A systems approach to bone remodeling mechanotransduction

A SYSTEMS APPROACH TO BONE REMODELING AND MECHANOTRANSDUCTION MYNAMPATI KALYAN CHAKRAVARTHY NATIONAL UNIVERSITY OF SINGAPORE 2007 A SYSTEMS APPROACH TO BONE REMODELING AND MECHANOTRANSDUCTION MYNAMPATI KALYAN CHAKRAVARTHY (B.Eng. (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAMME IN BIOENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGEMENTS I would like to express my deepest gratitude to the following: Associate Professor Peter Lee For introducing me to the field of systems biology, hearing patiently to my unceasing yapping, encouraging me in my ventures, and his invaluable support, advice and guidance all through out the project Associate Professor Toh Siew Lok For his support to this research, and providing me the resources to carry out the project Ms Ling Wen Wan and Mr Koh Geoffrey For their contribution to the parameter estimation part of this dissertation GPBE mates For their help, support and encouragement and all the fun and laughter we shared over the past 30 months And above all, To the Supreme Lord and His devotees for giving a purpose to my existence NOTE Due to the inputs from several sources to help shape up this project, first person plural is used in active voice all through out this dissertation, instead of first person singular. i TABLE OF CONTENTS Topic Page Acknowledgements i Summary v List of Tables vii List of Figures viii List of Abbreviations xi Chapter 1: Introduction 1.1 Motivation 1.2 Objectives of the project 1.3 Methodology 1.4 Overview of the thesis Chapter 2: Bone Remodeling 2.1 Synopsis 2.2 Bone 2.2.1 Bone: The Organ 2.2.2 Osseous tissue 2.2.3 Bone formation 2.3 Bone remodeling 2.3.1 Factors affecting Bone remodeling 11 2.3.2 Homeostatic Imbalances in Bone 13 2.4 Outstanding questions in Bone Remodeling 15 ii 2.5 Summary Chapter 3: Bone Mechanotransduction 16 17 3.1 Synopsis 17 3.2 Mechanotransduction 17 3.3 Bone Mechanotransduction 19 3.3.1 Signaling in Bone Mechanotransduction 22 3.4 Salient Issues 32 3.5 Summary 33 Chapter 4: Modeling 34 4.1 Synopsis 34 4.2 Modeling Cellular Dynamics 34 4.3 Potential Model 37 4.4 Summary 38 Chapter 5: Systems Modeling 39 5.1 Synopsis 39 5.2 ‘Systems-level’ Modeling 39 5.2.1 Implementation of the systems level modeling 5.3 Summary 43 66 Chapter 6: Results and Discussion 67 6.1 Synopsis 67 6.2 Boundary Conditions 67 6.3 SIMULINK block diagrams 68 6.4 Results and Discussion 71 iii 6.5 Significant Inferences 81 6.6 The Missing Link 82 6.7 Summary 84 Chapter 7: Conclusion 86 Chapter 8: Future Work 88 8.1 Synopsis 88 8.2 Experimentation 88 8.3 Refining the current computational model 91 8.4 Application #1: Osteoporosis Treatment 92 8.5 Application #2: Bone Tissue Engineering 93 8.6 Summary 95 Bibliography 96 Appendix A: Bone Mechanotransduction over the years 106 Appendix B: Networks 117 B.1 Osteoblast Signaling Network 117 B.2 Osteoblast-Osteoclast Interaction Network 118 B.3 Osteoclast Signaling Network 119 Appendix C: Analysis of the Parameter Estimation Algorithm 120 Appendix D: MATLAB files for Parameter Estimation 123 Appendix E: Profiles of Signaling Proteins 137 iv SUMMARY Bone remodeling refers to a fundamental homeostatic process in the body, which maintains bone strength by continuously replacing old bone with new bone. Disruption in the homeostasis leads to skeletal disorders like osteopetrosis or osteoporosis. Mechanical loading affects bone remodeling. Increased loading leads to increased bone mass, while reduced loading results in decreased bone mass. The underlying cellular dynamics for such an observation is not clearly understood. Hence, the main objective of this project is to investigate the affect of mechanical loading on bone remodeling at the cellular level. In this project, a novel