Osteoclasts- What Do They Do and How Do They Do It? Prof. Steven L. Teitelbaum

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1 Osteoclasts; What Do They Do 1 Steven L. Teitelbaum, M.D. Wilma and Roswell Messing Professor Department of Pathology and Immunology Washington University School of Medicine OSTEOCLASTS OSTEOBLASTS 2 Osteoclasts 3 1

2 Osteoporosis 4 Osteoporosis (2) 5 Bone remodeling 6 2

3 Bone remodeling (2) 7 Annu. Rev. Pathol. Mech. Dis : Bone remodeling (3) MSC OB atgfβ1 TGFβ1 BONE 8 Nat. Med : 757 Bone remodeling (4) 9 3

4 Osteoporosis 10 Osteopetrosis (Marble bone disease)

5 Increased bone mass 13 Clinical forms of osteopetrosis Benign - autosomal dominant Malignant - autosomal recessive 14 Malignant osteopetrosis Autosomal recessive Dense sclerotic skeleton Hematologic abnormalities Neurologic abnormalities Death in infancy or early childhood 15 5

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7 19 What is the osteoclast precursor? 20 Hematopoietic Precursor?? 21 Stromal Cell 7

8 Y-chromosome Y-body analysis of osteoclasts and osteoblasts Cell Before Analyzed Transplantation After Transplantation Osteoclasts weeks weeks weeks weeks weeks Cells (no.) Nuclei (no.) Y bodies (no.) Male nuclei (%) Osteoblasts Cells (no.) Y bodies (no.) Male cells (%) Coccia et al., NEJM, : BONE MATRIX Effector substances Effector substances (Cell interaction) 24 BONE MATRIX BONE MATRIX 8

9 Osteoclast differentiation Stromal Cell/Osteoblast? Stromal Cell/Osteoblast? Macrophage? Osteoclast 25 Bone 26 Osteoclast differentiation Stromal Cell/Osteoblast OPG RANK LIGAND RANK Macrophage Osteoclast 27 Bone 9

10 Wild type RANK ligand -/- 28 Stromal Cell/Osteoblast OPG RANKL RANK Macrophage c-fms M-CSF Osteoclast 29 Bone Osteoclasts generated in vitro 30 10

11 Osteoclastogenic signals RANK c-fms 31 Annu. Rev. Pathol. Mech. Dis : Molecular mechanisms of osteoclast differentiation and function M-CSF RANKL 32 How does the osteoclast degrade bone? 33 11

12 Osteoclast H + Bone

13 Molecular mechanisms of osteoclast differentiation and function M-CSF RANKL H + ATPase 37 Carbonic anhydrase Osteoclast H + +HCO C.A. - 3 H 2 O+CO2 H + Bone 38 Family with benign osteopetrosis 39 13

14 40 Molecular mechanisms of osteoclast differentiation and function M-CSF RANKL C.A.II H + ATPase 41 Cl - Osteoclast H + + HCO 3 - C.A. HCO 3 - H 2 O+CO2 H + Bone 42 14

15 In the presence of Cl - HCO - 3 Cl - Osteoclast HCO - 3 Cl - H + Bone 43 Cl - /HCO 3- exchange 44 J. Clin. Invest.1989; 83(1): Cl - /HCO 3- exchange (2) 45 J. Clin. Invest.1989; 83(1):

16 46 Molecular mechanisms of osteoclast differentiation and function M-CSF RANKL HCO 3- /Cl - C.A.II H + ATPase 47 HCO 3- Cl - Osteoclast Slc4a2 +/+ Slc4a2 -/- H+ Cl-?? H + Cl - Bone 48 16

17 49 HCO 3- Cl - Osteoclast H + HCO - 3 Cl - H + Cl - Bone

18 Molecular mechanisms of osteoclast differentiation and function M-CSF RANKL Cl - CHANNEL HCO 3- /Cl - C.A.II H + ATPase 52 HCl? Bone = mineral + organic matrix 53 HCO 3- Cl - Osteoclast H + HCO - 3 Cl - H + Cl - Bone 54 18

19 55 Pyknodysostosis 56 Molecular mechanisms of osteoclast differentiation and function M-CSF RANKL CATH. K Cl - CHANNEL HCO3 - /Cl - C.A.II H + ATPase 57 19

20 HCO 3 - Cl - Osteoclast Cath K H + HCO - 3 Cl - H + Cl - Bone 58 Integrin-matrix attachment 59 Integrin family α 3 Peyers Patch Addressin α 4 α 2 α 1 LM Col I FN Fn alt. Col I Col I Col IV Col IV LM LM β 1 α 5 FN RGD LM FN RGD β 5 α 6 β p β 4 VN RGD BM α v β 6 Osteoclast Integrin FB VWF VN OP BSP1 RGD β 3 β 2 C36I FX FB α IIb FB VWF VN FN RGD ICAM-1 ICAM-2 α X α M α L 60 20

21 β3-/- mice are osteopetrotic 61 J. Clin. Invest. 2000; 105(4): β3-/- mice are osteopetrotic (2) b 3 +/+ b 3 +/- b 3 -/- 62 J. Clin. Invest.2000; 105(4): Osteoclasts TRAP stain of BMM / ST2 cocultures 63 J. Clin. Invest.2000; 105(4):

22 Osteoclast cytoskeleton 64 Podosome Vinculin 65 Annu. Rev. Pathol. Mech. Dis : Actin ring αvβ3 66 Annu. Rev. Pathol. Mech. Dis :

23 Osteoclast E/M BL BL AR RB AR 67 BL= Basolateral Membrane RB= Ruffled Border AR= Actin Ring Non-polarized H + ATPase in non-attached osteoclast 68 Science245: 855, 1989 Osteoclast polarized H + ATPase (proton pump) 69 Science245: 855,

24 Formation of the ruffled border 70 Synaptotagmins (Syt) mediate vesicle/plasma membrane fusion 71 Does SytVII organize the ruffled border SytVII SZ SZ 72 Bone CTK HCl Dev. Cell 14: 914,

25 SytVII -/- osteoclasts have abnormal ruffled border SytVII +/+ SytVII -/- 73 Dev. Cell 14: 914, 2008 SytVII -/- OCs resorb bone poorly 74 Dev. Cell 14: 914, 2008 Integrin-matrix attachment 75 25

26 αvβ3 Integrin organizes the osteoclast cytoskeleton αv RGD β3 J. Cell Biol. 176: 877, 2007 Nature Med.11: 284, 2005 J. Immunol.183: 1804, 2009 J. Cell Sci. 123: 2955, 2010 p-y Syk SH2Y Y c-src p-416y ITAM RAC GDP VAV3 p-y RAC GTP 76 Stromal Cell/Osteoblast OPG RANKL RANK Macrophage c-fms M-CSF HCO 3 - Cl - Osteoclast Cath K H + HCO - 3 Cl - 77 H + Cl - Bone Molecular mechanisms of osteoclast differentiation and function PU.1 Therapeutic targets M-CSF MITF NFAT RANKL C-FOS NF-κB SytVII Syk Rac Rab3D TRAF6 C-SRC αvβ3 RANKL H + ATPase HCO3 - /Cl - CATH. K αvβ3 Cl - CHAN C.A.II

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