Matrix proteins of the skeleton

Matrix . proteins

of the skeleton

S.M. Seyedin and D.M. Rosen* Matrix

Biosystems

and *Celtrix

Current

Opinion

Laboratories, in Cell

Biology

Introduction

914

USA

2:914-919

matrix

Collagen makes up more than 90% of the organic matrix of bone and normal bone tissue is made up principally of all type I collagen. Fibrillar collagen is produced by boneforming cells called osteoblasts. Type I collagen exists as a triple helical molecule composed of two al chains and one a2 chain. These molecules are synthesized as propeptides and assembled into helical structures intracellularly, and then into thick fibrils extracellularly. Type I collagen is synthesized by a number of different cell

BMP-bone

1990,

California,

types and has been found in a number of tissues besides bone, most notably skin. Following the biosynthesis and secretion of collagen, stabilization of the newly assembled collagen fibrils is accomplished extracellularly by a complex series of spontaneous reactions that are initiated by a single enzymatic step; oxidation by lysyl oxidase of specific lysine or hydroxylysine residues situated within the collagen molecules (Eyre et al, Annu Rev Biochem 1984, 53717-748). The aldehydes produced react with the s-amino groups of lysine or hydroxylysine residues on adjacent molecules to form intermolecular, bifunctional crosslinks (Fig. 1). Whether the oxidized residues are derived from lysine or hydroxylysine residues results in important tissue-specific crosslinks (Fig. 1). Analysis of the tissue concentration of these pyridinium crosslinks has revealed that cartilage contains exclusively pyridinoline (Pyd) with negligible amounts of its analogue, deoxypyridinoline (Dpd). The amount of Pyd in bone is higher than in any other tissue, accounting for up to 80% of the crosslinks produced by lysyl ox&se. Bone collagen contains, in addition to Pyd, sign&ant amounts of Dpd (Robins and Duncan, B&him Biopbys Acta 1987, 914:233239; Eyre et al, Biocbem J 1988, 252:495-500). With the exception of dentin, Dpd formation appears to be highly specific to bone collagen. This type and pattern of crosslinking makes the bone collagen fibers highly in soluble. However, like other bone matrix proteins, collagen is turned over in the process of bone resorption by osteoclasts and replaced by newly synthesized collagen (produced by osteoblasts). Recent data have suggested that excretion of pyridinium crosslinks may be an index of bone resorption [ 11.

The skeleton is a complex system composed of independent components that together perform a number of lifesupporting functions. These include physical support, aid in locomotion, protection of vital organs, and the storage and release of many organic and inorganic substances. Hard tissue matrices are composed of three readily identiIiable phases. First is the macromolecular structure of the extracellular organic matrix, which is comprised principally of various structural protein components. Second is the inorganic phase containing calcium phosphate crystals (primarily hydroxyapatite) which permeates into the organic matrix. Third are the cellular components which include the nerves and vascular elements that provide nutrients, as well as the bone-specific supporting cells responsible for metabolic activity. These cells include osteoblasts, osteocytes, osteoclasts and their respective precursors. Bone is a highly active tissue which remodels and repairs itself throughout life. Remodelling is a continuous turnover process that results in complete removal and replacement of bone cells and the extracellular matrix components of bone. Bone formation and resorption is highly regulated and coupled. Bone remodelling is regulated by systemic hormones and by local factors which affect osteoblast and osteoclast proliferation and differentiation. Major advances have been made in the last year in studying the structures and biological activities of proteins associated with the organic matrix of bone and these will be reviewed in this article.

Bone collagen

Palo Alto,

morphogenetic OIF-osteoinductive

protein;

W-cartilage-inducing factor; OP-osteogenic

@

Current

Non-collagen

in bone

tie-dimensional electrophoretic analysis of the noncollagen proteins of bone suggests that there are at least 200 such proteins (Delmas et al. Gzkif Tiss Int 1984, 36:30&316). Some of them have been identified as originating from sources other than bone. The high affiity of hydroxyiapatite for many proteins presumably allows adsorption from blood which results in a concentration

Abbreviations factor; Dpd-deoxypyridinoline; protein; Pybpyridinoline;

Biology

proteins

Ltd

ISSN

0955-0674

Cla--y-carboxylated TCF-transforming growth

glutamyl factor.

residues;

Matrix

proteins

Cartilage, bone ttelopeptide hydroxylysine)

Skin (tendon) (telopeptide

Seyedin and Rosen

of the skeleton

lysine)

Aldol (intramolecular cross link) n r!J?

from

the helix

Tri- and tetrafunctional crosslinks with

after reduction borohydride

P - (CH,),

- CH=

N - CH,(Schiff

(CH,),

- P

P-

ICH,),-

CH,-

NH-

CH,-

OH I CH-

fCH&-

--iT==llNy2 - &I-

CH,

- NH - CH - (CH,), N-C1/

CH - COOH

- P CH,-

P HO

\\

iNHi”

H ti

CH - COOH

I

I

‘3’2

CH2

,

+/

N

CH,-

CH,-

CH - COOH I

NH,

H

HO

H ti

I

,

Cl-l,-

‘N’

H

CH2-

CH2- CH,-

specificity

and

maturational

changes

in crosslinking.

of a number of circulating proteins in bone. The capacity of bone to regenerate on injury may depend on the presence of some of these proteins. Major non-collagen proteins that are made by bone cells are listed in Table 1. One of the first bone proteins, other than collagen, to be characterized was a glycoprotein rich in sialic acid. To date, two distinct sialoproteins have been identilied (Prince et al, J Biol C&n 1987, 262:290&2907; Franzen et al, Biocbem J 1985, 232:715-724). Sialoproteins I and II have been renamed osteopontin and bone sialoprotein, respectively. Bone sialoprotein is made by osteoblasts and is a major noncollagenous protein in bone (making up approximately 12% of the total). The precise role of bone sialoprotein is not known. However, because its deduced amino acid sequence contains the cell attachment recognition tripeptide Arg-Gly-Asp, it is possible that this protein serves as a cell-adhesion molecule allowing osteoblasts to attach to the extracellular matrix. Furthermore, the high concentration and apparent tissue specificity of bone sialopro-

Production

Cli - COOH NH2

AH

1. Tissue

CH - COOH

LH2 I CH, - CH,-

CH, - CH - CH2- CH, - CH - COOH

Fig.

P

(Keto amine)

base)

4+ P-

C!-i - (CH,),

II C-

of principal

crosslinks

in mature

tissue

are shown.

tein may make this protein an excellent marker to study bone cell metabolism (Fisher et al, J Biol @em 1990, 265:2347-2354). Vitamin K promotes the post-translational modification of several proteins. One such activity was first found for blood-clotting factors, which contain y-carboxylation at glutamic acid residues. The y-carboxylated glutamyl residues (called Gla) facilitate low-afFmity calcium binding via their vicinal dicarboxcyclic side-chain groups. Bone contains two proteins with this particular modiIication, osteocalcin (or bone Gla protein; BGP) and matrix Gla protein (MGP; Price et al, In Calcium Regulation and Bone Metabolism: Basic and Clinical Aspects, edited by Cohn DV et al, 1987, 9419-425). The two proteins are related but are the products of two Werent genes. There is no doubt that BGP is the most widely studied of the non-collagenous bone proteins. A variety of immunoassays have been developed in various clinical settings (TriIfitt, Clin Sci 1987, 72:399-408). BGP is also synthesized by osteoblasts; however, not all the nwly synthe-

915

916

Cell-to-cell

Table

contact

1. Major

and extracellular

non-collagen

proteins

synthesized

Also referred Protein

matrix

by bone

cells.