computational modeling approach called ‘systems-level’ modeling is implemented to study the mechano-regulation of bone at cellular level. Specific issues addressed using this approach include determining the intra-cellular response of bone cells to mechanical stimulus, bone response to different mechanical loading conditions, the role of feedback regulation in bone remodeling, and the link between reduced mechanical loading and decreased bone mass. This computational modeling approach, implemented in SIMULINK® environment, derives concepts from the emerging field of systems biology, control theory, and computer science. The salient features of this modeling technique include – (i) Systems biology based network modeling: A system of differential equations is developed based on Michaelis-Menten enzyme kinetics to model the intracellular signaling networks of osteoblasts and osteoclasts. v (ii) Parameter estimation, based on evolutionary computing, is used to estimate the Michaelis-Menten rate constants of the kinetic models of the networks. (iii) Control systems theory is used to model feedback in the signaling networks. An inter-connected network of eight major signaling pathways in osteoblasts and seven in osteoclasts, which are initiated as part of the intra-cellular response of bone to mechanical stimulus, are identified for this dissertation, based on a comprehensive literature survey. The ‘systems-level’ computational models simulate the temporal dynamics of the signaling proteins in these two networks. The simulation studies indicate that the signaling networks cause unique physiological response in the bone cells with respect to different mechanical stimulus. Disruption of intra-cellular feedback regulation leads to decreased bone formation in osteoblasts and increased bone resorption in osteoclasts, a phenomenon generally observed in reduced loading conditions. The results of these simulation studies serve as useful guidelines for planning relevant experimental work to study the affect of mechanical loading on bone remodeling at cellular level. vi LIST OF TABLES Table Legend Page 5.1 Parameter values for the hypothetical pathway model 48 5.2 Constants Used for the Parameter Estimation Algorithm 63 5.3 Rate constants in the Osteoblast network 64 5.4 Rate constants in the Osteoclast network 65 6.1 Input stimuli for network perturbation 68 vii LIST OF FIGURES Figure Legend Page 2.1 The four different types of bone cells 2.2 The five phases of bone remodeling 10 2.3 Determinants of bone remodeling 11 2.4 Cross section of healthy bone vs. Osteoporotic bone 14 3.1 Model for the transduction of mechanical strain to osteocytes in bone 20 3.2 Osteocytes as mechanosensory cells 21 3.3 Mechanotransduction response in Osteoblast 23 3.4 Mechanotransduction response in Osteoclast 23 3.5 Block Diagram representation of the Osteoblast signaling network 24 3.6 Molecules involved in the Osteoblast-Osteoclast interactions 28 3.7 Block Diagram representation of the Osteoclast signaling network 29 viii Appendix E: Simulations Akt Pathway RANKL Æ RANK Æ PI3K Æ PIP3 Æ PDK1 Æ Akt PI3K Profile (step) PIP3 Profile (step) 80 80 Concentration (in % ) 100 Concentration (in % ) 100 60 40 20 60 40 20 12 23 34 45 56 67 78 89 100 111 122 133 144 155 166 177 188 199 210 Steady Steady Unsteady 100 80 80 Concentration (in % ) Concentration (in %) 100 40 20 60 40 20 77 153 229 305 381 457 533 609 685 761 837 913 989 1065 1141 1217 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115 Tim e (in units) Time (in units) Steady Unsteady Steady Unsteady PIP3 Profile (pulse) PI3K Profile (pulse) 100 100 80 Concentration (in % ) Concentration (in %) Unsteady PIP3 Profile (sine) PI3K Profile (sine) 60 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 Time (in units) Time (in units) 60 40 80 60 40 20 20 18 35 52 69 86 103 120 137 154 171 188 205 222 239 256 273 290 307 324 341 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115 Time (in units) Time (in units) Steady Steady Unsteady Unsteady Figure E28 Activation profiles of PI3-K and PIP3 160 Appendix E: Simulations PDK1 Profile (step) Akt Profile (step) Co n c en trat io n (in % ) Concentration (in %) 100 80 60 40 20 100 80 60 40 20 11 31 41 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 Unsteady Time (in units) Steady Steady Unsteady Akt Profile (sine) Con cen tratio n (in % ) PDK1 Profile (sine) 100 Concentration (in %) 21 Time (in units) 80 60 40 100 80 60 40 20 20 11 21 31 41 Time (in units) Unsteady 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 Steady Time (in units) Steady Unsteady Akt Profile (pulse) C o n c e n tra ti o n (i n % ) PDK1 Profile (pulse) Concentration (in %) 100 80 60 40 20 100 80 60 40 20 11 21 31 41 Time (in units) 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 Time (in units) Steady Unsteady Steady Unsteady Figure E29 Activation profiles of PDK1and Akt Figures E28 and E29 illustrate the activation profiles of the signaling factors involved in the AKT pathway for the three different input stimuli. The steady and unsteady profiles are the same for all the four downstream proteins because of lack of any feedback regulation. 161 Appendix E: Simulations PKC Pathway RANKLÆ RANK Æ PIPK1 Æ PIP2 Æ PLC Æ DAG Æ PKC PIP2 Profile (step) 100 80 80 Concentration (in %) Concentration (in %) PIPK1 Profile (step) 100 60 40 20 60 40 20 18 35 52 69 86 103 120 137 154 171 188 205 222 239 256 273 290 307 324 341 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115 Time (in units) Time (in units) Steady Unsteady Steady PIP2 Profile (sine) 100 80 80 Concentration (in %) Concentration (in %) PIPK1 Profile (sine) 100 60 40 20 60 40 20 122 243 364 485 606 727 848 969 1090 1211 1332 1453 1574 1695 1816 1937 17 25 33 41 49 57 Steady PIPK1 Profile (pulse) 80 Concentration (in %) 100 80 20 81 89 97 105 113 121 129 137 145 Unsteady PIP2 Profile (pulse) 100 40 73 Steady Unsteady 60 65 Time (in units) Time (in units) Concentration (in %) Unsteady 60 40 20 25 49 73 97 121 145 169 193 217 241 265 289 313 337 361 385 409 433 457 481 17 25 33 41 49 57 65 73 81 89 97 105 113 121 129 137 145 Time (in units) Tim e (in units) Steady Steady Unsteady Unsteady Figure E30 Activation profiles of PIPK1and PIP2 Figures E21 to E23 illustrate the activation profiles of the signaling factors involved in the PKC pathway for the three different input stimuli. As this pathway is not feedback regulated, the steady and unsteady profiles are same for all the five signaling factors. 162 Appendix E: Simulations PLC Profile (step) DAG Profile (step) 80 80 Concentration (in %) 100 Concentration (in %) 100 60 40 20 60 40 20 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 Time (in units) Steady Time (in units) Unsteady Steady DAG Profile (sine) 100 100 80 80 Concentration (in %) Concentrati on (in %) PLC Profile (sine) 60 40 20 60 40 20 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 Time (in units) Steady Time (in units) Steady Unsteady PLC Profile (pulse) Unsteady DAG Profile (pulse) 100 100 80 80 Concentration (in %) Concentration (in %) Unsteady 60 40 20 60 40 20 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 Time (in units) Time (in units) Steady Steady Unsteady Unsteady Figure E31 Activation profiles of PLC and DAG PKC Profile (sine) 100 80 60 40 20 11 21 31 41 51 100 80 60 40 20 11 21 31 41 51 100 80 60 40 20 61 Steady Unsteady 11 21 31 41 51 61 Time (in units) Time (in units) Time (in units) Unsteady 61 PKC Profile (pulse) Co n ce ntration (i n % ) C o n c e n tratio n (i n % ) Co n c e n tratio n (i n % ) PKC Profile (step) Steady Unsteady Steady Figure E32 Activation profiles of PKC 163 Appendix E: Simulations JNK Pathway RANKL Æ RANK Æ p21 Ras Æ MEKK4/7 Æ MKK4/7 Æ JNK MEKK4/7 Profile (step) 100 100 80 80 Concentration (in %) Concentration (in %) p21Ras Profile (step) 60 40 20 60 40 20 13 25 37 49 61 73 85 97 109 121 133 145 157 169 181 193 205 217 229 241 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 Time (in units) Steady Time (in units) Unsteady Steady MEKK4/7 Profile (sine) 100 100 80 80 Concentration (in %) Concentration (in %) p21Ras Profile (sine) 60 40 20 60 40 20 74 147 220 293 366 439 512 585 658 731 804 877 950 1023 1096 1169 Time (in units) Steady Steady 80 80 Concentration (in %) 100 40 20 Unsteady MEKK4/7 Profile (pulse) 100 60 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 Time (in units) Unsteady p21Ras Profile (pulse) Concentration (in %) Unsteady 60 40 20 20 39 58 77 96 115 134 153 172 191 210 229 248 267 286 305 324 343 362 381 400 Time (in units) Steady Unsteady 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115 Time (in units) Steady Unsteady Figure E33 Activation profiles of p21 Ras and MEKK4/7 164 Appendix E: Simulations MKK4/7 Profile (step) JNK Profile (step) C o n c e n tr a tio n (i n % ) Concentration (in %) 100 80 60 40 20 100 80 60 40 20 21 41 61 81 121 Unsteady Time (in units) Steady 101 141 161 181 Time (in units) 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 Steady Unsteady MKK4/7 Profile (sine) JNK Profile (sine) C o n c e n tra tio n (in % ) Concentration (in %) 100 80 60 40 20 100 80 60 40 20 21 41 61 81 17 25 33 41 49 57 65 73 81 89 97 105 113 121 129 137 145 C o n c e n tr a ti o n ( i n % ) Concentration (in %) 80 60 40 20 25 33 41 49 57 65 73 81 89 97 105 113 121 129 137 145 Time (in units) Steady 181 Steady 100 80 60 40 20 17 161 JNK Profile (pulse) 100 141 Unsteady MKK4/7 Profile (pulse) 121 Unsteady Time (in units) Steady 101 Time (in units) 21 41 61 81 101 121 141 161 181 Time (in units) Unsteady Steady Unsteady Figure E34 Activation profiles of MKK4/7 and JNK Figures E33 and E34 illustrate the activation profiles of the signaling factors involved in the JNK pathway for the three different input stimuli. In this pathway, feedback regulation affects MKK4/7 and JNK. As a result, the steady and unsteady profiles are different for these two signaling factors. 165 Appendix E: Simulations p38 MAPK Pathway RANKL Æ RANK Æ p38 Ras Æ MEKK3/6 Æ MKK3/6 Æ p38 MAPK Mekk3/6 Profile (step) 100 80 80 Concentration (in %) Concentration (in %) p38Ras Profile (step) 100 60 40 20 60 40 20 13 25 37 49 61 73 85 97 109 121 133 145 157 169 181 193 205 217 229 241 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 Time (in units) Steady Time (in units) Unsteady Steady Mekk3/6 Profile (sine) 100 100 80 80 Concentration (in %) Concentration (in %) p38Ras Profile (sine) 60 40 20 60 40 20 98 195 292 389 486 583 680 777 874 971 1068 1165 1262 1359 1456 1553 17 25 33 41 49 57 Time (in units) Steady 65 73 81 89 97 105 113 121 129 137 145 Time (in units) Unsteady Steady Unsteady Mekk3/6 Profile (pulse) p38Ras Profile (pulse) 100 100 80 Concentration (in %) Concentration (in %) Unsteady 60 40 20 80 60 40 20 20 39 58 77 96 115 134 153 172 191 210 229 248 267 286 305 324 343 362 381 400 Time (in units) Steady Unsteady 17 25 33 41 49 57 65 73 81 89 97 105 113 121 129 137 145 Time (in units) Steady Unsteady Figure E35 Activation profiles of p38 Ras and MEKK3/6 166 Appendix E: Simulations Mkk3/6 Profile (step) p38MAPK Profile (step) Co n cen trati o n (in % ) Concentration (in %) 100 80 60 40 20 100 80 60 40 20 11 16 26 31 Unsteady Time (in units) Steady 21 36 41 46 41 46 41 46 Time (in units) 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 Steady Unsteady Mkk3/6 Profile (sine) p38MAPK Profile (sine) Concentration (in % ) Concentration (in %) 100 80 60 40 20 100 80 60 40 20 11 16 26 31 Unsteady Time (in units) Steady 21 36 Time (in units) 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 Steady Unsteady Mkk3/6 Profile (pulse) p38MAPK Profile (pulse) Concentration (in % ) Concentration (in %) 100 80 60 40 20 100 80 60 40 20 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 Time (in units) Steady 11 16 21 26 31 36 Time (in units) Unsteady Steady Unsteady Figure E36 Activation profiles of MKK3/6 and p38 MAPK Figures E35 and E36 illustrate the activation profiles of the signaling factors involved in the p38 MAPK pathway for the three different input stimuli. As this pathway is not feedback regulated, the steady and unsteady profiles are same for all the five signaling factors. 167 Appendix E: Simulations ERK Pathway RANKL Æ RANK Æ Ras Æ MEKK1/2 Æ MEK1/2 Æ ERK Ras Profile (step) Mekk1/2 Profile (step) 80 80 Concentration (in %) 100 Concentration (in %) 100 60 40 20 60 40 20 18 35 52 69 86 103 120 137 154 171 188 205 222 239 256 273 290 307 324 341 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115 121 Time (in units) Time (in units) Steady Steady Unsteady Mekk1/2 Profile (sine) Ras Profile (sine) 80 Concentration (in %) 80 Concentration (in %) 100 100 60 40 20 60 40 20 95 189 283 377 471 565 659 753 847 941 1035 1129 1223 1317 1411 1505 17 25 33 41 49 57 Steady 65 73 81 89 97 105 113 121 129 137 145 Time (in units) Time (in units) Steady Unsteady Unsteady Mekk1/2 Profile (pulse) Ras Profile (pulse) 100 100 80 Concentration (in %) Concentration (in %) Unsteady 60 40 80 60 40 20 20 23 45 67 89 111 133 155 177 199 221 243 265 287 309 331 353 375 397 419 441 17 25 33 41 49 57 65 73 81 89 97 105 113 121 129 137 145 Time (in units) Time (in units) Steady Steady Unsteady Unsteady Figure E37 Activation profiles of Ras and MEKK1/2 168 Appendix E: Simulations Mek1/2 Profile (step) ERK Profile (step) C o n c e n tr a ti o n (i n % ) Concentration (in %) 100 80 60 40 20 100 80 60 40 20 11 21 31 41 51 61 71 81 91 101 111 121 131 141 17 25 33 41 49 57 65 73 81 89 Time (in units) 97 105 113 121 129 137 145 Unsteady Time (in units) Steady Steady Unsteady Mek1/2 Profile (sine) ERK Profile (sine) C o n c e n tr a ti o n ( i n % ) 100 Concentration (in % ) 100 80 60 40 20 80 60 40 20 11 21 31 41 51 61 71 81 91 101 111 121 131 141 17 25 33 41 49 57 65 73 Time (in units) 81 89 97 105 113 121 129 137 145 Unsteady Time (in units) Steady Steady Unsteady Mek1/2 Profile (pulse) ERK Profile (pulse) C o n c e n tr a t i o n ( i n % ) 100 Concentration (in % ) 100 80 60 40 20 80 60 40 20 11 21 31 41 51 61 71 81 91 101 111 121 131 141 17 25 33 41 49 57 65 73 81 89 97 105 113 121 129 137 145 Time (in units) Steady Time (in units) Unsteady Steady Unsteady Figure E38 Activation profiles of MEK1/2 and ERK Figures E37 and E38 illustrate the activation profiles of the signaling factors involved in the ERK pathway for the three different input stimuli. In this pathway, feedback regulation affects the downstream signaling factors - MEKK1/2, MEK1/2, and ERK. As a result, the steady and unsteady profiles are different for these three signaling factors. It can be observed that the response to the stimulus signal gets attenuated as we go down the cascade. 169 Appendix E: Simulations NFAT Pathway RANKLÆ RANK Æ PIPK1 Æ PIP2 Æ PLC Æ IP3 Æ Ca2+ Æ Calcineurin Æ NFAT PIP2 Profile (step) 100 80 80 Concentration (in %) Concentration (in %) PIPK1 Profile (step) 100 60 40 20 60 40 20 20 39 58 77 96 115 134 153 172 191 210 229 248 267 286 305 324 343 362 381 400 17 25 33 41 49 57 Time (in units) Steady Unsteady 73 81 Steady PIPK1 Profile (sine) 89 97 105 113 121 129 137 145 Unsteady PIP2 Profile (sine) 100 100 80 80 Concentration (in %) Concentration (in %) 65 Time (in units) 60 40 20 60 40 20 110 219 328 437 546 655 764 873 982 1091 1200 1309 1418 1527 1636 1745 Time (in units) 17 25 33 41 49 57 65 73 81 89 97 105 113 121 129 137 145 Time (in units) Steady Unsteady Steady Unsteady PIPK1 Profile (pulse) PIP2 Profile (pulse) 100 80 Concentration (in %) Concentration (in %) 100 60 40 20 80 60 40 20 25 49 73 97 121 145 169 193 217 241 265 289 313 337 361 385 409 433 457 481 Time (in units) Steady 11 21 31 41 51 61 71 81 91 101 111 121 131 141 151 161 171 181 191 Time (in units) Steady Unsteady Unsteady Figure E39 Activation profiles of PIPK1 and PIP2 170 Appendix E: Simulations IP3 Profile (step) 100 80 80 Concentration (in %) Concentration (in %) PLC Profile (step) 100 60 40 20 60 40 20 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 Time (in units) Steady Time (in units) Unsteady Steady IP3 Profile (sine) 100 100 80 80 Concentrati on (in %) Concentration (in %) PLC Profile (sine) Unsteady 60 40 20 60 40 20 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 Time (in units) 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 Time (in units) Steady Unsteady Steady IP3 Profile (pulse) 100 100 80 80 Concentration (in %) Concentrati on (in %) PLC Profile (pulse) 60 40 20 Unsteady 60 40 20 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 Time (in units) Steady Unsteady Time (in units) Steady Unsteady Figure E40 Activation profiles of PLC1 and IP3 Figures E39 to E42 illustrate the activation profiles of the signaling factors involved in the NFAT pathway for the three different input stimuli. As this pathway is not feedback regulated, the steady and unsteady profiles are same for all the seven signaling factors. 171 Appendix E: Simulations Ca/Calmodulin Profile (step) 100 80 80 Concentration (in %) Concentration (in %) Ca Profile (step) 100 60 40 20 60 40 20 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 Time (in units) Steady Time (in units) Unsteady Steady Ca/Calmodulin Profile (sine) 100 100 80 80 Concentration (in % ) Concentration (in %) Ca Profile (sine) 60 40 20 60 40 20 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 Time (in units) Time (in units) Steady Steady Unsteady Ca Profile (pulse) Unsteady Ca/Calmodulin Profile (pulse) 100 100 80 80 C o n ce n tra tio n (in % ) Concentration (in % ) Unsteady 60 40 20 60 40 20 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 Time (in units) Time (in units) Steady Unsteady Steady Unsteady Figure E41 Activation profiles of Ca and Calcineurin NFAT Profile (sine) 80 60 40 20 11 16 21 26 31 36 41 46 51 56 61 66 100 80 60 40 20 11 16 21 26 31 36 41 46 51 56 61 66 Steady Unsteady 100 80 60 40 20 11 16 21 26 31 36 41 46 51 56 61 66 Time (in units) Time (in units) Time (in units) Unsteady NFAT Profile (pulse) Concentration (in % ) 100 Concen tration (in % ) C o n c en tratio n (in % ) NFAT Profile (step) Steady Unsteady Figure E42 Activation profiles of NFAT 172 Steady Appendix E: Simulations NFkB Pathway RANKL Æ RANK Æ NIK Æ IKKα Æ NFκB/IκB Æ NFκB IKKa Profile (step) 100 100 80 80 Concentration (in %) Concentration (in %) NIK Profile (step) 60 40 20 60 40 20 16 31 46 61 76 91 106 121 136 151 166 181 196 211 226 241 256 271 286 11 21 31 41 51 61 71 81 91 101 111 121 131 141 151 161 171 181 191 Time (in units) Time (in units) Steady Unsteady Steady IKKa Profile (sine) 100 100 80 80 Concentrati on (in %) Concentration (in %) NIK Profile (sine) 60 40 20 60 40 20 158 315 472 629 786 943 1100 1257 1414 1571 1728 1885 2042 2199 2356 2513 16 31 46 61 76 91 106 121 136 151 166 181 196 211 226 241 256 271 286 Time (in units) Time (in units) Steady Unsteady Steady NIK Profile (pulse) 100 80 80 60 40 20 Unsteady IKKa Profile (pulse) 100 Concentration (in %) Concentration (in %) Unsteady 60 40 20 25 49 73 97 121 145 169 193 217 241 265 289 313 337 361 385 409 433 457 481 13 25 37 49 61 73 85 97 