Osteogenesis Imperfecta

Osteogenesis imperfecta is a condition inherited in mendelian fashion that illustrates

many principles of human genetics. It is a heterogeneous and pleiotropic group of

disorders characterized by a tendency toward fragility of bone. Advances in the last two

decades demonstrate that nearly every case is caused by a mutation of the COL1A1 or

COL1A2 genes, which encode the subunits of type I collagen, pro1(I) and pro 2(I),

respectively. More than 100 different mutant alleles have been described for

osteogenesis imperfecta; the relationships between different DNA sequence alterations

and the type of disease (genotype-phenotype correlations) illustrate several

pathophysiologic principles in human genetics.

Clinical Manifestations

The clinical and genetic characteristics of osteogenesis imperfecta are summarized in

Table 23, in which the timing and severity of fractures, radiologic findings, presence of

additional clinical features, and family history are used to discriminate among four

different subtypes. All forms of osteogenesis imperfecta are characterized by increased

susceptibility to fractures ("brittle bones"), but there is considerable phenotypic

heterogeneity, even within individual subtypes. Individuals with type I or type IV

osteogenesis imperfecta present in early childhood with one or a few fractures of long

bones in response to minimal or no trauma; x-ray films reveal mild osteopenia, little or

no bony deformity, and often evidence of earlier subclinical fractures. However, most

individuals with type I or type IV osteogenesis imperfecta do not have fractures in utero.

Type I and type IV osteogenesis imperfecta are distinguished by the severity (less in

type I than in type IV) and by scleral hue, which indicates the thickness of this tissue

and the deposition of type I collagen. Individuals with type I osteogenesis imperfecta

have blue scleras, whereas the scleras of those with type IV are normal or slightly gray.

In type I, the typical number of fractures during childhood is 1020; fracture incidence

decreases after puberty, and the main features in adult life are mild short stature, a

tendency toward conductive hearing loss, and occasionally dentinogenesis imperfecta.

Individuals with type IV osteogenesis imperfecta generally experience more fractures

than those with type I and have significant short stature caused by a combination of

long bone and spinal deformities, but they often are able to walk independently.

Approximately one fourth of the cases of type I or type IV osteogenesis imperfecta

represent new mutations; in the remainder, the history and examination of other family

members reveal findings consistent with autosomal dominant inheritance.

Table 23 Clinical and Molecular Subtypes of Osteogenesis Imperfecta.

Type Phenotype Genetics Molecular Pathophysiology

Type

I

Mild: Short stature, postnatal

fractures, little or no

deformity, blue scleras,

premature hearing loss

Autosomal

dominant

Loss-of-function mutation in

pro 1(I) chain resulting in

decreased amount of mRNA;

quality of collagen is normal;

quantity is reduced twofold

Type

II

Perinatal lethal: Severe

prenatal fractures, abnormal

bone formation, severe

deformities, blue scleras,

connective tissue fragility

Sporadic

(autosomal

dominant)

Structural mutation in pro 1(I) or

pro 2(I) chain that has mild

effect on heterotrimer assembly;

quality of collagen is severely

abnormal; quantity often

reduced also

Type

III

Progressive deforming:

Prenatal fractures,

deformities usually present at

birth, very short stature,

Autosomal

dominant1

Structural mutation in pro 1(I) or

pro 2(I) chain that has mild

effect on heterotrimer assembly;

quality of collagen is severely

usually nonambulatory, blue

scleras, hearing loss

abnormal; quantity can be

normal

Type

IV

Deforming with normal

scleras: Postnatal fractures,

mild to moderate deformities,

premature hearing loss,

normal or gray scleras,

dental abnormalities

Autosomal

dominant

Structural mutation in the pro

2(I), or, less frequently, pro 1(I)

chain that has little or no effect

on heterotrimer assembly;

quality of collagen is usually

abnormal; quantity can be

normal

1Autosomal recessive in rare cases.

Type II osteogenesis imperfecta presents at or before birth (diagnosed by prenatal

ultrasonography) with multiple fractures, bony deformities, increased fragility of nonbony

connective tissue, and blue scleras and usually results in death in infancy. Two typical

radiologic findings are the presence of isolated "islands" of mineralization in the skull

(wormian bones) and a beaded appearance to the ribs. Nearly all cases of type II

osteogenesis imperfecta represent a new dominant mutation, and there is no family

history. Death usually results from respiratory difficulties.

