LYSYL OXIDASE REGULATES TRANSFORMING GROWTH FACTOR-ß1 FUNCTION IN BONE Phimon Atsawasuwan A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment

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LYSYL OXIDASE REGULATES TRANSFORMING GROWTH FACTOR-ß1 FUNCTION IN BONE Phimon Atsawasuwan A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the curriculum of School of Dentistry (Oral Biology) Chapel Hill 2008 Approved by: Professor Professor Professor Mitsuo Yamauchi Phillip Trackman Timothy Wright Assistant Professor Yuji Mishina Assistant Professor Yoshiyuki Mochida 2008 Phimon Atsawasuwan ALL RIGHTS RESERVED ii ABSTRACT PHIMON ATSAWASUWAN: Lysyl Oxidase Regulates Transforming Growth Factor-ß1 Function in Bone (under the direction of Mitsuo Yamauchi) Lysyl oxidase (LOX), an amine oxidase critical for the initiation of collagen and elastin cross-linking, has recently been shown to regulate cellular activities possibly by modulating growth factor activity. In this study, we discovered that osteoblastic (MC3T3- E1) cell-derived clones expressing higher (S) levels of LOX exhibited smaller collagen fibrils and lower collagen production than controls (MC, EV) while the clones expressing lower (AS) levels of LOX exhibited larger collagen fibrils and higher amount of collagen leading to subsequent defective mineralization. In order to elucidate the mechanisms by which collagen synthesis is controlled through LOX, we investigated the potential role of LOX in regulating growth factors. We further investigated the interaction of LOX with TGF-ß1, a potent growth factor abundant in bone, and evaluated the effect of this interaction. The specific binding between LOX and TGF-ß1 was demonstrated both by immunoprecipitation and glutathione-s-transferase pull down assay. Both molecules were co-localized in the extracellular matrix in culture and the binding complex was identified in the mineral-associated fraction of bone matrix. Furthermore, LOX suppressed TGF-ß1 induced Smad3 phosphorylation and collagen (I/V) expression but the effects were nullified by ß-aminopropionitrile. The suppression of Smad3 phosphorylation was not affected by the presence of catalase. The data indicate that LOX may bind to mature TGF-ß1 and regulate its signaling via its amine oxidase activity in bone, thus, may play an important role in bone remodeling and mineralization. iii ACKNOWLEDGEMENTS I would like to express my gratitude to all those who gave me the opportunity to complete this dissertation. To graduate PhD study at UNC-Chapel hill is the most prestigious experience. I am so grateful and thankful for every help I received throughout my study. I am deeply indebted to my mentor Prof. Dr. Mitsuo Yamauchi whose help, stimulating suggestions and encouragement helped me in all the time of research and writing of this dissertation. He is a major key person for my doctoral student life from the beginning to the end. He has given me his precious time, guidance and constructive comments. His mentorship was paramount in providing a well-rounded experience consistent with my long-term career goals. He encouraged me to grow not only as a researcher but also as an independent thinker. I am not sure that many graduate students are given the opportunity to develop their own individuality and self-sufficiency by being allowed to work with such independence. He also provided me a financial support throughout my study. I would like to express my sincere gratitude and appreciation to him for giving me this honorable privilege to work in his laboratory and finish my research project. I would like to thank the Ph.D. program in Oral Biology at University of North Carolina at Chapel Hill for giving me a chance to pursue a doctoral degree and Professor Patrick Flood, an Oral Biology Program director, who gave me this chance. iv I would like to thank all members of my doctoral dissertation committee, Professor Timothy Wright, Professor Phillip Trackman, Assistant Professor Yuji Mishina and Assistant