Proteomic Alterations Associated with Biomechanical Dysfunction are Early Processes in the Emilin1 Deficient Mouse Model of Aortic Valve Disease.

Aortic valve (AV) disease involves stiffening of the AV cusp with progression characterized by inflammation, fibrosis, and calcification. Here, we examine the relationship between biomechanical valve function and proteomic changes before and after the development of AV pathology in the Emilin1-/- mouse model of latent AV disease. Biomechanical studies were performed to quantify tissue stiffness at the macro (micropipette) and micro (atomic force microscopy (AFM)) levels. Micropipette studies showed that the Emilin1-/- AV annulus and cusp regions demonstrated increased stiffness only after the onset of AV disease. AFM studies showed that the Emilin1-/- cusp stiffens before the onset of AV disease and worsens with the onset of disease. Proteomes from AV cusps were investigated to identify protein functions, pathways, and interaction network alterations that occur with age- and genotype-related valve stiffening. Protein alterations due to Emilin1 deficiency, including changes in pathways and functions, preceded biomechanical aberrations, resulting in marked depletion of extracellular matrix (ECM) proteins interacting with TGFB1, including latent transforming growth factor beta 3 (LTBP3), fibulin 5 (FBLN5), and cartilage intermediate layer protein 1 (CILP1). This study identifies proteomic dysregulation is associated with biomechanical dysfunction as early pathogenic processes in the Emilin1-/- model of AV disease.

Physical mapping of mouse collagen genes on chromosome 10 by high-resolution FISH.

Fluorescence in situ hybridization (FISH) on mechanically stretched chromosomes (MSCs) and extended DNA fibers enables construction of high-resolution physical maps by accurate ordering and orienting genomic clones as well as by measuring physical lengths of gaps and overlaps between them. These high-resolution FISH targets have hitherto been used mainly in the study of the human genome. Here we have applied both MSCs and extended DNA fibers to the physical mapping of the mouse genome. At first, five mouse collagen genes were localized by metaphase-FISH: Col10a1 to chromosomal bands 10B1-B3; Col13a1 to 10B4; and Col6a1, Col6a2, and Col18a1 to 10B5-C1. The mutual order of the genes, centromere--Col10a1--Col13a1--Col6a2--Col6a1--Col18a1--telomere, was determined by FISH on metaphase chromosomes, MSCs, and extended DNA fibers. To our knowledge, this is the first time mouse metaphase chromosomes have been stretched and used as targets for FISH. We also used MSCs to determine the transcriptional orientations, telomere--5'-->3'--centromere, of both Col13a1 and Col18a1. With fiber-FISH, Col18a1, Col6a1, and Col6a2 were shown to be in a head-to-tail configuration with respective intergenic distances of about 350 kb and 90 kb. Comparison of our physical mapping results with the homologous human data reveals both similarities and differences concerning the chromosomal distribution, order, transcriptional orientations, and intergenic distances of the collagen genes studied.

Mechanisms of transcriptional activation of the col6a1 gene during Schwann cell differentiation.

A transgenic mouse line expressing the lacZ reporter under the control of a regulatory region of the col6a1 gene has been used to investigate differentiation of Schwann cells. The data suggest that: (1) activation of col6a1 gene transcription in the peripheral nervous system is part of the differentiation program of Schwann cells from neural crest cells stimulated by neuregulins; (2) once the Schwann cell precursors have acquired the competence of transcribing the col6a1 gene, transcriptional regulation becomes independent from neuregulins and is modulated by different mechanisms, including cell cycle; (3) activation of transgene expression after birth in sciatic nerves corresponds to the time of withdrawal of immature Schwann cells from the cell cycle and the beginning of their differentiation into myelinating Schwann cells.

Elastic fiber proteins in the glomerular mesangium in vivo and in cell culture.