Type III osteogenesis imperfecta presents at birth or in infancy with progressive bony

deformities, multiple fractures, and blue scleras. It is intermediate in severity between

types II and IV; most affected individuals will require multiple corrective surgeries and

lose the ability to ambulate by early adulthood. Unlike other forms of osteogenesis

imperfecta, which are nearly always due to mutations that act dominantly, type III can

be inherited in either a dominant or (rarely) recessive fashion. From a biochemical and

molecular perspective, type III osteogenesis imperfecta is the least well understood

form.

Although different subtypes of osteogenesis imperfecta can often be distinguished

biochemically, the classification presented in Table 23 is clinical rather than molecular,

and the disease phenotypes for each subtype show a spectrum of severities that

overlap one another. For example, a few individuals diagnosed with type II

osteogenesis imperfecta based on the presence of severe bony deformities in utero will

survive for many years and thus overlap the type III subtype. Similarly, some individuals

with type IV osteogenesis imperfecta have fractures in utero and develop deformities

that lead to loss of ambulation. Distinguishing this presentation from type III

osteogenesis imperfecta may be possible only if other affected family members exhibit a

milder course.

Additional subtypes of osteogenesis imperfecta have been suggested for individuals

that do not match types IIV, and there are additional disorders associated with

congenital fractures that are usually not considered to be "classic" osteogenesis

imperfecta. In some cases, mutations of type I collagen genes have been excluded as

potential causes of these additional disorders. However, the approach to clinical

classification depicted in Table 23 is helpful for most affected individuals in predicting

the course and inheritance pattern of the illness. The classification also serves as an

important framework within which to correlate molecular abnormalities with disease

phenotypes.

Pathophysiology

Osteogenesis imperfecta is a disease of type I collagen, which constitutes the major

extracellular protein in the body. It is the major collagen in the dermis, the connective

tissue capsules of most organs, and the vascular and GI adventitia and is the only

collagen in bone. A mature type I collagen fibril is a rigid structure that contains multiple

type I collagen molecules packed in a staggered array and stabilized by intermolecular

covalent cross-links. Each mature type I collagen molecule contains two 1 chains and

one 2 chain, encoded by the COL1A1 and COL1A2 genes, respectively (Figure 22).

The COL1A1 and COL1A2 genes have 51 and 52 exons, respectively, of which exons

649 encode the entire triple-helical domain. The 1 and 2 chains are synthesized as

larger precursors with amino and carboxyl terminal "propeptide" extensions, assemble

with each other inside the cell, and are ultimately secreted as a heterotrimeric type I

procollagen molecule. During intracellular assembly, the three chains wind around each

other in a triple helix that is stabilized by interchain interactions between hydroxy proline

and adjacent carbonyl residues. There is a dynamic relationship between the

posttranslational action of prolyl hydroxylase and assembly of the triple helix, which

begins at the carboxyl terminal end of the molecule. Increased levels of hydroxylation

result in a more stable helix, but helix formation prevents further prolyl hydroxylation.

The nature of the triple helix causes the side chain of every third amino acid to point

inward, and steric constraints allow only a proton in this position. Thus, the amino acid

sequence of virtually all collagen chains in the triple-helical portion is (Gly-X-Y)n, where

Y is proline about one third of the time.

The fundamental defect in most individuals with type I osteogenesis imperfecta is

reduced synthesis of type I collagen resulting from loss-of-function mutations in

COL1A1. In most cases, the mutant COL1A1 allele gives rise to greatly reduced (partial

loss-of-function) or no (complete loss-of-function) mRNA. Because the nonmutant

COL1A1 allele continues to produce mRNA at a normal rate (ie, there is no dosage

compensation), heterozygosity for a complete loss-of-function mutation results in a 50%

reduction in the total rate of pro 1(I) mRNA synthesis, whereas heterozygosity for a

partial loss-of-function mutation results in a less severe reduction. A reduced

concentration of pro 1(I) chains limits the production of type I procollagen, leading to (1)

a reduced amount of structurally normal type I collagen and (2) an excess of

unassembled pro 2(I) chains, which are degraded inside the cell (Figure 23).