Professor Yoshiyuki Mochida for their input, valuable discussion and accessibility. In particular, Dr. Mochida who always gives me invaluable advices, intriguing analogy, and edutainment quizzes. I would like to thank my colleagues in collagen biochemistry laboratory in the past and present, Drs. Parisuthiman, Katafuchi, Pornprasertsuk, Sricholpech, Kaku, Nagaoka, Tokutomi, Kitamura, and Shiiba for their encouragement and friendship. In addition, I would like to express my appreciation to Mrs. Chandlers who passed away many years ago but her friendship is always around. I would like to gratefully and sincerely thank Dr. Si-urai for his guidance, suggestion and encouragement during the down-time in my graduate study at University of North Carolina at Chapel Hill, Dr. and Mrs. Maixner who always offer helps when I need and a group of Thai students at UNC-Chapel hill who accompany me to stay healthy in the badminton courts. Finally, and most importantly, I would like to thank my parents and family in Thailand. Their constant support, love, encouragement, quiet patience and faith in me allow me to be as ambitious as I want and pursue my goal. v TABLE OF CONTENTS Page LIST OF TABLES vii LIST OF FIGURES. viii LIST OF ABBREVIATIONS AND SYMBOLS. x Chapter I. Introduction.. 1 II. Hypothesis.. 36 III. Study I: Lysyl oxidase regulates collagen quality and quantity in osteoblasts Abstract. 38 Introduction.. 39 Experimental procedures Results.. 46 Discussion 55 IV. Study II: Lysyl oxidase regulates transforming growth factor-β1 function in bone via its amine oxidase activity.. 58 Abstract. 59 Introduction.. 60 Experimental procedures Results.. 74 Discussion 92 V. Concluding remarks 96 BIBLIOGRAPHY. 99 vi LIST OF TABLES Table Table 1.1 The various regulators and effects on LOX mrna and enzymatic activity 17 Table 1.2 The comparison of LOX family member.. 21 Table 2.1 The amount of total aldehydes and reducible and, non-reducible cross-links in each clone are expressed as mean+s.d Table 2.2 The fibril density (number fibrils per square micron). from each clone are expressed as mean+s.d Table 3.1 Primers list of constructs used in the study.73 vii LIST OF FIGURES Figure Figure 1.1 Schematic for the biosynthesis of type I collagen. 6 Figure 1.2 The sites and reaction of LOX on collagen molecules.. 7 Figure 1.3 Major cross-linking pathways in type I collagen. 9 Figure 1.4 Pathway for LOX biosynthesis Figure 2.1 The level of LOX protein expression in stable clones and controls Figure 2.2 Cell proliferation rate of S and AS clones Figure 2.3 Amounts of collagen cross-links and their precursor expressed in moles/ mole of collagen at 2 weeks of cultures.. 51 Figure 2.4 Total collagen content from culture matrix and medium at day 3 and 7 of the cultures 51 Figure 2.5 Cross-section of the collagen fibrils in the ECM at 3 weeks of cultures observed under TEM and their diameter Figure 2.6 Distribution of the collagen fibril diameter in the ECM at 3 weeks of cultures observed under TEM and their diameter distribution based on total numbers of 500 fibrils. 53 Figure 2.7 In vitro mineralization assay Figure 3.1 Binding of LOX to TGF-ß1/BMPs. 80 Figure 3.2 LOX constructs and their binding to TGF-ß1 by IP-WB. 81 Figure 3.3 Purity and activity of LOX-V5/His protein 82 Figure 3.4 Direct binding of LOX to TGF-ß Figure 3.5 Co-localization of LOX and TGF-ß1 in a MC cell culture system.. 84 Figure 3.6 Binding of LOX and TGF-ß1 in bone extracellular matrix. 85 Figure 3.7 Effect of LOX overexpression on TGF-ß signaling in osteoblasts.. 86 Figure 3.8 Effect of over/underexpression of LOX on TGF-ß signaling in MC3T3-E1 cells.. 87 viii Figure 3.9 Effect of LOX overexpression on BMP signaling in osteoblasts.. 88 Figure 3.10 Effect of exogenous LOX protein on TGF-ß signaling in osteoblasts.. 89 Figure 3.11 Effect of LOX on TGF-ß induced type I and type V collagen expression in osteoblasts.. 90 Figure 3.12 Effects of LOX suppression on TGF-ß signaling by RNA interference.. 