BACKGROUND: Glomerular capillaries of the mammalian kidney are exposed to high intraluminal hydrostatic pressures and require elastic constraint to maintain size, shape, and integrity. Previous morphological and functional studies indicated that the extracellular matrices of glomeruli, that is, basement membrane and mesangial matrix, contribute to glomerular resilience and mechanical stability. Immunofluorescence microscopy findings demonstrated elastic fiber components to be located in the renal vasculature, including glomeruli. The aim of this study was to clarify the exact glomerular localization, composition, and cellular production of these proteins. METHODS: We examined the renal distribution of the elastic fiber proteins fibrillin-1, emilin, microfibril-associated glycoproteins (MAGPs) 1 and 2, latent transforming growth factor-binding protein-1 (LTBP-1), and elastin using immunohistology and immunoelectron microscopy of human, rat, and mouse kidneys. In mesangial cell cultures, we also studied the expression and extracellular deposition of such proteins by use of Northern blotting and immunocytochemistry. RESULTS: Fibrillin-1, emilin, MAGPs 1 and 2, and LTBP-1 were present in glomeruli of mouse, rat, and human kidney, where they were located predominantly in the mesangial extracellular matrix underlying glomerular endothelium and basement membrane. Several of these proteins, as well as elastin, were also expressed in the renal vasculature. While elastin localized to the glomerular vascular pole in afferent and efferent arterioles extending to Bowman's capsule, it was not found in the glomerular capillary tuft. Cultured mesangial cells of rat, mouse, and human kidneys expressed mRNAs of fibrillin-1, emilin, MAGP-2, and elastin, and the respective proteins localized within and outside of mesangial cells, as shown by immunocytochemistry. mRNA expression of fibrillin-1, emilin, and elastin was strong in quiescent mesangial cells; their gene expression was further up-regulated by transforming growth factor-beta1, while it was transiently reduced when cells were exposed to mitogenic 10% fetal calf serum and platelet-derived growth factor. CONCLUSIONS: These findings demonstrate that specific elastic fiber proteins are produced and secreted by mesangial cells. This process is regulated by growth factors. Their abundance in the extracellular matrix of the mesangium is in keeping with the concept that elastic fiber proteins contribute to the mechanical stability and elastic strength of the glomerular capillary tuft.

Analysis of transcription of the Col6a1 gene in a specific set of tissues suggests a new variant of enhancer region.

The region extending from -5.4 to -3.9 kilobase pairs from the transcription start site of the Col6a1 gene has been previously shown to contain sequences activating tissue-specific transcription in articular cartilage, intervertebral disks, subepidermal, and vibrissae mesenchyme and peripheral nervous system (Braghetta, P., Fabbro, C., Piccolo, S., Marvulli, D., Bonaldo, P., Volpin, D., and Bressan, G. M. (1996) J. Cell Biol. 135, 1163-1177). Analysis of expression of deletions of this region in transgenic mice has identified the 383-base pair fragment E-L as the most active sequence of the region. Linker-scanning mutagenesis analysis of segment E-J, which spans the 5' 245 base pairs of E-L and is sufficient for high frequency expression in articular cartilage, showed that all the mutations reduced transcription considerably, suggesting that the integrity of the entire cluster of elements is necessary for enhancer activity. Electrophoretic mobility shift assays with nuclear extracts derived from various sources showed that fragment E-J binds numerous transcription factors (at least 22). These factors are present in most cells, expressing and nonexpressing alpha1(VI) collagen mRNA, but in different relative proportions, and none of them appears to be cell type-specific. Several lines of evidence indicate that sequence elements of the enhancer may have different functional roles in various cells. The data configure the -5.4/-3.9 region of the Col6a1 gene as a new type of tissue-specific enhancer, characterized by a variety of tissues supporting its activation and by the dependence of its function only on ubiquitous transcription factors. This type of enhancer is postulated to be particularly important for genes such as those of the extracellular matrix, which are often expressed with broad tissue specificity.

EMILIN, a component of the elastic fiber and a new member of the C1q/tumor necrosis factor superfamily of proteins.

