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PubMed1979

Partial characterization of a tropoelastin precursor isolated from chick aorta.

Evidence is presented that indicates tropoelastin is derived from a soluble elastin with a molecular weight of 95000. Tropoelastin and its proposed precursor were isolated from the aortas of copper-deficient chicks. Although it is doubtful that the proposed precursor is an initial product of elastin translation, i.e., a proelastin, it is proposed to be at least a truncated form of proelastin that is converted to tropoelastin. The key to its isolation was the presence of alpha 1-antitrypsin at each step in the purification procedure. The first 11 amino acid residues at the NH2 terminal of the proposed tropoelastin precursor (GGVPGVAVPGGV) are the same as those for tropoelastin. Its amino acid composition is similar to that of tropoelastin, except for higher amounts of acidic amino acid residues. Further, the proposed precursor contains a limited number of aldehydic functions, presumably in the form of peptidyl allysine. This was taken as an indication that the proposed precursor serves as a substract for lysyl oxidase. Under the conditions used for the isolation, the precursor appeared to be in higher concentrations than tropoelastin in aorta extracts from copper-deficient chicks.

Amino Acid Sequence↗

PubMed2002

Tropoelastin expression is up-regulated during activation of hepatic stellate cells and in the livers of CCl(4)-cirrhotic rats.

BACKGROUND/AIMS: Activated hepatic stellate cells (HSC) are regarded as the principal cells synthesizing extracellular matrix components in fibrotic liver. Elastin content is increased in cirrhotic livers, but the cellular source is not known. Contribution of HSC to the production of elastin was investigated. METHODS: Expression of elastin in CCl(4)-cirrhotic rat liver was studied by immunohistochemistry and in situ hybridization, liver myofibroblasts were identified in histological sections by alpha-smooth muscle actin (alpha-SMA) staining. LightCycler PCR and Northern blotting were used to detect tropoelastin mRNA in isolated HSC; tropoelastin protein was detected in the cells and in cell-conditioned medium by Western blotting. RESULTS: HSC, isolated from normal rat liver, displayed increasing tropoelastin mRNA expression during transdifferentiation in culture. Expression of tropoelastin mRNA was accompanied by the production of tropoelastin protein in vitro. Increased levels of tropoelastin transcripts were found in the connective tissue septa of CCl(4)-cirrhotic rats and co-localized with alpha-SMA positive cells. Immunohistochemistry demonstrated elastin presence in the septa. CONCLUSION: HSC express tropoelastin and its expression increases during transdifferentiation to myofibroblast-like cells.

Actins↗

PubMed1999

Fibroblast tropoelastin and alpha-smooth-muscle actin expression are repressed by particulate-activated macrophage-derived tumor necrosis factor-alpha in experimental silicosis.

Lung elastin synthesis is normally confined to periods of development, is maximal during alveolarization, and declines to low levels in mature lung. We have previously described an elastogenic response in the adult rat lung associated with experimental granulomatous disease induced by silica instillation. Reinitiated tropoelastin expression was identified throughout the lung in fibroblasts expressing alpha-smooth-muscle actin, whereas fibroblasts within the granulomatous lesions failed to express both tropoelastin and alpha-smooth- muscle actin (Mariani and colleagues, Am. J. Pathol. 1995;147:988-1000). We hypothesized that inflammatory cells within the granulomatous lesions produce factors that alter fibroblast phenotype. We found that macrophages accumulating within granulomatous lesions of silicotic rat lungs produce and secrete tumor necrosis factor (TNF)-alpha, a proinflammatory cytokine previously appreciated as a repressor of tropoelastin gene expression. In experimental cell systems, macrophages activated by particulates, either in vivo or in vitro, conditioned medium with a tropoelastin-repressing activity. This activity repressed both tropoelastin and alpha-smooth-muscle actin expression in primary cultures of rat lung fibroblasts in a time- dependent, transient manner. The particulate-activated macrophage-conditioned medium was found to contain TNF-alpha, which was both necessary and sufficient to induce these changes in lung fibroblast gene expression. These data indicate that macrophage-derived factors can modulate lung fibroblast tropoelastin expression in the diseased lung. Furthermore, the findings extend the association between expression by lung fibroblasts of tropoelastin and alpha-smooth-muscle actin.

Actins↗

PubMed1995

Accumulation of tropoelastin by a short-term ascorbic acid treatment in the culture medium of aortic smooth muscle cells in vitro.

Treatment of cultured smooth muscle cells with ascorbic acid resulted in an accumulation of tropoelastin in the culture medium in dose-dependent and exposure time-dependent manner under the condition in which collagen synthesis was stimulated 2-fold. The steady-state level of elastin mRNA was essentially unchanged, whereas collagen mRNA content increased 2-fold by ascorbic acid treatment. Newly synthesized tropoelastin was hydroxylated in the presence of ascorbic acid but was underhydroxylated in a scorbutic condition. Short pulse experiments showed that the secretion rate of tropoelastin was unaltered by ascorbic acid treatment. Pulse-chase experiment demonstrated that the level of fully hydroxylated tropoelastin in the medium of ascorbate-treated cells was greater than that of under-hydroxylated tropoelastin. These results indicate that accumulation of tropoelastin in the medium by ascorbic acid is related to an increased stability of hydroxylated tropoelastin and/or its impaired incorporation into insoluble elastin.

Actins↗

PubMed1991

Inhibition of tropoelastin expression by 1,25-dihydroxyvitamin D3.

Elastin production is modulated by steroid hormones and is dependent on calcium. Because vitamin D3 is involved in the regulation of calcium metabolism and influences the expression of various extracellular matrix proteins, we investigated whether vitamin D3 influences tropoelastin expression. Three elastin-producing, bovine cell types, auricular chondroblasts, nuchal ligament fibroblasts and arterial smooth muscle cells, were treated with the principal active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25[OH]2D3), and with 24,25 dihydroxyvitamin D3 (24,25[OH]2D3). Tropoelastin levels in culture media and cell layers, as measured by an enzyme-linked immunoassay, decreased in a dose and exposure dependent manner after treatment with 1,25(OH)2D3; 24,25(OH)2D3 had no effect on tropoelastin production relative to solvent-treated controls. The maximal effective dose of 1,25(OH)2D3 was 10(-7) M for 48 hr, which resulted in a severalfold reduction of tropoelastin production, and decreased tropoelastin levels were detected at 8 hr after treatment. Reduction of tropoelastin protein production was paralleled by a decrease of equal magnitude in the steady-state levels of tropoelastin mRNA. Vitamin D3 metabolites had no effect on DNA or total protein synthesis. These results suggest that vitamin D3 may be an important modulator of elastin expression.

Animals↗

PubMed2004

Tropoelastin inhibits vascular calcification via 67-kDa elastin binding protein in cultured bovine aortic smooth muscle cells.

In cases of vascular calcification, the expression of tropoelastin is down-regulated, which most likely decreases elastic fiber formation. However, the function of tropoelastin in vascular calcification remains unknown. We investigated whether tropoelastin affects the induction of vascular calcification. Calcification was induced using inorganic phosphate in cultured bovine aortic smooth muscle cells. The increase in tropoelastin due to the addition of recombinant bovine tropoelastin (ReBTE; 1 or 10 microg/ml) or beta-aminopropionitrile (25 microg/ml) significantly inhibited calcification at day 6, as assessed by the o-cresolphthalein complexone method. The addition of an elastin-derived peptide, VGVAPG peptide (0.1-1,000 nM), inhibited calcification at day 6 in a dose-dependent manner. In addition, these responses of beta-aminopropionitrile, ReBTE, and VGVAPG peptide were confirmed using von Kossa staining. To examine whether ReBTE inhibited calcium deposition via the elastin binding protein, lactose and elastin-specific antibody were used. The combination of lactose (20 mM) or this antibody (50 microg/ml) with ReBTE (10 microg/ml) attenuated the inhibition of calcification. These results suggest that increased tropoelastin inhibits vascular calcification in this model via the interaction between tropoelastin and elastin binding protein.

