COORDINATION OF CELL CYCLE AND CELL DIFFERENTIATION BY RECEPTOR ACTIVATOR OF NF-KAPPA-B LIGAND DURING OSTEOCLAST DIFFERENTIATION DISSERTATION Presented in Partial Fulfillment of the Requirements for The Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Uma Sankar ***** The Ohio State University 2003 Dissertation Committee: Approved by Professor Michael C. Ostrowski, Adviser Professor Gustavo W. Leone Adviser Professor Natarajan Muthusamy Molecular, Cellular and Professor Russell J. Hill Developmental Biology ABSTRACT Osteoclasts are bone resorbing multinuclear cells formed by the fusion of hematopoietic mononuclear precursor cells of the macrophage/monocyte lineage. Microphthalmia transcription factor (MITF) is a basic helix-loop-helix leucine zipper transcription factor that is important for the differentiation of many cell types, including osteoclasts and melanocytes. MITF regulates the expression of osteoclast-differentiation marker genes, Tartrate-resistant acid phosphatase (TRAP) and cathepsin K. Deletion in arginine 215 in the basic domain of MITF results in severe osteopetrosis in homozygous recessive mice (Mitfmi/mi). A substitution of arginine 216, in the basic domain, with lysine results in age resolving osteopetrosis in mice in the homozygous condition (Mitfor/or). However, mice that are homozygous recessive for a substitution of isoleucine to asparagine in the basic domain of this transcription factor (Mitfwh/wh) do not exhibit any osteopetrosis. We identified several novel genes regulated by Mitf with potential roles in osteoclast differentiation via microarray analysis of cDNA from WT and Mitfmi/mi osteoclasts. In particular, Eos, HOX11L2, Hematopoietic cell phosphatase (HCP) and p9 were confirmed to be expressed in lower levels in Mitfmi/mi osteoclasts compared to the WT. We also observed that while TRAP mRNA levels were upregulated in Mitfor/or, similar to the levels in WT, Cathepsin ii K levels were lower in both Mitfmi/mi and Mitfor/or osteoclasts. Thus, Mitf-regulated genes could be classified into two groups based on their expression pattern in the Mitfor/or mutant. Osteoclast progenitors shift from a population of actively proliferating cells to that of committed, post-mitotic mononuclear precursor cells prior to becoming multinuclear osteoclasts. We observed that the cytokine, receptor activator of NF-kB ligand (RANKL), induces wildtype (WT) osteoclast progenitors to withdraw from cell cycle within 24 hours of its application. This event coincides with elevation in p27KIP1 (via the p38 MAPK pathway) and p21CIP1 and with decreased CDK2 activity. We also observed that p27KIP1 is required by osteoclast progenitors to exit from the cell cycle in response to RANKL and that p27KIP1-/- osteoclasts express lower levels of TRAP mRNA. However, deficiency in p21CIP1 does not affect any of these phenomena. We also observed that removal of p27KIP1 or p21CIP1 alone does not affect osteoclast differentiation or function, in vitro or in vivo. Osteoclast progenitors from p27KIP1-/-p21CIP1-/- double knockout mice do not withdraw from cell cycle in response to RANKL and express significantly lower levels of TRAP and Cathepsin K mRNA. p27KIP1-/-p21CIP1-/- mice exhibit age resolving osteopetrosis. In addition, precursors from the double mutant mice form fewer multinuclear functional osteoclasts in vitro. These data suggest that that only p27KIP1 has a role in cell cycle withdrawal during osteoclast differentiation while both p21CIP1 and p27KIP1 might have additional, potentially redundant roles, during osteoclast differentiation. iii Dedicated to my parents and to Keith, my best friend .… iv ACKNOWLEDGMENTS I would like to thank my adviser, Dr. Michael C. Ostrowski, for his guidance, support and for the helpful scientific discussions. I would also like to thank Drs. Gustavo W. Leone, Raj Muthusamy and Russell J. Hill, members of my dissertation committee, for their helpful guidance and support. In addition, I would like to thank all the past and present members of the Ostrowski laboratory for their support and discussions. I would also like to thank Dr. Thomas Rosol for his help with the analysis of the osteopetrotic phenotype in the mutant mice. I would also like to thank Drs. Andrew Koff and Ming You for providing us with the p27KIP1 and p21CIP1 knockout mice. Special thanks to the staff at the ULAR facility at the Ohio State University for the help with maintaining the mouse colonies and to Evelyn at the College of Veterinary Biosciences for help with the histological sections. Also, special thanks to Krupen Patel for help with maintaining the mouse colonies and help with the histochemical analysis. Finally, I would like to thank my family for their love and support and my husband Keith for his love and support during my graduate school career. v VITA May 1, 1968 ……………………………… Born, Trivandrum, India 1992 ………………………………………. BSc. (Agriculture), Kerala Agricultural University, India 1997……………………………………….. MS. Department of Horticulture and Crop Sciences, The Ohio State University PUBLICATIONS 1. Mansky, K. C., Sankar, U., Han, J., and Ostrowski, M. C. (2002). Microphthalmia transcription factor is a target of the p38 MAPK pathway in response to receptor activator of NF-kappa B ligand signaling. J Biol Chem 277, 11077-11083. 