osteogenesis
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• Produced by adipocytes • ob/ob mice (no leptin): increased bone mass
Leptin and Bone
Ducy et coll., 2000
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•Leptin acts through its receptor (Ob-R)
•Ob-Rb (long form) : physiological form, present in hypothalamus.
•KO Leptin or KO leptin receptor: high bone mass
•Leptin negatively regulates bone formation by acting in hypothalamus
Ducy et coll. Cell 2000
Leptin regulates bone formation
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Leptin acts centrally to regulate osteoblasts
Takeda S., Cell, 2002
Bone
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Reid , Bone, 2004
Double control of bone formation by leptin
Leptin promotes osteoblast differentiation from mesenchymal cellsin vitro (Thomas et coll. 1999)
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Summary
- Bone formation is mainly dependent on the number ofactive osteoblasts
- Bone formation is controlled at multiple levels (central, hormonal, local) by activating multiple signallingpathways
- Some of these pathways can be targeted to increasebone formation in vivo