Up-regulation of the enzymes involved in prostacyclin synthesis via Ras induces vascular endothelial growth factor

Background & Aims: The constitutive activation of Ras is an important step in the development and progression of several different cancers and is known to increase the level of cyclooxygenase 2 (COX-2). Prostaglandins are the downstream bioactive lipid mediators produced by the COX-2 enzyme. We sought to determine the role of Ras-induced up-regulation of the enzymes involved in prostacyclin biosynthesis in nontransformed rat intestinal epithelial cells (IECs). Methods: Messenger RNA (mRNA) and protein expression were analyzed by Northern and Western analysis, respectively, to determine the level of enzymes induced by Ras. In vitro assays were used to determine the production of vascular endothelial growth factor (VEGF) and prostaglandins as well as the promoter and enzymatic activation of the rate-limiting enzyme in prostaglandin production (phospholipase A₂ [cPLA₂]). Results: The inducible expression of Ha-Ras^V12 increased the production of prostaglandin (PG)F_2α and prostacyclin by 2- and 13-fold, respectively. The induction of Ha-Ras^V12 also up-regulated the mRNA and protein levels of cPLA₂, COX-2, and prostacyclin synthase, as well as the promoter and enzyme activity of cPLA₂. Furthermore, oncogenic Ras increased the production of the pro-angiogenic factor VEGF. The increase of VEGF was abolished after treatment with celecoxib, a selective COX-2 inhibitor. The addition of PGI₂ alone also induced the expression of VEGF. Conclusions: Inducible Ha-Ras^V12 increases the production of PGI₂ through the coordinate up-regulation of cPLA₂, COX-2, and prostacyclin synthase (PGIS). The production of PGI₂ leads to an increase in the level of the pro-angiogenic factor VEGF, which is known to play a crucial role in the regulation of tumor-associated angiogenesis.