2. Bayoumi, M., Sankar, U., Muthusamy, N. (2002). Role of macrophage stimulating factor and osteoclast differentiation factor in osteoclastogenesis of bone marrow derived stem cells. Indian Journal of Experimental Biology. 40: 995-1000. 3. U. Sankar, K. D. Simcox, K. R. Davis and R. C. Pratt. (1997). "Detecting interactions of maize cDNA clones with MCDV proteins using the yeast Two- Hybrid system". Proceedings Annual Maize Genetics Conference, 39:87-87. FIELDS OF STUDY Major Field: Molecular, Cellular and Developmental Biology vi TABLE OF CONTENTS Page Abstract …………………...…………………………………………………………………… ii Dedication ……………….………………….………………………………………………… iv Acknowledgments ……...…..………………………………………………………………… v Vita ………………………..…………………………………………………………………… vi List of figures ……………..………….……………………….……………………………… xii List of tables …………….….……….……………………….………………………………. xv Abbreviations …………………………….………….……………………………………… xvi Chapters 1. Introduction ………………………………………………………………….………. 1 1.1 The Bone Tissue …………………………………………………….……… 2 1.1.1 Development of bone tissue ……………………………….……… 3 1.1.2 Structure of the long bone ……………………………….………. .3 1.1.3 Intramembraneous and endochondral ossifications ……………. 6 1.2 Cells in the Bone ……………………..………………….…………………11 1.2.1 Chrondrocytes .………………………………………………..….. 11 1.2.2 Osteoblasts ………….……………………………………………..12 1.2.3 Osteoclasts ………….………………………………….………… 13 1.2.3.1 Morphology of the osteoclast ..………...………………. 14 1.2.3.2 Mechanism of bone resorption by osteoclasts………... 16 1.2.3.2.1 Attachment to bone surface and consequent signaling events…………….……………... 16 1.2.3.2.2 Activation of osteoclasts…………………….... 19 1.2.3.2.3 Bone demineralization and resorption by osteoclasts………….…………………. 22 1.2.3.3 Stimulators and inhibitors of osteoclast activity…..….… 25 1.2.3.3.1 Stimulators of osteoclast activity………....….. 25 1.2.3.3.2 Inhibitors of osteoclast activity……………….. 27 1.2.3.4 Origin and differentiation of osteoclasts………………………………….……………….…….. 29 1.2.3.4.1 Origin of osteoclasts……………………….…..29 vii 1.2.3.4.2 Osteoclast precursors……………….……… 30 1.2.3.4.3 in vivo studies of osteoclast differentiation………………………..….….…………… 33 1.2.3.4.3 in vitro studies of osteoclast differentiation………………………………….……….… 34 1.2.3.5 Osteoclast differentiation and cell cycle arrest…….... 35 1.2.3.6 Molecules involved in osteoclast differentiation……... 37 1.2.3.6.1 M-CSF1……………………………………….. 37 1.2.3.6.2 OPG…………………………………………… 39 1.2.3.6.3 RANKL………………………………………... 39 1.2.3.6.4 RANK……………………………………….…. 41 1.2.3.6.5 TRAFs……………………………………….… 44 1.2.3.6.6 NFκB…………………………………………… 49 1.2.3.6.7 Activation of JNK, p38 and other signaling pathways………………………………….… 49 1.2.3.6.8 TNFa………………………………….……….. 53 1.2.3.7 Transcriptional control of osteoclast differentiation…………………………………………………… 53 1.2.3.8. Markers of osteoclast differentiation …………………. 57 1.2.3.8.1 MITF……………………………………………. 57 1.2.3.8.2 Regulation of Mitf……………………………... 66 1.2.3.8.3 Mitf and osteoclast-specific target gene expression……………………………………….. 68 1.2.3.8.4 Mutations in the Mitf locus………….………... 69 1.2.3.8.5 Osteopetrosis in Mitfmi/mi and Mitfor/or mutants…………………………………………….………. 79 1.3 Conclusion….……………………………...……………………………..… 83 2. Materials and Methods ……………….……………………………………………. 84 2.1 DNA Manipulations……………………………………….…………………84 2.1.1 Agarose gel electrophoresis…………………….……………….. 84 2.1.2 Polymerase chain reaction……………………………….………. 85 2.1.3 Cloning of DNA...………………………………….………………. 86 2.1.3.1 Restriction digests………………………..………..…….. 86 2.1.3.2 Alkaline phosphatase treatment and DNA ligation…… 86 2.1.3.3 Phenol-chloroform extraction and precipitation of DNA using ethanol…………….……….……………………….. 87 2.1.3.4 DNA transformation into E.coli competent cells…….... 88 2.1.3.4.1 Preparation of E.coli competent cells….……..88 2.1.3.4.2 DNA transformation into competent cells…….89 2.1.4 Plasmid mini and maxipreps………………………….…………...89 2.2 Preparation of recombinant RANKL and L-cell media containing CSF1………………………………………………………………………….91 2.2.1 Cloning of soluble RANKL cDNA……..…………………………. 91 2.2.2 Expression of pET32b-RANKL……………..……………………..92 2.2.3 Large scale purification of recombinant soluble RANKL……….93 2.2.4 Preparation of M-CSF1…………………………………………….95 2.3 Mouse lines and genotyping……………………………………………….95