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Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, Ninth Edition. Edited by John P. Bilezikian.
© 2019 American Society for Bone and Mineral Research. Published 2019 by John Wiley & Sons, Inc.
Companion website: www.wiley.com/go/asbmrprimer
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INTRODUCTION
Bone comprises the largest proportion of the body’s con- nective tissue mass. Unlike most other connective tissue matrices, bone matrix is physiologically mineralized and is unique in that it is constantly regenerated throughout life as a consequence of bone turnover. Bone as an organ is made up of the cartilaginous joints, the calcified cartilage in the growth plate (in developing individuals), the mar- row space, and the cortical and cancellous mineralized structures. Bone as a tissue consists of mineralized and nonmineralized (osteoid) components of the cortical and cancellous regions of long and flat bones. There are three cell types on (and in) bone tissue: (i) the bone‐forming osteoblasts, which when engulfed in mineral become (ii) osteocytes; and (iii) the bone‐destroying osteoclasts.
Each of these cells communicates with one another by either direct cell contact or through signaling molecules, and respond to each other. The detailed properties of these cells have been discussed in numerous publications; see for example the review by Teti [1] or the text by Bilezikian and colleagues [2]. This chapter focuses on the extracellu- lar matrix, which is synthesized primarily by osteoblasts and osteocytes but also contains proteins adsorbed from the circulation. The preponderance of bone is mineral and extracellular matrix (ECM). Information on the gene and protein structure and potential function of bone ECM constituents has exploded during the last few decades.
This information has been described in great detail in several recent texts [2,3], to which the reader is referred for specific references, which are too numerous to be listed
adequately here. This chapter summarizes the composi- tion of bone and the salient features of the classes of bone matrix proteins. The tables list specific details for the individual ECM components.
Bone as a Composite 85
mineral have been determined by a variety of techniques including chemical analyses, X‐ray diffraction, vibrational spectroscopy, energy dispersive electron analysis, nuclear magnetic resonance, small angle scattering, and transmis- sion and atomic force microscopy [8].
The functions of the mineral are to strengthen the col- lagen composite, providing more mechanical resistance to the tissue, and also to serve as a source of calcium, phosphate, and magnesium ions for mineral homeostasis.
For physicochemical reasons, usually it is the smallest mineral crystals that are lost during remodeling. Thus, in osteoporosis, it is not surprising that the larger, more perfect crystals persist within the matrix [9] contributing to the brittle nature of osteoporotic bone. When remode- ling is impaired, as in osteopetrosis, the mineral crystals remain small relative to age‐matched controls [9].
Collagen
The basic building block of the bone matrix fiber network is type I collagen, which is a triple helical molecule con- taining two identical αl(1) chains and a structurally simi- lar, but genetically different, αl(2) chain [2]. Collagen α chains are characterized by a Gly‐X‐Y repeating triplet (where X is usually proline, and Y is often hydroxyproline) and by several posttranslational modifications including:
(i) hydroxylation of certain lysyl or prolyl residues; (ii) gly- cosylation of the hydroxylysine with glucose or galactose residues or both; (iii) addition of mannose at the propep- tide termini; and (iv) formation of intra‐ and intermolecu- lar covalent cross‐links that differ from those found in soft connective tissues. Measurement of these bone‐
derived collagen cross‐links in urine has proven to be a
good measure of bone resorption [10]. Bone matrix proper consists predominantly of type I collagen; however, trace amounts of type III and V and fibril‐associated collagens (Table 11.1) may be present during certain stages of bone formation and may regulate collagen fibril diameter.
Noncollagenous proteins
Noncollagenous proteins (NCPs) compose 10% to 15% of the total bone protein content. NCPs are multifunctional, having roles in organizing the ECM, coordinating cell‐
matrix and mineral‐matrix interactions, and regulating the mineralization process. The multifunctionality can be attributed to their protein structure, as a large number of these proteins are intrinsically disordered (mostly random coils) and thus can bind to many partners [11]. Knowledge of their specific functions has come from studies of the iso- lated proteins in solution, from analyses of mice in which the proteins are ablated (knocked out [KO]), or overex- pressed, characterization of human diseases in which these proteins have mutations, and studies using appropriate cell cultures. Tables 11.2–11.7 summarize the gene and protein structures, and the functions of these protein families.
Serum‐derived proteins
Approximately one‐fourth of the total NCP content is exogenously derived (Table 11.2). This fraction is largely composed of serum‐derived proteins, such as albumin and α2‐HS‐glycoprotein, which are acidic in character and bind to bone matrix because of their affinity for hydroxyapatite. Although these proteins are not endoge- nously synthesized, they may exert effects on matrix Table 11.1. Characteristics of collagen‐related genes and proteins found in bone matrix.
Protein/Gene Function Disease/Animal Model/Phenotype
Type I: 17q21.23, 7q21.3‐22 [α1(I)2α2(I)]
[α1(I)3]
Serves as scaffolding, binds and orients other proteins that nucleate hydroxyapatite deposition
Human mutations: osteogenesis imperfecta
(OMIM #166210, 166200, 610854, 259420, 166220) Mouse models: oim mouse; mov 13 mouse; brittle
mouse; Amish mouse; bones hypermineralized and mechanically weak, mineral crystals small, some mineral outside collagen
Type X: 6q21‐22.3
[α1(X)3] Present in hypertrophic cartilage of the growth plate, but does not appear to regulate matrix mineralization
Human mutations: Schmid metaphyseal chondrodysplasia (OMIM #120110)
Knockout mouse: no apparent skeletal phenotype Type III: 2q24.3‐31
[α1(III)]3 Present in bone in trace amounts, may regulate collagen fibril diameter, its paucity in bone may explain the large diameter size of bone collagen fibrils
Human mutations in type III: different forms of vascular Ehlers‐Danlos syndrome and abnormal collagen type I folding (OMIM #130050)
Mouse model: disrupted trabecular bone formation Type V: 9q34.2‐
34.3;2q24.3‐31, 9q34.2‐34.3 [α1(V)2α2(V)]
[α1(V)α2(V)α3(V)]
Present in bone in trace amounts, may regulate collagen fibril diameter, their paucity in bone may explain the large diameter size of bone collagen fibrils
Mutations in type V α1 or α2 (OMIM #120215, 120190) Mouse model: disrupted fibril arrangement
mineralization and bone cell proliferation. For example, α2‐HS‐glycoprotein, the human analog of fetuin, when ablated in mice causes ectopic calcification [12] suggest- ing that the protein is a mineralization inhibitor. The remainder of the exogenous fraction is composed of growth factors and a large variety of other molecules pre- sent in trace amounts, which influence local bone cell activity [1,2].
On a mole‐to‐mole basis, bone‐forming cells synthe- size and secrete as many molecules of NCPs as of colla- gen. These molecules can be classified into four general (and sometimes overlapping) groups: (i) proteoglycans; (ii) glycosylated proteins; (iii) glycosylated proteins with potential cell attachment activities; and (iv) γ‐carboxy- lated (gla) proteins. The physiological roles for individual bone protein constituents are not well defined; however, they may participate not only in regulating the deposi- tion of mineral but also in the control of osteoblastic and osteoclastic metabolism.
Proteoglycans
Proteoglycans are macromolecules that contain acidic polysaccharide side chains (glycosaminoglycans) attached to a central core protein. Bone matrix contains several members of this family [2] (Table 11.3).
