Judith Ann Foster

The synthesis of connective tissue protein in smooth muscle cells

The synthesis of elastin by smooth muscle cells was clearly demonstrated by amino acid analyses and the presence of lysine-derived crosslinks. The values obtained were compatible with those found in amorphous elastin isolated from rabbit aortic tissue. Collagen synthesis by these same cells was monitored by the appearance of [14C] hydroxyproline when the cells were grown in the presence of [14C] proline. When the cells were pulsed with [14C] lysine, one could detect [14C] hydroxylysine and [14C] glucosylgalactosylhydroxylysine. Further evidence for the synthesis of elastin and collagen was the finding of radiolabelled epsilon-hydroxynorleucine and the reduced aldol condensate of two residues of allysine after reduction of [14C] lysine pulsed cells with NaBH4.

Insulin-like Growth Factor-I Regulates Transcription of the Elastin Gene through a Putative Retinoblastoma Control Element A ROLE FOR Sp3 ACTING AS A REPRESSOR OF ELASTIN GENE TRANSCRIPTION

Previous studies have demonstrated that insulin-like growth factor-I (IGF-I) increases elastin gene transcription in aortic smooth muscle cells and that this up-regulation is accompanied by a loss of protein binding to the proximal promoter. Sp1 has been identified as one of the factors whose binding is lost, and in the present study we show that Sp3 binding is also abrogated by IGF-I, but in a selected manner. In functional analyses using Drosophila SL-2 cells, Sp1 expression can drive transcription from the elastin proximal promoter, while co-expression of Sp3 results in a repression of Sp1 activity. Footprint and gel shift analyses position the IGF-I responsive sequences to a putative retinoblastoma control element (RCE). Mutation of the putative RCE sequence as assessed by transient transfection of smooth muscle cells results in an increase in reporter activity equal in magnitude to that conferred by IGF-I on the wild type promoter. Together these results support the hypothesis that IGF-I-mediated increase in elastin transcription occurs via a mechanism of derepression involving the abrogation of a repressor that appears to be Sp3 binding to the RCE.

Inhibition of histone acetyltransferase by glycosaminoglycans.

Histone acetyltransferases (HATs) are a class of enzymes that participate in modulating chromatin structure and gene expression. Altered HAT activity has been implicated in a number of diseases, yet little is known about the regulation of HATs. In this study, we report that glycosaminoglycans (GAGs) are potent inhibitors of p300 and pCAF HAT activities in vitro, with heparin and heparan sulfate proteoglycans (HSPGs) being the most potent inhibitors. The mechanism of inhibition by heparin was investigated. The ability of heparin to inhibit HAT activity was in part dependent upon its size and structure, as small heparin‐derived oligosaccharides (>8 sugars) and N‐desulfated or O‐desulfated heparin showed reduced inhibitory activity. Heparin was shown to bind to pCAF; and enzyme assays indicated that heparin shows the characteristics of a competitive‐like inhibitor causing an ∼50‐fold increase in the apparent Km of pCAF for histone H4. HSPGs isolated from corneal and pulmonary fibroblasts inhibited HAT activity with similar effectiveness as heparin. As evidence that endogenous GAGs might be involved in modulating histone acetylation, the direct addition of heparin to pulmonary fibroblasts resulted in an ∼50% reduction of histone H3 acetylation after 6 h of treatment. In addition, Chinese hamster ovary cells deficient in GAG synthesis showed increased levels of acetylated histone H3 compared to wild‐type parent cells. GAGs represent a new class of HAT inhibitors that might participate in modulating cell function by regulating histone acetylation. J. Cell. Biochem. 105: 108–120, 2008.

A new, improved technique for automated sequencing of non-polar peptides.

Abstract A method for the automated sequential degradation of non-polar peptides is reported. Both the polarity and film-forming properties of these peptides are increased by the attachment of 2-amino-1,5 napthalene disulfonic acid to the C-terminal residue via a water-soluble carbodiimide. The sequences of three individual peptides modified by the procedure are reported with high yields while these same peptides could not be successfully sequenced if no modification was made.

A high molecular weight species of soluble elastin.

Summary The isolation of a high molecular weight species (130–140,000 daltons) of soluble elastin from the aortas of lathyritic chicks is described. In comparison to chick tropoelastin, the higher molecular weight material possesses an increased amount of acidic and hydroxyl amino acids and in contrast to tropoelastin, contains histidine, methionine and cystine residues. This molecular weight species of soluble elastin is susceptible to proteolytic degradation and can be shown to readily breakdown to lower molecular weight components including tropoelastin.

