Extracellular matrix 4: the elastic fiber

#{149}.:;; L

Extracellular JOEL

matrix 4: The elastic fiber

ROSENBLOOM,’

Department Pennsylvania

WILLIAM

R. ABRAMS,

-J. Frederick Woessner, Jr. of Miami School of Medicine Miami, Florida 33101-6960, USA

ABSTRACT The elastic properties of many tissues such as the lung, dermis, and large blood vessels are due to the presence of elastic fibers in the extracellular space. These fibers have been shown by biochemical and ultrastructural analysis to be composed of two distinct components, a more abundant amorphous component and a 10-12 nm microfibrillar component, which is located primarily around the periphery of the amorphous component. The protein elastin makes up the highly insoluble amorphous component and is responsible for the elastic properties. Elastin is found throughout the vertebrate kingdom and possesses an unusual chemical composition rich in glycine, proline, and hydrophobic amino acids, consonant with its characteristic physical properties. The 72-kDa biosynthetic precursor, tropoelastin, is secreted into the extracellular space where it becomes highly crosslinked into a rubber-like network through the activity of the copper-requiring enzyme lysyl oxidase. Analysis of the elastin gene has demonstrated that hydrophobic and cross-linking domains are encoded in separate exons and that there is significant alternative splicing, resulting in multiple isoforms of tropoelastin. The elastin gene promoter contains many potential binding sites for various

modulating

factors

indicative

of a complex

pattern

MECHAM5

protein. Mutations in the fibrillin gene located on human chromosome 15 have been strongly implicated as the cause of the Marfan syndrome.-Rosenbloom, J., Abrams, W. R., Mecham, R. The elastic fiber. FASEB J. 1208-1218; 1993.

7:

Key Wordc: elastin tropoelostin jibril-associated proteins

cross-linking

fibrillin

micro-

PHYSIOLOGICFUNCTIONOF MANYTISSUES in multicellular organisms requires that they possess elastic properties. Thus, during systole the work of the heart is absorbed by expansion of the great vessels, which then elastically recoil during diastole, maintaining the blood pressure and assuring continuous perfusion of the tissues. Similarly, under normal circumstances inspiration is an active, energy-requiring process, whereas expiration is a passive one due to the elastic recoil of the respiratory tree. Several unrelated proteins have evolved to achieve the property of elasticity including resilin in arthropods (1), abductin in molluscs (2), an elastomer in octopus ‘(3), and elastin in vertebrates (4). Phylogenetic studies have shown that elastin appeared at some point after the divergence of the cyclostome and gnathostome lines and is found in all vertebrate species, including the cartilaginous fish, but not in invertebrates. Within vertebrate tissues, elastin is found in the extracellular matrix in elastic fibers, which may comprise a small (2-4%) but important fraction of the dry weight (as in the skin) or greater than 50% (as in large arteries). In the electron microscope (5-7), elastic fibers are seen to be composed of two morphologically distinguishable components: 1) an amorphous fraction lacking any apparent regular or repeating structure constitutes 90% of the mature fiber and is composed exclusively of elastin, and 2) a microfibrillar component consisting of 10 to 12 nm fibrils, which are located primarily around the periphery of the amorphous component, but also to some extent interspersed within it (Fig. 1). Although the exact composition of the microfibrils remains to be defined, they contain several glycoproteins (8), including fibrillin (9). Early work on the histochemistry and chemical and physical properties of elastin has been reviewed by Partridge (10). The present review will focus on current findings on the genes encoding components of the elastic fiber and the relationship of protein structure to function. THE

of

transcriptional regulation. The microfibrils contain several proteins, including fibrillin, and probably act as an organizing scaffold in the formation of the elastin network. There appears to be a fibrillin gene family in which each protein contains multiple repeats of a motif previously found in epidermal growth factor and a second motif observed in transforming growth factor (31-binding

1208

ROBERT

of Anatomy and Histology; School of Dental Medicine, University of Pennsylvania, Philadelphia, 19104, USA; and 5Depment of Cell Biology; Washington University, St. Louis, Missouri 63110, USA

This is the fourth in the Serial Reviews on the extracellular matrix, initiated in the June 1993 issue. The third review dealt with evolutionary aspects of the extracellular matrix (Tanzer and Har-El, Sept., F] 7, 1115-1123, 1993). Elastin is an anomaly in the evolutionary pattern of matrix development, in that nature has been able to find several solutions to the problem of making a resilient elastic material that can be used for elastic recoil after an energy-consuming step of tissue movement. Thus, resilin developed in arthropods and abductin in molluscs. Elastin is a relatively recent arrival on the scene, appearing first in the vertebrates. One of several interesting properties of this highly cross-linked hydrophobic protein is how such an amorphous material can be formed into sheets and fibers within the tissues. The present review addresses these issues and concludes with a brief explanation of the basis of the elastic property of elastin. University

AND

‘To whom correspondence should be sent, at: Department Anatomy and Histology, School of Dental Medicine, University Pennsylvania, Philadelphia, PA 19104, USA.

0892-663819310007-12081$01.50.

of of

© FASEB

REVIEWS

Figure 1. Electron micrographs of developing elastic fibers in fetal bovine ligamenturn nuchae. A) 180 day gestation. Linear elastic fibers (stained black) are just starting to form. The nucleus of a fibroblast appears in the upper portion of the photomicrograph. Many cotlagenous fibers cut in cross-section appear as small black dots in the extracellular matrix. 9,600 x. B) 270 day gestation. Note the size of the mature elastic fibers which are now quite large compared to those seen in A. 9,600 x; C) 200 day gestation. Longitudinal section. Elastin is stained black and is surrounded by microfibrils. 47,000 x. D) 200 day gestation. Cross-section of fiber similar to C. Microfibrils appear as tubules. 47,000 x.

ELASTIN Isolation

and

characterization

Because of extensive cross-linking, elastin can be purified fairly readily by rather harsh extraction procedures, such as 0.1 M NaOH at 98#{176}C for 60 mm, which solubilize most other proteins but leave an insoluble elastin residue (11). Other, somewhat milder methods involving extraction with concentrated guanidine solutions and digestion with bacterial collagenase are also effective with some tissues, and result in less nonspecific cleavage of peptide bonds in the elastin (12). The profound insolubility of elastin has precluded all but the most superficial type of analysis. Amino acid analysis of elastin from a wide variety of vertebrate Species has revealed a peculiar composition, consistent with its unique physical properties (4, 13). It is rich in glycine (33%) and proline (10-13%), and hydrophobic amino acids constitute approximately 44% of the total residues. Although elastin contains about 4% lysine, most of the lysines are incorporated into cross-links and there are very few charged residues in the elastin portion of the mature fiber. Changes in composition during evolution suggest that there has been a progressive increase in hydrophobicity. This trend may be related to a parallel change in systolic blood pressure, which also increases from a low of 30 mm Hg in fish and amphibians to 120 to 150 mm Hg in mammals and birds. Tropoelastin Animals placed on aneurysms of the aorta tributed to a decreased fibers. This observation

THE ELASTIC FIBER

a copper-deficient diet suffered and other defects that could be atcontent of elastin in their elastic suggested that the deficiency im-

paired the cross-linking of elastin, and further experiments led to the isolation of a soluble precursor, tropoelastin (Mr 72,000), from the aortas of copper-deficient animals (14). Peptides resulting from tryptic digestion of porcine tropoelastin were extensively sequenced and could be divided into two classes: 1) small ones rich in alanine derived from regions destined to form the cross-links, and 2) larger peptides rich in hydrophobic residues derived from the regions responsible for the elastic behavior (15).

