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P Bornstein

Publications and source records attributed to P Bornstein.

At least 19 recordsLinked to original sources
PubMed1992

Thrombospondin 3 (Thbs3), a new member of the thrombospondin gene family.

A third member of the thrombospondin gene family (Thbs3) has been partially characterized in the mouse. In both the mouse and humans, the Thbs3/THBS3 gene is located immediately upstream from the Muc1/MUC1 (episialin) gene; less than 3 kilobases separate the polyadenylation signal of one gene from the start of transcription of the other. The available coding sequence in Thbs3 shows a high degree of amino acid sequence identity to Thbs1 and Thbs2 (58 and 59%, respectively, in exons 15 and 16), but the exon/intron organization of Thbs3 appears to be more disparate than that of the two previously described members of the family. The shorter length of the Thbs3 mRNA (3.5 kilobases) can be attributed largely to a shorter 3'-untranslated region. The Thbs3 gene is expressed in a distinctive pattern in mouse tissues, with the highest level of expression in lung. This pattern suggests a unique function for the translation product of the Thbs3 gene.

Amino Acid Sequence↗
PubMed1992

Modulation of thrombospondin gene expression during osteoblast differentiation in MC3T3-E1 cells.

The levels of expression of two related extracellular matrix protein genes, thrombospondins 1 and 2 (TSP1 and TSP2), were analyzed in the mouse osteogenic cell line, MC3T3-E1. To monitor differentiation, we also measured two potential markers of the osteoblastic phenotype, alkaline phosphatase (ALP) activity, and alpha 1(I) collagen mRNA levels. TSP1 mRNA levels increased 10- to 15-fold during the first nine days of osteoblastic conversion, and then dropped to a level still significantly above baseline values. This increase in TSP1 mRNA closely paralleled that observed in ALP activity. In contrast, TSP2 mRNA levels were unchanged throughout the 21-day time course. These findings suggest that TSP1 is a marker for osteoblast differentiation and could play a role in the cellular changes that accompany acquisition of the osteoblastic phenotype in MC3T3-E1 cells.

Alkaline Phosphatase↗
PubMed1992

Thrombospondins: structure and regulation of expression.

Thrombospondin (TSP) is a large, trimeric, modular glycoprotein that is a major constituent of platelet alpha granules. TSP is also secreted by a wide variety of epithelial and mesenchymal cells in patterns that reflect developmental changes in the embryo and response to injury in the adult. In addition to its role in blood coagulation, TSP has been reported to serve both adhesive and anti-adhesive functions, to foster neurite outgrowth, stimulate and inhibit cell growth and migration, and inhibit angiogenesis. Although this diversity in apparent function can be attributed, in part, to the ability of a single TSP to interact with several different cell-surface receptors, it is now known that the TSPs are encoded by at least three homologous genes in both human and mouse. TSP1, the commonly recognized protein isolated from platelets, is similar to TSP2 in structure. Both proteins contain NH2-terminal, COOH-terminal, and procollagen homology domains, and type I (TSP or properdin), type II (EGF-like), and type III (Ca(2+)-binding) repeats. However, the two TSPs differ in amino acid sequence and in the regulation of their expression. TSP1 is rapidly induced by serum and growth factors. An SRE and a binding site for NF-Y have been shown to mediate the serum response of the human TSP1 gene. On the other hand, TSP2 is far less responsive to serum than TSP1 and lacks the promoter elements that mediate the serum responsiveness of TSP1. TSP3 resembles TSP1 and TSP2 in its COOH-terminal domain and type III repeats, but contains four rather than three type II repeats and lacks type I repeats and a procollagen homology. The NH2-terminal domain of TSP3 also differs from that of either TSP1 or TSP2. All three TSPs demonstrate characteristic patterns of expression in the developing and adult mouse. It is therefore likely that each protein subserves a discrete function. In the future it will be necessary to distinguish among the three TSPs in addressing the function of these proteins.

Amino Acid Sequence↗
PubMed1992

Regulation of collagen I gene expression by ras.