There are several potential molecular defects responsible for COL1A1 mutations in type

I osteogenesis imperfecta, including alterations in a regulatory region leading to reduced

transcription, splicing abnormalities leading to reduced steady state levels of RNA, and

deletion of the entire COL1A1 gene. However, in many cases, the underlying defect is a

single base pair change that creates a premature stop codon (also known as a

"nonsense mutation") in exons 649. In a process referred to as "nonsense-mediated

decay," partially synthesized mRNA precursors that carry the nonsense codon are

recognized and degraded by the cell. With collagen and many other genes, production

of a truncated protein (as might be predicted from a nonsense mutation) would be more

damaging to the cell than production of no protein at all. Thus, nonsense-mediated

decay, which has been observed to occur for mutations in many different multiexon

genes, serves as a protective phenomenon and is an important component of genetic

pathophysiology.

An example of these principles is apparent from considering type II osteogenesis

imperfecta, which is caused by structurally abnormal forms of type I collagen and is

more severe than type I osteogenesis imperfecta. Mutations in type II osteogenesis

imperfecta can be caused by defects in either COL1A1 or COL1A2 and usually are

missense alterations of a glycine residue that allow the mutant peptide chain to bind to

normal chains in the initial steps of trimer assembly (Figure 23). However, triple-helix

formation is ineffective, often because amino acids with large side chains are

substituted for glycine. Ineffective triple-helix formation leads to increased

posttranslational modification by prolyl hydroxylase and a reduced rate of secretion.

These appear to be critical events in the cellular pathogenesis of type II osteogenesis

imperfecta, because glycine substitutions toward the carboxyl terminal end of the

molecule are generally more severe than those at the amino terminal end.

These considerations help to explain why type II osteogenesis imperfecta is more

severe than type I and exemplify the principle of dominant negative gene action. The

effects of an amino acid substitution in a pro 1(I) peptide chain are amplified at the

levels of both triple-helix assembly and fibril formation. Because every type I

procollagen molecule has two pro 1(I) chains, only 25% of type I procollagen molecules

will contain two normal pro 1(I) chains even though only one of the two COL1A1 alleles

is mutated. Furthermore, because each molecule in a fibril interacts with several others,

incorporation of an abnormal molecule can have disproportionately large effects on fibril

structure and integrity.

Collagen mutations that cause type III and type IV osteogenesis imperfecta are diverse

and include glycine substitutions in the amino terminal portion of the collagen triple

helix, a few internal deletions of COL1A1 and COL1A2 that do not significantly disturb

triple helix formation, and some unusual alterations in the nontriple-helical extensions

at the amino and carboxyl terminals of pro chains.

Genetic Principles

As already described, most cases of type I osteogenesis imperfecta are caused by

partial or complete loss-of-function mutations in COL1A1. However, in approximately

one third of affected individuals, the disease is caused by a new mutation; in addition,

there are many ways in which DNA sequence alterations can reduce gene expression.

Consequently, there is a wide range of mutant alleles (ie, allelic heterogeneity), which

represents a challenge for the development of molecular diagnostic tests. In a family in

which type I osteogenesis imperfecta is known to occur clinically and a proband seeks a

diagnostic test for the purposes of reproductive planning, it is possible in most cases to

use linkage analysis at the COL1A1 locus. In this approach, one distinguishes between

chromosomes that carry the mutant and nonmutant COL1A1 alleles using closely linked

DNA-based polymorphisms, even though the causative molecular defect is not known.

Once this information is established for a particular family, inheritance of the mutant

allele can be predicted in future pregnancies.

For types III and IV osteogenesis imperfecta, mutations can occur in COL1A1 or

COL1A2 (ie, locus heterogeneity), and in this situation linkage analysis is more difficult

because one cannot be sure which locus is abnormal.

For both type I and type IV osteogenesis imperfecta, the most important question in the

clinical setting often relates to the natural history of the illness. For example,

reproductive decision making in families at risk for osteogenesis imperfecta is

influenced greatly by the relative likelihood of producing a child who will never walk and

will require multiple orthopedic operations versus a child whose major problems will be

a few long bone fractures and an increased risk of mixed sensorineural and conductive

hearing loss in childhood and adulthood. As evident from the prior discussion, both

different mutant genes and different mutant alleles, as well as other genes that modify

the osteogenesis imperfecta phenotype, can contribute to this phenotypic heterogeneity.

When allelic rather than locus heterogeneity is operative, as in type I osteogenesis

imperfecta, comparison of interfamilial with intrafamilial variability allows one to assess

the relative contribution of different mutant alleles to phenotypic heterogeneity. For most

genetic diseases, including type I osteogenesis imperfecta, intrafamilial variability is less

than interfamilial variability.