91 ix LIST OF ABBREVIATIONS AND SYMBOLS ACP ALP AS clone Asp Asn ATTC bfgf Bip BMP bp aldol condensation product alkaline phosphatase MC3T3-E1 cells derived clones expressing lower level of LOX aspartic acid asparagine American type culture collection basic fibroblast growth factor binding proteins bone morphogenetic protein basepair(s) C- carboxy- cdna COLI COL3A1 Cu DDW DEAE deh- DHLNL DHNL DTT ECM ER EV FACIT FBS FN Gly complimentary deoxyribonucleic acid type I collagen type III collagen alpha I copper distilled deionized water Diethylaminoethyl dehydrodihydroxylysinonorleucine dihydroxynorleucine dithiothreitol extracellular matrix endoplasmic reticulum empty vector fibril-associated collagen with interrupted triple helices fetal bovine serum fibronectin glycine x Glu GRP GGT GT H 2 O 2 HEK HHL HHMD His HLNL HNL HPLC HRP HSP Hyl Hyl ald Hyp IGF IFN-γ kda LAP LH LLC LOPP LOX LOXdm LOXL LTBP LTQ Lys glutamine glucose-regulated protein galactosylhydroxylysyl glucosyltransferase hydroxylysyl galactosyltransferase hydrogen peroxide human embryonic kidney histidinohydroxylysinonorleucine histidinohydroxymerodesmosine histidine hydroxylysinonorleucine hydroxynorleucine high performance liquid chromatography horseradish peroxidase heat shock protein hydroxylysine hydroxylysine aldehyde hydroxyproline insulin-like growth factor interferon-gamma kilodalton latency-associated peptide lysyl hydroxylase large latent complex lysyl oxidase propeptide lysyl oxidase Lysyl oxidase with Lys314 and Tyr349 mutated lysyl oxidase-like latent TGF-β binding proteins lysyl tyrosylquinone lysine xi Lys ald M Man n lysine aldehyde molar high-manose N-linked oligosaccharide MC, MC3T3-E1 osteoblastic cell line from mouse calvaria mrna mtld mtll messenger ribonucleic acid mammalian tolloid mammalian tolloid-like N- amino- NaB 3 H 4 NF- B NH 3 OPG PAGE PBS PCP PCR PDI PH pi PNP PPI Prl Pro Pyr RANK RANKL res rrg S clone Ser tritiated sodium borohydride nuclear factor-kappa B ammonia osteoprotegerin polyacrylamide gel electrophoresis phosphate buffered saline procollagen C-proteinase polymerase chain reaction protein disulfide isomerase prolyl hydroxylase Isoelectric point procollagen N-proteinase peptidyl-prolyl cis-trans isomerase pyrrole proline pyridinoline receptor/activator of NF- B receptor/activator of NF- B ligand residue ras recision gene MC3T3-E1 cells derived clones expressing higher level of LOX serine xii SLC Smad SRCR small latent complex small mothers against decapentaplegic protein scavenger receptor cysteine-rich region TGF-ß1 transforming growth factor-beta 1 TNF-α -MEM ßAPN α ß γ m n tumor necrosis factor-alpha alpha minimum essential medium ß-aminopropionitrile alpha beta delta gamma epsilon micro milli nano +/+ homozygous wildtype +/- heterozygous -/- homozygous deficiency C D E G L K P Q R S W Y cysteine aspartic acid glutamic acid glycine leucine lysine proline glutamine arginine serine tryptophan tyrosine xiii CHAPTER I Introduction Biology of bone Bone is a specialized form of connective tissue in vertebrates. It serves both mechanical and metabolic functions and is composed of two components, cellular and matrix components. The cellular components include bone lining cells, osteoblasts, osteocytes and osteoclasts while its matrix components contain organic and inorganic components (1, 2). Morphologically, bone is characterized either as cortical (compact) or as cancellous (spongy, trabecular) bone. Functionally, cortical bone provides mechanical resistance and strength while cancellous bone serves for mineral homeostasis and mechanical strength. The bone homeostatic events include bone formation, resorption and remodeling. Cellular components of bone The cellular components of bone are osteoblasts, osteocytes, bone lining cells and osteoclasts (1, 3). Osteoblasts, osteocytes and bone lining cells are derived from mesenchymal stem cells known as osteoprogenitor cells, whereas osteoclasts are derived from hematopoietic stem cells. Osteoblasts, osteocytes and bone lining cells