EMILIN (elastin microfibril interface located protein) is an extracellular matrix glycoprotein abundantly expressed in elastin-rich tissues such as blood vessels, skin, heart, and lung. It occurs associated with elastic fibers at the interface between amorphous elastin and microfibrils. Avian EMILIN was extracted from 19-day-old embryonic chick aortas and associated blood vessels and purified by ion-exchange chromatography and gel filtration. Tryptic peptides were generated from EMILIN and sequenced, and degenerate inosine-containing oligonucleotide primers were designed from some peptides. A set of primers allowed the amplification of a 360-base pair reverse transcription polymerase chain reaction product from chick aorta mRNA. A probe based on a human homologue selected by comparison of the chick sequence with EST data base was used to select overlapping clones from both human aorta and kidney cDNA libraries. Here we present the cDNA sequence of the entire coding region of human EMILIN encompassing an open reading frame of 1016 amino acid residues. There was a high degree of homology (76% identity and 88% similarity) between the chick C terminus and the human sequence as well as between the N terminus of the mature chick protein where 10 of 12 residues, as determined by N-terminal sequencing, were identical or similar to the deduced N terminus of human EMILIN. The domain organization of human EMILIN includes a C1q-like globular domain at the C terminus, a collagenous stalk, and a longer segment in which at least four heptad repeats and a leucine zipper can be identified with a high potential for forming coiled-coil alpha helices. At the N terminus there is a cysteine-rich sequence stretch similar to a region of multimerin, a platelet and endothelial cell component, containing a partial epidermal growth factor-like motif. The native state of the recombinantly expressed EMILIN C1q-like domain to be used in cell adhesion was determined by CD spectra analysis, which indicated a high value of beta-sheet conformation. The EMILIN C1q-like domain promoted a high cell adhesion of the leiomyosarcoma cell line SK-UT-1, whereas the fibrosarcoma cell line HT1080 was negative.

Cell type-specific transcription of the alpha1(VI) collagen gene. Role of the AP1 binding site and of the core promoter.

Analysis of the chromatin of different cell types has identified four DNase I-hypersensitive sites in the 5'-flanking region of the alpha1(VI) collagen gene, mapping at -4.6, -4.4, -2.5, and -0.1 kilobase (kb) from the RNA start site. The site at -2.5 kb was independent from, whereas the other three sites could be related to, alpha1(VI) mRNA expression. The site at -0.1 kb was present in cells expressing (NIH3T3 and C2C12) but absent in cells not expressing (EL4) the mRNA; the remaining two sites were apparently related with high levels of mRNA. DNase I footprinting and gel-shift assays with NIH3T3 and C2C12 nuclear extracts have located a binding site for transcription factor AP1 (activator protein 1) between nucleotides -104 and -73. When nuclear extracts from EL4 lymphocytes were used, the AP1 site-containing sequence was bound by proteins not related to AP1. The existence of the hypersensitive site at -0.1 kb may be related to the binding of AP1 and of additional factors to the core promoter (Piccolo, S., Bonaldo, P., Vitale, P., Volpin, D., and Bressan, G. M. (1995) J. Biol. Chem. 270, 19583-19590). The function of the AP1 binding site and of the core promoter in the transcriptional regulation of the Col6a1 gene was investigated by expressing several promoter-reporter gene constructs in transgenic mice and in cell cultures. The results indicate that regulation of transcription of the Col6a1 gene by different cis-acting elements (core promoter, AP1 binding site and enhancers) is not completely modular, but the final output depends on the specific interactions among the three elements in a defined cell type.

Collagen VI deficiency induces early onset myopathy in the mouse: an animal model for Bethlem myopathy.

To gain insight into the function of type VI collagen, the col6a1 gene was inactivated by targeted gene disruption in the mouse. The homozygous mutants lacked collagen VI in the tissues and showed histological features of myopathy such as fiber necrosis and phagocytosis and a pronounced variation in the fiber diameter. Muscles also showed signs of stimulated regeneration of fibers. Necrotic fibers were particularly frequent in the diaphragm at all ages examined. Similar, although milder, alterations were detected in heterozygous mutant mice, indicating haploinsufficiency of the col6a1 gene function. The data led us to conclude that collagen VI is necessary for maintenance of the integrity of muscle fibers and that the col6a1 -deficient mouse can be considered an animal model of Bethlem myopathy.

Tissue-specific expression of promoter regions of the alpha1(VI) collagen gene in cell cultures and transgenic mice.