Although transformation of rodent fibroblasts can lead to dramatic changes in expression of extracellular matrix genes, the molecular basis and physiological significance of these changes remain poorly understood. In this study, we have investigated the mechanism(s) by which ras affects expression of the genes encoding type I collagen. Levels of both alpha 1(I) and alpha 2(I) collagen mRNAs were markedly reduced in Rat 1 fibroblasts overexpressing either the N-rasLys-61 or the Ha-rasVal-12 oncogene. In fibroblasts conditionally transformed with N-rasLys-61, alpha 1(I) transcript levels began to decline within 8 h of ras induction and reached 1 to 5% of control levels after 96 h. In contrast, overexpression of normal ras p21 had no effect on alpha 1(I) or alpha 2(I) mRNA levels. Nuclear run-on experiments demonstrated that the transcription rates of both the alpha 1(I) and alpha 2(I) genes were significantly reduced in ras-transformed cells compared with those in parental cells. In addition, the alpha 1(I) transcript was less stable in transformed cells. Chimeric plasmids containing up to 3.6 kb of alpha 1(I) 5'-flanking DNA and up to 2.3 kb of the 3'-flanking region were expressed at equivalent levels in both normal and ras-transformed fibroblasts. However, a cosmid clone containing the entire mouse alpha 1(I) gene, including 3.7 kb of 5'- and 4 kb of 3'-flanking DNA, was expressed at reduced levels in fibroblasts overexpressing oncogenic ras. We conclude that oncogenic ras regulates the type I collagen genes at both transcriptional and posttranscriptional levels and that this effect, at least for the alpha 1(I) gene, may be mediated by sequences located either within the body of the gene itself or in the distal 3'-flanking region.

3T3 Cells↗
PubMed1992

Elements in the first intron of the alpha 1(I) collagen gene interact with Sp1 to regulate gene expression.

Sequences within the first intron of the alpha 1(I) collagen gene act both positively and negatively to regulate expression of the gene. We have further characterized a 274 bp intronic sequence that contains an orientation-specific inhibitory activity and represents a constitutive DNase I-hypersensitive site in the gene. We show that this sequence contains two tandem, unique binding elements for the transcription factor Sp1. In addition, an Sp1-like site, capable of competing for protein binding to the intronic elements, resides in the distal promoter of the collagen gene. The results of experiments with site-directed mutations that abolish binding to the intronic elements indicate that these protein-DNA interactions have an inhibitory effect on the transcriptional efficiency of alpha 1(I) collagen-reporter gene constructs in transient transfection analysis. These data support our conclusion that the first intron plays a complex role, involving multiple protein-DNA binding interactions, in the regulation of expression of the alpha 1(I) collagen gene.

Animals↗
PubMed1991

Feedback regulation of collagen gene expression: a Trojan horse approach.

The mechanisms involved in feedback regulation of type I procollagen synthesis by the N-terminal propeptide of the pro alpha 1(I) chain, termed Col 1, are poorly understood. We have constructed a metallothionein-human collagen chimeric minigene (pMTCol) that codes for a Col 1 fusion protein but lacks a signal peptide sequence and, therefore, would be expected to direct the synthesis of the fusion protein to the cytosol. Baby hamster kidney cells and fetal calf ligament cells, transfected with pMTCol, transcribed the gene and synthesized an intracellular antigen that was identified as the fusion protein with a monospecific antibody. Transfected fetal calf ligament fibroblasts showed significantly reduced levels of endogenously produced type I collagen, as determined by imaging and digital quantitation of immunofluorescence by confocal microscopy; synthesis of fibronectin, thrombospondin, and SPARC (secreted protein, acidic and rich in cysteine) was unchanged or increased in these cells. This recombinant approach offers the potential for a systematic analysis of feedback regulation of collagen synthesis.

Amino Acid Sequence↗
PubMed1991

A second thrombospondin gene in the mouse is similar in organization to thrombospondin 1 but does not respond to serum.