Molecular

to as

matrix. However, recent studies have shown that these molecules may also bind to and regulate growth factor activity (see below).

mass

(SDS gels)

Growth Sialoprotein

I

Osteopontin

50kD

Sialoprotein

II

Bone sialoprotein

75 kD

Osteocalcin Matrix

Bone Cla protein

Cla

Matrix

Osteonectin

Cla protein

KICP)

6kD

(MGP)

9kD

SPARC, BM40

35 kD

43 kD protein Proteoglycan

I

Proteoglycan

II

PC-I, PC-Sm-1

170kD

PC-II, PC-SM-2

75kD

proteodermatan

Gb,

y-carboxylated

glutamyl

sulphate

residues.

sized BGP is incorporated into bone. The concentration of intact BGP in serum has been used as index of bone formation, A clear biological role for BGP has not been established, although it has been implicated in calcium binding and bone turnover. Osteonectin is one of the most abundant non-collagen proteins produced by bone cells, accounting for approximately 2% of the total protein of developing bone in most species. However, the protein has also been found in platelets (Kelm and Mann, Blood 1!990, 75105-113). Osteonectin mRNA appears to be widely distributed in several developing tissues. It is believed that circulating osteonectin is primarily derived from platelets. This protein has a high a5nity for hydroxyapatite, type I collagen, and thrombospondin and it has been speculated that the protein is involved in bone matrix mineralization. Osteonectin biosynthesis is also upregulated in rapidly regenerating and remodelling tissues, such as a healing wound.Therefore, another potential activity of this protein in bone may be associated with bone growth, repair, or remodelling. Bone also contains two small proteoglycans, PG-I and PG-II which are products of two distinct genes (Fisher et al, J Biol cbem 1987,262:97029708) [ 21. These proteins were lirst identiIied in fetal bone extracts. Messenger RNAs for both PG-I and PG-II have been found in a variety of different connective tissues. Thus, although they are abundant in bone, they do not appear to be speciIic to that tissue. PG-I core protein contains two glycosaminoglycan chains and therefore it has been named biglycan [ 21. PG-II has a single chondroitin sulphate/dermatan sulphate chain and is also known as decorin. It is believed that these proteoglycans may interact with growing collagen molecules to regulate the precise organization and maturation of the collagen fibers within the extracellular

and differentiative

factors

Perhaps the greatest advances in hard tissue research have been in the isolation of new growth and diiferentiative factors. It has long been known that bone has the ability to repair itself without leaving a scar. In fact, bone undergoes constant remodelling. The local (and systemic) factors involved in these processes have been sought after for many years. Several systemic hormones involved in bone metabolism have been identified (e.g. parathyroid hormone, calcitonin, vitamin D metabolites, and estrogens). Research in the area of local growth and differentiation factors has undergone a virtual explosion over the past 5-10 years. The search for these factors in bone has resulted in the discovery of many new proteins described below. Much of this work stemmed from observations by Urist (Science 1965, 150:893-899) that demineralized bone matrix can induce mesenchymal cells to form cartilage, which is eventually replaced by bone. The sequential cascade (Table 2) was also found to be replicated with soluble proteins extracted from demineralized bone matrix.

Table

2. Cascade

Chemotaxis

in ectopic

bone

induction.

of polymorphonuclear

Chemotaxis

neutrophils

4 and proliferation

Differentiation

and monocytes

of mesenchymal

4 of mesenchymal

cells

cells, cartilage

formation Vascular

1 and calcification

invasion

of cartilage

matrix 4 Bone formation Bone remodelling

and

marrow

formation

To date, there are a number of factors reported to be involved in cartilage/bone formation. Almost all of these were isolated using 4M guanidine-HCl extracts derived from bovine demineralized bone. Because cartilage precedes bone formation in this process in viva, an in vitro assay was developed to identify cartilageInducing factor(s) from bone matrix. The culture conditions of this in vitro assay enabled expression of a chondroblastic phenotype as measured by cartilage proteoglycan and type II collagen expression (Seyedin et al, J cell Bioll983, 97:1950-1953). Embryonic rat muscle mesenchymal cells were embedded in agarose gels and treated with bone matrix extracts. Such cultures undergo morphological changes and begin to assume a

Matrix

chondro-b&tic phenotype, including the production of cartilage-specific macromolecules. Using this type of in vitro assay, two cartilage-inducing factors (CIF-A and CIFB) were isolated and characterized (Seyedin et al, Proc NatlAud Sci USA 1985,82:2267-2271). Subsequently, sequencing data indicated that CIF-A is identical to transforming growth factor (TGF)Pl isolated from human platelets whereas CIF-B is a homologous, but unique protein which is now called TGFP2. In vivo, however, ectopic administration of TGFPl or TGFP2 does not result in cartilage induction. One of the most readily apparent in llivo responses to a subcutaneous injection of TGFPl or TGFP2 is the induction and formation of a collagenous granulation tissue (Roberts et al, Prcc Nat1 Acud Sci USA 1986, 83:4167-4172). However, daily injection of TGFPl or TGFP2 into the subperiosteal region of newborn rat femurs resulted in localized intramembranous bone formation and chondrogenesis [3]. Similar injections into the parietal bone resulted only in intramembranous bone formation [ 4,5]. These data demonstrate that mesenchymal precursor cells in the periosteum are stimulated by TGFP to proliferate and differentiate in a manner similar to that observed during fracture healing. These data are in agreement with in vivo data indicating that TGFP stimulates proliferation of periosteal cells (Centrella et al, J Biol Ckm 1987, 262128692875). Using an in vivo model described by Sampath and Reddi (Proc Nat1 Acud Sci USA 1981, 78:75+7603), Wang et al. (Proc Nat1 Acud Sci USA 1988, 85:9484-9488) isolated several bone morphogenetic proteins (BMPs) with molecular weights of about 30kD. Using sequences of ttyptic peptides, four cDNAs were initially cloned by Wozney et al. (Science 1988, 242:152%1534). The corresponding peptides were originally named BMP-1, BMP2A, BMP-2B, and BMP-3; BMPJA, 2B and BMP-3 are all members of the TGFP superfamily. Recent studies with highly purified human recombinant BMP-2A (now called BMP-2) have shown that bone formation occurs, suggesting that a single protein may elicit all the activity originally ascribed to demineralized bone matrix [6]. Additional

Table

3. Reported

osteoinductive

Protein

M, (kD)

BMP BMP-1 BMP-2 BMP-3 BMP-4 BMP-5 BMP-6 BMP-7 OP-1 OP-2

-18.5 -30 -30 -30 -30 -30 -30 -30 -30 -30

BMP, bone

morphogenetic

of the skeleton

Seyedin and Rosen

members of the BMP family have been identified recently, including BMP-5, BMP-6 and BMP-7 [7]. Iast year, a protein called osteogenin was also purified [B] from bovine bone. Sequencing data revealed that osteogenin appears to be identical to BMP3 (see Table 3 for summary). One area of interest has been the question of whether the BMPs exist naturally as homodimers or heterodimers. AU recombinant BMPs reported to date have been homodimers. Recently, work by Sampath and colleagues described the isolation [9] and cloning [lo] of a heterodimeric osteogenic protein (OP) composed of one chain of BMP-2 and one chain of OP-2. This molecule reportedly has a greater specific activity than any of the other recombinantly produced homodimers. Purified recombinant BMP-2 induces endochondral bone formation in a manner similar to a crude bone extract and the quantity of bone relative to cartilage formed is directly related to amount of BMP-2 implanted. Similar responses have been observed with crude bone extracts suggesting that this response can be modulated by more than one factor. It has been observed that TGFPs can modulate this response under some conditions [ 111. A high BMP content of implants leads predominantly to bone formation, whereas addition of exogenous TGFP leads to more cartilage formation (Fig. 2). Thus, in vivo, the regulation of bone and cartilage formation appear to be influenced by both the site and multiple protein factors. During the past year, a unique glycoprotein was isolated from bovine bone and termed osteoinductive factor or OIF [ 121. This molecule was originally isolated using an ectopic bone-forming assay. Subsequent cloning [13] and refined purification of the molecule indicated that it did not possess the osteoinductive activity originally ascribed to it. However, complete sequencing of the molecule, including its precursor, revealed that the molecule is a member of the leucine-rich family of molecules (H Benz, AY Thompson, RA Armstrong, R-J Chang, KA Piez and DM Rosen, submitted). Other members of this family include the bone proteoglycans I and II discussed previously [ 21. A very exciting piece of in-

proteins.