Cis-acting regions regulating transcription of the alpha1(VI) collagen chain have been investigated in vitro by transfection of promoter-CAT (where CAT is chloramphenicol acetyltransferase) constructs in different types of cultured cells and in vivo in transgenic mice carrying the same CAT constructs or minigenes derived from the fusion of genomic and cDNA sequences in which small deletions of the collagenous domain had been engineered. 215 bp of 5'-flanking sequence showed promoter activity in vitro, yet were not expressed in any tissue of six transgenic lines, indicating that this fragment contains the basal promoter, but not activator sequences. Constructs with 0.6 and 1.4 kb of the 5'-flanking region produced significantly higher CAT activity in transfected cells and were expressed in tissues of about 30% of transgenic lines. Although CAT activity was totally unrelated to the pattern of expression of the alpha1(VI) mRNA, these results suggest the presence of an activator(s) between -0.2 and -0.6 kb from the transcription start site. When the promoter size was increased to 5.4 or 6.5 kb, CAT activity was stimulated severalfold relative to the construct p1.4CAT and p4.0CAT in NIH3T3 fibroblasts and chick embryo chondroblasts. This stimulation was, however, not observed in C2C12 myoblasts. Transgenic mice generated with 6.5CAT construct or minigenes, containing 6.2 kb of promoter, exhibited very high levels of expression, which was similar to the relative amount alpha1(VI) mRNA in the majority of tissues, with the exception of lung, adrenal gland and uterus. CAT activity in tissues was 100-1000-fold higher than that measured in transgenic mice with shorter promoter (0.6 or 1.4 kb). Since expression of minigenes was determined by RNase protection assay, the levels of mRNA per transgene copy were compared to those of the chromosomal gene and found to be always less than one quarter. These data suggest that the region -4.0/-5.4 contains an important activator(s) sequence which induces transcription in several, but not all, type VI collagen-producing tissues. Finally, analysis with the longest promoter fragment (7.5 kb) revealed a complex effect of the region -6.5/-7.5 on alpha1(VI) chain transcription. The sequence was inhibitory in NIH3T3 cells, indifferent in myoblasts and activating in chondroblasts in vitro, whereas transgenic animals generated with 7.5CAT construct produced a pattern of expression comparable to that of 6.5CAT and minigenes. During postnatal development transcription from both the endogenous gene and the transgenes decreased. However, the ratio of transgene/chromosomal gene expression was not constant, but varied in a way dependent on the tissue. This observation suggests that the fragment studied contains key sequences for the age-dependent regulation of the alpha1(VI) gene. No phenotypic alterations were induced by the presence of mutations in the minigenes.

Distinct regions control transcriptional activation of the alpha1(VI) collagen promoter in different tissues of transgenic mice.

To identify regions involved in tissue specific regulation of transcription of the alpha1(VI) collagen chain, transgenic mice were generated carrying various portions of the gene's 5'-flanking sequence fused to the E. coli beta-galactosidase gene. Analysis of the transgene expression pattern by X-gal staining of embryos revealed that: (a) The proximal 0.6 kb of promoter sequence activated transcription in mesenchymal cells at sites of insertion of superficial muscular aponeurosis into the skin; tendons were also faintly positive. (b) The region between -4.0 and -5.4 kb from the transcription start site was required for activation of the transgene in nerves. It also drove expression in joints, in intervertebral disks, and in subepidermal and vibrissae mesenchyme. (c) The fragment comprised within -6.2 and -7.5 kb was necessary for high level transcription in skeletal muscle and meninges. Positive cells in muscle were mostly mononuclear and probably included connective tissue elements, although staining of myoblasts was not ruled out. This fragment also activated expression in joints, in intervertebral disks, and in subepidermal and vibrissae mesenchyme. (d) beta-Galactosidase staining in vibrissae induced by the sequences -4.0 to -5.4 and -6.2 to -7.5 was not coincident: with the latter sequence labeled nuclei were found mainly in the ventral and posterior quadrant, and, histologically, in the outer layers of mesenchyme surrounding and between the follicles, whereas with the former the remaining quadrants were positive and expressing cells were mostly in the inner layers of the dermal sheath. (e) Other tissues, notably lung, adrenal gland, digestive tract, which produce high amounts of collagen type VI, did not stain for beta-galactosidase. (f) Central nervous system and retina, in which the endogenous gene is inactive, expressed the lacZ transgene in most lines. The data suggest that transcription of alpha1(VI) in different tissues is regulated by distinct sequence elements in a modular arrangement, a mechanism which confers high flexibility in the temporal and spatial pattern of expression during development.

Spatial and temporal changes of type VI collagen expression during mouse development.

The expression of type VI collagen has been studied in mouse tissues. By Northern blotting, the mRNA for the alpha 1 (VI) chain was detectable in whole embryos at 10.5 days postcoitum and steeply increased afterward. The messenger levels were high at birth, but decreased rapidly in the following days, reaching low levels in adult animals. In 2-month-old mice, lung, skin, adrenal gland, heart, skeletal muscle and tail and fat were among the most active producers of alpha 1 (VI) mRNA. In situ hybridization first identified mRNA for alpha 1 (VI) collagen in mesenchymal cells of 10.5-day embryos in various locations, including serosae, branchial arches, large blood vessels and the cephalic mesenchyme. Staining increased at later stages of development and most connective tissues were positive at 16.5 days and later. Strongly staining tissues were joints, intervertebral disks, perichondrium, periostium, dermis, skeletal muscle and heart valves, whereas cartilage and bone were very poorly labelled. Epithelia and the central nervous system were completely negative. In several organs, notably lung, salivary glands and the digestive tract, staining was concentrated underneath epithelia. This staining pattern was different from that for collagen type I, which was evenly distributed in the subepithelial mesenchyme. The pattern of distribution of the protein, revealed by immunocytochemistry, was coincident with that of the alpha 1 (VI) mRNA. In addition, the results confirmed that type VI collagen is preferentially deposited in the pericellular environment. This was particularly evident in skeletal muscle. The data show that type VI collagen is mainly produced by mesenchymal cells and suggest a role for the protein in delineating the boundary of distinct domains in connective tissue.

Identification of a recognition element for CAAT-enhancer binding proteins (C/EBPs) in the elastin promoter.

DNase I footprinting experiments with a DNA fragment of the human elastin promoter have revealed a protected segment comprised between -156 and -172 nucleotides from the translation start site. Various types of gel retardation experiments indicate that the protected element binds different members of the C/EBP family of transcription factors. CAT (chloramphenicol acetyltransferase) fusion constructs carrying the wild type or a mutated promoter sequence were transfected into NIH3T3 and chick embryo aorta cells. The mutation significantly lowered CAT expression in NIH3T3 cells, but was ineffective in aorta cells. Cotransfection of the CAT promoter constructs with eucaryotic vectors expressing C/EBPs, did not affect the production of the reporter gene in NIH3T3 cells; on the contrary a several-fold increase of CAT activity was observed in aortic cells. This increase, however, was identical for the wild type and the mutated constructs. Taken together the data indicate that the elastin promoter contains a recognition site for proteins of the C/EBP family and that the function of this cis-acting element on basal elastin transcription varies with the cell type.

Transcriptional activation of the alpha 1(VI) collagen gene during myoblast differentiation is mediated by multiple GA boxes.