A second, expressed thrombospondin (TSP) gene, Thbs2, has been identified in the mouse. The exon/intron organization of Thbs2 is highly conserved in comparison with Thbs1 in that exon size and the pattern of interruption of the reading frame by introns are preserved, but there is a marked divergence in coding sequence, primarily in the first 7 exons. On the other hand, the DNA and translated amino acid sequences of exons coding for the type I, II, and III repeats in the two TSPs are far better conserved. Thbs2 is located on chromosome 17, band A3, whereas Thbs1 was found on chromosome 2, band F. In marked contrast to Thbs1, the Thbs2 gene is not induced by serum in NIH 3T3 cells; promoter sequences in the two genes are also very different. It is therefore likely that the two TSPs perform related but distinct functions.

Animals↗
PubMed1991

Transcriptional activity of the alpha 1(I)-collagen promoter is correlated with the formation of capillary-like structures by endothelial cells in vitro.

Bovine aortic endothelial (BAE) cells spontaneously form structures in vitro that resemble capillary-like cords or tubes. This process is associated with changes in the expression of certain extracellular matrix proteins that include type I collagen. BAE cells exhibiting angiogenesis in vitro were transfected with plasmids containing either chloramphenicol acetyltransferase or human growth hormone genes directed by promoter sequences from the human alpha 1(I)-collagen gene. Immunostaining for chloramphenicol acetyltransferase demonstrated that collagen promoter activity was restricted to cells involved in the formation of endothelial cords. In comparison to transfected monolayers of BAE cells, the transcriptional activity of the alpha 1(I)-collagen promoter increased by 7-fold in cultures undergoing angiogenesis in vitro. The selective ability of angiogenic endothelium to utilize the alpha 1(I)-collagen promoter is consistent with previous studies showing high levels of alpha 1(I)-collagen mRNA in BAE cells actively engaged in the formation of tubes (Iruela-Arispe, L., Hasselaar, P., and Sage, H. (1991) Lab. Invest. 64, 174-186). We conclude that transcriptional activation of the alpha 1(I)-collagen gene is closely linked to the morphologic alterations in cellular phenotype that accompany the transition of quiescent endothelial monolayers to the angiogenic state.

Animals↗
PubMed1991

A second, expressed thrombospondin gene (Thbs2) exists in the mouse genome.

The diverse and occasionally conflicting properties described for the extracellular, cell surface-associated protein thrombospondin (TSP) have raised the possibility that functionally distinct forms of the protein exist in the same organism. We have isolated and characterized a partial cDNA clone for mouse TSP that is clearly homologous to, but distinct from, the coding sequence for mouse TSP deduced from a mouse genomic clone (Bornstein, P., Alfi, D., Devarayalu, L., Framson, P., and Li, P. (1990) J. Biol. Chem. 265, 16691-16698). This second TSP, which we term thrombospondin 2, is the product of a separate gene (Thbs2) and is expressed in a variety of mouse tissues in a pattern that differs from that for TSP1. Based on their translated amino acid sequences, it seems likely that TSP1 and TSP2 will be found to have both common and unique properties and that the functional consequences of TSP production will reflect the ratio of the levels of these two related proteins.

Amino Acid Sequence↗
PubMed1991

Thrombospondin exerts an antiangiogenic effect on cord formation by endothelial cells in vitro.

The response of endothelial cells to angiogenic stimuli has been shown to be influenced by the extracellular microenvironment. We tested whether thrombospondin, an extracellular matrix protein, modulated the spontaneous formation of cords by endothelial cells in vitro. Despite continued proliferation, a decrease in secreted thrombospondin was detected in cord-containing, as compared with subconfluent, cultures of both aortic and microvascular endothelial cells. Consistent with this trend, mRNA levels of thrombospondin decreased by factors of 16 in aortic and 60 in microvascular cultures that contained endothelial cords. Since thrombospondin was immunolocalized to fibrillar arrays that appeared to be associated with endothelial cords, we added anti-thrombospondin IgG to cord-forming cultures to limit the availability of the protein during this process. In the presence of anti-thrombospondin antibodies, there was a 33-50% increase in cord formation. These results suggest that thrombospondin is an inhibitor of angiogenesis in vitro and are consistent with its proposed roles as a destabilizer of endothelial cell focal contacts and as an inhibitor of endothelial cell proliferation.