In type II osteogenesis imperfecta, a single copy of the mutant allele causes the

abnormal phenotype and, therefore, has a dominant mechanism of action. Although the

type II phenotype itself is never inherited, there are rare situations in which a

phenotypically normal individual harbors a COL1A1 mutant allele among his or her

germ cells. These individuals with so-called gonadal mosaicism can produce multiple

offspring with type II osteogenesis imperfecta (Figure 24), a pattern of segregation that

can be confused with recessive inheritance. In fact, many other mutations, including

Duchenne's muscular dystrophy, which is X linked, and type 1 neurofibromatosis, which

is autosomal dominant, also occasionally show unusual inheritance patterns explained

by gonadal mosaicism.

DISEASES ASSOCIATED WITH DEFECTS IN EXTRACELLULAR STRUCTURAL PROTEINS

The interaction of the organic components of bone matrix is complex and a focus of intense

scientific investigation. The importance of the structural bone proteins is exemplified by the

diseases associated with deranged metabolism of the collagens important in bone and cartilage

formation (types 1, 2, 9, 10, and 11). Their clinical manifestations are highly variable, ranging

from lethal disease to premature osteoarthritis.

Type 1 Collagen Diseases (Osteogenesis Imperfecta)

Osteogenesis imperfecta, or brittle bone disease, is a phenotypically diverse disorder caused by

deficiencies in the synthesis of type 1 collagen. It is the most common inherited disorder of

connective tissue. It principally affects bone, but also impacts other tissues rich in type 1

collagen (joints, eyes, ears, skin, and teeth). Osteogenesis imperfecta usually results from

autosomal dominant mutations (over 800 have been identified) in the genes that enco de the1 and 2 chains of collagen.[19] Many of these mutations involve the substitution of glycine residues in the triple-helical domain. The genotypephenotype relationship underlying

osteogenesis imperfecta is based on the location of the mutation within the protein. Mutations

resulting in decreased synthesis of qualitatively normal collagen are associated with mild skeletal

abnormalities. More severe or lethal phenotypes have abnormal polypeptide chains that cannot

be arranged in the triple helix. Recently, mutations in the genes for cartilage-associated protein

(CRTAP) and leucine proline-enriched proteoglycan 1 (LEPRE1) have been shown to be

responsible for three rare variants of the disease.[20]

Clinically, osteogenesis imperfecta is separated into four major subtypes that vary widely in

severity ( Table 26-3 ). The type II variant is at one end of the spectrum and is uniformly fatal in

utero or during the perinatal period. It is characterized by extraordinary bone fragility with

multiple intrauterine fractures ( Fig. 26-6 ). In contrast, individuals with the type I form have a

normal life span but experience childhood fractures that decrease in frequency following

puberty. Other findings include blue sclerae caused by decreased collagen content, making the

sclera translucent and allowing partial visualization of the underlying choroid; hearing loss

related to both a sensorineural deficit and impeded conduction due to abnormalities in the bones

of the middle and inner ear; and dental imperfections (small, misshapen, and blue-yellow teeth)

secondary to a deficiency in dentin. The basic abnormality in all forms of osteogenesis

imperfecta is too little bone, thus constituting a type of osteoporosis with marked cortical

thinning and attenuation of trabeculae.

TABLE 26-3 -- Subtypes of Osteogenesis Imperfecta

Subtype Inheritance Collagen Defect Major Clinical Features

OI type I Compatible

with survival

Autosomal

dominant

Decreased synthesis

of pro-1(1) chain Postnatal fractures, blue

sclera

Abnormal pro-1(1) or pro-2(1) chains

Normal stature

Skeletal fragility

Dentinogenesis imperfecta

Hearing impairment

Joint laxity

Blue sclerae

OI type

II

Perinatal lethal Most autosomal

recessive

Abnormally short

pro-1(1) chain Death in utero or within

days of birth

Some

autosomal

dominant

Unstable triple helix Skeletal deformity with

excessive fragility and

multiple fractures

?New mutations Abnormal or

insufficient pro-

Blue sclera

Subtype Inheritance Collagen Defect Major Clinical Features

2(1)

OI type

III

Progressive,

deforming

Autosomal

dominant (75%)

Altered structure of

pro-peptides of pro-

2(1)

Compatible with survival

Autosomal

recessive (25%)