During initial stages of bone formation, the large chon- droitin sulfate proteoglycan, versican, and the glycosamino- glycan, hyaluronan (which is not attached to a protein core), are highly expressed and may delineate areas that will become bone. With continued osteogenesis, versican is replaced by two small chondroitin sulfate proteoglycans, decorin and biglycan, composed of tandem repeats of a leu- cine‐rich repeat (LRR) sequence. Decorin has been impli- cated in the regulation of collagen fibrillogenesis and is distributed predominantly in the ECM space of connective tissues and in bone, whereas biglycan tends to be found in pericellular locales. A heparan sulfate proteoglycan, perle- can, is involved in limb patterning, and is found surround- ing chondrocytes in the growth plate, whereas the glypican family of cell surface‐associated heparan sulfate proteogly- cans also affect skeletal growth. In addition, there are other small leucine‐rich proteoglycans (SLRPs) in bone [13],
including osteoglycin (mimecan),keratocan, osteoadherin, lumican, asporin, and fibromodulin. Although their exact physiological functions are not known, these proteoglycans are assumed to be important for the integrity of most con- nective tissue matrices. Deletion of the biglycan gene, for example, leads to a significant decrease in the development of trabecular bone, indicating that it is a positive regulator of bone formation. Deletion of the epiphican gene, or the epiphican and biglycan genes together, causes shortening of the femur during growth and early onset osteoarthritis [14].
Other functions might arise from the ability of these pro- teoglycans to bind and modulate the activity of the growth factors in the extracellular space, thereby influencing cell proliferation and differentiation [1].
Glycosylated proteins
Glycosylated proteins with diverse functions abound in bone. One of the hallmarks of bone formation is the synthe- sis of high levels of alkaline phosphatase (Table 11.4).
Alkaline phosphatase, a glycoprotein enzyme, is primar- ily bound to the cell surface through a phosphoinositol linkage, but is cleaved from the cell surface and found within mineralized matrix. The function of alkaline phosphatase in bone cell biology has been the matter of much speculation and remains undefined. Mice lacking tissue nonspecific alkaline phosphatase have impaired mineralization, suggesting the importance of this enzyme for mineral deposition [15].
The most abundant NCP produced by bone cells is osteonectin [16,17], a phosphorylated glycoprotein accounting for ~2% of the total protein of developing bone in most animal species. Osteonectin is transiently produced in nonbone tissues that are rapidly proliferat- ing, remodeling, or undergoing profound changes in tissue architecture, and is also found constitutively expressed in certain types of epithelial cells, cells associated with the skeleton, and in platelets. Osteonectin, along with thrombospondin‐2 (TSP‐2) and periostin are members of the class of “matricellular proteins,” each of which has a role in bone cell proliferation and dif ferentiation;
with some role in regulating mineralization. Tetranectin (which is important for wound healing), tenascin Table 11.2. Gene and protein characteristics of serum proteins found in bone matrix.
Protein/Gene Function Disease/Animal
Model/Phenotype Albumin: 2q11‐13
69 kDa, nonglycosylated, one sulfhydryl, 17 disulfide bonds, high affinity hydrophobic binding pocket
Inhibits hydroxyapatite crystal growth
α2HS glycoprotein: 3q27‐29
Precursor protein of fetuin, cleaved to form A and B chains that are disulfide linked, Ala‐Ala and Pro‐Pro repeat sequences, N‐linked oligosaccharides, cystatin‐like domains
Promotes endocytosis, has opsonic
properties, chemoattractant for monocytic cells, bovine analog (fetuin) is a growth factor; inhibits calcification
Knockout mouse:
adult ectopic calcification
Table 11.3. Gene and protein characteristics: glycosaminoglycan‐containing molecules in bone.
Protein/Gene Function Disease/Animal Model/Phenotype
Aggrecan: 15q26.1
~2.5 × 106 intact protein,
~180–370,000 kDa core,
~100 CS chains of 25 kDa, and some KS chains of similar size, G1, G2, and G3 globular domains with
hyaluronan‐binding sites, EGF and CRP‐like sequences
Matrix organization, retention of water and ions, resilience to mechanical forces
Human mutation: spondyloepiphyseal dysplasia (OMIM #155760, 608361) and premature growth cessation (OMIM #165800) Mouse models: brachymorphic mouse;
accelerated growth plate calcification, cartilage matrix deficiency, shortened stature Nanomelic chick (mutation): abnormal
bone shape Versican (PG‐100): 5q12‐14
~1 × 106 intact protein, ~360 kDa core,
~12 CS chains of 45 kDa, G1 and G3 globular domains with hyaluronan binding sites, EGF and CRP‐like sequences
Regulates chondrogenesis; may
“capture” space that is destined to become bone
Human mutation: Wagner syndrome (an ocular disorder) (OMIM #143200)
Decorin (class 1 LRR): 12q21.33
~130 kDa intact protein, ~38–45 kDa core with 10 leucine‐rich repeat sequences, 1 CS chain of 40 kDa
Binds to collagen and may regulate fibril diameter, binds to TGF‐β and may modulate activity, inhibits cell attachment to fibronectin
Human mutation: congenital stromal corneal dystrophy (OMIM #610048)
Mouse knockout: no apparent skeletal phenotype although collagen fibrils are abnormal; Decorin (DCN)/Biglycan (BGN) double knockout – progeroid form of Ehlers‐Danlos syndrome
Biglycan (class 1 LRR): Xq27
~270 kDa intact protein, ~38–45 kDa core protein with 12 leucine‐rich repeat sequences, 2 CS chains of 40 kDa
Binds to collagen, TGF‐β, and other growth factors;
pericellular environment, a genetic determinant of peak bone mass
Human mutation: thoracic aortic aneurysms and dissections (OMIM #615291)
Knockout mouse: osteopenia; thin bones, decreased mineral content, increased crystal size; short stature
Asporin (class 1 LRR): 9q22.31
67 kDa, most likely few GAG chains Regulates collagen structure Human polymorphisms: associated with osteoarthritis (OMIM #608135) and intervertebral disk degeneration (OMIM #603932)
Fibromodulin (class 2 LRR): 1q32.1 59 kDa intact protein, 42 kDa core
protein, one N‐linked KS chain
Binds to collagen, may regulate
fibril formation, binds to TGF‐β Mouse model: fewer tendon fibril bundles Fmod/Bgn double knockout mice: joint laxity
and formation of supernumery sesmoid bones
Osteoadherin (class 2 LRR)/
osteomodulin
85 kDa intact protein, 47 kDa core protein, rich in KS, RGD sequence
Expression restricted to mineralized tissues, may mediate cell attachment and play a role in endochondral bone formation
Lumican (class 2 LRR): 12q21.33 70–80 kDa intact protein, 37 kDa core
proetin
Binds to collagen, may regulate
fibril formation and growth Lum/Fmod double knockout mouse: ectopic calcification and a variant of Ehlers‐Danlos syndrome (OMIM #130000)
Perlecan: 1p36.12
Five domain heparan sulfate proteoglycan, core protein 400 kDa
Interacts with matrix components to regulate cell signaling; cephalic
development
Human mutations: short stature, dystrophy of epiphyseal cartilage; dyssegmental dysplasia, Silverman‐Handmaker type Transgenic mice with mutated perlecan:
Schwartz‐Jampel syndrome (OMIM
#142461); mice have impaired
mineralization and misshapen skeletons and joint abnormalities
Knockout mice: phenotype resembling thanatophoric dysplasia (TD) type I (OMIM #187600)
Glypican: Xq26
Lipid‐linked heparan sulfate
proteoglycan, 14 conserved cysteine residues
Regulates BMP‐SMAD signaling; regulates cell development
Human mutation: Simpson‐Golabi‐Behmel syndrome (OMIM #300037)
Knockout mouse: delayed endochondral ossification, impaired osteoclast development Osteoglycin/mimecan (class 3
LRR): 9q22
299 aa precursor, 105 aa mature protein, no GAG in bone, keratan sulfate in other tissues
Binds to TGF‐β, regulates collagen fibrillogenesis
Hyaluronan: multi‐gene complex Multiple proteins associated outside of
the cell, structure unknown
May work with versican molecule to capture space destined to become bone CRP = C‐reactive protein; EGF = epidermal growth factor; TGF = transforming growth factor.
(which regulates organization of ECM) [18], and secreted phosphoprotein 24 (which regulates bone morphogenetic protein expression) along with periostin [19] are other gly- coproteins found in the bone matrix.
