Abstract
Thrombospondin-1 (TSP-1), a multifunctional extracellular matrix protein, inhibits neovascularization and is implicated in the regression of tumor growth and metastasis. We found that the synthesis of TSP-1 in porcine aortic endothelial (PAE) cells was decreased in a dose-dependent manner by phorbol 12-myristate 13-acetate (PMA) treatment in porcine aortic endothelial (PAE) cells. In this study, a responsive site on the TSP-1 promotor affected by PMA treatment in PAE was characterized. The level of TSP-1 mRNA was also decreased by PMA after 1 h and persisted that way for at least 24 h. PMA treatment and c-Jun overexpression suppressed the transcription of TSP-1 promotor-luciferase reporter gene. A deletion between -767 and -657 on the TSP-1 promotor neutralized the PMA-induced down-regulation. In addition, oligo a (-767 approximately -723) was responsive to PMA-induced repression, while oligo b (-734 approximately -689) and c (-700 approximately -656) was not. Electrophoretic mobility shift assays showed that this PMA responsive element specifically bound a nuclear protein and that the binding activity was diminished by PMA treatment in PAE cells but not in Hep 3B cells. In supershift assay, potential regulatory elements in this region, SP1 and GATA-1, were not responsive to the inhibition of TSP-1 expression by PMA. Our results suggest that the repression of TSP-1 synthesis by PMA is mediated by blocking a particular unknown nuclear protein binding to the responsive site (-767 approximately -735), which is regulated by c-Jun.
Highlights
Thrombospondin-1 (TSP-1) is a homotrimeric extracellular matrix glycoprotein of 450 kD (Lawler, 1986; Bornstein, 1992)
In order to confirm the phorbol 12myristate 13-acetate (PMA) effect on endothelial cells, the level of TSP-1 synthesis in porcine aortic endothelial (PAE) cells was investigated and found that TSP-1 synthesis was inhibited in a dose-dependent manner upon treatment with PMA (Figure 1A)
When the PAE cells were treated with 100 nM PMA, the level of TSP-1 mRNA started to decrease after 30 min and steadily decreased after 6 h
Summary
Thrombospondin-1 (TSP-1) is a homotrimeric extracellular matrix glycoprotein of 450 kD (Lawler, 1986; Bornstein, 1992). Each monomer (180 kD) has functional domains capable of binding to many extracellular matrices and a variety of other proteins including several receptors, enzymes, and cytokines (Bornstein, 1995). Because of these multiple interactions, TSP-1 has been implicated in a number of biological processes such as development, inflammation, wound healing, tumor growth and metastasis (Mosher, 1990; Bornstein, 1992; Lahav, 1993). AP-1 is activated following stimulation by protein kinase C (PKC), and it regulates the expression of AP-1 responsive target genes
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