Secretion of endothelin-1 in human endothelial cell line but not in B cell line by transfection of preproendothelin-1 cDNA.
Stable transformants with preproendothelin-1 (preproET-1) cDNA were established for the study of the regulation of endothelin-1 (ET-1) biosynthesis in human cells. ET-1, a potent vasoconstrictor peptide, is produced by endothelial cells and is secreted into the blood at a low level. Human preproET-1 cDNA was introduced into two immortal human cell lines, t-HUE2, an endothelial cell line, and Raji, a B cell line, with Ecogpt selection. Several stable transformants of t-HUE2 expressed extraordinarily high levels of preproET-1 specific mRNA and secreted ET-1 into serum-free culture medium, while the transformants of Raji cells expressed high levels of ET-1 mRNA, but secreted a negligible amount of ET-1. Immunocytochemical studies of intracellular ET-1 content revealed that there were some defects in the translation or processing of preproET-1 in the B cell line transformants. In addition, the ratio of ET-1 to ET-1 precursor (big ET-1) was much higher in the t-HUE2 transformants than in normal endothelial cells, suggesting that t-HUE2 transformants (for example t-HUE2-1) possess high levels of endothelin converting enzyme (ECE). The establishment of stable transformants producing high levels of ET-1 in serum-free medium will be useful for the study of cell-type-specific translation and processing to mature ET-1, and of the regulatory factors of ECE.
Kondo
M
M
Cell Science and Technology Division, Agency of Industrial Science and Technology, Tsukuba Science City, Ibaraki, Japan.
Ishida
N
N
Kobayashi
M
M
Mitsui
Y
Y
D001402:B-Lymphocytes / Q000378:metabolism*; D015139:Blotting, Southern; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D019332:Endothelin-1; D016232:Endothelins / Q000235:genetics* / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D011863:Radioimmunoassay; D014162:Transfection / Q000235:genetics
D019332:Endothelin-1; D016232:Endothelins; D011498:Protein Precursors