Induction of cPLA(2) in lung epithelial cells and non-small cell lung cancer is mediated by Sp1 and c-jun

Activating mutations in ras genes are frequently associated with non-small cell lung cancer cells (NSCLC) and contribute to transformed growth in thes e cells. Expression of oncogenic forms of Ras in these cells is associated with increased expression and activity of cytosolic phospholipase A(2) (cPL A(2)) and cyclooxygenase-2 (COX-2), leading to constitutively elevated leve ls of prostaglandin production. Expression of oncogenic Ras is sufficient t o induce these enzymes in normal lung epithelial cells. We have previously reported that the JNK and ERK pathways are necessary for induction of cPLA( 2) and have defined a minimal region of the cPLA(2) promoter from -58 to -1 2 that is required for Ha-Ras-mediated induction. To further characterize t he cis-regulatory elements within this region involved in this response, si te-directed mutagenesis was used to make mutations at various sites. Three cis-regulatory elements were identified: regions -21/-18, -37/-30, and -55/ -53. Mutations in any of these elements decreased basal and Ha-Ras-induced cPLA(2) promoter activity in both normal lung epithelial cells, as well as steady state promoter activity in A549 cells, with a mutation in element -2 1/-18 completely eliminating all promoter activity. Overexpression studies and gel shift assays indicated that Spl may serve as a transcription factor functionally regulating promoter activity by directly interacting with two of the cis-regulatory elements, -21/-18 and -37/-30. Expression of Ha-Ras led to induction of e-Jun protein, which showed functional cooperation with Sp1 in driving promoter activity. