Lecture 3: Skeletogenesis and diseases

Transcription

1 Jilin University School of Stomatology Skeletogenesis Lecture 3: Skeletogenesis and diseases Aug. 21, 2015 Yuji Mishina, Ph.D.

2 Bone Development Mouse embryo, E14.5 Mouse embryo, E18.0 First, cartilage primordia are formed (blue). Then, osteoblasts replace cartilage (red). Dr. Yuji Mishina

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4 Bones are formed through two distinct processes Intramembranous Ossification Endochondral Ossification

5 Growth Plate Development Determines the Size of Long Bones Resting Resting zone Proliferative zone Prehypertrophic zone Hypertrophic zone Proliferative Prehypertrophic Hypertrophic (early, late)

6 Clinical Examples

7 How Does Development of Growth Plate Influence Size of Long Bones? 1. Chondrocytes need to proliferate. Resting Proliferative Prehypertrophic Hypertrophic If suppressed, bones become shorter. FGF, Indian Hedgehog 2. Chondrocytes need to differentiate. If differentiate too quickly, less time to proliferate. PTHrP, Indian Hedgehog

8 Over 20 FGF ligands interact with 4 FGF receptors.

9 FGF Signaling In Growth Plate FGF signaling negatively regulates chondrocyte proliferation Over activation of FGF signaling causes chondrodysplasia (short-limbed dwarfism). Achondroplasia: most common, caused by point mutations in FGF receptor 3 (FGFR3). Nature Vol May

10 Clinical Example of Achondroplasia FGFR3

11 PTHrP Signaling In Growth Plate Resting chondrocytes PTHrP is produced in the resting zone. PTHrP signaling negatively regulate chondrocyte differentiation (#1 arrow). Morphogen gradient! Nature Vol May

12 PTHrP Knockout Mice Wild Type PTHrP-/- Genes Dev :

13 PTHrP Knockout Mice gp: Growth plate Wild Type PTHrP-/- Genes Dev :

14 Indian Hedgehog Signaling In Growth Plate Resting chondrocytes Indian Hedgehog (Ihh) is produced in the prehypertrophic/early hypertrophic zone. Ihh acts on proliferating chondrocytes to keep dividing (#2 arrow). Ihh stimulates PTHrP production (#3 arrow), thus negatively regulate chondrocyte differentiation Nature Vol May

15 Ihh Knockout Mice Wild Type Ihh-/- Genes Dev :

16 Ihh Knockout Mice Wild Type Ihh-/- Genes Dev :

17 Ihh Knockout Mice Chondrocytes never displayed a clear stacked columnar organization Hypertrophic cells were observed in abnormal positions, close to the ends of the skeletal elements Wild Type (E18.5) Ihh-/- Fraction of chondrocytes that are hypertrophic increased Genes Dev :

18 Outline Introduction: bone and growth factors Growth Factors in Bone Development a) FGF b) PTHrP/Ihh Growth Factors in Bone Remodeling a) Coupling (bone formation and bone resorption) b) WNT c) BMP

19 Bone Development chondrocytes <--> osteoblasts FGF, PTHrP, Ihh Bone Remodeling osteoblasts <--> osteoclasts

20 Four phases Bone Remodeling Activation - initiating event that converts a quiescent bone surface into a remodeling one - recruitment, penetration, fusion Resorption - fully differentiated osteoclasts acidify the area bordered by the sealing zone Reversal - resorption lacunae is occupied by mononuclear cells, coupling signals are initiated Formation - osteoblasts synthesize the organic matrix and then mineralize it

21 Bone Remodeling Cycle

22 Bone Remodeling

23 Bone remodeling coupling of formation and resorption 1. Osteoclasts chew up bone matrixes to release growth factors (TGF-b). (Yellow) 2. Osteoblasts produce factors to regulate osteoclasts (RANKL/OPG). (Orange)

24 How are bone formation and resorption coupled? Osteoblast (bone formation) OPG/RANKL Bone resorption RANKL OPG Osteoclast (bone resorption)

25 Physiological: Normal bone mass Bone Formation = Bone Resorption (osteoblasts) (osteoclasts)

26 Why is the coupling so important? Bone mass & Bone quality Bone Formation <----> Bone Resorption Bone mass normal, but Dr. Yuji Mishina

27 Post-menopausal Osteoporosis

28 Canonical Wnt signaling Wnt1 Wnt3 Nucleus *adapted from Montcouquiol M, et al , Annu. Rev. Neurosci. 29:363-86

29 _ + + Mesenchymal progenitors Pre-chondrocytes Proliferating chondrocytes + + Hypertrophic chondrocytes Runx2 + Osx + Ocn + Osteoblast progenitors Osteoblast progenitors Osteoblasts OPG/RANKL + _ HSC CFU-GM Pre-osteoclasts Osteoclasts

30 High Bone Mass diseases are caused by high Wnt signaling Mutations in an Wnt co-receptor, LRP5, increase Wnt signaling. (Gain-of-function type mutations)

31 Wnt Receptor Wnt Signaling In Osteoblasts Can Regulate Osteoclast Through OPG and RANKL Wnt1 Wnt3 Sclerostin OPG up RANKL down Osteoclast Osteoblast

32 Wnt Receptor Wnt Signaling In Osteoblasts Can Regulate Osteoclast Through OPG and RANKL Wnt1 Wnt3 Sclerostin OPG up RANKL down Osteoclast Osteoblast Dr. Yuji Mishina

33 Wnt Receptor Wnt Signaling In Osteoblasts Can Regulate Osteoclast Through OPG and RANKL Wnt1 Wnt3 Sclerostin OPG down RANKL up Osteoclast Osteoblast Dr. Yuji Mishina

34 N Engl J Med 2014;370:412-20

35 BMP was found by Marshall Urist as a potent bone inducer. Dr. M. Urist and Dr. Yuji Mishina? years ago

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37 BMPs (BMP2 and 7) have been clinically used to accelerate fracture healing.

38 BMP2 and Dentistry Supraalveolar critical-size peri-implant defects (Hound Labrador) Implants coated with BMP2 induced bone formation in alveolar bones (right). J. Clin. Periodontol., 2008, 35,

39 Key features of BMPs - found by their ectopic bone formation abilities - induce proliferation / differentiation of osteoblasts - are approved by FDA for assisting fracture healing - No / reverse effects of BMPs in some clinical cases - May have synergistic or antagonistic functions with other growth factors / signaling pathways

40 Hyper-calcification on spine, ribs, and sternum when BMP signaling was shut off in osteoblasts. 34 weeks, Male Ribs/Sternum Spine WT cko WT cko cko; conditional knockout. In this case, one BMP receptor (Bmpr1a) is knocked out only in osteoblasts. Dr. Yuji Mishina

41 More bone volume when BMP signaling is off in osteoblasts. 34 weeks old, H&E staining, spine (Lumbar) WT cko 200 mm A lot more bones are formed in trabecular bone area in cko. Dr. Yuji Mishina

42 Why is the coupling so important? Bone mass & Bone quality Bone Formation <----> Bone Resorption Bone mass normal Dr. Yuji Mishina

43 BMP Receptor Wnt Receptor BMP signaling in osteoblasts is important for both bone formation and resorption via the regulation of Wnt signaling BMP Sclerostin Wnt1 Wnt3 sclerostin expression OPG up RANKL down Osteoclast Osteoblast Dr. Yuji Mishina

44 BMP Receptor Wnt Receptor BMP signaling in osteoblasts is important for both bone formation and resorption via inhibition of Wnt signaling Wnt1 Wnt3 BMP Sclerostin sclerostin expression OPG up RANKL down Osteoclast Osteoblast Dr. Yuji Mishina