Animals↗

PubMed2004

[Modulation of tropoelastin expression in cultured human dermal fibroblasts by heat shock].

OBJECTIVE: To observe the effect of heat shock on tropoelastin mRNA and protein expression in cultured human dermal fibroblasts and to elucidate the possible role of heat shock in the development of solar elastosis in human skin. METHODS: Primary cultured human dermal fibroblasts were incubated in 43 degrees C water bath for 30 minutes, then returned to 37 degrees C, 5%(volume fraction) CO(2) atmosphere normal culture condition. The cells were harvested at hour 24 and 48 after heat treatment respectively. Total RNA was extracted from cell lysis and tropoelastin mRNA level was measured by Northern blot assay. Tropoelastin protein level in cultured supernatant was measured by Western blot assay. RESULTS: At hour 24 after heat treatment, the level of tropoelastin mRNA was enhanced to (163+/-12)% of control group (P<0.05). It was increased further to (221+/-22)% of control group at hour 48 after heat treatment (P<0.05). Similarly, the tropoelastin protein level was also increased to (149+/-12)% (P<0.05) at hour 24, and to (783+/-10)% (P<0.05) at hour 48 after heat shock as compared with control group. CONCLUSION: Heat shock could up-regulate tropoelastin mRNA and protein expression in primary cultured human dermal fibroblasts, and the over-expression of tropoelastin might play some roles in the development of solar elastosis in photoaged skin.

Blotting, Western↗

PubMed1992

1,25-Dihydroxyvitamin D3 represses tropoelastin expression by a posttranscriptional mechanism.

Tropoelastin expression is down-regulated by exposure to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), and we present data indicating that this repression is primarily controlled by a posttranscriptional mechanism. Steady-state and functional levels of tropoelastin mRNA were coordinately repressed by 10(-7) M 1,25 (OH)2D3 in fetal bovine chondrocytes, but transcription, as determined by nuclear runoff assay, was not appreciably influenced in fetal bovine chondrocytes or in rat lung fibroblasts. Similarly, exposure to 1,25(OH)2D3 did not influence chloramphenicol acetyl-transferase activity expressed by a human tropoelastin promoter-expression construct in either cell type. Exposure to cycloheximide had little effect on tropoelastin mRNA levels in control cells but partially restored tropoelastin mRNA levels in cells pretreated with 1,25(OH)2D3 and prevented repression when added together with 1,25(OH)2D3. Similarly, simultaneous exposure to actinomycin D and 1,25(OH)2D3 attenuated the down-regulation of tropoelastin. These data indicate that repression of tropoelastin steady-state mRNA levels by 1,25(OH)2D3 is primarily mediated by a posttranscriptional mechanism that requires both transcription and protein synthesis for full effect.

Animals↗

PubMed1995

Recycling of the 67-kDa elastin binding protein in arterial myocytes is imperative for secretion of tropoelastin.

We have shown previously that the 67-kDa elastin binding protein (EBP) colocalizes intracellularly and extracellularly with tropoelastin in fetal sheep aorta, suggesting that these two proteins associate along the secretory pathway. Moreover, we have established that association with EBP protects tropoelastin from serine proteinases and from intracellular coacervation, and is necessary for its proper extracellular assembly. Since the production of tropoelastin by aortic smooth muscle cells (Ao SMC) exceeds production of the EBP, we speculated that this binding protein might recycle back into the cell, associating again with newly synthesized tropoelastin. In this report we labeled cultured Ao SMC externally with the F(ab')2 fragments of immunoglobulin which recognizes sheep EBP and followed trafficking of EBP by immunofluorescence and electron microscopy. Our results indicate that the majority of the EBP residing on the cell surface can be internalized to endocytic compartments (but not to lysosomes) and recycled back to the plasma membrane within 45-60 min. We have also determined that reagents disturbing pH of distinct endocytic compartments (chloroquine and bafilomycin A1, but not ammonium chloride) arrest recycling of the EBP and, at the same time, strongly inhibit deposition of insoluble elastin in cultures of sheep Ao SMC and in organ cultures of chicken aorta. In contrast, neither chloroquine nor bafilomycin A1 inhibit total protein synthesis or synthesis of tropoelastin. Our results suggest that the EBP serves as a reusable shuttle protein for tropoelastin and that its recycling is essential for effective deposition of insoluble elastin.

