Expression of thrombospondin-1 in human hepatocarcinoma cell lines and its regulation by transcription factor Jun/AP-1.
Thrombospondin-1 (TSP-1) is a homotrimeric glycoprotein synthesized in a variety of normal and transformed cells, and secreted into the extracellular matrix. Based on its known effects on the tumor and endothelial cells, TSP-1 was implicated in the tumor growth and metastasis. In the present study, we have demonstrated the expression of TSP-1 in the human hepatocarcinoma cell lines. TSP-1 was detected in human hepatocarcinoma SK-HEP-1, Hep 3B and immortalized human liver Chang cells. Using two different cell lines, SK-HEP-1 and Hep 3B cells, we have studied effects of phorbol 12-myristate 13-acetate (PMA) on TSP-1 expression. TSP-1 synthesis was stimulated by PMA in both cell lines. When the cells were treated with PMA, the TSP-1 mRNA started to increase at 30 min and reached the maximal level at 6 h. TSP-1 induction by PMA was completely inhibited by the pre-treatment of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a potent protein kinase C inhibitor. A TSP-1 promoter-luciferase reporter gene was transcriptionally activated by PMA, as well as by the expression of c-Jun. Among three putative AP-1 recognition sites on the TSP-1 promoter, a deletion of the 1st and 2nd sites caused loss of PMA-induced upregulation, while the 3rd site deletion showed no effect. In subsequent experiments, both the recombinant c-Jun and nuclear proteins induced by PMA have a stronger binding affinity for the 2nd AP-1 recognition site than the 1st and 3rd ones. Our study demonstrated that TSP-1 could be expressed and secreted by human hepatoma cell lines and its expression could be effectively regulated by PMA. We also suggest that AP-1 binding activity through the protein kinase C activation is a critical event for the TSP-1 gene expression and consequently affects production and processing of the protein.
Kim
S A
SA
Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul.
Um
S J
SJ
Kang
J H
JH
Hong
K J
KJ
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D002273:Carcinogens; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017353:Gene Deletion; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000494:pharmacology; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate; D019700:Thrombospondin 1 / Q000096:biosynthesis* / Q000235:genetics*; D013997:Time Factors; D018808:Transcription Factor AP-1 / Q000096:biosynthesis*; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D002273:Carcinogens; D004791:Enzyme Inhibitors; D016755:Proto-Oncogene Proteins c-jun; D019700:Thrombospondin 1; D018808:Transcription Factor AP-1; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D008156:Luciferases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate