OSTEOBLASTSPierre J Marie, Ph.D.

Inserm Unit 606 & University Paris 7,Hopital Lariboisière, Paris, France

Osteoblasts

Bone marrow

Calcifiedbone

Unmineralizedmatrix

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

Bone

MesenchymalCells

Proliferation Zone(chondrocytes)

Calcification

Reserve Zone (chondroblasts)

Hypertrophic Zone

Osteoblasts

Membranous Bones (Cranial Vault)Long Bones (Humerus)

Bone Formation during Growth

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

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

Organ CultureAllows analysis of bone formation

(cranial & long bone)

Osteoblast proliferationMatrix formationLong bone

Cranial bone

Organ Culture

Prelabeling with3H-Thymidine/Proline

Primary Osteoblast CulturesIsolated from cranial & long bone, or

bone marrow stroma

Osteoblast Cultures

Osteoblast proliferation, differentiation & apoptosis

Long bone

Cranial bone

Digestion/Migration

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

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

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

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

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

Osteoblasts : Origin and Plasticity

Pluripotent cell (Mesenchyme)

Chondrocyte Adipocyte

Fibroblast

Osteoblasts

Osteoprogenitor

?

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

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

Ducy et al , Genes & Dev, 1999

Runx2 is Also Involved in Postnatal Osteogenesis

3 weeks mice

Ducy and Karsenty, 1997; Komori, 1997

Runx2 Controls Osteoblast Differentiation

Pratap et al., Cancer Res. 2003Galindo et al., JBC 2005

Runx2 Controls Osteoblast Proliferation

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

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

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

PPARγ ControlsOsteoblast Plasticity

Osteoblast

Adipocyte

MesenchymalStem Cell PPARγ2

RUNX2

Ex: Invalidation of PPARγ2 in Mice: Increased bone formation with age

PPARγ2

MesenchymalCell

Runx2

Akune et al, J Clin Invest 2004

IncreasedBone formation

DecreasedAdipogenesis

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)

• 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

• 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

•• 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

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)

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

-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)

Krishnan et al. J. Clin. Invest. 2006

Wnt/β-catenin signaling regulates osteogenesis

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

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

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

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

Osteoblast Apoptosis

Dying OsteoblastDyingOsteoblast

x 250 x 1000

5-10 % osteoblasts and osteocytes die by apoptosis

Tunel staining, rat bone

Dying Osteoblast

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

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

Regulation of osteoblast apoptosis

Apoptosis Survival

TNFalpha, IL1BMP-2Glucocorticoids

-+

PTHEstrogens

Wnt/β-cateninFGF

IGF, TGFβStrain

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

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

Hormonal Regulation

Hormones(PTH, GC, Estrogens,

GH, Vit.D…) Bone Growth

Bone Formation

Bone Mass

Main Hormones Regulating Bone Formation

Osteoblast Number Osteoblast Activity

1,25 (OH)2 Vitamin D −

Estrogens

Growth Hormone

Glucocorticoids

Parathyroid hormone (PTH)

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

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

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+

+ ++

Estrogens and Bone Formation

- Important role in the acquisition of peakbone mass during skeletal growth

- Act on osteoblasts through genomic andnongenomic effects

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…)

Estrogens Promote Bone Formation ThroughGrowth Factor Expression

IGF-ITGFß

COLIA1

E+

+

-

Cell proliferation

Collagen synthesis

Apoptosis

E

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

Growth factors

Local regulation of Osteogenesis

Bone Growth

Bone Formation

Bone Mass

• 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

Main growth factorsregulating bone formation

Number ofosteoblasts

Osteoblastactivity

PDGF

EGF

IGF

FGF

TGFβ

BMP

-

-

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

BMP Signaling is Important in Post-Natal BoneFormation

Invalidation of BMP-RIB: Decreased bone formation and bone loss

Zhao et al., JCB 2002

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

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

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

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

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

Zhang, M. et al. J. Biol. Chem. 2002

Disruption of the Igf1 receptor reduces bone formation rate

Fibroblast Growth Factors (FGFs)

-FGF-2,-9,-18:Expressed by osteoblasts

Complex effects:

- FGF2 promotes osteoblast proliferation

- Modulates osteoblast apoptosis

- Promotes osteogenesis in vivo

Montero et al., J Clin Invest, 2000FGF2-/-

FGF2+/+8 months mice: bone loss

FGF2 -/-FGF2 +/+

FGF2 Invalidation Reduces Bone Formation

Defective bone formation

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

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

Hypothalamus(leptin, …)

Bone Formation

Bone mass

Central Regulation of Osteogenesis

• Produced by adipocytes • ob/ob mice (no leptin): increased bone mass

Leptin and Bone

Ducy et coll., 2000

•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

Leptin acts centrally to regulate osteoblasts

Takeda S., Cell, 2002

Bone

Reid , Bone, 2004

Double control of bone formation by leptin

Leptin promotes osteoblast differentiation from mesenchymal cellsin vitro (Thomas et coll. 1999)

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