Animals↗
PubMed1991

Highly conserved sequences in the 3'-untranslated region of the COL1A1 gene bind cell-specific nuclear proteins.

Sequencing of the 3' untranslated region (3'-UTR) of the human COL1A1 gene revealed numerous putative regulatory motifs and two highly conserved regions flanking the two polyadenylation sites. The conserved regions were separated by about 700 bp of less conserved sequences. The first region consists of almost all the 3'-UTR of the shorter (4.8 kbp) COL1A1 transcript. The second conserved domain includes a motif shared with several collagen genes. Both conserved domains bind cell-specific nuclear proteins suggesting that the 3'-UTR is important for cell specific expression of the COL1A1 gene.

Amino Acid Sequence↗
PubMed1991

An upstream regulatory region mediates high-level, tissue-specific expression of the human alpha 1(I) collagen gene in transgenic mice.

Studies in vitro have not adequately resolved the role of intronic and upstream elements in regulating expression of the alpha 1(I) collagen gene. To address this issue, we generated 12 separate lines of transgenic mice with alpha 1(I) collagen-human growth hormone (hGH) constructs containing different amounts of 5'-flanking sequence, with or without most of the first intron. Transgenes driven by 2.3 kb of alpha 1(I) 5'-flanking sequence, whether or not they contained the first intron, were expressed at a high level and in a tissue-specific manner in seven out of seven independent lines of transgenic mice. In most tissues, the transgene was expressed at levels approaching that of the endogenous alpha 1(I) gene and was regulated identically with the endogenous gene as animals aged. However, in lung, expression of the transgene was anomalously high, and in muscle, expression was lower than that of the endogenous gene, suggesting that in these tissues other regions of the gene may participate in directing appropriate expression. Five lines of mice were generated containing transgenes driven by 0.44 kb of alpha 1(I) 5'-flanking sequence (with or without the first intron), and expression was detected in four out of five of these lines. The level of expression of the 0.44-kb constructs in the major collagen-producing tissues was 15- to 500-fold lower than that observed with the longer 2.3-kb promoter. While transgenes containing the 0.44-kb promoter and the first intron retained a modest degree of tissue-specific expression, those without the first intron lacked tissue specificity and were poorly expressed in all tissues except lung. These results contribute to our understanding of the role of the first intron in regulating alpha1(I) gene expression and identify a region, upstream of the basal alpha1(I) promotor, which is necessary for full tissue-specific, developmentally regulated expression of the alpha1(I) collagen gene.

Animals↗
PubMed1990

Characterization of the mouse thrombospondin gene and evaluation of the role of the first intron in human gene expression.

We have isolated the mouse thrombospondin (TS) gene and determined the DNA sequence of the first nine exons and eight introns. Comparison with the human cDNA sequence reveals a high degree of conservation in coding sequences. Exon 3 of the mouse gene, which encodes the heparin-binding domain of TS, has a higher degree of nucleotide substitution than the other exons, but the distribution of charged and hydrophobic amino acids found in the human protein is generally conserved. DNA and protein sequences in exons 6-9, which encode a procollagen homology and motifs very similar to those found in at least two malarial parasite proteins, are highly conserved. The first two of the three malarial homologies in TS, which are also found in properdin and in components C6-9 of the lytic complement complex, are each encoded by a separate exon (8 and 9) in the mouse gene. Since the sequence data did not reveal substantial similarity in sequence between intron I in the human and mouse genes, we have reexamined the role of the first intron in the transcriptional regulation of the human TS gene. In accord with published studies (Laherty, C.D., Gierman, T.M., and Dixit, V.M. (1989) J. Biol. Chem. 264, 11222-11227), we find that deletion of some intronic segments from TS-chloramphenicol acetyltransferase (CAT) constructs reduces CAT activity in NIH 3T3 cells. However, deletion of the same sequences from TS-bovine growth hormone constructs does not affect the expression of bovine growth hormone in these cells. We conclude that differences in the activity of TS-CAT constructs reflect post-transcriptional differences that are peculiar to the resulting chimeric transcripts and that there is currently no evidence for a transcriptional enhancer in the first intron of the human TS gene.

