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P Bornstein

Publications and source records attributed to P Bornstein.

At least 91 records · Page 5Linked to original sources
PubMed1988

A highly conserved, 5' untranslated, inverted repeat sequence is ineffective in translational control of the alpha 1(I) collagen gene.

An inverted repeat sequence, extending from the 5' untranslated region of the first exon through the translation initiation codon, is highly conserved in the alpha 1(I), alpha 2(I) and alpha 1(III) collagen genes of mammals and birds. It has been suggested that this sequence functions in translational control of collagen gene expression. When the upstream axis of the dyad of symmetry was deleted, the efficiency of translation of transcripts from a human alpha 1(I) collagen-bovine growth hormone fusion gene was unchanged in either transiently or stably transfected cells. Furthermore, mRNA levels were not affected when the same deletion was transferred to a collagen-human growth hormone fusion gene in which the collagen sequence retained the first intron. Examination of human alpha 1(I) DNA, extending from the start of transcription to the start of translation, by the DNAse I protection procedure revealed evidence for protein binding to a sequence just upstream of the inverted repeat sequence but not to the inverted repeat itself. Our studies therefore indicate that this highly conserved DNA sequence does not function generally in translational or transcriptional control of type I procollagen synthesis.

Animals↗
PubMed1988

The first intron of the alpha 1(I) collagen gene contains several transcriptional regulatory elements.

The first intron of the human alpha 1(I) collagen gene contains a negatively acting element that inhibits transcription of the chloramphenicol acetyltransferase gene driven by either a collagen or an SV40 basal promoter (Bornstein, P., McKay, J., Morishima, J., Devarayalu, S., and Gelinas, R. E. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, in press). We now find that this element is flanked by sequences that both neutralize the inhibitory effect and impart a net positive effect on transcription. A collagen-human growth hormone minigene was constructed in which varying lengths of the collagen intron were retained. Plasmids were transfected into chick tendon fibroblasts, and transcriptional activity was measured by solution hybridization with an antisense RNA probe. The presence of the intact intronic sequence stimulated transcription by a factor of 2-3-fold in comparison with intron-deleted plasmids. However, the isolated negatively acting element inhibited transcription by a factor of 15-20-fold. Surprisingly, this effect was markedly orientation-dependent. Intronic segments flanking the negatively acting element stimulated transcription both when cloned 5' to the collagen promoter in chloramphenicol acetyltransferase-based plasmids and 3' in collagen-human growth hormone constructions. We conclude that expression of the alpha 1(I) collagen gene is controlled by several intronic elements that function coordinately with 5'-flanking and promoter elements.

Acetyltransferases↗
PubMed1988

Transforming growth factor beta increases mRNA for matrix proteins both in the presence and in the absence of changes in mRNA stability.

Transforming growth factor-beta (TGF-beta) has been shown to stimulate synthesis of extracellular matrix proteins, both in animals and in cell culture. We found that mRNAs for alpha 1(I) collagen, fibronectin, and thrombospondin were markedly increased in TGF-beta-treated 3T3 (mouse) cells. For collagen and fibronectin this increase was 10-to 20-fold, as measured by quantitative blot hybridization analysis. A maximal value was reached at 16-24 hr, with a subsequent gradual decline. Concomitant treatment with cycloheximide prevented the stimulation observed with TGF-beta. Under conditions of confluent growth a clear increase in alpha 1(I) collagen mRNA stability was observed, whereas in subconfluent cells no change in mRNA half-life was found, despite an equally large increase in mRNA levels. We suggest that the mode of action of TGF-beta varies with the target cell and depends on the interplay of a number of complex cellular factors.

Animals↗
PubMed1988

Type VIII collagen has a restricted distribution in specialized extracellular matrices.

A pepsin-resistant triple helical domain (chain 50,000 Mr) of type VIII collagen was isolated from bovine corneal Descemet's membrane and used as an immunogen for the production of mAbs. An antibody was selected for biochemical and tissue immunofluorescence studies which reacted both with Descemet's membrane and with type VIII collagen 50,000-Mr polypeptides by competition ELISA and immunoblotting. This antibody exhibited no crossreactivity with collagen types I-VI by competition ELISA. The mAb specifically precipitated a high molecular mass component of type VIII collagen (EC2, of chain 125,000 Mr) from the culture medium of subconfluent bovine corneal endothelial cells metabolically labeled for 24 h. In contrast, confluent cells in the presence of FCS and isotope for 7 d secreted a collagenous component of chain 60,000 Mr that did not react with the anti-type VIII collagen IgG. Type VIII collagen therefore appears to be synthesized as a discontinuous triple helical molecule with a predominant chain 125,000 Mr by subconfluent, proliferating cells in culture. Immunofluorescence studies with the mAb showed that type VIII collagen was deposited as fibrils in the extracellular matrix of corneal endothelial cells. In the fetal calf, type VIII collagen was absent from basement membranes and was found in a limited number of tissues. In addition to the linear staining pattern observed in the Descemet's membrane, type VIII collagen was found in highly fibrillar arrays in the ocular sclera, in the meninges surrounding brain, spinal cord, and optic nerve, and in periosteum and perichondrium. Fine fibrils were evident in the white matter of spinal cord, whereas a more generalized staining was apparent in the matrices of cartilage and bone. Despite attempts to unmask the epitope, type VIII collagen was not found in aorta, kidney, lung, liver, skin, and ligament. We conclude that this unusual collagen is a component of certain specialized extracellular matrices, several of which are derived from the neural crest.

Animals↗
PubMed1988

Interactions between the promoter and first intron are involved in transcriptional control of alpha 1(I) collagen gene expression.

The first intron of the human collagen alpha 1(I) gene contains several positively and negatively acting elements. We have studied the transcription of collagen-human growth hormone fusion genes, containing deletions and rearrangements of collagen intronic sequences, by transient transfection of chick tendon fibroblasts and NIH 3T3 cells. In chick tendon fibroblasts, but not in 3T3 cells, inversion of intronic sequences containing a previously studied 274-base-pair segment, A274, resulted in markedly reduced human growth hormone mRNA levels as determined by an RNase protection assay. This inhibitory effect was largely alleviated when deletions were introduced in the collagen promoter of plasmids containing negatively oriented intronic sequences. Evidence for interaction of the promoter with the intronic segment, A274, was obtained by gel mobility shift assays. We suggest that promoter-intron interactions, mediated by DNA-binding proteins, regulate collagen gene transcription. Inversion of intronic segments containing critical interactive elements might then lead to an altered geometry and reduced activity of a transcriptional complex in those cells with sufficiently high levels of appropriate transcription factors. We further suggest that the deleted promoter segment plays a key role in directing DNA interactions involved in transcriptional control.

Chromosome Deletion↗
PubMed1987

Regulatory elements in the first intron contribute to transcriptional control of the human alpha 1(I) collagen gene.

Several lines of evidence have suggested that the regulation of type I collagen gene transcription is complex and that important regulatory elements reside 5' to, and within, the first intron of the alpha 1(I) gene. We therefore sequenced a 2.3-kilobase HindIII fragment that encompasses 804 base pairs of 5' flanking sequence, the first exon, and most of the first intron of the alpha 1(I) human collagen gene. A 274-base-pair intronic sequence, flanked by Ava I sites (A274), contained a sequence identical to a high-affinity decanucleotide binding site for transcription factor Sp1 and a viral core enhancer sequence. DNase I protection experiments indicated zones of protection that corresponded to these motifs. When A274 was cloned 5' to the chloramphenicol acetyltransferase (CAT) gene, driven by an alpha 1(I) collagen promoter sequence, and expression was assessed by transfection, significant orientation-specific inhibition of CAT activity was observed. This effect was most apparent in chicken tendon fibroblasts, which modulate their level of collagen synthesis in culture. We propose that normal regulation of alpha 1(I) collagen gene transcription results from an interplay of positive and negative elements present in the promoter region and within the first intron.

Amino Acid Sequence↗
PubMed1986

Partial amino acid sequence of human thrombospondin as determined by analysis of cDNA clones: homology to malarial circumsporozoite proteins.

A lambda gt 11 library prepared from human umbilical vein endothelial cell RNA was screened for cDNAs encoding thrombospondin. Reagents included a monospecific antibody to human thrombospondin and a mixture of four synthetic oligodeoxyribonucleotides derived from an amino acid sequence near the NH2 terminus of mature human thrombospondin. Two series of cDNA clones coding for sequences at the 5' and 3' ends of thrombospondin mRNA, respectively, were isolated. The nucleotide sequence of a 1.3-kilobase (kb) 5' clone (lambda TS-33) coded for 99 bases of 5' untranslated RNA, a signal peptide of 18 amino acids, and the first 379 amino acids of thrombospondin. Northern blot analysis with lambda TS-33 detected a single mRNA species of approximately 6.0 kb in rat aortic smooth muscle cell RNA. Thrombospondin mRNA levels increased rapidly, but transiently, in quiescent smooth muscle cells treated with platelet-derived growth factor. The kinetics of this response were very similar to those of the thrombospondin protein to this growth factor. There was significant homology in amino acid sequence between thrombospondin and a conserved region in the circumsporozoite protein of two malarial sporozoites. This region of thrombospondin may therefore represent a potential recognition site for a cell surface thrombospondin receptor.