Animals↗

PubMed2004

Expression of tropoelastin in human periodontal ligament fibroblasts after simulation of orthodontic force.

The ability of elastic fibers to respond to mechanical stimuli suggests that they play a central role in physiological adaptation to external stimuli including application of orthodontic force. The purpose of this study was to examine the effect of external pressure simulating orthodontic force on tropoelastin gene expression in cultured human periodontal ligament fibroblasts (hPDLF). External pressure simulation was achieved by centrifugation for 10, 30, 60, 90 and 120 min of hPDLF in a horizontal microplate rotor. Semi-quantitative RT-PCR analysis of tropoelastin mRNA was performed and beta-actin was used as an internal invariant control. While centrifugal force on mRNA levels of beta-actin showed almost no change, the mRNA levels of tropoelastin increased significantly to a peak level of more than four-fold after 30 min. Thereafter, at 60 min, the mRNA levels remained at more than three-fold. After 90 min, mRNA levels decreased to control levels. The finding that no changes in mRNA levels of beta-actin occurred during the first 90 min of centrifugation validates its use as an invariant control gene in such an experimental model. This study demonstrated that tropoelastin is expressed in hPDLF and that the pressure caused significant time-dependent upregulation of the tropoelastin gene. The responsiveness of the tropoelastin gene to force shows its possible clinical importance in orthodontic tooth movement. Further studies, however, are essential in order to learn whether the high expression of the gene in vitro will also be followed by corresponding protein synthesis and deposition in vivo in the extracellular matrix (ECM) of the periodontal ligament (PDL).

Actins↗

PubMed2004

Deposition of tropoelastin into the extracellular matrix requires a competent elastic fiber scaffold but not live cells.

The initial steps of elastic fiber assembly were investigated using an in vitro assembly model in which purified recombinant tropoelastin (rbTE) was added to cultures of live or dead cells. The ability of tropoelastin to associate with preexisting elastic fibers or microfibrils in the extracellular matrix was then assessed by immunofluorescence microscopy using species-specific tropoelastin antibodies. Results show that rbTE can associate with elastic fiber components in the absence of live cells through a process that does not depend on crosslink formation. Time course studies show a transformation of the deposited protein from an initial globular appearance early in culture to a more fibrous structure as the matrix matures. Deposition required the C-terminal region of tropoelastin and correlated with the presence of preexisting elastic fibers or microfibrils. Association of exogenously added tropoelastin to the cellular extracellular matrix was inhibited by the addition of heparan sulfate but not chondroitin sulfate sugars. Together, these results suggest that the matrix elaborated by the cell is sufficient for the initial deposition of tropoelastin in the extracellular space and that elastin assembly may be influenced by the composition of sulfated proteoglycans in the matrix.

Animals↗

PubMed2006

Differential expression of two tropoelastin genes in zebrafish.

Elastin is the extracellular matrix protein responsible for properties of extensibility and elastic recoil in large blood vessels, lung and skin of most vertebrates. Elastin is synthesized as a monomer, tropoelastin, but is rapidly transformed into its final polymeric form in the extracellular matrix. Until recently information on sequence and developmental expression of tropoelastins was limited to mammalian and avian species. We have recently identified and characterized two expressed tropoelastin genes in zebrafish. This was the first example of a species with multiple tropoelastin genes, raising the possibility of differential expression and function of these tropoelastins in elastic tissues of the zebrafish. Here we have investigated the temporal expression and tissue distribution of the two tropoelastin genes in developing and adult zebrafish. Expression was detected early in skeletal cartilage structures of the head, in the developing outflow tract of the heart, including the bulbus arteriosus and the ventral aorta, and in the wall of the swim bladder. While the temporal pattern of expression was similar for both genes, the upregulation of eln2 was much stronger than that of eln1. In general, both genes were expressed and their gene products deposited in most of the elastic tissues examined, with the notable exception of the bulbus arteriosus in which eln2 expression and its gene product was predominant. This finding may represent a sub-specialization of eln2 to provide the unique architecture of elastin and the specific mechanical properties required by this organ.