Amino Acid Sequence↗
PubMed1990

Structural and functional analysis of the first intron of the human alpha 2(I) collagen-encoding gene.

The nucleotide (nt) sequence of the first intron of the human alpha 2(I) collagen-encoding gene (COL1A2) has been determined from its 5' terminus (nt 207) to nt 2045 with respect to the transcription start point. Although the first intron contains elements known to function in transcriptional regulation of other genes (two AP1-binding sites and an alternating GT stretch), comparison of this sequence with that of the mouse COL1A2 first intron revealed a low degree of nt sequence identity and very few common DNA-protein binding motifs. In keeping with this structural analysis, the human intron was found to inhibit COL1A2 promoter activity in transfection experiments, whereas a strong enhancer was reported to be present in the first intron of mouse COL1A2 [Rossi and deCrombrugghe, Proc. Natl. Acad. Sci. USA 84 (1987) 5590-5594]. We conclude that the high degree of nt sequence conservation existing in the promoter and first exons of human, mouse and chicken COL1A2 does not extend to the first introns of these genes but that the promoter activity of COL1A2 is strongly influenced by the presence of the first intron.

Animals↗
PubMed1990

Transcriptional control of the alpha 1(I) collagen gene involves orientation- and position-specific intronic sequences.

The 3' half of the first intron of the human alpha 1(I) collagen gene interacts with the promoter to regulate transcription. We questioned whether this intronic sequence also exerted its effect when placed 5' to the promoter. In transient transfection assays using several cell lines, little or no stimulation of alpha 1(I)-driven chloroamphenicol acetyltransferase transcription was observed. We conclude that transcriptional control of the alpha 1(I) gene is dependent on a complex series of interactions that require both orientation and position specificity of the intronic segment.

Chloramphenicol O-Acetyltransferase↗
PubMed1990

Thrombospondin gene expression is associated with mitogenesis in 3T3 cells: induction by basic fibroblast growth factor.

Growth factor-depleted Swiss 3T3 cells responded to basic fibroblast growth factor (bFGF) with a burst of mitogenesis and with a rapid and marked increase in thrombospondin (TS) mRNA levels. mRNA levels for the alpha 1 chain of type I collagen and for fibronectin were unaffected. At early times following stimulation (0-2 h), "superinduction" of TS mRNA by inhibition of protein synthesis with cycloheximide was not observed, and the increase in TS mRNA could be attributed primarily to an increase in transcription rate of the TS gene. However, at later times (4-8 h) the combination of cycloheximide and bFGF superinduced TS mRNA levels, suggesting the existence of a labile inhibitor of transcription or a short-lived RNase that might be produced in response to prolonged treatment with bFGF. In contrast to its stimulatory effect on 3T3 cells, bFGF did not stimulate the proliferation of mouse muscle BC3H1 cells nor did it cause an increase in TS mRNA levels, but BC3H1 cells do respond to bFGF by inhibition of myogenic differentiation. We propose, on the basis of these and other findings, that TS facilitates the progression of some anchorage-dependent cells through the cell cycle.

Animals↗
PubMed1990

Mapping of the thrombospondin gene to human chromosome 15 and mouse chromosome 2 by in situ hybridization.

We have mapped the thrombospondin gene (THBS1) to a single locus on human chromosome 15 (band q15) and on mouse chromosome 2 (region F). Thrombospondin has been implicated in a variety of cell-matrix and cell-cell interactions. The finding of a single locus suggests that the different functions of thrombospondin are not due to a closely related family of genes. These results also confirm a region of homology between the proximal part of human chromosome 15 and region F of mouse chromosome 2.

Animals↗