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PubMed2000

Mechanical strain and dexamethasone selectively increase surfactant protein C and tropoelastin gene expression.

Physical forces derived from fetal breathing movements and hormones such as glucocorticoids are implicated in regulating fetal lung development. To elucidate whether the different signaling pathways activated by physical and hormonal factors are integrated and coordinated at the cellular and transcriptional levels, organotypic cultures of mixed fetal rat lung cells were subjected to static culture or mechanical strain in the presence and absence of dexamethasone. Tropoelastin and collagen type I were used as marker genes for fibroblasts, whereas surfactant protein (SP) A and SP-C were used as marker genes for distal epithelial cells. Mechanical strain, but not dexamethasone, significantly increased SP-C mRNA expression. Tropoelastin mRNA expression was upregulated by both mechanical strain and dexamethasone. No additive or synergistic effect was observed when cells were subjected to mechanical stretch in the presence of dexamethasone. Neither mechanical strain nor dexamethasone alone or in combination had any significant effect on the expression of SP-A mRNA. Dexamethasone decreased collagen type I mRNA expression, whereas mechanical strain had no effect. The increases in tropoelastin and SP-C mRNA levels induced by mechanical strain and/or dexamethasone were accompanied by increases in their heterogeneous nuclear RNA. In addition, the stretch- and glucocorticoid-induced alterations in tropoelastin and SP-C mRNA expression were abrogated with 10 microg/ml actinomycin D. These findings suggest that tropoelastin and SP-C genes are selectively stimulated by physical and/or hormonal factors at the transcriptional level in fetal lung fibroblasts and distal epithelial cells, respectively.

Animals↗
PubMed1991

Developmental changes in tropoelastin mRNA levels in rat lung: evaluation by in situ hybridization.

Alveolarization of the immature lung is thought to be influenced by the presence of elastic fibers that could provide structural support for developing septa. Although morphometric studies have established that alveolar septal development occurs from days 4 to 13 in the neonatal rat, the precise time period over which elastin synthesis occurs has proved difficult to determine. We have evaluated the usefulness of in situ hybridization techniques to follow tropoelastin message expression in parenchymal tissue, small vessels, and bronchioles in the developing rat lung from days 4 through 18. This method proved to be sufficiently sensitive to detect differences in rates of tropoelastin message expression from days 4 through 18 (P less than 0.0001). Peak tropoelastin message expression was observed in the small vessels on day 4 and in parenchymal tissue on days 9 through 11. Because the time course of tropoelastin message expression in small vessels differs from that in parenchymal tissue, the use of lung extracts to analyze rates of tropoelastin synthesis in the developing lung may be in error.

Animals↗
PubMed1991

Developmental changes in tropoelastin gene expression in the rat lung studied by in situ hybridization.

Gene expression for tropoelastin, the proprotein for elastin, was examined in the rat lung from 17 days of gestation (pseudoglandular stage) to adulthood by in situ hybridization using a rat-specific 35S-radiolabeled riboprobe. The tropoelastin message was present in vascular and airway smooth muscle, endothelial, septal interstitial, alveolar wall, and mesothelial cells but not in epithelial cells. With alveolar septal formation, the message in the interstitium increased progressively from 17 days of gestation, reaching a peak at 7 to 11 days postnatal. The signal in the arterial walls, in contrast, peaked between 19 days of gestation to 1 day postnatal and thereafter declined first from the outer media. The signal in general declined significantly by 21 days postnatal, and elastogenesis was virtually absent in the adult. These results support the idea that tropoelastin gene expression in the interstitium is closely associated with the centripetal progression of alveolarization, and the early postnatal decrease of tropoelastin expression in blood vessels corresponds with the sudden postnatal changes in the pulmonary hemodynamics. Furthermore, in the rat fetus and neonate, endothelial cells expressed the gene for tropoelastin and hence probably play a significant role in the formation of internal elastic lamina in vivo.

Animals↗
PubMed1994

Increased mesenchymal cell density accompanies induction of tropoelastin expression in developing elastic tissue.