Impaired formation

of triple helix

Growth retardation

Multiple fractures

Progressive kyphoscoliosis

Blue sclera at birth that

become white

Hearing impairment

Dentinogenesis imperfecta

OI type

IV

Compatible

with survival

Autosomal

dominant

Short pro-2(1) chain

Postnatal fractures, normal

sclerae

Unstable triple helix Moderate skeletal fragility

Short stature

Sometimes dentinogenesis

imperfecta

Extracellular Matrix and Cell-Matrix Interactions

Tissue repair and regeneration depend not only on the activity of soluble factors, but also on

interactions between cells and the components of the extracellular matrix (ECM). The ECM

regulates the growth, proliferation, movement, and differentiation of the cells living within it. It

is constantly remodeling, and its synthesis and degradation accompanies morphogenesis,

regeneration, wound healing, chronic fibrotic processes, tumor invasion, and metastasis. The

ECM sequesters water, providing turgor to soft tissues, and minerals that give rigidity to bone,

but it does much more than just fill the spaces around cells to maintain tissue structure. Its

various functions include:

Mechanical support for cell anchorage and cell migration, and maintenance of cell

polarity

Control of cell growth. ECM components can regulate cell proliferation by signaling

through cellular receptors of the integrin family.

Maintenance of cell differentiation. The type of ECM proteins can affect the degree of

differentiation of the cells in the tissue, also acting largely via cell surface integrins.

Scaffolding for tissue renewal. The maintenance of normal tissue structure requires a basement membrane or stromal scaffold. The integrity of the basement membrane or the

stroma of the parenchymal cells is critical for the organized regeneration of tissues. It is

particularly noteworthy that although labile and stable cells are capable of regeneration,

injury to these tissues results in restitution of the normal structure only if the ECM is not

damaged. Disruption of these structures leads to collagen deposition and scar formation

(see Fig. 3-2 ).

Establishment of tissue microenvironments. Basement membrane acts as a boundary between epithelium and underlying connective tissue and also forms part of the filtration

apparatus in the kidney.

Storage and presentation of regulatory molecules. For example, growth factors like FGF and HGF are secreted and stored in the ECM in some tissues. This allows the rapid

deployment of growth factors after local injury, or during regeneration.

The ECM is composed of three groups of macromolecules: fibrous structural proteins, such as

collagens and elastins that provide tensile strength and recoil; adhesive glycoproteins that

connect the matrix elements to one another and to cells; and proteoglycans and hyaluronan that

provide resilience and lubrication. These molecules assemble to form two basic forms of ECM:

interstitial matrix and basement membranes. The interstitial matrix is found in spaces between

epithelial, endothelial, and smooth muscle cells, as well as in connective tissue. It consists mostly

of fibrillar and nonfibrillar collagen, elastin, fibronectin, proteoglycans, and hyaluronan.

Basement membranes are closely associated with cell surfaces, and consist of nonfibrillar

collagen (mostly type IV), laminin, heparin sulfate, and proteoglycans.[71]

We will now consider the main components of the ECM.

COLLAGEN

Collagen is the most common protein in the animal world, providing the extracellular framework

for all multicellular organisms. Without collagen, a human being would be reduced to a clump

of cells, like the Blob (the gelatinous horror from outer space of 1950s movie fame), interconnected by a few neurons. Currently, 27 different types of collagens encoded by 41 genes

dispersed on at least 14 chromosomes are known[72]

( Table 3-2 ). Each collagen is composed of

three chains that form a trimer in the shape of a triple helix. The polypeptide is characterized by

a repeating sequence in which glycine is in every third position (Gly-X-Y, in which X and Y can

be any amino acid other than cysteine or tryptophan), and it contains

FIGURE 3-12 Main components of the extracellular matrix (ECM), including collagens, proteoglycans, and adhesive glycoproteins.

Both epithelial and mesenchymal cells (e.g., fibroblasts) interact with ECM via integrins. Basement membranes and interstitial ECM

have different architecture and general composition, although there is some overlap in their constituents. For the sake of

simplification, many ECM components (e.g., elastin, fibrillin, hyaluronan, and syndecan) are not included.

the specialized amino acids 4-hydroxyproline and hydroxylysine. Prolyl residues in the Y-

position are characteristically hydroxylated to produce hydroxyproline, which serves to stabilize

the triple helix. Types I, II, III and V, and XI are the fibrillar collagens, in which the triple-

helical domain is uninterrupted for more than 1000 residues; these proteins are found in

extracellular fibrillar structures. Type IV collagens have long but interrupted triple-helical

domains and form sheets instead of fibrils; they are the main components of the basement

membrane, together with laminin. Another collagen with a long interrupted triple-helical domain

(type VII) forms the anchoring fibrils between some epithelial and mesenchymal structures, such

as epidermis and dermis. Still other collagens are transmembrane and may also help to anchor

epidermal and dermal structures.

TABLE 3-2 -- Main Types of Collagens, Tissue Distribution, and Genetic Disorders

Collagen

Type Tissue Distribution Genetic Disorders

FIBRILLAR COLLAGENS

I Ubiquitous in hard and soft

tissues

Osteogenesis imperfecta; Ehlers-Danlos

syndromearthrochalasias type I

II Cartilage, intervertebral disk,

vitreous

Achondrogenesis type II, spondyloepiphysea

dysplasia syndrome

III Hollow organs, soft tissues Vascular Ehlers-Danlos syndrome

V Soft tissues, blood vessels Classical Ehlers-Danlos syndrome

Collagen

Type Tissue Distribution Genetic Disorders

IX Cartilage, vitreous Stickler syndrome

BASEMENT MEMBRANE COLLAGENS

IV Basement membranes Alport syndrome

OTHER COLLAGENS

VI Ubiquitous in microfibrils Bethlem myopathy

VII Anchoring fibrils at dermal-

epidermal junctions

Dystrophic epidermolysis bullosa

IX Cartilage, intervertebral disks Multiple epiphyseal dysplasias

XVII Transmembrane collagen in

epidermal cells

Benign atrophic generalized epidermolysis

bullosa

XV and

XVIII

Endostatin-forming collagens,

endothelial cells

Knobloch syndrome (type XVIII collagen)

Courtesy of Dr. Peter H. Byers, Department of Pathology, University of Washington, Seattle,

WA.

The messenger RNAs transcribed from fibrillar collagen genes are translated into pre-pro- chains that assemble in a type-specific manner into trimers. Hydroxylation of proline and lysine

residues and lysine glycosylation occur during translation. Three chains of a particular collagen

type assemble to form the triple helix ( Fig. 3-15 ). Procollagen is secreted from the cell and

cleaved by proteases to form the basic unit of the fibrils. Collagen fibril formation is associated

with the oxidation of lysine and hydroxylysine residues by the extracellular enzyme lysyl

oxidase. This results in cross-linking between the chains of adjacent molecules, which stabilizes

the array, and is a major contributor to the tensile strength of collagen. Vitamin C is required for

the hydroxylation of procollagen, a requirement that explains the inadequate wound healing in

scurvy ( Chapter 9 ). Genetic defects in collagen production (see Table 3-2 ) cause many

inherited syndromes, including various forms of the Ehlers-Danlos syndrome and osteogenesis

imperfecta[73]

( Chapters 5 and 26 .

FIGURE 3-15 Angiogenesis by mobilization of endothelial precursor cells (EPCs) from the bone marrow and from preexisting

vessels (capillary growth). A, In angiogenesis from preexisting vessels, endothelial cells from these vessels become motile and

proliferate to form capillary sprouts. Regardless of the initiating mechanism, vessel maturation (stabilization) involves the

recruitment of pericytes and smooth muscle cells to form the periendothelial layer. B, EPCs are mobilized from the bone marrow and

may migrate to a site of injury or tumor growth. At these sites, EPCs differentiate and form a mature network by linking to existing

vessels. (Modified from Conway EM et al: Molecular mechanisms of blood vessel growth. Cardiovasc Res 49:507, 2001.)

ELASTIN, FIBRILLIN, AND ELASTIC FIBERS

Tissues such as blood vessels, skin, uterus, and lung require elasticity for their function. Proteins

of the collagen family provide tensile strength, but the ability of these tissues to expand and

recoil (compliance) depends on the elastic fibers. These fibers can stretch and then return to their

original size after release of the tension. Morphologically, elastic fibers consist of a central core

made of elastin, surrounded by a peripheral network of microfibrils. Substantial amounts of

elastin are found in the walls of large blood vessels, such as the aorta, and in the uterus, skin, and

ligaments. The peripheral microfibrillar network that surrounds the core consists largely of

fibrillin, a 350-kD secreted glycoprotein, which associates either with itself or with other

components of the ECM. The microfibrils serve, in part, as scaffolding for deposition of elastin

and the assembly of elastic fibers. They also influence the availability of active TGF in the ECM. As already mentioned, inherited defects in fibrillin result in formation of abnormal elastic

fibers in Marfan syndrome, manifested by changes in the cardiovascular system (aortic

dissection) and the skeleton[74]

( Chapter 5 ).