Amino Acid Sequence↗

PubMed1988

In vitro processing of tropoelastin: investigation of a possible transport function associated with the carboxy-terminal domain.

In vitro translation systems were used to characterize the processing of bovine tropoelastin isoforms and to investigate the possibility that the carboxy-terminal 19 amino acids of tropoelastin encode a molecular domain that directs intracellular transport. Immunoprecipitation with domain-specific antibodies demonstrated that multiple tropoelastin isoforms corresponding to those identified in tissue and cell culture studies were correctly translated and were processed by dog pancreas microsomes. Our results demonstrate that all tropoelastin isoforms are translocated completely into the microsomal vesicle and do not remain associated with the microsomal membrane. These results exclude the possibility that the carboxy-terminal domain of tropoelastin functions as a trafficking signal by effecting an association between tropoelastin and an intracellular membraneous compartment.

Amino Acid Sequence↗

PubMed1999

Increased tropoelastin and procollagen expression in the lung of nitrofen-induced diaphragmatic hernia in rats.

BACKGROUND/PURPOSE: Collagen and elastin, the predominant components of the lung connective tissue network, have been suggested to have an important influence on lung compliance and maximal expansion. Decrease in lung compliance and distensibility often is seen in human congenital diaphragmatic hernia (CDH) lung as well as in experimentally produced CDH lung. The aim of this study was to investigate mRNA levels of tropoelastin and alpha1 (I) procollagen, the precursors of elastin, and type I collagen, respectively, in CDH lung and to determine whether antenatal dexamethasone treatment has any effect on the production of these extracellular matrix proteins. METHODS: CDH model was induced in pregnant rats after administration of 100 mg nitrofen on day 9.5 of gestation (term, 22 days). Dexamethasone (0.25 mg/kg) was given on day 18.5 and 19.5. Cesarean section was performed on day 21. The fetuses were divided into three groups: group I, normal controls; group II, nitrofen-induced CDH; and group III, nitrofen-induced CDH with antenatal dexamethasone treatment. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate relative amounts of tropoelastin and alpha1 (I) procollagen mRNA. RESULTS: Levels of both tropoelastin and alpha1 (I) procollagen mRNA were significantly increased in group II compared with group I (P< .05). Neither tropoelastin nor alpha1 (I) procollagen mRNA levels were significantly different between group II and III. CONCLUSIONS: The increased local synthesis of tropoelastin and type I procollagen in CDH lung may be responsible for the increased rigidity and decreased compliance observed in the CDH hypoplastic lung. Glucocorticoids have no effect on pulmonary tropoelastin and alpha1 (I) procollagen gene expression in CDH lungs.

Animals↗

PubMed1992

In-situ hybridization of tropoelastin mRNA during the development of the multilayered neonatal rat aortic smooth muscle cell culture.

Cultured neonatal rat aortic smooth muscle cells are active in synthesizing and depositing large amounts of elastin in their extracellular matrix, making this an ideal system for studying elastogenesis. In this study, the ability of individual cells to synthesize tropoelastin was examined by in-situ hybridization methods. One-micron semi-thin epoxy resin-embedded transverse sections of cells cultured 1, 2, 3 and 4 weeks showed an increase with time in both the number of cells with hybridization signal and the signal intensity; tropoelastin mRNA hybridization signal intensity decreased thereafter up to 8 weeks in culture. In longitudinal sections through the early cultures (1-week), we observed mitotic cells with no detectable hybridization signal, and non-mitotic cells with either no, little or high signal intensity. These data suggest that mitotic cells do not synthesize tropoelastin, and that there is a strong correlation between the hybridization signal intensity and the rate of tropoelastin synthesis. These data also suggest in-situ hybridization methods can detect which cell(s) contain tropoelastin mRNA, their location in the multilayer, and variations in signal intensity. We conclude it is possible to correlate hybridization signal intensity with variations of tropoelastin mRNA levels within individual cells of the cultured smooth muscle cell multilayer.