CHARACTERIZATION cDNA

OF

THE

ELASTIN

GENE

Analysis

Although early work yielded only partial cDNA clones encoding chick and sheep elastin (16, 17), improved techniques have permitted the construction of cDNA clones encompassing the entire length of human, bovine, chick, and rat elastin mRNA (18-22). Homologous sequences to the tryptic peptides of porcine tropoelastin were also identified. Further, all the cDNAs encoded an unusual, highly conserved carboxylterminal segment, GGACLGLACGRKRK, which was not previously identified by protein sequencing. The highly basic character of the carboxyl terminus suggests that this portion of tropoelastin may interact strongly with acidic microfibrillar proteins. The eDNA analyses demonstrated that tropoelastin consists predominantly of alternating hydrophobic and lysine-rich domains (Fig. 2), in which the lysines usually occur in pairs. This arrangement supports the suggestion that a given desmosine/isodesmosine serves to join only two tropoelastin molecules rather than the four that are theoretically possible. It is also apparent that these potential cross-linking sequences are not uniformly dis-

REVIEWS 200

100 2

300

500

400

600

700

ResidueNumber

1111111

liii

2 >1

-1

Poly A

-2

J

11111111 liii LI 4

[12 11

3

7

5

6

8

9

10 11

13 14

12

19

16

15

22

20

17 18

23

21

I

24

0

0.5

L__ 1.0

26A

30 31 29

1

I 1.5 Nucleotides

27 28

26

111 I

‘

liii 25

33 36

Poly A

C

5

55 ‘Ii

Exon

#

32

55 U,

11 I 2.0

3.5

(kb)

2. Diagrammatic representation of human elastin cDNA and hydropathy analysis of tropoelastin by the method of Kyte and Doolittle. The eDNA is divided into exons, which are numbered. In order to preserve a numbering system consistent with homologous sequences in the bovine gene, the most 3’ exon has been designated 36 in the human gene. Exons 34 and 35, found in the bovine gene, are absent in the human gene. Exons encoding hydrophobic sequences (0);exons encoding potential cross-linking sequences (U); exon 1 encoding most of the signal sequence (); exon encoding the carboxylterminus (U); 3’ untranslatedregion (D); exon encoding 26A (). A I symbol marks the exons known to be subject to alternative splicing in the human and bovine transcripts. Figure

tributed and occur at shorter intervals in the first 200 residues, resulting in an asymmetry to the molecule. In general, there is good agreement at the nucleotide and encoded amino acid sequence levels among the mammalian elastins, which differ, however, in some segments from those of the chicken. Among mammalian elastins, most amino acid substitutions are of a conservative nature, but some significant differences do exist. For example, near the center of bovine and porcine tropoelastins a pentapeptide, GVGVP, is repeated 11 times, but this repeat segment is considerably different and more irregular in human tropoelastin and is replaced in rat tropoelastin by GVGIP. Similarly, in human tropoelastin, a hexapeptide, GVGVAP, is repeated seven times, but only five times with conservative substitutions in bovine tropoelastin, and it is absent altogether in rat tropoelastin. These observed variations suggest that a particular number of amino acids and a precise sequence in a given hydrophobic region are not critical to the adequate functioning of the molecule, although there appears to be a strong tendency to conserve the size of the total polypeptide chain to 750-800 residues. There is rather extensive (80%) nucleotide homology in the 3’ untranslated region, suggesting that this region may have a function either in stabilizing the mature mRNA or even in modulating translation. Structure

of elastin

gene

All experiments carried out to date, including Southern blot hybridization analyses of bovine, ovine, and human genomic DNA, indicate that the elastin gene exists as a single copy. The human gene has been localized to chromosome 7q11.l-

1210

Vol. 7

October

1993

21.1 (23). The entire bovine and human elastin genes have been isolated and extensively sequenced (20, 21, 24). Rather small exons (27-186 bp) are interspersed between large introns resulting in a low coding ratio, about 1:20. Another important characteristic is that hydrophobic and cross-link domains of the protein are encoded in separate exons, so that the domain structure of the protein is a reflection of the exon organization of the gene. Although exons are all multiples of three nucleotides and glycine is usually found at the exonhintron junctions, the exons do not exhibit any regularity in size as is found in the fibrillar collagen genes. Exon/intron borders always split codons in the same way in the elastin gene. Thus, the second and third nucleotides of a codon are included in the 5’ exon border whereas the first nucleotide of a codon is found at the 3’ border of the previous exon. This consistent structure permits extensive alternative splicing of the primary transcript in a cassette-like fashion while maintaining the reading frame. Sequences homologous to exons 34 and 35 in other species have not been found in the human gene despite extensive searches. It is not presently known whether this difference has any functional significance. In the human genome, short interspersed elements of approximately 300 bp containing an Alul restriction site (Alu repeat) constitute 3-6% of the total mass of DNA (25), but such sequences are found at a frequency of about four times the expected value in the introns of the elastin gene. In addition to Mu repeats, rather long stretches composed of either alternating purines or alternating pyrimidines occur. The function, if any, of these repetitive elements remains to be determined. In other human genes, deletions apparently mediated by recombination between repetitive sequences

The FASEB Journal

ROSENBLOOM

Et AL.

REVIEWS have occurred resulting in hereditary diseases, and evidence for genomic instability in regions of human DNA enriched in Alu repeat sequences has been presented (26), but no mutations in the human elastin gene, possibly mediated by similar mechanisms, have been found. However, very recently a disruption of the elastin gene through a translocation in which the breakpoint occurred within exon 28 has been strongly linked to supravalvular aortic stanosis, an inherited disorder that causes hemodynamically significant narrowing of large arteries (27).

Alternative

splicing

of elastin

mRNA

Analysis of bovine, human, and rat elastin cDNAs have clearly demonstrated alternative splicing of the primary transcripts (19-22). In most cases, splicing occurs in a cassette-like fashion in which an exon is either included or deleted, but rarely a splicing event may divide an exon, as in the case of exons 24 and 26. Both hydrophobic and crosslink domains are affected, so that two cross-link domains may be brought into apposition (deletion of exon 22) or the interval between crosslink domains may be increased (deletion of exon 23). It is not known whether these variations have any functional significance, although clearly a tighter or looser fiber network could be produced. Si mapping experiments using elastin mRNA isolated from the developing bovine nuchal ligament have demonstrated that the alternative splicing of some exons occurs frequently but in the majority of cases it is infrequent. These experiments also suggest that the alternative splicing pattern in the adult may differ significantly from that occurring during development. Translation of the alternatively spliced transcripts would result in significant primary sequence variation among individual tropoelastin molecules, and it is likely that such differences explain the finding of variant isoforms of tropoelastin in several species (28, 29). It remains to be determined whether the splicing pattern is tissue-specific or whether variation in splicing occurs in disease situations. The possible variable expression of exon 26A in human elastin is particularly intriguing, as this segment, which is highly hydrophilic and atypical in amino acid sequence for elastin, rarely appears to be expressed under normal circumstances. Inclusion of this sequence might substantially alter the properties of the elastic fiber.

REGULATION Sequence initiation

OF

analysis

ELASTIN of the

GENE

promoter

and

EXPRESSION transcription

DNA sequencing of the 51 region flanking the start of the translation codon of the human and bovine genes revealed extremely strong conservation of portions of the sequence (94% homology from -ito -192 and 86% from -193 to -588), suggesting that these segments may have important functional roles (21, 24). No canonical TATA boxes were found and although two CAAT sequences were found in the human gene, their locations (-57 to -61 and -599 to -603) suggested that they are not functionally significant. The 5’ flanking region is generally G+C-rich (66%) with a high frequency of CpG dinucleotides. Many of these features have been previously associated with promoters of so-called ‘housekeeping genes,” but more tissue-specific genes are being found to possess them as well, so that the distinction between these classes is now breaking down (30). As is becoming apparent for a number of genes, the elastin promoter

THE ELASTICFIBER

contains a remarkable constellation of potential binding sites for transcription regulatory factors indicative of complex regulation. These binding sites include multiple SP1 and AP2 binding sites, glucocorticoid responsive elements, and TPA and cyclic AMP responsive elements (C RE)2 (for review of such elements, see ref 31). There is also an extended sequence of alternating guanine and pyrimidine residues (-225 to -275), a type of sequence that may be associated with Z DNA and may be involved in transcriptional regulation. The absence of a TATA box in the putative promoter region suggested that there may be multiple sites of transcription initiation. Sl protection and primer extension analyses using human fetal aorta RNA consistently identified three major clusters of transcription initiation centered around nucleotides -[7, 8], -[15, 16], and -[32, 33]. Five other minor initiation sites were observed between -45 and -195 (24). It remains to be determined whether a similar pattern is found in other tissues, whether the multiple initiation has any physiologic significance, and whether there is any relationship between the position of transcript initiation and the pattern of alternative splicing.

Functional

analysis

of the human

elastin

promoter

A panel of promoter/reporter gene constructs was used in transient transfection assays to determine the functional characteristics of the promoter (32). These experiments demonstrated the presence of multiple up- and downregulatory elements within the 2.2 kbp 5’ flanking region tested, and indicated that the core promoter necessary for basal expression was contained within the region -128 to -1, as removal of this segment abolished all promoter activity. The positive regulatory and core promoter activity may be explained, at least in part, by the presence of multiple SP-i and AP2 binding sites within these regions, which may act as general enhancer elements, and preliminary DNAse footprinting experiments have indicated that SP-i and AP2 sites interact with their respective trans factors. This notion is supported by the observation that deletion of the segment -134 to -87 containing three putative SP-1 binding sites reduced the activity to 10-20% of the reference -475 to -1 construct. Most of the detailed analyses were performed with rat aortic smooth muscle cells, but qualitatively similar results were obtained with NIH-3T3 cells, human skin fibroblasts, human HT-1080 fibrosarcoma cells, and HeLa cells. The human skin fibroblasts and fibrosarcoma HT-1080 cells express the endogenous elastin gene albeit at a low level, but HeLa and NIH-3T3 cells do not. Although gel mobility shift assays with segments of the elastin promoter (+2 to -195) have shown that nuclear extracts from elastin-producing cells (smooth muscle) give a different gel-shift pattern than extracts from cells that do not produce elastin (HeLa), collectively these observations suggest that all elements for tissue and development-specific expression of the elastin gene may not reside within the 2.26 kb promoter region tested. It has been observed in a number of genes, including three collagen genes, that the first intron contains segments that act as en-

2Abbreviations: IGF-I, insulin-like growth factor-I; TGF-f3;il, transforming growth factor-fl; TNF-a, tumor necrosis factor-a; MAGP, microfibril-associated glycoprotein; FLP, fibrillin-like protein; CRE, cyclic AMP responsive elements; TPA, 12-0tetradecanoylphorbol 13-acetate.

1211

REVIEWS hancer elements tron sequences vealed regions

but enhancer

of promoter activity. Comparison of the inof the bovine and human elastin genes reof strong homology only in the first intron,

activity

yet been

demonstrated.

Hormone,

vitamin,

of the homologous

growth

factor,

and

segments

has not

cytokine

effects

Analysis of the elastin promoter region revealed the presence of putative giucocorticoid and cAMP responsive elements, and several earlier observations suggest that these elements may be functionally active. For example, injection of dexamethasone into developing chick embryos increased the rate of elastin accumulation in the aorta (33), and incubation of fetal bovine nuchal ligament fibroblasts with dexamethasone increased elastin gene expression (34), whereas CAMP abrogated the cGMP stimulation of elastin production by the ligament fibroblasts (35). However, direct proof that any of these effects are regulated at the transcriptional level is still lacking. Insulin-like growth factor-i (IGF-i) has also been shown to enhance elastin gene expression. Incubation of rat neonatal aortic smooth muscle cells with 20-80 ng/ml of IGF-i produced a dose-dependent increase (to a maximum of fourfold) in the synthesis of tropoelastin and a corresponding increase in the steady-state levels of tropoelastin mRNA. Transient transfection with a 500-bp elastin promoter! reporter gene construct suggested that the IGF-I was acting at the transcriptional level and that the responsive element was within the 500 bp (36). In contrast, recombinant tumor necrosis factor-a (TNF-a) markedly suppressed elastin mRNA levels in a time- and dose-dependent manner by up to 91% in cultured human skin fibroblasts and rat aortic smooth muscle cells (37). TNF-a also suppressed the expression of elastin promoter/reporter constructs by up to 70% in transiently transfected cells, again indicating regulation at the transcriptional level. Detailed analyses of the mechanisms involved strongly suggested that the down-regulatory effect of TNF-a was mediated through jun/fos binding to an AP-1 site located at -223 to -229 in the elastin promoter. Interleukin-1f3 has also been shown to inhibit the synthesis of elastin and decreased elastin mRNA steady-state levels in cultures of a neonatal rat lung subtype that contained large amounts of intracellular lipid (38). However, the mechanisms involved in the interleukin inhibition are not presently

known. Although it is quite possible that general transcription factors interact with other factors yet to be identified to achieve regulation of elastin expression, other mechanisms must be considered. Presently, there is no evidence for translational control of elastin mRNA, but strong evidence for either upor down-regulation of tropoelastin expression through alteration of elastin mRNA stability by several modulators has been presented recently. Elastin production, as determined by ELISA, was increased approximately threefold when porcine smooth muscle cells were incubated with 1tF-i (39). Examination of the elastin gene, from -2,260 to -1, did not reveal the presence of putative NF-l binding sites, which have previously been shown to mediate the up-regulatory effects of TGF-f31 in the expression of the pro a2(I) collagen gene (40), suggesting that the TGF-fll responsive elements in the elastin gene may reside outside the region, differ from NF-1, or that the TGF-/3 effects are mediated at the posttranscriptional level, possibly through stabilization of elastin mRNA as has been reported with cultured human skin fibroblasts incubated with TGF-/3 (41). When cultures of elastogenic fetal bovine chondrocytes were exposed to 10 M 12-O-tetradecanoylphorbol 13-acetate (TPA), tropoelastin

1212

Vol. 7

October

1993

mRNA

paralleled

levels

decreased

by a decline

greater

than

in the production

10-fold

and

this was

of tropoelastin (42). and transient trans-

As determined by nuclear-runoff assay fection with a human gene promoter-CAT construct, tropoelastin transcription was unaffected by exposure to TPA. When actinomycin D was used to inhibit transcription, the half-life of tropoelastin mRNA in control cells was estimated to be about 20 h, but exposure to TPA reduced the half-life to 2.2 h. Similarly, i0 M 1,25-dihydroxyvitamin D3, a rather high unphysiologic dose, produced a marked decrease in steady-state and functional levels of tropoelastin mRNA, but transcription was not affected (43). Collectively, these data indicate that tropoelastin expression can be controlled by posttranscriptional mechanisms.

MICROFIBRILS On electron microscopic examination, the microfibrils are seen to consist of apparently tubular structures, about 1012 nm in diameter, that possess different staining properties and susceptibilities to enzymatic digestion from the amorphous elastin (Fig. 1) (8). At high resolution, the microfibrils appear in cross section as an outer electron-dense shell surrounding an inner lucid core, and in longitudinal section as a beaded chain, suggesting that they may be composed of more than one protein. Apparently similar microfibrils are present in many tissues, including those that contain abundant elastin as well as those in which there is no visible or immunoreactive elastin, such as the ocular ciliary zonule and the periodontal ligament. In the dermis, the microfibrillary bundles that connect the deep dermal elastic plexus with the dermoepidermal junction region are seen to consist of microfibrils indistinguishable from those associated with elastic fibers. In their superficial distribution, no immunoreactive elastin is associated with them, but as they traverse the dermis they are associated with an increasing amount of amorphous elastin. Microfibrillar

proteins

Because of their insolubility and apparent complexity, chemical characterization of the microfibrils has progressed slowly. Although there had been several earlier reports provisionally identifying different proteins as components of the microfibrils, in most cases this identification has subsequently proved to be incorrect. For example, a 150-kDa polypeptide synthesized by bovine ligamentum nuchae fibroblasts in culture, and once thought to be microfibril-related, has been shown to be an a-chain of type VI collagen, which is found in other matrix fibrils but not in elastin-associated microfibrils (44). The problem of identifying structurally important components is compounded by the observation that small amounts of several proteins, including amyloid P and decay-accelerating factor, have been localized to the elastic microfibrils by immunoelectron microscopy. Although they may have some functional role,itisunlikely that any one of them is a major structural component of the microfibrils. Nevertheless, within the last few years significant progress has been made in identifying certain proteins as structural Components. The largest of these and possibly the most important is fibrillin, a 350-kDa glyprotein reported to form an integral part of the microfibril structure (9). Electron microscopic images of monomeric fibrillin prepared from human fibroblast cultures reveal an extended flexible molecule approximately 148 nm long and 2.2 nm wide. Multiple fibrillin molecules may align in a parallel head-to-tail fashion

The FASEBJournal

ROSENBLOOM El AL.

REVIEWS to form a major portion of the microfibrils. Molecular cloning studies have demonstrated that there are at least two distinct homologous human genes encoding fibrillin proteins, one located on chromosome 15q-21 and the other on chromosome 5q23-31 (45, 46). Recently a third protein, provisionally named fibrillin-like protein (FLP), was isolated and shown to contain the same repeating domains as Fib 5 and Fib 15 (M. A. Gibson, M. Mariencheck, E. Davis, E. Baker, G. R. Sutherland, and R. P. Mecham, unpublished results). Immunohistological studies localized FLP to microfibrils in elastic fibers, suggesting that FLP is a third member of the fibrillin family of proteins. Analysis of the amino acid sequences deduced from the cloned cDNAs for Fib 5, Fib 15, and FLP (although still incomplete) demonstrated that the proteins contain multiple repeats of a sequence motif previously observed in epidermal growth factor, having six conserved cysteines (47). Many of these repeats contain consensus sequences that have been associated with calcium binding through hydroxlation of asparagine or aspartic acid residues. Also, several of the EGF-like repeats have homology to the Notch gene in Drosophila and lin-12 in Caenorhabditis elegans. A second motif containing eight cysteines, found in transforming growth factor 131-binding protein (48), has also been identified in all three fibrillins (Fig. 3). It appears from the partial information available on the structure of the genes that they are likely to be quite large and contain relatively small, widely dispersed exons. An exciting finding is the unequivocal genetic linkage of the fibrillin gene on chromosome 15 to the Marfan syndrome (45, 49). Inherited as an autosomal dominant disorder with complete penetrance, this disease is characterized by a complex and variable phenotype in which disorders in the skeletal, ocular, and cardiovascular systems predominate (50). Skeletal features include tall stature, long extremities (dolichostenomelia), arachnodactyly, joint laxity, and chest and spine deformities (pectus excavatum and carinatum, scoliosis). Ocular findings can include subluxation of the lens (ectopia lentis), retinal detachment, and myopia. The cardiovascular features of progressive aortic root dilation and mitral valve prolapse, if left untreated, can lead to more

Domain Alignment

I

I iI

I litiI

-

Ii

ii

of Fib-15, Fib-5,

Ii IiIil1

Ii i II

II II

III

1111 liii

ii t8

11I1I1 Iliii

.11

OEGF-IIke

II’I

FLP and TGF-BP

I

-

I1

lit

domain

ITGF-BP.Iike domain Cys-Cys domain #{149} Cys-fr.. domain - -- Unknown sequence Potential Olycosylation SIte

Figure 3. Domain map of fibrillin gene family. Schematic representation of domain structure of fibrillin 15 (Fib 15), fibrillin 5 (Fib 5), fibrillin-like protein (FLP), and transforming growth factor beta-i binding protein (TGF-BP). cDNA analysis indicates that FLP is alternatively spliced at the carboxyl terminus.

THE ELASTICFIBER

severe and potentially life-threatening complications such as aortic and mitral regurgitation, aortic aneurysm, and aortic dissection. Cardiovascular events account for approximately 85% of patient deaths, and the average life expectancy of affected individuals is reduced by one-third to one-half (51). Immunofluorescent studies using monoclonal antibodies to fibrillin provided the first evidence of decreased or abnormal microfibrils in the skin of Marfan patients. In addition, fibroblasts cultured from skin biopsies of affected individuals were defective in their ability to produce a normal microfibrillar network (52). In the sequence cloned to date, a variety of mutations in the fibrillin 15 gene of Marfan patients have been identified, and fall into two broad categories (53). The first are the exon deletion events. These include gross exon deletions as well as exon skipping caused by splicing errors. The second group of mutations are centered on the EGF-like domains of fibrillin, with the cysteine residues in these motifs being particularly vulnerable to mutation. Because each of the six cysteines in the repeat participate in disulfide bonds that connect the f-turns of the EFG module, substitutions or deletions of even one cysteine could disrupt the structure of the molecule as a whole. In addition, mutations that affect the calcium binding consensus sequence of the EGF repeats have been reported. The gene located on chromosome 5 was tightly linked genetically to a second condition, congenital contractural arachnodactyly (45). The cloning of these genes promises to facilitate greatly the determination of the function of the fibrillin proteins and their role in the pathophysiology of the elastic fiber. A number of other proteins described as microfibril constituents appear unrelated to the fibrillins. When bovine nuchal ligament, a tissue rich in microfibrils, was extracted with reductive saline, five major bands of 340, 78, 70, 31, and 25 kDa were identified upon gel electrophoresis (54). The 340-kDa component is the bovine homologue of human fibrillin 15. Immunoelectron microscopy using affinitypurified antibodies demonstrated that the 78-kDa (called microfibrillar protein 78 or MP78) and the 31-kDa protein, termed microfibril associated glycoprotein (MAGP), were components of microfibrils. The relationship of the 25-kDa and 70-kDa species to the microfibrils remains to be established. MAGP has been cloned and found to be distinct from fibrillin. The deduced primary structure indicates that MAGP contains two structurally distinct regions, the aminoterminal half of the protein is rich in glutamine, proline, and acidic amino acids whereas the carboxyl-terminal half contains all 13 of the cysteine residues and most of the basic amino acids (55). The observation that MAGP can be extracted from tissues by several solvents provided they contain a strong reducing agent suggests that intermolecular disulfide bonding is an important feature of the association of the polypeptide chains in the fibrils. MAGP contains no Nlinked carbohydrate consensus sequences (Asn-X-Ser/Thr). Thus, the carbohydrate components of MAGP are most likely (0)-linked through serine or threonine for which there are no known consensus sequences. Northern blot analysis identified a 1.1 kb mRNA encoding a protein with a calcu lated molecular weight of only 21,000. The discrepancy between this value and that observed (31 kDa) for the isolated protein is due to anomalous behavior of the protein upon gel electrophoresis. Other candidate microfibril components include the enzyme lysyl oxidase (56), glycoproteins of 36 kDa (57), 115 kDa (58), and a 32-kDa glycoprotein termed associated microfibril protein (59). The 32-kDa associated microfibril protein was cloned from a chick embryo library and the encoded protein was remarkable in that it was extremely acidic with glutamic acid com-

1213

n

V

prising 23% and aspartic acid 6% of the residues. Antiserum elicited to a 14 amino acid synthetic peptide sequence of the protein was shown by immunoelectron microscopy to localize specifically to ultrastructurally definable microfibrils in tissue sections from chick aortae, bovine nuchal ligament, and human ocular zonules. The extremely acidic nature of the protein suggests that it may have an important function in the assembly of the very basic tropoelastin molecules. In summary, the available evidence indicates that microfibrillar structures with apparently similar morphology are widely distributed in many tissues in the body. However, despite the characterization and cloning of several putative microfibril proteins, a number of questions regarding microfibril structure and function remain unresolved. The precise relationships between the fibrillins and other proposed components of the microfibril remain obscure. Moreover, although apparently identical microfibrils have been identified ultrastructurally both with and without associated elastin, it has yet to be determined whether structural or compositional differences exist between these two groups or among microfibrils in different tissues. In particular, the emergence of a family of fibrillins raises the possibility of tissue-specific or temporally regulated expression of the different fibrillin genes. Biosynthesis

of elastin

and

fiber

assembly

Under normal circumstances, elastin is synthesized in quantity only by embryonic and rapidly growing tissues, and cells derived from them. Studies with such systems have shown that approximately 20 mm are required to synthesize tropoelastin and secrete it into the extracellular matrix (60). The intracellular biosynthetic pathway appears to be the classical one followed by several secreted proteins (61), and colchicine, which depolymerizes microtubules, inhibits secretion significantly (62). Although some of the prolyl residues in tropoelastin are hydroxylated posttranslationally, unlike the case of collagen in which hydroxyproline stabilizes the triple helix and inhibition of hydroxylation inhibits secretion, inhibition of hydroxylation of tropoelastin does not inhibit the rate of tropoelastin secretion, and the function of hydroxyproline, if any, in tropoelastin is unknown. Several reports have suggested that ascorbic acid, a cofactor in the hydroxylation reaction, does not alter the synthesis of tropoelastin, but the accumulation of insoluble elastin is markedly affected in concentrations as low as 2 tg/ml (64). It is conceivable that ascorbic acid mediates an increase in the degree of prolyl hydroxylation of tropoelastin, which interferes with fibrillogenesis and/or stabilization of elastin fibers. Elastin isolated from aortas of Marfan syndrome patients shows considerably increased levels of hydroxyproline. It has been suggested that this may explain, at least in part, some of the compromised tensile strength properties in the aortas of Marfan patients (64), but the relation of this finding to mutations in fibrillin is not clear. Also unlike the case of some fibrillar collagens in which cleavage of amino and carboxyl propeptides occurs, it is probable that tropoelastin is incorporated without proteolytic cleavage into the insoluble fiber. In several developing embryonic systems, including chick aorta and in sheep nuchal ligament and lung, a strong correlation between messenger RNA levels and the rate of elastin synthesis has been demonstrated, which suggests that the rate of synthesis is controlled by the mRNA steady-state level (16, 65). Cross-linking A critical function, 1214

feature of the elastic fiber, crucial to its proper is the high degree of cross-linking of tropoelastin

Vol. 7

October

1993

that takes place in the extracellular space. The enzyme that catalyzes the oxidative deamination and condensation of lysyl side chains, lysyl oxidase (for review, see ref 66), is the same enzyme involved in collagen cross-link formation and it may be directly associated with microfibrils. Some of the final difunctional products, such as lysinonorleucine and allysine aldol, are also found in collagen fibers, but the tetrafunctional ones, the desmosines, are found exclusively in elastin. The lysine residues that serve as cross-links in elastin occur as pairs in polyalanine sequences or, to a lesser extent, embedded in hydrophobic amino acids and separated by a proline residue and an alanine or a glycine. In contrast to the ability of tropoelastin to tolerate sequence variability in the hydrophobic domains, there appears to be conservation of the cross-linking domains, especially in the numbers of residues between lysines. The reason for this motif preservation becomes obvious when the conformational requirements of cross-linking are considered. In the polyalanine regions of elastin, the lysine residues are always separated by two or three alanines, never only one or more than three. The conformation of the alanine-rich cross-linking domains is essentially a-helical (67). In an ahelix, each residue is related to the next one by a translation of 1.5 A along the helix axis and a rotation of 100#{176}, forming a rodlike structure with side chains extended outward in a helical array. Thus, the side chains of amino acids separated by two or three residues in the linear sequence are spatially close to one another on the same side of the helix whereas side chains of amino acids separated by one or four residues are situated on opposite sides of the helix and are unlikely to make contact (Fig. 4). Gray (68) has proposed that a critical step in the cross-linking pathway is the formation of a bifunctional “within chain” cross-link intermediate, which then condenses with another bifunctional intermediate on a second chain to form the tetrafunctional desmosine cross-links. If the elastin sequences are considered in this context, the lysine side chains in the sequences . - .Lys-Ala-Ala-Lys.. and ...Lys-Ala-Ala-Ala-Lys... are positioned on the same side of the helix where they can interact one with another to form a bifunctional intermediate after oxidation by lysyl oxidase. Separation by one or four alanines would position the side chains on opposite sides of the helix, too far apart for subsequent condensation reactions.

A

Figure 4. Cross-sectional view of the cross-linking region of elastin drawn as an a-helix. The polypeptide main chain forms the inner part of the helix with side chains extending outward in a helical array. When the sequences found in elastin are substituted into the side chain positions beginning with position 1, it can be seen that lysine side chains in the 4 or 5 position are close to the side chain of lysine 1, able to form the “within chain” cross-link that is the precursor to the tetrafunctional desmosine cross-link. Lysines in positions 3 and 6 are on the opposite side of the helix from lysine 1, and hence cannot interact.

The FASEBJournal

ROSENBLOOM E AL.

REVIEWS All but about 5 of the 34 lysine residues of tropoelastin participate in some form of cross-link. The net result is a highly insoluble polymer in which some type of interchain lysine-derived cross-link occurs at approximately every 65-70 residues.

posed that the carboxyl-terminal domain is cleaved from the protein after it serves its alignment function. Other laboratories, however, have detected the carboxyl-terminal sequence in insoluble elastin (29, 75), suggesting that tropoelastin does not undergo cleavage before cross-linking.

Fiber

BASIS

assembly

Relatively little is known concerning the detailed mechanisms involved in fiber assembly, but the notion that tropoelastin molecules are simply secreted and then diffuse onto the surface of growing fibers where they bind and become cross-linked seems inadequate to explain the efficiency of the assembly process and the variable form of elastic fibers in different tissues. There is increasing evidence that tropoelastin secretion and assembly require helper proteins both inside and outside the cell. Elastin fibrillogenesis takes place at unique sites close to the cell membrane, generally in infoldings of the cell surface (69). Microfibrils are the first visible components of elastic fibers and . are found grouped in small bundles near the plasma membrane. As the fiber develops, elastin appears as an amorphous material in discrete loci within each microfibrillar bundle. These amorphous areas gradually coalesce and generate the central core of elastin. The majority of microfibrils are progressively displaced to the outer aspect of the fiber, a position they retain in the mature tissue. The observation that microfibrillar aggregates take the form and orientation of presumptive elastic fibers suggest that they direct the morphogenesis of elastic fibers by acting as a ‘scaffold’ on which elastin is deposited. It is likely that microfibrils serve to align tropoelastin molecules in precise register so that cross-linking regions are juxtaposed before oxidation by lysyl oxidase. How secretion of tropoelastin is targeted to sites of elastic fiber assembly remains unknown. There is some evidence, however, that a 67-kDa protein binds tropoelastin in the endoplasmic reticulum and directs its secretion to assembly sites on the cell surface (70). The intracellular motor that transports the tropoelastin-67 kDa complex appears to be intracellular actin (71), which may be directed to membrane sites by cell-matrix receptors that interact with microfibrillar proteins. At the plasma membrane, tropoelastin remains bound to the 67-kDa protein until an interaction with the microfibril induces the transfer of tropoelastin to the growing fiber. Biochemical studies have shown that the 67-kDa elastin-binding protein is a galactoside lectin and that its affinity for elastin is greatly diminished by interactions with carbohydrate. It is possible that galactose sugars on microfibrillar glycoproteins provide specific signals for release of tropoelastin at sites of fiber assembly. Supporting a matrix-assembly function for the 67-kDa protein are studies showing inhibition of elastin fiber assembly by the addition of lactose or galactose sugars to the culture medium of elastin-producing cells (72). In the presence of lactose, the majority of newly synthesized tropoelastin is released directly into the medium with only low levels remaining associated with the cell layer. These results suggest that lactose causes premature release of tropoelastin before it can be transferred to acceptor sites on microfibrils. The domain on tropoelastin that mediates interactions with microfibrils is unknown, although the carboxyl-terminal end of the protein has been suggested to serve such a function (73, 74). The only two cysteine residues in elastin are found in this part of the molecule where they form a disulfide bond that stabilizes a positively charged pocket suitable for binding acid microfibrils (74). Franzblau et al. (73) have pro-

THE ELASTIC FIBER

OF

ELASTIC

PROPERTIES

Elastic recoil is a critical property of several tissues and organ systems. For example, during an average lifetime the elastic fibers in the aortic arch undergo more than a billion stretch/relaxation cycles. As the turnover of elastin appears to be quite low (10), individual fibers can conceivably last a lifetime. The rather amazing durability of the elastic fiber suggests that the elastomeric force results not from the stressing of chemical bonds that could result in progressive deterioration, but rather results, upon stretching, from a decrease in the number of conformations accessible to the cross-linked polypeptide chains. The increase in the number of available conformational states upon removal of the stretching force provides the free energy for elastic recoil. That is, like a true rubber, the elasticity is due to an increase in entropy upon relaxation (Fig. 5). This view has been essentially proved by a number of physical studies (76). Structural

models

Determination of the structure of elastin, other than the primary amino acid sequence, has proved difficult. Elastic fibers yield only broad rings upon X-ray diffraction, indicating little short-range order (77), and by polarized light microscopy they appear optically isotropic. Nuclear magnetic resonance studies of elastin have clearly shown that the backbone chain of the protein is highly mobile, with individual residues in the chain able to rotate freely in three dimensions (78). Although these observations have indicated that elastin exists predominantly in a kinetically free, largely random coil network, some observations suggest regions of local order in the molecule. For example, circular dichroic spectra indicate that the alanine-rich sequences in the crosslink regions are in an a-helix (67). In addition, some electron microscopy studies have suggested the presence of a filamentous substructure. Whereas freeze-fracture electron microscopy of unstretched elastin revealed an isotropic structure, samples that had been stretched 150-200% appeared to contain ordered filaments approximately 13 nm in diameter. Negative staining of insoluble elastin revealed a filamentous network with the filaments having a diameter of 3-5 nm. Urry and co-workers (79) have carried out a variety of physical/chemical studies of a-elastin (peptides solubilized by hydrolysis of insoluble elastin with oxalic acid solutions) as well as of a number of synthetic polypeptides, some of which are found as limited repeating units in elastin, for example VPGVG and VAPGVG (79). Based on these studies and the observation that under certain circumstances elastin polypeptides can be found in thin filaments, Urry (79) has proposed that a significant proportion of the hydrophobic segments of elastin are found in a structure designated a spiral, a loose water-containing helical structure in which 13turns act as spacers between suspended segments of the helix. From these rather different views of the elastin molecule, one in which the polypeptide chain is largely a random coil and the second in which it is a f-spiral, two alternative structural models have been developed to explain the elastic force: 1) In the first model, elastin is a network of largely random chains within the elastic fibers, which behave according to

13-

1215

REVIEWS

Relaxed

Figure 5. Diagrammatic representation of cross-linked elastin in of which are probably in an a-helical conformation, are in bold. or bifunctional (I) cross-links are shown. The hydrophobic limited portions may exist in a f-spiral conformation (see text). alignment and limits their conformationai freedom.

(

1216

)

Vol. 7

October

1993

relaxed and stretched states. The potential cross-linking domains, some For clarity of presentation only some of the tetrafunctional desmosine domains in the relaxed state are probably largely random coils, although In the stretched state, the imposed force brings the chains into relative

The FASEBJournal

ROSENBLOOM Er AL.

REVIEWS the classical theory of rubber elasticity (76). In this model, the collection of chains have a random distribution of end-toend chain lengths and displacement from this position of highest entropy provides the source of the restoring elastic force (Fig. 5). 2) In contrast, for the second model Urry (79) has proposed that the entropic elasticity derives from the f-spiral structure, with essentially fixed end-to-end chain lengths. The peptide segments suspended between the f-turns are free to undergo large-amplitude, low-frequency rocking motions called librations. Upon stretching, there is a decrease in amplitude of the librations that results in a large decrease in the entropy of the segment, and this provides the driving elastomeric force for return to the relaxed state. It should be emphasized that although these models differ in their molecular conformational details, both rely on an entropy-driven mechanism to provide the elastomeric force. Further studies are required to prove whether either of these models is uniquely valid or whether the structure of elastin incorporates some features of both. The present description has also failed to consider the role of water and hydrophobic interactions as factors in the elastic reaction. Such interactions are undoubtedly significant, but relatively minor components of the overall process. Supported by National Institutes AR20553, HL26499, and HL41926.

of Health

grants

AR41474,

18. Bressan, G. M., Argos, P., and Stanley, K. K. (1987) Repeating structure of chick tropoelastin revealed by complementary DNA cloning. Biochemistry 26, 1497-1503 19. Raju, K., and Anwar, R. A. (1987) Primary structures of bovine elastin a, b, and c deduced from the sequences of eDNA clones. J. Biol. Chem. 262, 5755-5762 20. Indik, Z., Yah, H., Ornstein-Goldstein, N., Sheppard, P., Anderson, N., Rosenbloom, J. C., Peltonen, L., and Rosenbloom, J. (1987) Alternative splicing of human elastin mRNA indicated by sequence analysis of cloned genomic and complementary DNA. Proc. NatI. Acad. Sci. USA 84, 5680-5684 21. Yeh, H., Anderson, N., Ornstein-Goldstein, N., Bashir, M. M., Rosenbloom,J. C., Abrams, W., Indik, Z., Yoon, K., Parks, W., Mecham, R., and Rosenbloom, J. (1989) Structure of the bovine elastin gene and SI nuclease analysis of alternative splicing of elastin mRNA in the bovine nuchal ligament. Biochemistry 28, 2365-2370 22. Pierce, R. A., Deak, S. B., Belsky, S. A., Stolle, C. A., and Boyd, C. D. (1990) Heterogeneity of rat tropoelastin mRNA revealed by eDNA cloning. Biochemistry 29, 9677-9683 23. Fazio, M. J., Mattei, M. -G., Passage, E., Chu, M. -L., Black, D., Solomon, E., Davidson, J. M., and Uitto, J. (1991) Human elastin gene: new evidence for localization to the long arm of chromosome 7. Am. j Hum. Genet. 48, 696-703 24. Bashir, M. M., Indik, Z., Yeh, H., Ornstein-Goldstein, N., Rosenbloom, J. C., Abrams, W., Fazio, M., Uitto, J., and Rosenbloom, J. (1989) Characterization of the complete human elastin gene. Delineation of unusual features in the 5-flanking region. J. Biol. Chem. 264, 8887-8891 25. Schmid,C. W., and Jelinek, W. R. (1982) The Alu family of dispersed repetitive sequences. Science 216, 1065-1070 26. Calabretta, B., Robberson, D. L., Berrera-Saldana, H. A., Lambrou, T. P., and Saunders, G. P (1982) Genome instability in a region of human DNA enriched in Alu repeat sequences. Nature (London) 296,

219-225

REFERENCES 1. Andersen, S. 0. (1971) Resilin. In Comprehensive Biochemistry (Florkin, M., Stotz, E. H., eds) p. 633, Elsevier, Amsterdam 2. Kelly, R. E., and Rice, R. V. (1967) Abductin: a rubber-like protein from the internal triangular hinge ligament of pecten. Science 155, 208-210 3. Shadwick, R. E., and Gosline, J. M. (1981) Elastic arteries in invertebrates: mechanics of the octopus aorta. Science 213, 759-761 4. Sage, H., and Gray, W. R. (1979) Studies on the evolution of elastin. I. Phylogenetic distribution. Comp. Biochem. PhysioL [B] 64, 313-327 5. Fahrenbach, W. H., Sandberg, L. B., and Cleary, E. G. (1966) Ultrastructural studies on early elastogenesis. Anal Rec. 155, 563-576 6. Greenlee, T. K., Jr., Ross, R., and Hartman, J. L. (1966) The fine structure of elastic fibers. J. Cell Biol. 30, 59-71 7. Karrer, H. E., and Cox, J. (1961) Electronmicroscope study of developing chick embryo aorta. j Ultrastruct. Res. 4, 420-454 8. Cleary, E. G. (1987) The microfibrillar component of the elastic fibers. Morphology and biochemistry. In Connective Tissu#{128} Disease. Molecular Pathology of the Extracellular Matrix (Uitto, J., Perejda, A. J., eds) pp. 55-81, Marcel Dekker, New York 9. Sakai, L. Y., Keene, D. R., and Engvall, E. (1986) Fibrillin, a new 350-kD glycoprotein, is a component of extracellular microflbrils.J. Cell BioL 103, 2499-2509 10. Partridge, S. M. (1962) Elastin. Ado. Protein Chem. 17, 227-302 11. Lansing, A. I., Rosenthal, T. B., Alex, M., and Dempsey, E. (1952) The structure and chemical characterization of elastic fibers as revealed by elastase and by electron microscopy. Anal Rec. 114, 555-575 12. Ross, R., and Bornstein, P. (1969) The elastic fiber. I. The separation and partial characterization of its macromolecular components. J. Cell Biol. 40, 366-381 13. Starcher, B. C., and Galione, M. J. (1976) Purification and comparison of elastins from different animal species. Anal. Biocliem. 74, 441-447 14. Sandberg, L. B., Weissman, N., and Smith, D. W. (1969) The purification and partial characterization of a soluble elastin-like protein from copper-deficient porcine aorta. Biochemistry 8, 2940-2945 15. Foster,J. A., Bruenger, E., Gray1 W. R., and Sandberg, L. B. (1973) Isolation and amino acid sequences of tropoelastin peptides. j Biol. Chem. 248, 2876-2879 16. Burnett, W., Finnigan-Bunick, A., Yoon, K., and Rosenbloom, J. (1982) Analysis of elastin gene expression in the developing chick aorta using cloned elastin eDNA. j Biol. Chem. 257, 1569-1572 17. Yoon, K., May, M., Goldstein, N., Indik, Z. K., Oliver, L., Boyd, C., and Rosenbloom, J. (1984) Characterization of a sheep elastin eDNA clone containing translated sequences. Biochem. Biophys. Res. Commun. 118, 261-269

THE ELASTIC FIBER

27. Curran, M. E., Atkinson, D. L., Ewart, A. K., Morris, C. A., Leppert, M. F., and Keating, M. T. (1993) The elastin gene is disrupted by a translocation associated with supravalvular aortic stenosis. Cell 73, 159-168 28. Rich, C. B., and Foster, J. A. (1984) Isolation of tropoelastin from lathyritic chick aortae. Biochem. J. 217, 581-584 29. Wrenn, D. S., Parks, W. C., Whitehouse, L. A., Crouch, E. C., Kucich, U., Rosenbloom, J., and Mecham, R. P (1987) Identification of multiple tropoelastins secreted by bovine cells.j Biol. Chem. 262, 2244-2249 30. Gardiner-Garden, M., and Frommer, M. (1987) CpG islands in vertebrate genomes. j Mol. Biol. 196, 261-282 31. Wingender, E. (1990) Transcription regulating proteins and their recognition sequences. Crit. Rev. Eukaryotic Gene Exp. 1, 11-48 32. Kahari, V. -M., Fazio, M. J., Chen, Y. Q., Bashir, M. M., Rosenbloom, J., and Uitto, J. (1990) Deletion analyses of 5-flanking region of the human elastin gene: delineation of functional promoter and regulatory ciselements. J. Biol. Chem. 265, 9485-9490 33. Eichner, R., and Rosenbloom, J. (1979) Collagen and elastin synthesis in the developing chick aorta. Arch. Biochem. Biophys. 198, 414-423 34. Mecham, R. P., Morris, S. L., Levy, B. D., and Wrenn, D. S. (1984) Glucocorticoids stimulate elastin production in differentiated bovine ligament fibroblasts but do not induce elastin synthesis in undifferentiated cells. j Biol. Chem. 259, 12414-12420 35. Mecham, R. P., Levy, B. D., Morris, S. L., Madaras, J. G., and Wrenn, D. S. (1985) Eincreased cyclic GMP levels lead to a stimulation of elastin production in ligament fibroblasts that is reversed by cyclic AMP. j Biol. Chem. 260, 3255-3261 36. Rich, C. B., Ewton, D. Z., Martin, B. M., Florini, J. R., Bashir, M., Rosenbloom, J., and Foster, J. A. (1992) IGF-i regulation of elastogenesis: comparison of aortic and lung cells. Am. j Phys. 263, L276-L282 37. Kahari, V. -M., Chen, Y. Bashir, M., Rosenbloom, J., and Uitto, J. (1992)Tumor necrosis factor-a down-regulates human elastin gene expression. j Biol. Chem. 267, 26134-26141 38. Berk, J. L., Franzblau, C., and Goldstein, R. H. (1991) Recombinant interleukin-lf inhibits elastin formation by a neonatal rat lung fibroblast subtype. j Biol. Chem. 266, 3192-3197 39. Liu, J., and Davidson, J. M. (1989) The elastogenic effect of recombinant transforming growth factor-f on porcine aortic smooth muscle cells. Biochem. Biophys. Res. Commun. 154, 895-899 40. Rossi, P., Karsenty, G., Roberts, A. B., Roche, N. S., Sporn, M. S., and de Crombrugghe, B. (1988) A nuclear factor I binding site mediates the transcriptional activation of a type I collagen promoter by transforming growth factor-f. Cell 52, 405-414 41. Kahari, V.-M., Olsen, D. R., Rhudy, R. W., Carrillo, P., Chen, Y. and Uitto, J. (1992) Transforming growth factor-f upregulates elastin gene expression in human skin fibroblasts. Lab. Invest. 66, 580-588 42. Parks, W. C., Kolodziej, M. E., and Pierce, R. A. (1992) Phorbol estermediated downregulation of tropoelastin expression is controlled by a posttranscriptional mechanism. Biochemistry 31, 6639-6645

Q,

Q,

1217

REVIEWS 43.

Pierce, R. A., Kolodziej, M. E., and Parks, W. C. (1992) 1,23Dihydroxyvitamin D5 represses tropoelastin expression by a posttranscriptional mechanism. j Biol. Chem. 267, 11593-11599 44. Gibson, M. A., and Cleary, E. G. (1985) CL glycoprotein is the tissue form of type VI collagen. j BioL Chem. 260, 11149-11159 45. Lee, B., Godfrey, M., Vitale, E., Hori, H., Mattei, M. -G., Sarfarazi, M., Tsipouras, F, Ramirez, F., and Hollister, D. W. (1991) Linkage of Marfan syndrome and a phenotypically related disorder to two different fibrillin genes. Nature (London) 352, 330-334

46. Maslen,

D. L., Corson,

G. M., Maddox,

Sakai, L.

47. 48.

49.

50. 51.

52.

53. 54.

55.

56.

57.

58.

59.

60.

61.

B. K., Glanville,

R. W., and

Y. (1991) Partial sequence of a candidate gene for the Marfan syndrome. Nature (London) 352, 334-337 Carpenter, C., and Cohen, S. (1990) Epidermal growth factor. j BioL Chem. 265, 7709-7712 Kanzaki, T, Olofsson, A., Moren, A., Wernstedt, C., Hellman, U., Miyazono, K., Claesson-Welsh, L., and Heldin, C. -H. (1990) IUF.f 1 binding protein: a component of the large latent complex of 113Ff-i with multiple repeat sequences. Cell 61, 1051-1061 Dietz, H. C., Cutting, G. R., Pyeritz, R. E., Maslen, C. L., Sakai, L. Y., Corson, C. M., Puffenberger, E. G., Hamosh, A., Nanthakumar, E. J., Curristin, S. M., Stetten, G., Meyers, D. A., and Francomano, C. A. (1991) Marfan syndrome caused by a recurrent de novo missense mutation in the fibrillin gene. Nature (London) 352, 337-339 Pyeritz, R. E., and McKusick, V. A. (1979) The Marfan syndrome: diagnosis and management. N EngI. J. Med. 300, 772-777 Godfrey, M., Menashe, V., Weleber, R. G., Koler, R. D., Bigley, R. H., Lovrien, E., Zonana, J., and Hollister, D. W. (1990) Cosegregation of elastin-associated microfibrillar abnormalities with the Marfan phenotype in families. Am. j Hum. Genet. 46, 652-660 Hollister, D. W., Godfrey, M., Sakai, L. Y., and Pyeritz, R. (1990) Immunohistologic abnormalities of the microfibrillar-fiber system in the Marfan syndrome. N Engi. j Med. 323, 152-159 Sykes, B. (1993) Marfan gene dissected. Nature Genet. 3, 99-100 Gibson, M. A., Kumaratilake, J. S., and Cleary, E. G. (1989) The protein components of the 12-nanometer microfibrils of elastic and nonelastic tissues. j Biol. Chem. 264, 4590-4598 Gibson, M. A., Sandberg, L. B., Grosso, L. E., and Cleary, E. G. (1991) Complementary DNA cloning establishes microfibril-associated glycoprotein (MAGP) to be a discrete component of the elastinassociated microfibrils. j Biol. Chem. 266, 7596-7601 Serafini-Fracassini, A., Ventrella, G., Field, M. J., Hinnie, J., Onyezili, N. I., and Griffiths, R. (1981) Characterization of a structural glycoprotein from bovine ligamentum nuchae exhibiting dual amine oxidase activity. Biochemistry 20, 5424-5431 Kobayashi, R., Tashima, Y., Masuda, H., Shozawa, T., Numata, Y., Miyauchi, K., and Hayakawa, T. (1989) Isolation and characterization of a new 36-kDa microlIbril-associated glycoprotein from porcine aorta. j Biol. Chem. 264, 17437-17444 Bressan, G. M., Castellani, I., Colombatti, A., and Volpin, D. (1983) Isolation and characterization of a 115,000 Da matrix-associated glycoprotein from chick aorta. J. Biol. Chem. 258, 13262-13267 Horrigan, S. K., Rich, C. B., Streeten, B. W., Li, Z.-Y., and Foster,J. A. (1992) Characterization of an associated microfibril protein through recombinant DNA techniques. j Biol. Chem. 267, 10087-10095 Rosenbloom, J., and Cywinski, A. (1976) Biosynthesis and secretion of tropoelastin by chick aorta cells. Biochem. Biophys. Res. Commun. 69, 613-620 Damiano, V., Tsang, A.-L., Weinbaum, G., Christner, P., and Rosenbloom, J. (1984) Secretion of elastin in the embryonic chick aorta as visualized by immunoelectron microscopy. Collagen Relat. Ret. 4, 153-164

1218

Vol.

7

October

1993

62. Uitto, J., Hoffman, H. -P., and Prockop, D. J. (1976) Synthesis of elastin and procollagen by cells from embryonic aorta. Differences in the role of hydroxyproline and the effects of proline analogs on the secretion of the two proteins. Arch. Biochem. Biophys. 173, 187-200 63. Barone, I. M., Fans, B., Chipman, S. D., Toselli, P., Oakes, B. W., and Franzblau, C. (1985) Alteration of extracellular matrix of smooth muscle cells by ascorbate treatment. Biochem. Biophys. Acta 840, 245-254 64. Perejda, A. F., Abraham, P. A., Carnes, W. H., Coulson, W. F., and Uitto, J. (1985) Marfan’s syndrome: structural, biochemical, and mechanical studies of the aortic media. j Lab. Clin. Med. 106, 3 76-382 65. Davidson,J. M., Shibahara, S., Boyd, C., Mason, M. L., Tolstoshev, P., and Crystal, R. G. (1984) Elastin mRNA levels during fetal development of sheep nuchal ligament and lung. Hybridization to complementary eDNA. Biochem. J. 220, 653-663 66. Kagan, H. M. (1986) Characterization and regulation of lysyl oxidase. In Regulation

of Matrix

Accumulalion

(Mecham,

R.

P., ed)

pp.

321-398,

Academic Press, Orlando, Florida 67. Foster, J. A., Bruenger, E., Rubin, L., Imberman, M., Kagan, H., Mecham, R., and Franzblau, C. (1976) Circular dichroism studies of an elastin cross-linked peptide. Biopolymers 15, 833-841 68. Gray, W. R. (1977) Some kinetic aspects of crosslink biosynthesis. Adv. Exp. Med. Biol. 79, 285-290 69. Serafini-Fracassini, A. (1984) Elastogenesis in embryonic and post-natal development. In Ultro.structure of the Connective Tissue Matrix (Ruggeri, A., and

Motta,

P. M.,

eds)

pp.

140-150,

Nijhoff,

The

Hague

70. Mecham, R. P., Hinek, A., Entwistle, R., Wrenn, D. S., Griffin, G. L., and Senior, R. M. (1989) Elastin binds to a multifunctional 67kilodalton peripheral membrane protein. Biochemistry 28, 3716-3722 71. Mecham, R. P., Whitehouse, L., Hay, M., Hinek, A., and Sheetz, M. (1991)Ligandaffinity ofthe67 kDa elastinllaminin binding protein is modulated by the protein’s lectin domain: nun receptor complexes with gold-tagged

visualization ligands. j

of elastinllami-

Cell BioL 113, 187-194 72. Hinek, A., Wrenn, D. S., Mecham, R. P., and Barondes, S. H. (1988) The elastin receptor: a galactoside-binding protein. Science 239, 15391541 73.

Franzblau, C., Pratt, C. A., Fans, B., Colannino, N. M., Offner, G. D., Mogayzel, P. J. J., and Troxler, R. F. (1989) Role of tropoelastin fragmentation in elastogenesis in rat smooth muscle cells.j BioL Chem. 264, 15116-15119.

74.

Brown, P. L., Mecham, L., Tisdale, C., and Mecham, R. P. (1992) The cysteine residues in thecarboxy-terminal domain of tropoelastin form an intrachain disulfide bond that stabilizes a loop structure and positively charged pocket. Biocliem. Biophys. Res. Commun. 186, 549-555 Rosenbloom, J., Abrams, W., Ornstein, G. N., Indik, Z., and Kucich, U. (1986) Newly determined carboxy terminal sequences in tropoelastin: immunologic identification in insoluble elastin. Collagen Relat. Res. 6, 423-433

75.

76.

Hoeve, C. A. J., and Flory, Biopolymers 13, 677-686

P.

J.

(1974)

The

elastic

properties

of elastin.

77. Gotte, L., Mammi, M., and Pezzin, G. (1967) Some structural aspects of elastin revealed by X-ray diffraction and other physical methods. In Symposium on Fibrous Proteins (Creuther, W. G., ed) pp. 236-245, Butter-

worths,Australia 78. Fleming,W. W., Sullivan, zation

C. E., and

Torchia,

D. A. (1980)

Characteri-

in ‘5C-labeled aortic elastin by ‘3C-’H magnetic double resonance. Biopolymers 19, 597-617 79. Urry, D. W. (1984) Protein elasticity based on conformations of sequential polypeptides: the biological elastic fiber. j Protein Chem. 3, 403-436

The FASEBJournal

of molecular

motions

ROSENBLOOM ET AL.