-

?

? ? ? I

TCFfl TGFP TGFB TCFfl TCFB TCFP TGFP TGFP

Vgr-1 (I) OP-1 BMP-7 BMP-2

-8 osteogenic

? ?

OP-2, BMP-2A Osteogenin BMP-2B -

-8 >8 -8

OP,

Sequence relation

Synonym

P’ -5

protein;

proteins

protein;

TGF, transforming

growth

factor.

Active recombinant

? (+) + (+) (+) ? I ? + +

917

918

CellTto-cell

contact

and extracellular

matrix

Annotated reading l

*a 1. 0

Y

Fibrosis

references

Of interest Of outstanding UEBELHART

and recommended

interest D, GINEYI-E

excretion of pyridiniurn resorption in metabolic 8:87-96. Using a specific high performance shown that Pyd and Dpd excretion of bone resorption.

M-C, DELMAS PD: Urinary crosslinks: a new marker of bone bone disease. Bone Miner 1990,

E, Cww

liquid chromatography assay it was represents the first sensitive marker

2.

FISHER LW, TERMINE JD, YOUNG MP: Deduced protein sequence of bone smaII proteoglycan I (biglycan) shows homology with proteoglycan II (decorin) and several nonconnective tissue proteins in a variety of species. J Biol them 1989, 264:457l-i576. This paper reports the cloning of PG.1 and PG-II and their sequence relationship to the leucine-rich family of proteins. l e

Fig. 2. Dose-dependent

effect

of TCFP

in bone

induction 3. 00

formation is that bone proteoglycan II, also known as decorin, has recently been shown to bind and neutralize TGFB activity [14]. Many other leucine-rich proteins are also though to be binding proteins. In view of the sequence relation between OIF and decorin and the difficulty encountered in separating the OIF and BMP molecules, it is interesting to speculate that OIP may serve as a binding protein for some of the BMPs. This possibility is currently under evaluation. Alternatively, molecules such as PGII (decorin), or OJF may serve as molecules to store cytokines in matrices, or perhaps to present them in an effective manner to responding cells. A similar situation is thought to occur with the matrix (and factors within) laid down by bone marrow stromal cells and the subsequent differentiation of lymphoid cells (Roberts, Nature 1988, 332:371-379).

Conclusion The extracellular matrix of bone provides not only structural function for this tissue but also serves as a reservoir for number of potent growth and differentiative factors. Many of the growth factors that influence osteoblasts are probably deposited in bone by osteoblasts themselves. ln the process of collagen synthesis and subsequent mineralization of extracellular matrix, protein Factors are sequestered in mineratized tissue. As osteoclas tic activity is followed by osteoblastic activity, the current belief is that osteoclasts may release the sequestered growth/diEerentiative factors during the bone resorption process. The released factors can then stimulate local osteoprogenitor or mesenchymal cells to proliferate and differentiate into mature osteoblasts thereby preserving bone mass. Clearly, the remodelling process must be in fluenced by the composition and the architecture of the bone matrix. Inappropriate regulation of bone resorption or formation will result in the formation of too little (e.g. osteoporosis) or too much (e.g. osteopetrosis) bone.

Aq SPORN MB, BOL~NDER ME: Transforming growth factor J3 and the initiation of chondrogenesis and osteogenesis in the rat femur. J Cell Biof 19$X1, 110:21952207. A demonstration that mesenchymal precursor cells in the periosteum are stimulated by TGFP to proliferate and dilferentiate into bone and cartilage in a manner similar to that observed during fracture healing. JOYCE MF, ROBERT

4. NODA M. CAMIUERE J: In o&o stimulation of bone formation .a by TGF-8. Endocrinology 1989, 124:28@2875. A description of the elects of lo&y injected TGFJ31 in the periosteum of newborn rats. There is a dramatic increase in trabecular bone. EJ, TRECHSEL V: Stimulation of bone formation in uiuo by TGF-P. RemodeIling of woven bone and lack of inhibition by indomethacin. Bone 1990, 11:295-300. Similar observations as [4], but showing similar effects by TGF81 and TGFP2. The most signikcant observation is that this process appears to be independent of prostaglandin.