are located along the surface of bone while osteocytes are located in lacuna inside the bone matrix (2, 3). Osteoblasts are derived from undifferentiated mesenchymal cells or preosteoblasts that are located in the bone marrow, endosteum and periosteum (see review in (4)). Bone lining cells cover most surfaces in the mature bone. They are inactive and sometimes called resting osteoblasts (1, 3). The third cell type, osteocyte, is estimated to make up more than 90% of the bone cells in an adult bone. Immature osteocytes are surrounded in shallow bone matrix and closely resemble osteoblasts. As these cells mature and more matrices are laid down, they become located deeper within the bone matrix and lose their cytoplasm. They are located within a space or lacuna and have long cytoplasmic processes that project through canaliculi within the matrix and that contact processes of adjacent cells. The processes are thought to be for cellular communication and nutrition within a mineralized matrix (1, 2, 5). The fourth cellular component is the osteoclast. It is a multinucleated giant cell responsible for bone resorption under normal and pathological conditions. It contains many lysosomal vacuoles exhibiting the common description of the foamy cytoplasm. The plasma membrane of the active osteoclast has an infolded appearance known as a ruffled border. It works as a seal to create microenvironment for bone resorption (3, 6). For the development of osteoclast, crosstalk between osteoblasts and osteoclasts must exist to coordinate the process of bone formation and resorption. Osteoprotegerin (OPG) was discovered to be secreted by osteoblasts and acts as a soluble competitive binding partner for RANKL (receptor/activator of NF- B ligand), which inhibits osteoclast formation and consequently bone resorption. Both OPG and RNAKL can bind to RANK (receptor/activator of NF- B); a transmembranous receptor expressed on osteoclast precursor cells. Interaction between RANKL and RANK initiates a signaling and gene expression cascade resulting in the promotion of osteoclast formation from the precursor pool (7-9). 2 Extracellular components of bone The extracellular matrix (ECM) in bone is composed of organic and inorganic components. The organic matrix accounts for approximately 35% of the total weight of bone tissue compared with 65% for the inorganic part (3). The inorganic component is generally referred to as hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ), a plate-like crystal nm in length and 2-5 nm thick. Because bone apatite is four times smaller than naturally occurring apatites and less perfect in structure, it is more reactive and soluble and facilitates chemical turnover (10). The freshly synthesized matrix prior to its mineralization, osteoid, consists primarily (approximately 94%) of collagen type I and is secreted by osteoblasts. Major non-collagenous proteins in bone consist of proteoglycans. In addition to their role in defining the spatial organization of the ECM, type I collagen interacts with growth factors during the development (11). Osteocalcin, osteopontin, osteonectin and matrix-gla protein play roles during the mineralization process (12, 13). Other proteins such as bone morphogenic proteins, growth factors, cytokines, and adhesion molecules also play roles in bone homeostasis (11). Collagens in bone Collagens are a large family of structurally related proteins that assemble in the ECM and contain one or more domain(s) of unique triple helices (collagenous domain). This collagenous domain, the hallmark of these proteins, is a coiled-coil right-handed triple helix composed of three polypeptide chains, called chains. The non-triple helical domains are called non-collagenous domains. Each chain in the molecule is coiled into an extended left-handed polyproline II-type helix and then the three left-handed helical 3 chains are intertwined to one another and folded into a ropelike right-handed triple helix structure (14). The triple helical