109 121 133 145 157 169 181 193 205 217 229 241 Time (in units) Steady Unsteady Time (in units) Steady Unsteady Figure E43 Activation profiles of NIK and IKKα 173 Appendix E: Simulations NFkB/IkB Profile (step) NFkB Profile (step) Concentration (in % ) Concentration (in %) 100 80 60 40 20 100 80 60 40 20 20 39 58 77 96 115 134 153 172 191 210 229 248 267 286 Time (in units) 34 67 100 133 166 199 232 265 298 331 364 397 430 463 496 529 562 595 628 661 694 Time (in units) Steady Unsteady NFkB Profile (sine) C o n cen tratio n (in % ) NFkB/IkB Profile (sine) 100 Concentration (in %) Steady Unsteady 80 60 40 20 100 80 60 40 20 20 39 58 77 96 115 134 153 172 191 210 229 248 267 286 Time (in units) 20 39 58 77 96 115 134 153 172 191 210 229 248 267 286 305 324 343 362 381 400 Unsteady Time (in units) Steady Steady Unsteady NFkB/IkB Profile (pulse) NFkB Profile (pulse) Co n cen tratio n (in % ) Concentration (in % ) 100 80 60 40 20 100 80 60 40 20 20 39 58 77 96 115 134 153 172 191 210 229 248 267 286 Time (in units) 30 59 88 117 146 175 204 233 262 291 320 349 378 407 436 465 494 523 552 581 Time (in units) Steady Unsteady Steady Unsteady Figure E44 Activation profiles of NFκB/IκB and NFκB Figures E43 and E44 illustrate the activation profiles of the signaling factors involved in the NFκB pathway for the three different input stimuli. In this pathway, feedback regulation from NFAT pathway affects NFκB/IκB. Hence, its steady and unsteady profiles are different, which translate into different activation profiles for NFκB transcription factor as well. 174 Appendix E: Simulations The activation profiles of the signaling factors in the osteoclast network (Figures E27 to E44) indicate that – (iii) The intra-cellular signaling network is sensitive to perturbation, and hence causes unique physiological response in the cells. (iv) The activation rate is higher in unsteady state (no feedback regulation) when compared to steady state (feedback regulated). In the context of bone remodeling, this might translate as increased bone resorption in osteoclasts, as discussed in Chapter 6. Summary Simulation studies of osteoblast and osteoclast signaling networks are discussed in this appendix chapter. These simulations corroborate with the inferences made in Chapter of this thesis - The signaling networks cause unique physiological response in the bone cells with respect to different mechanical stimulus. Disruption of intra-cellular feedback leads to decreased bone formation in osteoblasts and increased bone resorption in osteoclasts, a phenomenon generally observed in reduced loading conditions. Hence, it is deduced that reduced mechanical loading causes disruption in the feedback regulation to result in low bone mass. The results of these simulation studies serve as useful guidelines for planning relevant experimental work (discussed in Chapter 8) to study the effect of mechanical loading on bone remodeling at cellular level. 175 [...]... Stimulating Factor MMP Family of extracellular matrix metalloproteinases Nemo Regulatory noncatalytic subunit of IKK Nf-kB Nuclear factor kappa B NOS Nitric Oxide Synthase OA Osteoarthritis OCN Osteocalcin xii Abbreviation Expansion PI3K Phosphoinositide 3-kinase PIP3 Phosphatidylinositol (3,4,5)-trisphosphate PKA/PKC Protein Kinase A/ C RA Rheumatoid arthritis RANK Receptor activator of Nf-kB RANKL RANK... matrix, it leaves the bone as a whole unchanged, including its shape, internal architecture, and mineral content In healthy young adults, total bone mass remains constant, indicating that the rates of bone formation and resorption are equal Remodeling is vital for bone health, for a variety of reasons [Surgeon2004] It repairs the damage to the skeleton that can result from repeated stresses by replacing... four types of bone cells (Adapted from [Martini2006]) 6 Chapter 2: Bone Remodeling (i) Osteocytes are mature bone cells that account for most of the cell population Each osteocyte occupies a lacuna, a pocket sandwiched between layers of matrix (called lamellae) Narrow passageways called canaliculi penetrate the lamellae, radiating through the matrix and connecting lacunae with one another and with sources... low bone mass, or inadequate mineralization Osteomalacia is a 13 Chapter 2: Bone Remodeling disease characterized by inadequate mineralization Although osteoid is produced, calcium salts are not deposited So, bone become softened and weakens Paget’s disease is characterized by excessive bone deposit and resorption [Marieb2004] This along with reduced mineralization causes a spotty weakening of the bones... associated with an increase of growth factors and with a decrease in the apoptosis of the osteoblasts (c) Calcitonin - A polypeptide, reduces osteoclast function to inhibit bone resorption 12 Chapter 2: Bone Remodeling (d) Vitamin D - A steroid hormone that acts as a local regulator of osteoclast differentiation, and influences matrix mineralization (e) Androgens - Have an anabolic effect on bone through... Osteoprogenitor cells are mesenchymal stem cells which divide into daughter cells that differentiate into osteoblasts Osteoprogenitor cells maintain populations of osteoblasts and are important in the repair of a fracture They are located in the inner layers that line marrow cavities and in the linings of passageways, containing blood vessels that penetrate the matrix of compact bone 7 Chapter 2: Bone Remodeling. .. Legend Page 4.1 Block diagram model of the ERK signaling pathway 35 4.2 Mechanistic model of the MAPK signaling cascade, interacting with another pathway 36 5.1 Architecture of systems- level’ modeling 39 5.2 A hypothetical signaling pathway, including a positive and a negative feedback loop 43 5.3 Pathway map of the hypothetical signaling cascade 45 5.4 SIMULINK block diagram of the pathway (No feedback... 2.3), as explained below – Figure 2.3 Determinants of bone remodeling (Adapted from [Harada2003]) (i) Genetic factors – The maximum bone mass is controlled by genetic determinants Genes also determine bone s response to other factors influencing bone remodeling (ii) Mechanical factors –Mechanical environment and the gene expression patterns at various stages of ossification are observed to be intimately... termination of matrix synthesis The rate of matrix apposition is rapid initially, but it slows down after the termination of matrix synthesis, and continues until the bone surface returns to its original resting state 2.3.1 Factors affecting Bone Remodeling Numerous factors [Fernández2006 and Bonewald2003], including genetic, mechanical, vascular, nutritional, hormonal and local, affect the bone remodeling. .. adult body, rate of bone formation equals rate of bone resorption to maintain the homeostasis Unequal rates of bone formation and resorption result in diseased conditions like osteopetrosis or osteoporosis The exact cellular mechanisms for homeostasis disruption are still not clearly understood It has been observed that increased mechanical loading enhances bone mass indicating increased bone formation, . A SYSTEMS APPROACH TO BONE REMODELING AND MECHANOTRANSDUCTION MYNAMPATI KALYAN CHAKRAVARTHY NATIONAL UNIVERSITY OF SINGAPORE 2007 A SYSTEMS APPROACH TO BONE REMODELING. formation and resorption, popularly known as bone remodeling. In a healthy adult body, rate of bone formation equals rate of bone resorption to maintain the homeostasis. Unequal rates of bone. c-Jun N-terminal kinase MAP Mitogen Activated Protein MAPK MAP kinase MCSF Macrophage Colony Stimulating Factor MMP Family of extracellular matrix metalloproteinases Nemo