Basic Fibroblast Growth Factor Decreases Elastin Gene Transcription through an AP1/cAMP-response Element Hybrid Site in the Distal Promoter

Previous studies demonstrated that basic fibroblast growth factor (bFGF) decreases elastin gene transcription in pulmonary fibroblasts. In this study we pursue the identification of the element and the trans-acting factors responsible. Gel shift analyses show that bFGF increases protein binding to a sequence located at −564 to −558 base pairs (bp), which possesses homology to both AP1 and cAMP-response consensus elements yet displays a unique affinity for heterodimer binding. Site-directed mutation of the −564- to −558-bp sequence results in an increase in promoter activity and abrogates the effect of bFGF. Western blot analysis shows that bFGF induces a sustained increase in the steady-state levels of Fra 1, and co-transfection of a Fra 1 expression vector with an elastin promoter reporter construct results in an inhibition of elastin promoter activity. Overall the results suggest that bFGF represses elastin gene transcription by increasing the amount of the Fra 1 that subsequently binds to the −564- to −558-bp as a heterodimer with c-Jun to form an inhibitory complex. We propose that the identified bFGF response element can serve to down-regulate elastin transcription in elastogenic cells and, conversely, can serve to up-regulate elastogenesis in cells where endogenous bFGF signaling is attenuated or altered.

[30] Elastin structure and biosynthesis: An overview

Publisher Summary It is found that isolation and purification of insoluble elastin is an initial problem in structural and comparative studies. Recently, there has been a concerted effort to develop nondegradative methodologies to isolate elastin. As a result of these studies, insoluble elastin can be isolated using a combination of denaturants, enzymes, and hydrolytic agents. The available amino acid compositions of elastin isolated from a variety of organs and species closely resembles hot alkali-prepared elastin with the exception that more polar amino acids and less detectable NH2-terminal amino acids are present when milder isolation procedures are used. Several laboratories have reported on the synthesis of soluble elastin by homogeneous cultures of aortic smooth muscle cells. Utilizing either [2H]proline or [3H]valine labels, the identification of soluble elastin of 72,000 Mr was accomplished by copurification with added purified tropoelastin isolated from copper-deficient piglets. In addition, [3H]lysine-labeled tropoelastin was isolated from pig aorta organ cultures and added to the medium of confluent smooth muscle cell cultures. Further examination have shown the presence of two distinct species of tropoelastin. The two elastin proteins, referred to as tropoelastins a and b, differ in molecular weight, isoelectric point, cysteine content, and primary structure.

Heparan sulfate depletion within pulmonary fibroblasts: Implications for elastogenesis and repair

We investigated the role of sulfated proteoglycans in regulating extracellular matrix (ECM) deposition in pulmonary fibroblast cultures. Fibroblast cultures were subject to pharmacologic and enzymatic interventions to modify sulfated proteoglycan levels. Native and proteoglycan‐depleted fibroblasts were treated with porcine pancreatic elastase at 2–4‐day intervals and the elastase‐mediated release of fibroblast growth factor 2 (FGF‐2) and glycosaminoglycans was determined. Elastase treatment released significantly less FGF‐2 and glycosaminoglycans (GAG) from PG‐depleted fibroblasts with respect to native cells. Equilibrium ligand binding studies indicated that 125I FGF‐2 binding at both cell surface receptor and heparan sulfate proteoglycan sites was reduced to different extents based on the method of proteoglycan depletion. Quantitation of elastin protein and message levels indicated that biological sulfation is required for the proper incorporation of tropoelastin into the extracellular matrix. These results suggest that sulfated proteoglycans play a central role in modulating pulmonary fibroblast extracellular matrix composition and are important mediators of elastolytic injury.

Basic fibroblast growth factor decreases elastin gene transcription in aortic smooth muscle cells

The extracellular matrix (ECM) protein elastin plays an essential role in the cardiovascular system by imparting elasticity to blood vessel wall. In this study, we examined the effect of basic fibroblast growth factor (bFGF) on the expression of elastin in aortic smooth muscle cells (SMC) to gain insight into events associated with cardiovascular diseases. The results show that bFGF treatment of SMC causes a significant decrease in elastin mRNA and secreted tropoelastin levels. Nuclear run‐on analyses demonstrate that the downregulation is due to a decrease in the level of elastin gene transcription. Transient transfections of SMC with wild‐type and mutated elastin gene promoter/chloramphenicol acetyl transferase (CAT) constructs show that a previously identified activator protein‐1‐cAMP response element (AP1/CRE) (−564 to −558‐bp) within the elastin promoter mediates the bFGF‐dependent downregulation of elastin gene transcription in SMC. Addition of bFGF to SMC activates the extracellular signal‐regulated kinases 1/2 (ERK1/2) resulting in their translocation into the nucleus and subsequent induction of Fra‐1. The addition of PD‐98059, an inhibitor of ERK1/2 kinase, abrogates the bFGF‐dependent decrease of elastin mRNA in SMC. The described inhibitory effect of bFGF on elastin gene expression in SMC may significantly contribute to the inefficient repair of elastin in early stages of vascular wall injury. J. Cell. Biochem. 85: 592–600, 2002.

Intrinsic enzyme activity associated with tropoelastin.

The presence of an enzyme(s) associated with purified tropoelastin has been established. Results indicate that the enzyme(s) remains closely associated with the soluble elastin throughout the entire purification procedure suggesting that it is very tightly bound. Enzymatic activity is optimum through the pH range 7-9 (37 degrees C) and can be inhibited by disodium ethylenediaminetetraacetate, N-ethylmaleimide, sulfite, soybean trypsin inhibitor and human alpha-1-antitrypsin. The fragmentation pattern appears to be specific and reproducible.