Up-regulation of the enzymes involved in prostacyclin synthesis via Ras induces vascular endothelial growth factor.
OBJECTIVE
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 A(2) [cPLA(2)]).


RESULTS
The inducible expression of Ha-Ras(V12) increased the production of prostaglandin (PG)F(2alpha) and prostacyclin by 2- and 13-fold, respectively. The induction of Ha-Ras(V12) also up-regulated the mRNA and protein levels of cPLA(2), COX-2, and prostacyclin synthase, as well as the promoter and enzyme activity of cPLA(2). 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 PGI 2 alone also induced the expression of VEGF.


CONCLUSIONS
Inducible Ha-Ras(V12) increases the production of PGI(2) through the coordinate up-regulation of cPLA(2), COX-2, and prostacyclin synthase (PGIS). The production of PGI(2) 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.
Buchanan
F Gregory
FG
Department of Medicine, Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
Chang
Woogki
W
Sheng
Hongmiao
H
Shao
Jinyi
J
Morrow
Jason D
JD
DuBois
Raymond N
RN
D000818:Animals; D002460:Cell Line; D051546:Cyclooxygenase 2; D011464:Epoprostenol / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D007413:Intestinal Mucosa / Q000166:cytology / Q000502:physiology*; D007527:Isoenzymes / Q000235:genetics*; D010741:Phospholipases A / Q000235:genetics; D011401:Promoter Regions, Genetic; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D051381:Rats; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis*; D018631:ras Proteins / Q000502:physiology*
D007527:Isoenzymes; D042461:Vascular Endothelial Growth Factor A; D011464:Epoprostenol; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D010741:Phospholipases A; D018631:ras Proteins