5. 00

MACKIE

6.

WANG EA, ROSEN V, D ‘~LF+%NDRO JS, BAIJDW M, CORDES P, HARA~A T, ISRAEL DI, HEWICK RM, KEXNS KM, IAPAN P, LOXENBERG DP, MCQUAID D, Momwrsos IK, NOVE J,

00

WOZNEY JM: Recombinant human bone morphogenetic protein induces bone formation. Pm Nat1 Acad Sci USA 1990, 87:222&2224. A TGFB-iike molecule, purified recombinant human BMP-2, induces ectopic bone in rat with a coUagenous matrix carrier. 7. WOZNEY JM: Bone morphogenetic proteins. Progr Growth 00 actor Res 1990, 1:267-280. An overview of bone-inducing factors and the reports of the new members of the BMP famiily. 8. 0

LUYI-EN FP, CUNNINGHAM NS, MA S, HAMMONDS RG, NEVINS WB, WOOD WI,

MLJTHUKUMAF~AN N, REDD AH: PuriIica-

tion and partial amino acid sequences of osteogenin, a protein initiating bone differentiation. J Eiol @em 1989, 264:133T7-13380, Osteogenin induces bone formation in the presence of bone coIIagen matrix. Osteogenin appears to be BMP-3 described by Wozney [7]. 9. a.

SAMPATH TK, COUGHLIN JE, W~ONE RM, BANACH D, CORBETT C, RIDGE RJ, OZKA~NAK E, OPPERMANN H AND RUEGER

DC: Bovine osteogenic protein is composed of dimers of OP-1 and BMP -2A, two members of the TGF-P superfamily. J Biol c&m 1990, 65:1319%13205. The isolation of an osteogenic protein from bone, presumably composed of heterodimers as weli as homodimers, is described. 10. a

OZKA~NAK E, RUEGER DC, DRIER & RJ, SAMPATH TK, OPPERMANN H: OP-1

COR~ERT

cDNA

C,

encodes

RIDGE

an

Matrix osteogenic 9:2085-2093. This paper BMP-7.

11. l

protein

in

the

TGF-fl

family.

EMBO

/

1990,

l

describes

the

cloning

of OP-I,

presumably

equivalent

transforming

growth

factor-p.

Ann

NY Acud

Sci 1990.

593:9%106. A description

of the role of TGFB H,

CHANG

in bone

12.

BENIZ

l

Complete amino acid sequence factor (OIF). / Biol cbem 1990.

R.J, THOMPSON

The entire amino acid sequence mapping studies is presented.

of bovine

induction. AY,

GIASER

C.

ROSEN DM:

of bovine osteoinductive 265:5024-5029. OIF

obtained

by peptide

of the

skeleton

Seyedin

and Rosen

MADISEN L, NEURAUERE M, PKXVMAN G, ROSEN DM, SEGARINI PR, DAXH JR, THOMPWN AY, Zw J, BENIZ H. PURCH~O AF:

Molecular cloning of a novel bone. DNA 19!X, 9:303-30.

to

ROSEN DM, NATHAN R, AI&WRONG RA, BENIZ H, THOMPSON A. DELEON E, BUCKMAN E. FIEDIIR I SEEDIN SM: gone induction

and

13.

proteins

osteoinductive

factor

from

of bovine and human as a large precursor.

OIF

cDNAs

MANN DM, RUOSLWTI E: Negative by the proteoglycan decorin. N&we

regula-

Thee authors describe the cloning and demonstrate that OIF is made 14.

YAMACUCHI

00

tion

Y,

of TGF-P

346:281-284. Evidence that decorin is able activity of TGFPI in vitro.

to bind

to and neutralize

1990,

the biological