Small integrin‐binding ligand, N‐glycosylated proteins, and other glycoproteins with cell attachment activity All connective tissue cells interact with their extracel- lular environment in response to stimuli that direct or coordinate (or both) specific cell functions, such as migration, proliferation, and differentiation (Tables 11.5 and 11.6). These particular interactions involve cell attachment through transient or stable focal adhesions to extracellular macromolecules, which are mediated by cell surface receptors that subsequently transduce intra- cellular signals. Bone cells synthesize at least 12 proteins that may mediate cell attachment: members of the small integrin‐binding ligand, N‐glycosylated protein (SIBLING) family (osteopontin [OPN], bone sialoprotein, dentin matrix protein‐1, dentin sialophosphoprotein,
and matrix extracellular phosphoprotein [MEPE]), type I collagen, fibronectin, thrombospondin(s) (predominantly TSP‐2 with lower levels of TSP‐1, ‐3, and ‐4 and cartilage oligomeric matrix protein [COMP]), vitronectin, fibril- lin, BAG‐75, and osteoadherin (which is also a proteogly- can). Many of these proteins are phosphorylated and/or sulfated, and all contain RGD (Arg‐Gly‐Asn), the cell attachment consensus sequence that binds to the integ- rin class of cell surface molecules. However, in some cases, cell attachment seems to be independent of RGD, indicating the presence of other sequences or mechanisms of cell attachment [2]. Thrombospondin(s), fibronectin, vitronectin, fibrillin, and osteopontin are expressed in many tissues. Certain types of epithelial cells synthesize bone sialoprotein, and it is highly enriched in bone and is expressed by hypertrophic chon- drocytes, osteoblasts, osteocytes, and osteoclasts. In bone, the expression of bone sialoprotein correlates with the appearance of mineral [20]. The bone sialoprotein knockout at an early age has impaired new bone forma- tion, while adult knockout mice have shorter stature, Table 11.4. Gene and protein characteristics of glycoproteins in bone matrix.
Protein/Gene Function Disease/Animal Models/Phenotype
Alkaline phosphatase (bone‐liver‐kidney isozyme): 1p34‐36.1
Two identical subunits of ~80 kDa, disulfide bonded, tissue‐specific posttranslational modifications
Potential Ca2+ carrier, hydrolyzes inhibitors of mineral deposition such as pyrophosphates, increases local phosphate concentration
Human mutations: hypophosphatasia (OMIM #171760) (decreased activity) TNAP knockout mouse: growth
impaired; decreased mineralization Osteonectin: 5q31.3‐q32
~35–45 kDa, intramolecular disulfide bonds, α helical amino terminus with multiple low‐affinity Ca2+ binding sites, two EF hand high‐affinity Ca2+ sites, ovomucoid homology, glycosylated, phosphorylated, tissue‐specific modifications
Regulates collagen organization;
may mediate deposition of hydroxyapatite, binds to growth factors, may influence cell cycle, positive regulator of bone formation
Knockout mouse: severe osteopenia, decreased trabecular connectivity;
decreased mineral content;
increased crystal size
Tetranectin: 3p22‐p21.3
21 kDa protein composed of four identical subunits of 5.8 kDa, sequence homologies with a sialoprotein receptor and G3 domain of aggrecan
Binds to plasminogen, may regulate
matrix mineralization Knockout mouse: no long bone phenotype, spinal deformity (kyphosis, increased curvature of the thoracic spine), increased
mineralization in implant model Tenascin‐C: 9q33.1
Hexameric structure, six identical chaines of 320 kDA, Cys rich, EGF‐like repeats, FN type III repeats
Interferes with cell–‐FN
interactions Knockout mouse: no apparent skeletal phenotype
Tenascin‐X: 6p21.33
Hexameric with 5 N‐linked glycosylation sites and multiple EGF and 40 FN type III repeats
Regulates cell‐matrix interactions Human mutation and knockout mouse: Ehlers‐Danlos II phenotype with hyperextensible skin, hypermobile joints, and tissue fragility (OMIM #600985) Secreted phosphoprotein 2: 2q37.1
24‐kDa secreted phosphoprotein, shares sequence homology with members of the cystatin family of thiol protease
inhibitors
Associates with regulators of mineralization in serum, may regulate thiol proteases in bone, may have a role in inhibiting calcification
EGF = epidermal growth factor; FN = fibronectin;
Table 11.5. Gene and protein characteristics of SIBLINGs (small integrin‐binding ligands, N‐glycosylated proteins).
