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

ABSTRACT: 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 three-dimensional 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 steady-state 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.

SUBMITTER: Canfield AE

PROVIDER: S-EPMC1131416 | biostudies-other | 1990 May

REPOSITORIES: biostudies-other

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Project description:ZBTB46 is a transcription factor identified in classical dendritic cells and keeps dendritic cells in a quiescent state. Chromatin immunoprecipitation sequencing in dendritic cells has identified over 1300 potential gene targets of ZBTB46, affecting many processes including cell cycle. Endothelial cells (ECs) also express ZBTB46 and are mostly in a quiescent non-proliferative state. While EC proliferation is a critical process in development, dysregulation of EC proliferation as seen in areas of disturbed flow play an important role in many disease processes such as atherosclerosis, pulmonary hypertension, transplant vasculopathy, neointimal hyperplasia, and in-stent restenosis. We studied the role of ZBTB46 in ECs, hypothesizing that it inhibits EC proliferation. Using a model of disturbed flow in mice, we found that ZBTB46 is expressed in murine arterial ECs in vivo, and is downregulated by disturbed flow. In vitro results using HAECs showed that cell confluence and laminar shear stress, both known physiological conditions promoting EC quiescence, led to upregulation of ZBTB46 expression. Adenoviral-mediated overexpression of ZBTB46 in vitro caused reduced EC proliferation, and increased number of cells in the G0/G1 phase of cell cycle, without affecting apoptosis or senescence, while siRNA knockdown of ZBTB46 negated the known inhibitory role of unidirectional laminar shear stress on EC proliferation. ZBTB46 overexpression also led to a broad suppression of genes involved in cell cycle progression including multiple cyclins and cyclin-dependent kinases, but an increase in the CDK inhibitor CDKN1A. Phosphorylation of the retinoblastoma protein was also decreased as assessed by Western blot. Tube formation on Matrigel was reduced, suggesting an inhibitory role for ZBTB46 in angiogenesis. Further research is required to investigate the potential role of ZBTB46 in specific pathologic conditions and whether it can be targeted in a therapeutic manner.

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

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