CELL ADHESION PROTEINS

Most adhesion proteins, also called CAMs (cell adhesion molecules), can be classified into four

main families: immunoglobulin family CAMs, cadherins, integrins, and selectins. These proteins

function as transmembrane receptors but are sometimes stored in the cytoplasm.[75]

As receptors,

CAMs can bind to similar or different molecules in other cells, providing for interaction between

the same cells (homotypic interaction) or different cell types (heterotypic interaction). Selectins

have been discussed in Chapter 2 in the context of leukocyte/endothelial interactions. Selected

aspects of other cell adhesion proteins are described here. Integrins bind to ECM proteins such as

fibronectin, laminin, and osteopontin providing a connection between cells and ECM, and also to

adhesive proteins in other cells, establishing cell-to-cell contact. Fibronectin is a large protein

that binds to many molecules, such as collagen, fibrin, proteoglycans, and cell surface receptors.

It consists of two glycoprotein chains, held together by disulfide bonds. Fibronectin messenger

RNA has two splice forms, giving rise to tissue fibronectin and plasma fibronectin. The plasma

form binds to fibrin, helping to stabilize the blood clot that fills the gaps created by wounds, and

serves as a substratum for ECM deposition and formation of the provisional matrix during

wound healing (discussed later). Laminin is the most abundant glycoprotein in the basement

membrane and has binding domains for both ECM and cell surface receptors. In the basement

membrane, polymers of laminin and collagen type IV form tightly bound networks. Laminin can

also mediate the attachment of cells to connective tissue substrates.

Cadherins and integrins link the cell surface with the cytoskeleton through binding to actin and

intermediate filaments. These linkages, particularly for the integrins, provide a mechanism for

the transmission of mechanical force and the activation of intracellular signal transduction

pathways that respond to these forces. Ligand binding to integrins causes clustering of the

receptors in the cell membrane and formation of focal adhesion complexes. The cytoskeletal

proteins that co-localize with integrins at the cell focal adhesion complex include talin, vinculin,

and paxillin. The integrin-cytoskeleton complexes function as activated receptors and trigger a

number of signal transduction pathways, including the MAP kinase, PKC, and PI3K pathways,

which are also activated by growth factors. Not only is there a functional overlap between

integrin and growth factor receptors, but integrins and growth factor receptors interact (cross-talk) to transmit environmental signals to the cell that regulate proliferation, apoptosis, and differentiation ( Fig. 3-13 ).

FIGURE 3-13 Mechanisms by which ECM components and growth factors interact and activate signaling pathways. Integrins bind

ECM components and interact with the cytoskeleton at focal adhesion complexes (protein aggregates that include vinculin, -actin, and talin). This can initiate the production of intracellular messengers or can directly mediate nuclear signals. Cell surface receptors

for growth factors may activate signal transduction pathways that overlap with those activated by integrins. Signaling from ECM

components and growth factors is integrated by the cell to produce various responses, including changes in cell proliferation,

locomotion, and differentiation.

The name cadherin is derived from the term calcium-dependent adherence protein. This family contains almost 90 members, which participate in interactions between cells of the same type.

These interactions connect the plasma membrane of adjacent cells, forming two types of cell

junctions called (1) zonula adherens, small, spotlike junctions located near the apical surface of

epithelial cells, and (2) desmosomes, stronger and more extensive junctions, present in epithelial

and muscle cells. Migration of keratinocytes in the re-epithelialization of skin wounds is

dependent on the formation of dermosomal junctions. Linkage of cadherins with the cytoskeleton

occurs through two classes of catenins. -catenin links cadherins with -catenin, which, in turn, connects to actin, thus completing the connection with the cytoskeleton. Cell-to-cell interactions

mediated by cadherins and catenins play a major role in regulating cell motility, proliferation,

and differentiation and account for the inhibition of cell proliferation that occurs when cultured

normal cells contact each other (contact inhibition). Diminished function of E-cadherin contributes to certain forms of breast and gastric cancer. As already mentioned, free -catenin acts independently of cadherins in the Wnt signaling pathway, which participates in stem cell

homeostasis and regeneration. Mutation and altered expression of the Wnt/-catenin pathway is implicated in cancer development, particularly in gastrointestinal and liver cancers ( Chapter 7 ).

In addition to the main families of adhesive proteins described earlier, some other secreted

adhesion molecules are mentioned because of their potential role in disease processes: (1)

SPARC (secreted protein acidic and rich in cysteine), also known as osteonectin, contributes to

tissue remodeling in response to injury and functions as an angiogenesis inhibitor; (2) the

thrombospondins, a family of large multifunctional proteins, some of which, similar to SPARC,

also inhibit angiogenesis; (3) osteopontin (OPN) is a glycoprotein that regulates calcification, is a

mediator of leukocyte migration involved in inflammation, vascular remodeling, and fibrosis in

various organs[76,][77]

(discussed later in this chapter); and (4) the tenascin family, which consist

of large multimeric proteins involved in morphogenesis and cell adhesion.

GLYCOSAMINOGLYCANS (GAGS) AND PROTEOGLYCANS

GAGs make up the third type of component in the ECM, besides the fibrous structural proteins

and cell adhesion proteins. GAGs consist of long repeating polymers of specific disaccharides.

With the exception of hyaluronan (discussed later), GAGs are linked to a core protein, forming

molecules called proteoglycans.[78]

Proteoglycans are remarkable in their diversity. At most sites,

ECM may contain several different core proteins, each containing different GAGs.

Proteoglycans were originally described as ground substances or mucopolysaccharides, whose

main function was to organize the ECM, but it is now recognized that these molecules have

diverse roles in regulating connective tissue structure and permeability ( Fig. 3-14 ).

Proteoglycans can be integral membrane proteins and, through their binding to other proteins and

the activation of growth factors and chemokines, act as modulators of inflammation, immune

responses, and cell growth and differentiation.

FIGURE 3-14 Proteoglycans, glycosaminoglycans (GAGs), and hyaluronan. A, Regulation of FGF-2 activity by ECM and cellular

proteoglycans. Heparan sulfate binds FGF-2 (basic FGF) secreted into the ECM. Syndecan is a cell surface proteoglycan with a

transmembrane core protein, extracellular GAG side chains that can bind FGF-2, and a cytoplasmic tail that binds to the actin

cytoskeleton. Syndecan side chains bind FGF-2 released by damage to the ECM and facilitate the interaction with cell surface

receptors. B, Synthesis of hyaluronan at the inner surface of the plasma membrane. The molecule extends to the extracellular space,

while still attached to hyaluronan synthase. C, Hyaluronan chains in the extracellular space are bound to the plasma membrane

through the CD44 receptor. Multiple proteoglycans may attach to hyaluronan chains in the ECM. (B and C, modified from Toole

KR: Hyaluronan: from extracellular glue to pericellular cue. Nat Rev Cancer 4:528, 2004.)

There are four structurally distinct families of GAGs: heparan sulfate, chondroitin/dermatan

sulfate, keratan sulfate, and hyaluronan (HA). The first three of these families are synthesized

and assembled in the Golgi apparatus and rough endoplasmic reticulum as proteoglycans. By

contrast, HA is produced at the plasma membrane by enzymes called hyaluronan synthases and

is not linked to a protein backbone.

HA is a polysaccharide of the GAG family found in the ECM of many tissues and is abundant in

heart valves, skin and skeletal tissues, synovial fluid, the vitreous of the eye, and the umbilical

cord.[79]

It is a huge molecule that consists of many repeats of a simple disaccharide stretched

end-to-end. It binds a large amount of water (about 1000-fold its own weight), forming a viscous

hydrated gel that gives connective tissue the ability to resist compression forces. HA helps

provide resilience and lubrication to many types of connective tissue, notably for the cartilage in

joints. Its concentration increases in inflammatory diseases such as rheumatoid arthritis,

scleroderma, psoriasis, and osteoarthritis. Enzymes called hyaluronidases fragment HA into

lower molecular weight molecules (LMW HA) that have different functions than the parent

molecule. LMW HA produced by endothelial cells binds to the CD44 receptor on leukocytes,

promoting the recruitment of leukocytes to the sites of inflammation. In addition, LMW HA

molecules stimulate the production of inflammatory cytokines and chemokines by white cells

recruited to the sites of injury. The leukocyte recruitment process and the production of pro-

inflammatory molecules by LMW HA are strictly regulated processes; these activities are

beneficial if short-lived, but their persistence may lead to prolonged inflammation.