Thrombospondin gene expression by endothelial cells in culture is modulated by cell proliferation, cell shape and the substratum.
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.
Canfield
A E
AE
Department of Medical Oncology, Christie Hospital, Manchester, U.K.
Boot-Handford
R P
RP
Schor
A M
AM
D000818:Animals; D001011:Aorta; D002417:Cattle; D002455:Cell Division; D002478:Cells, Cultured; D003094:Collagen; D003470:Culture Media; D015342:DNA Probes; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D005780:Gelatin; D005782:Gels; D005786:Gene Expression Regulation; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000378:metabolism; D019699:Thrombospondins
D003470:Culture Media; D015342:DNA Probes; D005782:Gels; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D019699:Thrombospondins; D005780:Gelatin; D003094:Collagen