Animals↗

PubMed1996

Tropoelastin gene expression in optic nerve heads of normal and glaucomatous subjects.

Elastic fibers are a major component of the extracellular matrix in the optic nerve head (ONH) and undergo marked morphological changes during primary open angle glaucoma (POAG). Previous findings indicated that there is reactivation of tropoelastin mRNA synthesis in glaucoma. In this study, we sought to determine the alternative splicing pattern of tropoelastin in the human optic nerve head and in cultured laminar astrocytes. Furthermore, we compared the alternative splicing pattern of normal elastogenesis with that of reactivation of elastin synthesis in patients with primary open angle glaucoma. Our results demonstrate that exons 23 and 32 of tropoelastin are alternatively spliced in the normal ONH as well as in tissue from glaucomatous patients. There are no qualitative differences. We also demonstrated that astrocytes from the ONH synthesize tropoelastin in vitro. In conclusion, we have demonstrated a tropoelastin alternative splicing pattern in the human optic nerve head and laminar astrocytes. Abnormalities in elastic fibers in the ONH of patients with POAG are not due to an aberrant splice variant of tropoelastin. Astrocytes grown from ONH explants may serve as an in vitro model to study extracellular matrix changes in glaucoma.

Adult↗

PubMed1979

Additional evidence for a proform to tropoelastin from chick aorta.

Evidence is presented for the presence of precursor to tropoelastin in chick arterial extracts. The precursor is approx. 100 000 daltons in size. It is suggested to be a precursor to tropoelastin (72 000 daltons). This protein may be observed in culture in vitro if appropriate precautions are taken to inhibit proteolysis. Once synthesized, it appears to be converted into tropoelastin within 10--20 min. The protein may also be detected in vivo. When 1-day-old cockerels were fed on a copper-deficient diet (less than 1 p.p.m. to inhibit cross-linking) containing epsilon-aminohexanoic acid (0.2%) to retard proteolysis and then injected wiht [3H]valine, extraction of arterial proteins 12h after injection resulted in detection of two major peaks of [3H]valine-labelled protein with pI values of pH 7.0 and 5.0 respectively. The protein that focused at pH 7.0 was estimated to be about 100 000 daltons in size and could be shown to be converted into a more basic protein with the properties of tropoelastin. It is speculated that the protein with pI 5.0 may be yet another extension peptide. The data appear to be in keeping with similar observations by ourselves and others that a proform of tropoelastin exists, and, in at least one step before conversion into tropoelastin, exists as a 100 000-dalton protein subunit.

Animals↗

PubMed1984

Elastin biosynthesis in chick-embryo arteries. Studies on the intracellular site of synthesis of tropoelastin.

Electrophoretic analyses of the products of cell-free translation of elastin mRNA isolated from 17-day chick-embryo thoracic arteries have demonstrated that the elastin mRNA codes for polypeptides that are slightly larger than the cellular tropoelastin polypeptides synthesized and secreted by matrix-free artery cells. Pulse-chase experiments with cells labelled with [3H]proline established that newly synthesized tropoelastin polypeptides were associated solely with membrane-bound particulate fractions. Cell-free translation of membrane-bound and free polyribosomes isolated from artery cells revealed that the tropoelastin mRNA was associated predominantly with the membrane-bound fraction. When rough-microsomal fractions, isolated from cells labelled with [3H]proline for 10 min, were treated with proteinases in the presence and in the absence of detergent, the nascent tropoelastin polypeptides were shown to be susceptible to proteolysis only when the integrity of the membranes was destroyed by detergent treatment. In similar experiments tropoelastin polypeptides synthesized by membrane-bound polyribosomes in the nuclease-treated reticulocyte lysate were also resistant to the proteolytic-enzyme treatment. The results suggest that tropoelastin polypeptides are synthesized on membrane-bound polyribosomes and discharged into the lumen of the endoplasmic reticulum with co-translational removal of a signal peptide.

Animals↗