Canfield A E, Boot-Handford R P, Schor A M
Department of Medical Oncology, Christie Hospital, Manchester, U.K.
Biochem J. 1990 May 15;268(1):225-30. doi: 10.1042/bj2680225.
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.
接种在二维基质表面（明胶包被的培养皿、天然I型胶原包被的培养皿或胶原凝胶）的内皮细胞在汇合时形成鹅卵石样单层，而接种在三维凝胶基质中的细胞则伸长成芽状形态并自组装成管状结构。在本研究中，我们比较了静止内皮细胞在（a）不同基质（明胶和胶原）上呈现相同形态表型（鹅卵石样）以及（b）在相同基质（胶原）上呈现不同形态表型（鹅卵石样和芽状）时血小板反应蛋白的合成情况。我们证明，血小板反应蛋白是接种在明胶或胶原包被培养皿上的汇合静止细胞的主要生物合成产物，当细胞接种在三维胶原凝胶上或凝胶内时，该蛋白的合成显著减少。此外，我们证明接种在凝胶内（芽状）的细胞比接种在凝胶表面（鹅卵石样）的细胞分泌的血小板反应蛋白更少。血小板反应蛋白合成的调节是可逆的，且发生在转录水平，因为血小板反应蛋白的稳态mRNA水平以与这些细胞分泌的蛋白水平相当的方式降低。我们还表明，与接种在明胶包被培养皿上相比，当细胞在胶原凝胶上和凝胶内培养时，层粘连蛋白B2链的mRNA水平升高，这表明血小板反应蛋白和层粘连蛋白的合成受不同机制调节。当细胞在明胶或胶原包被的培养皿上培养时，血小板反应蛋白基因表达与培养物的增殖状态直接相关。相比之下，无论接种在胶原凝胶上的细胞在稀疏且快速增殖时还是在汇合且静止时进行标记，其血小板反应蛋白的合成均维持在同样低的水平。发现纤连蛋白的合成随着接种在所有三种基质上的细胞汇合度增加而增加。这些结果表明，血小板反应蛋白基因表达受细胞形状、细胞增殖及用于细胞培养的基质性质的调节。