We studied the differentiation of elastin-producing fetal bovine chondrocytes to understand the regulatory processes associated with induction of elastin expression. Analysis of auricular elastic cartilage development in vivo indicated that differentiation of the prechondrogenic blastema to an elastogenic phenotype was preceded and accompanied by condensation of the mesenchymal cells. In addition, induction of elastin production was temporally and spatially linked to expression of type II collagen and proteoglycans. We assessed the influence of cell density on the induction of tropoelastin expression in pre-elastogenic cells from developing ear buds. Tropoelastin expression was induced in prechondrogenic mesenchymal cells only if the cells were maintained at a high cellular density. In addition, high density culture upregulated tropoelastin expression in fully differentiated chondrocytes. Together these data suggest that high cell density facilitates cell:cell interactions that affect cell proliferation and influence tropoelastin expression.

Animals↗
PubMed1997

Smooth muscle cell proliferation, elastin formation, and tropoelastin transcripts during the development of intimal thickening in rabbit carotid arteries after endothelial denudation.

Extracellular matrix formation and smooth muscle cell proliferation are two major factors contributing to the development of intimal thickening after arterial injury. We investigated the elastin formation, tropoelastin transcripts, and proliferation of smooth muscle cells during the development of intimal thickening in rabbit carotid arteries after balloon endothelial denudation. Tropoelastin transcripts, identified by in situ hybridization using a digoxigenin-labeled probe, and elastin staining in the thickened intima were minimal 1 week after endothelial denudation when smooth muscle cell proliferation appeared throughout the thickened intima. A strong signal for tropoelastin transcripts was seen in the basal layer of the thickened intima 2 weeks after endothelial denudation, and then in the surface layer of the thickened intima 4 weeks after endothelial denudation. Immunohistochemistry for proliferating cell nuclear antigen and Ki-67, both markers for proliferating cell nuclei, showed that tropoelastin transcripts and elastin formation increased when smooth muscle cells enter quiescence after the end of the proliferative phase in the intima. Our findings strongly suggest that elastin synthesis and smooth muscle cell proliferation are tightly regulated during the repair of arterial wall injury.

Animals↗
PubMed2005

Structure and modeling studies of the carboxy-terminus region of human tropoelastin.

Elastin macromolecular assembly is a highly complex mechanism involving many steps including coacervation, cross-linking, and probably other (not known) phenomena. In past studies, it has been proposed that the C-terminal part of tropoelastin is also involved in this process and may play a key role in tropoelastin interactions with other proteins of the final elastic fibres scaffold. Presented here are the results of the biophysical studies (biospectroscopy, bioinformatics) of the C-terminal domain of tropoelastin. We report the detailed structures adopted by the oxidized (native) and reduced forms of the free synthetic peptide with sequence encoded by exon 36 of human tropoelastin (GGACLGKACGRKRK) and propose a dynamical interpretation of which structures may be involved in interactions with other extra-cellular matrix proteins. We also suggest that these structures may be retrieved in other proteins sharing a consensus sequence; however no definitive conclusion can be drawn here on a possible structure-function relationship.

Amino Acid Sequence↗
PubMed1999

Tropoelastin binding to fibulins, nidogen-2 and other extracellular matrix proteins.

Elastic fibers in vessel walls and other tissues consist of cross-linked tropoelastin in association with several microfibrillar proteins. In order to understand the molecular basis of these structures, we examined the binding of recombinant human tropoelastin to other extracellular matrix ligands in solid phase binding and surface plasmon resonance assays. These studies demonstrated a particularly high affinity (K(d) about 1 nM) of tropoelastin for microfibrillar fibulin-2 and the recently described nidogen-2 isoform. More moderate affinities were observed for fibulin-1, laminin-1 and perlecan, while several other ligands such as collagens, nidogen-1, fibronectin and BM-40 showed little or no binding. In immunogold staining of mouse aortic media, elastic fibers were heavily decorated with tropoelastin, fibulin-2 and nidogen-2, while the reaction with fibulin-1 was lower. The colocalization of these proteins emphasizes the potential for in vivo interactions.

Animals↗
PubMed1995

Extensive alternate exon usage at the 5' end of the sheep tropoelastin gene.

Several overlapping cDNA clones were isolated from a lambda gt10 cDNA library constructed using poly A+ RNA from neonatal sheep lung. DNA sequence analysis of these cDNA recombinants revealed the complete derived amino acid sequence of sheep tropoelastin. A comparison of DNA sequences from individual sheep tropoelastin cDNA also confirmed the presence of several tropoelastin mRNA isoforms in neonatal lung tissue. Coding domains corresponding to exons 13, 14 and 33 were present in several of the sheep tropoelastin cDNA fragments but absent in others. The relative amount of alternate usage of these exons was quantitated by polymerase chain amplification. In confirmation of previous studies in other mammalian species, extensive alternate usage of exon 33 was observed in total RNA isolated from aorta, nuchal ligament and pulmonary artery from neonatal sheep. In striking contrast to all previous studies, however, exons 13 and 14 were shown to be subject to almost the same level of alternate usage as exon 33 in all three neonatal sheep tissues examined.

Alternative Splicing↗
PubMed1993

Peptide sequences selected by BA4, a tropoelastin-specific monoclonal antibody, are ligands for the 67-kilodalton bovine elastin receptor.

A 67-kDa cell-surface elastin/laminin receptor is expressed by fetal bovine ligamentum nuchae fibroblasts and neutrophils. Two hexapeptides, VGVAPG and PGAIPG, contained within hydrophobic domains of tropoelastin are binding sites for this receptor. Studies of recombinant tropoelastin proteins and synthetic peptides demonstrated that a monoclonal antibody, BA4, recognized peptide sequences similar to those recognized by the 67-kDa receptor. Taking advantage of this similarity, an "epitope library" containing random hexapeptides was screened with BA4. Four BA4-selected peptides (VGAMPG, VGMAPG, VGSLPG, and VGLSPG) were synthesized; studies of fibroblast and neutrophil migration support the hypothesis that these peptides are ligands of the 67-kDa receptor present on ligamentum nuchae fibroblasts and neutrophils. Two additional, physically similar tropoelastin peptides,AGAIPG and PGAVGP, were also identified as peptide ligands, and hence potential binding sites within tropoelastin, of the elastin receptor. These data suggest that the 67-kDa elastin/laminin receptor may interact with a wide range of structurally similar peptides containing amino acid substitutions involving small nonpolar and uncharged amino acids.

Amino Acid Sequence↗
PubMed2002

Expression of fibrillins and tropoelastin by human gingival and periodontal ligament fibroblasts in vitro.

The elastic system fibers consist of three different types, oxytalan, elaunin and elastic fibers, which differ in the relative content of microfibrils and elastin. In periodontal tissues, oxytalan fibers are known to be distributed in the periodontal ligament and gingiva, while elaunin and elastic fibers are present only in the gingiva. We examined the in vitro synthesis of microfibrils and elastin by human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (HPLF). The two kinds of HGF and HPLF were cultured in MEM containing 10% newborn calf serum for 30 days. Since fibrillin-1 and fibrillin-2 are the major components of microfibrils involved in elastogenesis, we investigated the synthesis of fibrillins and tropoelastin in the conditioned medium of HGF and HPLF. Western blot analysis revealed fibrillin-1 and fibrillin-2 to occur in the HGF and HPLF culture medium, HGF exhibiting a higher level of synthesis than HPLF. Tropoelastin, on the other hand, was detected only in the medium of HGF after day 24. In addition, analysis of RNA extracted from HGF and HPLF on day 30 showed that only HGF expressed mRNA encoding tropoelastin. Immunohistochemically, accumulation of tropoelastin in the perinuclear area was found only in HGF. These results show that HGF expressed microfibrils and elastin, while HPLF expressed only microfibrils for the experimental period, and suggest a biochemical basis for the different distribution of elastic system fibers of the gingiva and periodontal ligament in vivo.

Blotting, Northern↗
PubMed1984

Isolation of tropoelastin a from lathyritic chick aortae.

Tropoelastin a was isolated from lathyritic chick aortae by using severe denaturing conditions for the initial extraction. The amino acid composition of this new species of tropoelastin is elastin-like in its high proportion of proline, glycine, alanine and valine. However, it differs from authentic tropoelastin b in containing a higher percentage of polar amino acids and cysteine residues. In addition, the amount of proline hydroxylation is 3 times higher than that found in chick tropoelastin b.

Amino Acids↗
PubMed1991

Fibroblast adhesion to recombinant tropoelastin expressed as a protein A-fusion protein.

A bovine tropoelastin cDNA encoding exons 15-36 that includes the elastin-receptor binding site was expressed in Escherichia coli as a fusion protein with Protein A from Staphylococcus aureus. After isolation of the fusion protein by affinity chromatography on Ig-Sepharose, the tropoelastin domain was separated from plasmid-pR1T2T-encoded Protein A (Protein A') by CNBr cleavage. Cell-adhesion assays demonstrated specific adhesion to the recombinant tropoelastin. Furthermore, the data indicate that interactions involving the bovine elastin receptor mediate nuchalligament fibroblast adhesion to the recombinant protein. In agreement with earlier studies of fibroblast chemotaxis to bovine tropoelastin, nuchal-ligament fibroblast adhesion demonstrated developmental regulation of the elastin receptor.

Amino Acid Sequence↗
PubMed2001

Ultraviolet radiation increases tropoelastin mRNA expression in the epidermis of human skin in vivo.

Photoaged skin contains elastotic materials in the upper reticular dermis. This phenomenon is commonly known as solar elastosis. Little is known about the mechanisms leading to the accumulation of elastotic materials in photoaged skin, however. In this study, it was demonstrated that ultraviolet irradiation induced tropoelastin mRNA expression in the keratinocytes of human skin in vivo and also in cultured human keratinocytes by in situ hybridization and reverse transcriptase polymerase chain reaction. It was also shown by northern blot analysis (n = 5) that there were increased tropoelastin mRNA levels in the forearm (sun-exposed) skin of elderly persons, compared with upper-inner arm (sun-protected) skin of the same individuals. As demonstrated by in situ hybridization compared to sun-protected skin (upper-inner arm) (n = 5), tropoelastin mRNA expression in photoaged skin was higher in keratinocytes as well as in fibroblasts. Therefore, our results suggest that keratinocytes are another source of tropoelastin production after acute and chronic ultraviolet irradiation in human skin in vivo.

Epidermis↗
PubMed2004

Molecular basis of elastic fiber formation. Critical interactions and a tropoelastin-fibrillin-1 cross-link.

We have investigated the molecular basis of elastic fiber formation on fibrillin microfibrils. Binding assays revealed high affinity calcium-independent binding of two overlapping fibrillin-1 fragments (encoded by central exons 18-25 and 24-30) to tropoelastin, which, in microfibrils, map to an exposed "arms" feature adjacent to the beads. A further binding site within an adjacent fragment (encoded by exons 9-17) was within an eight-cysteine motif designated TB2 (encoded by exons 16 and 17). Binding to TB2 was ablated by the presence of N-terminal domains (encoded by exons 1-8) and reduced after deleting the proline-rich region. A novel transglutaminase cross-link between tropoelastin and fibrillin-1 fragment (encoded by exons 9-17) was localized by mass spectrometry to a sequence encoded by exon 17. The high affinity binding and cross-linking of tropoelastin to a central fibrillin-1 sequence confirm that this association is fundamental to elastic fiber formation. Microfibril-associated glycoprotein-1 showed calcium-dependent binding of moderate affinity to fibrillin-1 N-terminal fragment (encoded by exons 1-8), which localize to the beads. Microfibril-associated glycoprotein-1 thus contributes to microfibril organization but may also form secondary interactions with adjacent microfibril-bound tropoelastin.

Amino Acid Sequence↗
PubMed2005

Tropoelastin interacts with cell-surface glycosaminoglycans via its COOH-terminal domain.

Using a biochemical and cell biological approach, we have identified a cell interaction site at the carboxyl terminus of tropoelastin. Cell interactions with the COOH-terminal sequence are not through the elastin-binding protein (EBP67) because neither VGVAPG-like peptides nor galactoside sugars altered adhesion. Our results also show that cell adhesion to tropoelastin is not promoted by integrins. Through the use of mutant Chinese hamster ovary cell lines defective in glycosaminoglycan biosynthesis, as well as competition studies and enzymatic removal of specific cell-surface glycosaminoglycans, the tropoelastin-binding moieties on the cell surface were identified as heparan and chondroitin sulfate-containing glycosaminoglycans, with heparan sulfate being greatly preferred. Heparin affinity chromatography combined with cell adhesion assays identified the last 17 amino acids as the sequence element at the carboxyl terminus of tropoelastin responsible for the adhesive activity.