Protein/Gene Function Disease/Animal Models/Phenotype
Osteopontin: 4q21
~44–75 kDa, polyaspartyl stretches, no disulfide bonds, glycosylated, phosphorylated, RGD located 2/3 from the N terminal
Binds to cells, may regulate mineralization, may regulate proliferation, inhibits nitric oxide synthase, may regulate resistance to viral infection
Knockout mouse: decreased crystal size; increased mineral content; not subject to osteoclastic remodeling
Bone sialoprotein: 4q21
~46–75 kDa, polyglutamyl stretches, no disulfide bonds, 50%
carbohydrate, tyrosine‐sulfated, RGD near the C terminus
Binds to cells, may initiate mineralization, regulates turnover
Knockout mouse: delayed endochondral ossification, adults have shorter stature, lower bone turnover and higher trabecular bone mass DMP‐1: 4q21
513 amino acids predicted; serine‐
rich, acidic, RGD 2/3 from N terminus
Regulator of biomineralization;
regulates osteocyte function Human mutations: dentinogenesis imperfecta and hypophosphatemia (OMIM #600980)
Knockout mouse: undermineralized with craniofacial and growth plate abnormalities and defective osteocyte function
Dentin sialophosphoprotein: 4q21.3 Gene produces three proteins, dentin
sialoprotein, dentin phosphophoryn, and dentin glycoprotein. All have RGD sites;
dentin phosphophoryn is highly phosphorylated
Regulation of biomineralization Human mutations: dentinal dysplasias and dentinogenesis imperfecta; no bone disease (OMIM #125485) Knockout mouse: thinner bones at 9
months, no significant other bone phenotype, and severe dentin abnormalities
MEPE: 4q21.1
525 amino acids, two N‐glycosylation motifs, a glycosaminoglycan‐
attachment site, an RGD cell‐
attachment motif, and phosphorylation motifs
Regulation of biomineralization;
regulation of PHEX (phosphaturic hormone) activity
Humans: association with oncogenic osteomalacia
Knockout mouse: increased bone mass and resistance to ovariectomy‐
induced bone loss
Table 11.6. Gene and protein characteristics of other RGD‐containing glycoproteins.
Protein/Gene Function Disease/Animal Models
Thrombospondins (1‐4, COMP): 15Q‐1, 6q27, 1q21‐24, 5q13, 19p13.1
~450 kDa molecules, three identical disulfide linked subunits of
~150–180 kDa, homologies to fibrinogen, properdin, EGF, collagen, von Willebrand, Plasmodium falciparum and calmodulin, RGD at the C terminal globular domain
Cell attachment (but usually not spreading), binds to heparin, platelets, types I and V collagens, thrombin, fibrinogen, laminin, plasminogen and plasminogen activator inhibitor, histidine‐rich glycoprotein
Human COMP mutation:
pseudoachondroplasia (OMIM
#600310)
TSP‐2 knockout mouse: large collagen fibrils, thickened bones;
spinal deformities
Fibronectin: 2q34
~400 kDa with two nonidentical subunits of ~200 kDa, composed of type I, II, and III repeats, RGD in the 11th type III repeat 2/3 from N terminus
Binds to cells, fibrin, heparin,
gelatin, collagen Knockout mouse: lethal prior to skeletal development
Vitronectin: 17q11
~70 kDa, RGD close to N terminus, homology to somatomedin B, rich in cysteines, sulfated, phosphorylated
Cell attachment protein, binds to collagen, plasminogen, and plasminogen activator inhibitor, and to heparin
Fibrillin 1 and 2: 15q21.1, 5q23‐q31 350 kDa, EGF‐like domains, RGD,
cysteine motifs
May regulate elastic fiber formation Human fibrillin 1 mutations: Marfan syndrome (OMIM #134797) Human fibrillin 2 mutations:
congenital contractural
arachnodactyly (OMIM #121050).
EGF = epidermal growth factor.
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