45 BMP Receptor Wnt Receptor BMP signaling in osteoblasts is important for both bone formation and resorption via inhibition of Wnt signaling Wnt1 Wnt3 BMP Sclerostin sclerostin expression OPG down RANKL up Osteoclast Osteoblast Dr. Yuji Mishina

46 Summary Basic concepts in bone development and bone remodeling Bone development (growth plate as example) is regulated by FGF, PTHrP, and Ihh through different mechanisms The coupling of bone formation and bone resorption is critical in bone remodeling The regulation of bone remodeling by Wnt and BMP signaling

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49 Disorders of the skull vault in human Craniosynostosis caused by premature fusion of sutures Parietal foramina Wilkie AO & Morriss-Kay GM, Nature reviews, Genetics, 2001

50 Craniosynostosis presents clinical problems -- If untreated; chronic headache, vision loss, deafness, mental retardation. -- Reconstitution surgery is the only way. Sutures will be fused again. -- Very variable symptoms; positions, degrees various underneath molecular reasons Understanding the developmental mechanisms of craniosynostosis is necessary. Craniosynostosis affects one in 2,500 live births.

51 Clinical symptoms of Craniosynostosis are viable. Sagittal synostosis/ scaphocephaly Metopic synostosis/ trigonocephaly Coronal synostosis/ lambdoid synostosis (Plagiocephaly) Lambdoid suture Sagittal suture Coronal sutures Metopic suture

52 Causative genes for Craniosynostosis Gene mutation suture FGF receptors GOF Coronal suture MSX2 GOF Coronal suture TWIST1 Het Coronal suture TCF12 LOF Coronal suture BBS9 SNP (GOF?) Sagittal suture Axin1 and Fgfr1 Compound LOF Sagittal suture BMP2 enhancer SNP (GOF?) Sagittal suture ERF LOF All sutures Bmpr1a GOF Metopic Lambdoid suture Sagittal suture Coronal sutures Metopic suture

53 Facial Bone Sutures zygomatico-frontal suture zygomatico-maxillary suture midpalatal suture Midfacial growth occurs by bone deposition at bony sutures and by periosteal apposition (bone deposition) and bone remodeling Facial growth requires freedom of movement of facial sutures

54 Molecular Genetics of Apert Syndrome Molecular Genetics of Crouzon Syndrome Mutation: Activating mutation in FGFR2 gene Mutation is in area of FGF binding site Two mutations only Molecular phenotype: Enhanced FGFR to ligand binding FGFR2 binding to wrong ligands Mutations can arise spontaneously Risk of mutation increases with paternal age Mutation: Activating mutation in FGFR2 gene Mutation is in area of FGF binding site Two common mutations with numerous additional less common mutations. Molecular phenotype: Ligand independent FGFR signaling Mutations can arise spontaneously Risk of mutation increases with paternal age

55 FGFR2 Mutations extracellular intracellular Ig1 Ig2 Ig3 SP TM TK1 TK2 FGF binding Apert mutations Crouzon common mutations

56 Apert Syndrome: Full penetrance, variable expression Prenatal Coronal Synostosis Increased intracranial pressure NL to diminished IQ Brain abnormalities (defects in septum pellucidum) Proptosis, hypertelorism Midface hypoplasia (facial sutures) Class III malocclusion Progressive cervical spine fusions Tracheal abnormalities Digit abnormalities: syndactyly Higher risk with higher paternal age

57 Crouzon Syndrome: Full penetrance, variable expression Coronal Synostosis, rare pansynostosis Increased intracranial pressure Brain abnormalities (defects in corpus callosum) NL intelligence Proptosis, hypertelorism Midface hypoplasia (facial sutures) Class III malocclusion Vertebral fusions Stylohyoid ligament calcification Tracheal abnormalities Digit abnormalities: None Higher risk with higher paternal age

58 Summary Basic concepts in bone development and bone remodeling Bone development (growth plate as example) is regulated by FGF, PTHrP, and Ihh through different mechanisms The coupling of bone formation and bone resorption is critical in bone remodeling The regulation of bone remodeling by Wnt and BMP signaling

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