Amino Acid Sequence↗
PubMed1994

67-kD elastin-binding protein is a protective "companion" of extracellular insoluble elastin and intracellular tropoelastin.

The 67-kD elastin-binding protein (EBP) mediates cell adhesion to elastin and elastin fiber assembly, and it is similar, if not identical, to the 67-kD enzymatically inactive, alternatively spliced beta-galactosidase. The latter contains an elastin binding domain (S-GAL) homologous both to the aorta EBP and to NH2-terminal sequences of serine proteinases (Hinek, A., M. Rabinovitch, F. W. Keeley, and J. Callahan. 1993. J. Clin. Invest. 91:1198-1205). We now confirm the functional importance of this homology by showing that elastolytic activity of a representative serine elastase, porcine pancreatic elastase, was prevented by an antibody (anti-S-GAL) and by competing with purified EBP or S-GAL peptide. Immunohistochemistry of adult aorta indicates that the EBP exists as a permanent component of mature elastic fibers. This observation, together with the in vitro studies, suggests that the EBP could protect insoluble elastin from extracellular proteolysis and contribute to the extraordinary stability of this protein. Double immunolabeling of fetal lamb aorta with anti-S-GAL and antitropoelastin antibodies demonstrated, under light and electron microscopy, intracellular colocalization of the proteins in smooth muscle cells (SMC). Incubation of SMC with galactosugars to dissociate tropoelastin from EBP caused intracellular aggregation of tropoelastin. A tropoelastin/EBP complex was extracted from SMC lysates by coimmunoprecipitation and cross-linking, and its functional significance was addressed by showing that its dissociation by galactosugars caused degradation of tropoelastin by endogenous serine proteinase(s). This suggests that the EBP may also serve as a "companion" to intracellular tropoelastin, protecting this highly hydrophobic protein from self-aggregation and proteolytic degradation.

Alternative Splicing↗
PubMed1998

Tropoelastin and elastin degradation products promote proliferation of human astrocytoma cell lines.

Expression of tropoelastin, the precursor of insoluble elastin and a major component of elastic fibers, has not yet been demonstrated in astrocytomas nor has it been linked to their proliferation. Here we report that human astrocytoma cell lines (U87 MG, U251 MG, U343 MG-A, U373 MG, SF 126, SF188, SF 539), as well as surgical specimens of malignant human astrocytomas, express intracellular tropoelastin. The tropoelastin produced by astrocytoma cells is, however, susceptible to proteolytic trimming to the extent that it cannot be assembled into extracellular elastic fibers. Astrocytoma cells also express the cell surface 67-kDa elastin binding protein (EBP), which binds elastin degradation products, leading to the upregulation of cyclin A and cdk2 and increased incorporation of [3H]-thymidine. The elastin-dependent mitogenic response of astrocytoma cells is abolished by lactose and chondroitin sulfate, factors which cause shedding of this 67-kDa elastin receptor from the cell surface and by blocking anti-EBP antibody. We therefore suggest that, in astrocytomas, endogenous tropoelastin degradation products bind to EBP and generate signals leading to cell cycle progression in an autocrine or paracrine manner. This is the first report implicating elastin-derived peptides as possible mitogens in malignant astrocytomas.

Astrocytoma↗
PubMed1981

Interaction of a tropoelastin model with connective tissue components.

It has been suggested that tropoelastin interacts with macromolecular components of connective tissue during the formation of elastic fibers. alpha-Elastin, used traditionally as a model of tropoelastin and mature elastin, is not suitable for the monitoring of this interaction in vitro. Therefore a new model of tropoelastin, the methyl ester of alpha-elastin, was synthesized and the interaction of this modified elastin with hyaluronic acid, chondroitin sulfate, proteoglycan subunits, solubilized structural glycoproteins and collagen was studied by turbidimetric measurement. Results indicate that alpha-elastin methyl ester is a better model of tropoelastin than alpha-elastin especially in those experiments where ionic interactions are believed to occur. It was also found that alpha-elastin methyl ester formed aggregates with hyaluronic acid, chondroitin sulfate, proteoglycan subunits and solubilized structural glycoproteins at room temperature and under conditions close to physiological ones. Alpha-elastin does not interact with these compounds under similar conditions. The character of the bonds involved in the interaction and the possible biological significance of the experiments are discussed.

Animals↗