During differentiation of ClC12 myoblasts in vitro, expression of alpha 1(VI) collagen mRNA was transiently stimulated severalfold. Promoter assays on cells transfected with chloramphenicol acetyltransferase (CAT) chimeric constructs have identified a region of the alpha 1(VI) a collagen promoter that increases CAT activity about 8-fold during differentiation. The region, which overlaps with transcription initiation sites, was shown to contain three protected segments (A, B, and C) in DNase I footprinting assays. The contact points between nuclear factors and the protected segments were determined by methylation interference assay and included the sequence GGGAGGG (GA box) in all segments. Experiments in which CAT constructs were cotransfected with double-stranded oligonucleotides containing the GA box suggested that this motif was necessary for induction. Transfections with deletion constructs of the natural promoter and with minipromoters made of three copies of A, B, or C showed that the elements have inducing activity and that elements C and, to a lower extent, B are stimulatory for basal transcription, whereas the contribution of A in this process is limited. Electrophoretic mobility shift assays with nuclear extracts from C2C12 cells indicated that the three GA box-containing elements bound several transcription factors, including Sp1. Comparison of the properties of the bands shifted under different experimental conditions (presence of 10 mM EDTA, heating of the nuclear extracts, addition of different concentrations of competitor oligonucleotides) established that A, B, and C probes form nine, eight and five main retarded complexes, respectively, and indicated that nuclear factors binding to C and B are subsets of proteins binding to A. UV cross-linking assays identified several peptides (seven with probe A, six with B, And five with C) in the range of 150-32 kDa. Comparison of the gel retardation pattern obtained with nuclear extracts from proliferating and differentiating cells revealed a particular increased intensity of two retarded bands. The data establish that multiple GA boxes mediate induction of the alpha 1(VI) collagen promoter during myoblast differentiation and suggest the attractive hypothesis that the effect may be related to variations of expression of transcription factors binding to these motifs.

Identification of a TGF-beta responsive element in the human elastin promoter.

In a previous report (Marigo, V., Volpin, D., and Bressan, G. M. (1993) Biochim. Biophys. Acta 1172, 31-36) it was shown that the elastin promoter contains a region mediating transcriptional activation by TGF-beta in aorta cells, but not in tendon fibroblasts from chick embryos. In this paper we have identified the sequence responsible for this effect by a combination of CAT assays with mutant constructs, DNase I footprinting and electrophoretic mobility shift assays. This TGF-beta responsive element binds different nuclear proteins in chick embryo aorta and tendon cells. Whereas association of the aorta protein(s) to the element is necessary for TGF-beta activation, binding of the tendon protein(s) has apparently no effect on promoter stimulation by the cytokine.

Murine alpha 1(VI) collagen chain. Complete amino acid sequence and identification of the gene promoter region.

The entire primary structure of the murine alpha 1(VI) collagen chain was deduced from cloned cDNA. The predicted polypeptide consists of 1025 amino acids and shows extensive homology with the corresponding human and chicken chains. A genomic clone isolated with a cDNA probe was found to contain about 13 kilobases of the 5'-flanking region and the first and second exon, coding for the 5'-untranslated sequence and signal peptide and part of the N-terminal portion of the mature protein, respectively. Polymerase chain reaction and primer extension analyses revealed two major and several minor transcription start sites distributed over 76 base pairs (bp). The region just upstream of the transcription initiation sites lacks canonical TATA and CAAT boxes and Sp1 binding sites, but contains putative binding sites for other transcription factors and a 90-bp polypyrimidine tract with elements of dyad symmetry. Chimeric constructs were derived from different fragments of the 5'-flanking genomic region and the chloramphenicol acetyltransferase (CAT) gene and expression of the reporter gene was assayed following transfection of various cell types. A construct containing sequences extending from -215 to +41 directed high levels of CAT expression. The data indicate that this region harbours a functional promoter.

Emilin, a component of elastic fibers preferentially located at the elastin-microfibrils interface.

The fine distribution of the extracellular matrix glycoprotein emilin (previously known as glycoprotein gp115) (Bressan, G. M., I. Castellani, A. Colombatti, and D. Volpin. 1983. J. Biol. Chem. 258: 13262-13267) has been studied at the ultrastructural level with specific antibodies. In newborn chick aorta the protein was exclusively found within elastic fibers. In both post- and pre-embedding immunolabeling emilin was mainly associated with regions where elastin and microfibrils are in close contact, such as the periphery of the fibers. This localization of emilin in aorta has been confirmed by quantitative evaluation of the distribution of gold particles within elastic fibers. In other tissues, besides being associated with typical elastic fibers, staining for emilin was found in structures lacking amorphous elastin, but where the presence of tropoelastin has been demonstrated by immunoelectron microscopy. This was particularly evident in the oxitalan fibers of the corneal stroma, in the Descemet's membrane, and in the ciliary zonule. Analysis of embryonic aorta revealed the presence of emilin at early stages of elastogenesis, before the appearance of amorphous elastin. Immunofluorescence studies have shown that emilin produced by chick embryo aorta cells in culture is strictly associated with elastin and that the process of elastin deposition is severely altered by the presence of antiemilin antibodies in the culture medium. The name of the protein was derived from its localization at sites where elastin and microfibrils are in proximity (emilin, elastin microfibril interface located protein).

Regulation of the human elastin promoter in chick embryo cells. Tissue-specific effect of TGF-beta.

The function of the 5' flanking region of the human elastin gene on transcription regulation has been investigated in chick embryo aorta cells by transient DNA transfer experiments with elastin-chloramphenicol acetyltransferase (CAT) fusions. The results have shown that the region comprised within -129 and -12 bp from the translation start site is essential for transcription and probably contains different control sequences. Expression of the reporter gene was increased 2-4-fold by addition of TGF-beta to the cell cultures. Analysis of CAT expression from different deletion constructs suggests that sequences in the region -196 to -12 play a major role in TGF-beta induction. The stimulating effect of the growth factor could not be observed when transfections were performed with chick embryo tendon fibroblasts. This suggests that transcriptional regulation of elastin by TGF-beta is tissue specific.

Effect of monoclonal antibodies to defined regions of tropoelastin on elastogenesis in vitro.

Primary cultures of chick embryo aorta cells were grown for one week in the presence of mouse monoclonal antibodies directed against defined regions of chick tropoelastin. This treatment did not significantly alter cell proliferation, cell viability and incorporation of labeled amino acids into total protein or tropoelastin compared with control cultures in which antibodies were either omitted or substituted with an unrelated monoclonal antibody. Tropoelastin-reactive material in the cell layer was revealed by immunologic staining with rabbit antibodies against the chick protein both at the optical and ultrastructural level. Immunofluorescence of control cultures showed that tropoelastin was incorporated into thin and straight fibrils which were sometimes associated with spot-like elements. In the electron microscope tropoelastin-reactive sites were found mainly on the amorphous core of typical, small elastic fibers. The morphological picture of tropoelastin deposits in cultures exposed to anti-tropoelastin monoclonal antibodies depended on the molecular form (whole antibody or Fab fragments) and the binding specificity of the antibody used. Although alterations common to different antibodies were observed, the main structural features were peculiar for each antibody. Two antibodies which bound epitopes present in two regions of tropoelastin grossly altered the formation of amorphous elastin. Moreover, two antibodies directed against the region of tropoelastin containing the polypentapeptide-repeat (VPGVG)n stimulated the deposition of the protein into the amorphous core of normal-looking elastic fibers and disorganized the compact bundles of parallel microfibrils seen in controls. Finally, one antibody which recognized a unique epitope close to the carboxy-terminal end of tropoelastin and Fab fragments from all antibodies apparently inhibited the formation of the amorphous nuclei of elastic fibers, but not the association of tropoelastin with microfibrils. The data suggest that the association of tropoelastin molecules during fiber assembly is not random, but follows an ordered alignment process which the antibodies alter by imposing a different molecular packing.

Mapping of binding sites for monoclonal antibodies to chick tropoelastin by recombinant DNA techniques.

A fusion molecule consisting of the entire coding sequence of mature chicken tropoelastin preceded by 14 amino acids of the signal peptide and 9 amino acids of vector origin has been expressed in a recombinant bacterial system and purified. The molecule has been used as immunogen for the production of hybridomas. Monoclonal antibodies which bound specifically the immunogen were also reactive with tropoelastin purified from chick aorta and stained elastic fibers in aorta sections by immunofluorescence. The region of tropoelastin containing the antigenic determinant recognized by each antibody has been identified by a recombinant DNA expression strategy based on the use of cDNA clones spanning different portions of the coding sequence. It could be shown that several antibodies were directed against unique epitopes; among these, a group of antibodies bound specifically to the sequence (PGVGV)n. Other antibodies were found to recognize antigenic determinants present more than once in the molecule. The monoclonal antibodies thus characterized will be useful reagents in studying the function of the different domains of tropoelastin.