structure is stabilized by the high content of imino acids, i.e. proline (Pro) and hydroxyproline (Hyp) and the presence of Hyp is essential for interchain hydrogen bonds that further stabilize the triple helical structure. Collagen molecules consist of the repetitive sequences of amino acids [Gly(glycine)-XY] n (X and Y can be any amino acid but often X is Pro and Y Hyp) are required. Every third amino acid is situated in the center of the triple helix in a very restricted space where only Gly, the smallest amino acid, can fit (15). Collagen is the most abundant protein in vertebrates accounting for about 30% of the body s total proteins and is present in essentially all tissues and organs of the body. The collagen superfamily consists of 27 different genetic types and these distinct types of collagen show marked diversity and complexity in the structure, their biological function and tissue distribution. The collagen superfamily can be divided roughly into 3 groups: fibril forming (type I, II, III, V, XI, XXIV and XXVII), fibril-associated collagen with interrupted triple helices (FACIT) (type IX, XII, XIV, XVI, XIX, XX, XXI, XXII and XXVI), and non-fibril forming (type IV, VI, VII, VIII, X, XIII, XV, XVII, XVIII, XXIII, XXV and XXVI)(15-19). In bone, type I collagen is the most predominant type of collagen and type V collagen is present as a minor type. Type I collagen is a heterotrimeric molecule composed of two 1 chains and one 2 chain, [ 1(I)] 2 2(I), although a homotrimeric form of 1 chains [ 1(I)] 3, does exist as a minor form. The precursor molecule called tropocollagen consists of three domains: the NH 2 - terminal nontriple helical (N-telopeptide), the central triple helical and the COOH-terminal nontriple helical (C-telopeptide) domains. The procollagen is secreted outside the cell as its precursor form. Proteolytic cleavage of the propeptides results in mature collagen molecules and can assemble into fibrils (20-22). The biosynthesis of procollagen is a complex process in which several enzymes and molecular chaperones assist its folding 4 and trimerization (23, 24). A number of posttranslational modifications occur both at intra- and extracellular locations. Protein disulphide isomerase (PDI) induces the formation of inter- and intrachain disulphide bonds within the C-propeptide, allowing the association between procollagen chains (25, 26). The C-propeptide ensures the association between monomeric and heteromeric procollagen chains. Newly synthesized procollagen chains are associated in trimers through their C-propeptides, leading to nucleation and folding in a C-to-N direction to form a triple helix. The biosynthesis of procollagen involves different posttranslational modifications that occur in the endoplasmic reticulum: peptidylproline cis-trans isomerase is required to convert the proline residues to the trans form (27, 28), and prolyl 4-hydroxylase is required to convert proline into hydroxyproline residues (29, 30). The family of lysyl hydroxylase (LH) contributes to the formation of hydroxylysine, which specific residues at telopeptide can subsequently be further modified by lysyl oxidases (LOXs). The collagen chaperone heat shock protein (HSP) 47 is also required for the folding of the collagen (31, 32). All of the enzymes responsible for these modifications work in a coordinated fashion to ensure the folding and assembly of a correctly aligned and thermally stable triple-helical molecule. During the secretion of these molecules into the ECM, propeptides are removed by procollagen N- and C- proteinases (NCP and PCP), thereby allowing spontaneous self-assembly of collagen molecules into fibrils (33). Finally, the triplehelical structure is stabilized by an important posttranslational modification that allows intra/intermolecular cross-links to take place as a result of the catalysis of lysyl oxidase (LOX), which oxidizes the specific lysine (Lys) and hydroxylysine (Hyl) residues at
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