Amino Acid Sequence↗
PubMed1986

Type VIII collagen from bovine Descemet's membrane: structural characterization of a triple-helical domain.

Bovine corneal Descemet's membrane (DM) was subjected to limited pepsin digestion. Soluble native collagens were fractionated by differential salt precipitation, and a mixture of type V collagen and collagenous fragments with a chain Mr of 50,000 (50K) was obtained at a concentration of 1.5 M NaCl. Further purification of the 50K collagen by molecular sieve and high-performance liquid chromatography resulted in the isolation of two-non-disulfide-bonded polypeptides, 50K-A and 50K-B, which were susceptible to several neutral proteases, including bacterial collagenase. By the criteria of peptide mapping, amino acid composition, and N-terminal sequence analysis, 50K-A and 50K-B were structurally dissimilar, although both chains contained Gly-X-Y repeats. 50K-A and 50K-B were immunologically and structurally distinct from collagen type I, III, IV, V, and VI. Immunohistochemical studies of bovine ocular tissue showed preferential distribution of the collagen containing the 50K fragment in the DM, with a more disperse arrangement of apparently interconnecting fibrils in the corneal stroma. Type VIII collagen isolated from the culture medium of metabolically radiolabeled bovine corneal endothelial (BCE) cells and its pepsin-resistant Mr 50 000 domain(s) both cross-reacted with antisera to 50K polypeptides from the corneal DM. Additionally, the CNBr peptide maps of pepsin-resistant Mr 50 000 polypeptides of type VIII collagen isolated from BCE cells and bovine corneal DM were highly similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Regulation of type I collagen mRNA levels in fibroblasts.

Type I procollagen mRNA levels, as well as total RNA and poly(A)-rich mRNA, remain constant when rapidly growing human fetal lung fibroblasts (HFL-1 cells) are compared with quiescent cells. Polysome profiles of cells in both growth states revealed that the distribution of type I collagen mRNA in the mRNP fraction and in polysomes also remained constant even though total RNA and poly(A)-rich mRNA were shifted from polysomes to the mRNP pool in resting cells. Similar results were obtained when RNA fractions in polysomes associated with the cytoskeletal framework were examined. It is known that procollagen production is unaffected by the growth state of cells [Breul, S. D., Bradley, K. H., Hance, A. J., Schafer, M. P., Berg, R. A. and Crystal, R. G. (1980) J. Biol. Chem. 255, 5250-5260] although total protein synthesis is markedly decreased in resting cells. It would therefore appear that the translational control responsible for reduced synthesis of non-collagenous proteins in resting cells does not extend to procollagen and that transcriptional control can account for levels of type I procollagen produced by cultured human fibroblasts.

Collagen↗
PubMed1986

Control of smooth muscle cell growth by components of the extracellular matrix: autocrine role for thrombospondin.

Addition of platelet-derived growth factor (PDGF) to growth-arrested cultured smooth muscle cells (SMC) induces the synthesis and secretion of thrombospondin (TS), a glycoprotein component of the SMC extracellular matrix in vitro. This induction occurs at PDGF concentrations that are suboptimal for a mitogenic response. In this study we examined the effect of TS on the proliferation of SMC, using a serum-free mitogenesis assay. Addition of either epidermal growth factor (EGF) or purified human platelet TS to quiescent rat vascular SMC did not substantially stimulate mitogenesis; the 30-hr nuclear labeling index increased from a mean of 7% in control cells to 20% for EGF-treated SMC and 17% for cells exposed to TS alone. However, TS and EGF acted synergistically to stimulate DNA synthesis by SMC, increasing the labeling index to 47%. The facilitative effect of TS on EGF-mediated mitogenesis was inhibited by heparin, a known inhibitor of SMC growth and migration that also blocks incorporation of TS into the SMC extracellular matrix. The effect was specific for EGF; TS did not augment the response of cells to insulin or insulin-like growth factor 1. These data establish a functional role for cell-derived TS and provide evidence for the presence of an autocrine, growth-supportive mechanism involving the extracellular matrix. In addition, our experiments support the existence of a novel, heparin-sensitive SMC mitogenic pathway and suggest a mechanism whereby heparin-like molecules may inhibit SMC proliferation.

Animals↗
PubMed1985

Characterization of a type VI collagen-related Mr-140 000 protein from cutis-laxa fibroblasts in culture.

