Abstract
Activation of Ras signaling by growth factors has been associated with gene regulation and cell proliferation. Here we characterize the contributory role of cytosolic phospholipase A(2) in the oncogenic Ha-Ras(V12) signaling pathway leading to activation of c-fos serum response element (SRE) and transformation in Rat-2 fibroblasts. Using a c-fos SRE-luciferase reporter gene, we showed that the transactivation of SRE by Ha-Ras(V12) is mainly via a Rac-linked cascade, although the Raf-mitogen-activated protein kinase cascade is required for full activation. In addition, Ha-Ras(V12)-induced DNA synthesis was significantly attenuated by microinjection of recombinant Rac(N17), a dominant negative mutant of Rac1. To identify the mediators downstream of Rac in the Ha-Ras(V12) signaling, we investigated the involvement of cytosolic phospholipase A(2). Oncogenic Ha-Ras(V12)-induced SRE activation was significantly inhibited by either pretreatment with mepacrine, a phospholipase A(2) inhibitor, or cotransfection with the antisense oligonucleotide of cytosolic phospholipase A(2). We also found cytosolic phospholipase A(2) to be situated downstream of Ha-Ras(V12) in a signal pathway leading to transformation. Together, these results are indicative of mediatory roles of Rac and cytosolic phospholipase A(2) in the signaling pathway by which Ha-Ras(V12) transactivates c-fos SRE and transformation. Our findings point to cytosolic phospholipase A(2) as a novel potential target for suppressing oncogenic Ha-Ras(V12) signaling in the cell.
Highlights
Ras is a 21-kDa guanine nucleotide-binding protein that functions as a molecular switch linking upstream activators, such as growth factor receptors and nonreceptor tyrosine kinases, to several downstream effectors [1, 2]
The plates were incubated at 37 °C for 10 days, and the colonies were counted by staining them with p-iodonitro tetrazolium violet dye as described previously [30]
We showed that the Rac-linked cascade apparently plays a crucial role in Ha-RasV12 signaling leading to transactivation of c-fos serum response element (SRE) and transformation
Summary
Chemicals and Reagents—Antisense cPLA2 oligonucleotide (GsTsGCTGGTAA GGATCTsAsT) is directed against codons 4 –9 of the human cytosolic, Ca2ϩ-dependent PLA2 gene; two linkages are phosphothiolated at both the 5Ј and 3Ј ends. Transient transfection was carried out by plating ϳ5 ϫ 105 cells in 100-mm dishes for 24 h, after which calcium phosphate:DNA precipitates prepared with a total of 20 g of DNA, including 3 g of pSRE-Luc and 5 g of a GTPase expression vector (e.g. pEXV-RacN17), were added to each dish [29]. After incubating 6 h with the calcium phosphate:DNA precipitates, the cells were rinsed twice with phosphate-buffered saline before incubating in fresh DMEM supplemented with 0.5% FBS for an additional 36 h. After 4 h, 2 Ci/ml [3H]AA (Amersham Pharmacia Biotech) was added to each well and incubated for an additional 36 h, after which the cells were washed at least three times with medium. Leukotriene LTC4/D4/E4 Assays—Rat-2 and Rat2-HO6 cells (3 ϫ 105) were plated on 60-mm dishes and incubated in DMEM supplemented with 10% FBS for 24 h. The statistical significance of LTC4/D4/E4 assays was assessed with analysis of variance (ANOVA) (p Ͻ 0.01)
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