Identification and partial characterization of two major proteins of <i>M</i>r 47,000 synthesized by bovine retinal endothelial cells in culture

Canfield, Ann E.; Schor, Ana M.; West, David C.; Schor, Seth L.; Grant, Michael E.

doi:10.1042/bj2460121

Cited by 8 publications

(13 citation statements)

References 41 publications

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“…We have examined the proteins secreted by cultured bovine retinal capillary endothelial cells. A major component secreted into the medium was a non-collageneous glycoprotein of M, 47000, which was composed of nine related polypeptides of PI 4.65.5 [12], a pattern similar to that of Lincoln et al and also to that of an endothelial cell inhibitor of plasminogen activators [44]. From these results, it appears that the presence of such a charge train is a useful indication that cultured cells are endothelial, at least for those of bovine origin.…”

Section: Purified Tumoursupporting

confidence: 62%

Heterogeneity in endothelial cells from large vessels and microvessels

1987

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Section: Purified Tumoursupporting

confidence: 62%

Heterogeneity in endothelial cells from large vessels and microvessels

1987

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“…NRK fibroblast p52 is similar in overall abundance (at least in the medium fraction where it composes approximately 20% of the total 35S-methionine-labeled protein species), PI microheterogeneity, size of tunicamycin-derived "core" peptide, and ammonium sulfate solubility (data not shown) to medium Gp47 of bovine retinal endothelial cells (Canfield et al, 1987). In retinal cells, however, the complementing M, 47,000 protein found in the cell layer/matrix (p47) was clearly distinct from Gp47 both in tunicamycin sensitivity and peptide-mapping profile (Canfield et al, 1987). NRK medium p52, in contrast, is equivalent to its matrix counterpart from which source it is likely derived.…”

Section: Discussionmentioning

confidence: 88%

“…In retinal cells, however, the complementing M, 47,000 protein found in the cell layer/matrix (p47) was clearly distinct from Gp47 both in tunicamycin sensitivity and peptide-mapping profile (Canfield et al, 1987). NRK medium p52, in contrast, is equivalent to its matrix counterpart from which source it is likely derived.…”

Section: Discussionmentioning

confidence: 94%

Cytochalasin D‐mediated hyperinduction of the substrate‐associated 52‐kilodalton protein p52 in rat kidney fibroblasts

Higgins

Ryan

Chaudhari

1989

Journal Cellular Physiology

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Regulation of certain differentiated and housekeeping functions in cultured mammalian cells is significantly influenced by cell shape. The shape-modulating agent cytochalasin D (CD) was used, therefore, to elucidate potential cytoarchitectural influences affecting synthesis of a major 52 kDa secreted/substrate-associated protein (p52) of normal rat kidney (NRK) fibroblasts. Biosynthetic labeling experiments indicated that treatment of NRK cells with CD increased, by 10-18-fold, the medium content of an Mr 52,000 protein. Two-dimensional gel electrophoresis and peptide fragment mapping confirmed that the 52 kDa protein produced in abundance as a consequence of CD treatment was identical to p52 constitutively expressed by NRK cells. A lower mw protein (p50; Mr 50,000) was also resolved which, based on pl microheterogeneity, protease fragmentation profile, and sensitivity to tunicamycin, could be identified as a less-glycosylated form of p52. p50 and p52 were both detected in the matrix and medium compartments of NRK and NRK/CD cells. The matrix p52 content of CD-induced and uninduced cells, however, was significantly greater (by 200-500-fold) than the corresponding medium levels. This differential compartmentalization, the time course of p52 accumulation in the matrix of NRK/CD cells compared to its appearance in the medium, and the kinetics of p52 pulse-chase from the matrix collectively indicated that the matrix is the initial site of p52 deposition. Low levels of CD (1 microM) produced extensive disruptions of cellular microfilaments but did not result in an overall cell shape change nor a hyperinduction of p52. Morphologic rounding (seen in 10-100 microM CD) coincided with augmented p52 production. Transition from a flat to a round phenotype in NRK cells, or at least the generation of sufficient microfilament fragmentation to compromise cell-substrate adhesivity, appears to be an essential aspect of CD-mediated p52 hyperinduction.

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“…Newly synthesized thrombospondin and fibronectin in the culture medium were identified by (a) immunoprecipitation with specific antibodies and analysis by SDS/PAGE and (b) affinity to heparin-Sepharose (thrombospondin and fibronectin) and gelatin-Sepharose (fibronectin) as previously described (Canfield et al, 1986(Canfield et al, , 1987. The levels of mRNA for thrombospondin and laminin were determined by Northern blot hybridization as described below.…”

Section: Introductionmentioning

confidence: 99%

Thrombospondin gene expression by endothelial cells in culture is modulated by cell proliferation, cell shape and the substratum

Canfield

Boot-Handford

Schor

1990

Biochemical Journal

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Endothelial cells plated on the surface of a two-dimensional substratum (gelatin-coated dishes, dishes coated with native type I collagen or collagen gels) form a cobblestone monolayer at confluence, whereas cells plated within a threedimensional gel matrix elongate into a sprouting morphology and self-associate into tube-like structures. In this study, we have compared the synthesis of thrombospondin by quiescent endothelial cells displaying (a) the same morphological phenotype (cobblestone) on different substrata (gelatin and collagen) and (b) different morphological phenotypes (cobblestone and sprouting) on the same substratum (collagen). We demonstrate that thrombospondin is a major biosynthetic product of confluent, quiescent cells cultured on dishes coated with either gelatin or collagen, and that the synthesis of this protein is markedly decreased when cells are plated on or in three-dimensional collagen gels. Moreover, we demonstrate that cells plated in gel (sprouting) secrete less thrombospondin than do cells plated on the gel surface (cobblestone). The regulation of thrombospondin synthesis is reversible and occurs at the level of transcription, as steadystate mRNA levels for thrombospondin decrease in a manner comparable with the levels of protein secreted by these cells. We also show that mRNA levels for laminin B2 chains are increased when cells are cultured on and in collagen gels compared with on gelatin-coated dishes, suggesting that the syntheses of thrombospondin and laminin are regulated by different mechanisms. When cells are cultured on gelatin-or collagen-coated dishes, thrombospondin gene expression is directly proportional to the proliferative state of the cultures. By contrast, the synthesis of thrombospondin by cells cultured on collagen gels remains at equally low levels whether they are labelled when they are sparse and rapidly proliferating or when they are confluent and quiescent. Fibronectin synthesis was found to increase with increasing confluency of the cells plated on all three substrata. These results demonstrate that thrombospondin gene expression is modulated by cell shape, cell proliferation and the nature of the substratum used for cell culture.

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Identification and partial characterization of two major proteins of Mr 47,000 synthesized by bovine retinal endothelial cells in culture

Cited by 8 publications

References 41 publications

Heterogeneity in endothelial cells from large vessels and microvessels

Heterogeneity in endothelial cells from large vessels and microvessels

Cytochalasin D‐mediated hyperinduction of the substrate‐associated 52‐kilodalton protein p52 in rat kidney fibroblasts

Thrombospondin gene expression by endothelial cells in culture is modulated by cell proliferation, cell shape and the substratum

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