The precise biochemical defects in connective-tissue metabolism that are responsible for the laxity of skin seen in the syndrome of cutis laxa are largely unknown. We have studied fibroblasts cultured from skin explants of a 2-year-old male with the syndrome. Electron-microscopic examination of this skin revealed decreased amounts of amorphous elastin and an increase in elastin-associated microfibrils. Although the cultured fibroblasts were similar to control skin fibroblasts in morphology, growth rate and total protein synthesis, there was a 4-6-fold increase in accumulation of a collagenous protein of Mr 140 000 in both the culture medium and in the cell layer. This protein was structurally distinct from collagen types I, III, IV, V and VIII. It was found to be related to a cell-surface-associated glycoprotein, GP140, by both antigenic cross-reactivity and peptide mapping. Our data support observations that GP140 is a precursor of at least one form of pepsin-extracted type VI collagen.

Cells, Cultured↗
PubMed1985

Heparin regulates the collagen phenotype of vascular smooth muscle cells: induced synthesis of an Mr 60,000 collagen.

The effect of heparin on the biosynthetic phenotype of rat vascular smooth muscle cells (SMC) was investigated in vitro. Addition of heparin to the culture medium of early passage rat SMC resulted in a marked (3-15-fold) increase of a cell layer-associated Mr 60,000 protein that was sensitive to digestion by purified bacterial collagenase and contained significant amounts of hydroxyproline. Pulse-chase analysis of heparin-treated SMC revealed that the Mr 60,000 collagen was a primary and abundant product of these cells and was not processed extracellularly to a smaller form. The inductive effect of heparin could be mimicked by iota carrageenan or dextran sulfates but not by hyaluronic acid, dermatan sulfate, or chondroitin sulfates. The induction was concentration dependent with a maximal effect observed at a heparin concentration of 10 micrograms/ml. Synthesis of the Mr 60,000 collagen increased 18-24 h after addition of heparin to the cultures. Following induction and subsequent removal of heparin, synthesis of the protein remained maximal for at least 12 h and required 72 h to return to a basal level. These data demonstrate that the biosynthetic phenotype of vascular SMC in vitro can be controlled, at least in part, by heparin and related polyanions and suggest a role for similar molecules endogenous to the vessel wall in the regulation of SMC function.

Animals↗
PubMed1985

Platelet-derived growth factor and heparin-like glycosaminoglycans regulate thrombospondin synthesis and deposition in the matrix by smooth muscle cells.

Platelet-derived growth factor (PDGF), a smooth muscle cell (SMC) mitogen, and heparin-like glycosaminoglycans, known inhibitors of SMC growth and migration, were found to regulate thrombospondin synthesis and matrix deposition by cultured rat aortic SMC. The synthesis and distribution of thrombospondin was examined in growth-arrested SMCs, in PDGF-stimulated SMCs, and in heparin-treated SMCs using metabolic labeling and immunofluorescence techniques. Thrombospondin synthesis in response to purified PDGF occurred within 1 h after addition of growth factor to growth-arrested SMCs, peaked at 2 h, and returned to baseline levels by 5 h. The induction of synthesis of thrombospondin by PDGF was dose dependent, with a maximal effect observed at 2.5 ng/ml. Actinomycin D (2 micrograms/ml) inhibited thrombospondin induction by PDGF, suggesting a requirement for new RNA synthesis. In the presence of heparin and related polyanions, the incorporation of thrombospondin into the SMC extracellular matrix was markedly reduced. This effect was dose dependent with a maximal effect observed at a heparin concentration of 1 microgram/ml. Heparin did not affect the ability of SMCs to synthesize thrombospondin in response to PDGF. We interpret these data to suggest a role for thrombospondin in the SMC proliferative response to PDGF and in the regulation of SMC growth and migration by glycosaminoglycans.

Animals↗
PubMed1985

Light microscopic immunolocation of thrombospondin in human tissues.

Affinity-purified antisera against thrombospondin were used to locate the presence of this glycoprotein in frozen sections of several human tissues by immunofluorescence techniques. Immunostaining was observed in the peritubular connective tissue and in basement membrane regions beneath glandular epithelium in skin and lung. Intense immunostaining was observed at the dermal-epidermal junction in skin and in small blood vessels throughout this tissue. Skeletal muscle exhibited positive staining with anti-thrombospondin antisera within interstitial areas. Immunostaining was confined to the luminal portions of large blood vessels such as aorta. In large blood vessels that contained lesions of atherosclerosis, immunostaining was observed throughout the lesion area and was especially prominent surrounding some of the lesion cells. These results indicate that thrombospondin is located within the matrix of a variety of human tissues and supports the suggestion that this glycoprotein is an endogenous component of some extracellular matrices.

Aorta↗