Expression of thrombospondin-1 in human hepatocarcinoma cell lines and itsregulation by transcription factor Jun/AP-1

Thrombospondin-1 (TSP-1) is a homotrimeric glycoprotein synthesized in a va riety 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 c ell 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-H EP-1 and Hep 3B cells, we have studied effects of phorbol 12-myristate 13-a cetate (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 start ed to increase at 30 min and reached the maximal level at 6 h. TSP-1 induct ion by PMA was completely inhibited by the pre-treatment of 1-(5-isoquinoli nylsulfonyl)-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 re cognition sites on the TSP-1 promoter, a deletion of the 1st and 2nd sites caused loss of PMA-induced up-regulation, while the 3rd site deletion showe d no effect. In subsequent experiments, both the recombinant c-Jun and nucl ear 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 eve nt for the TSP-1 gene expression and consequently affects production and pr ocessing of the protein.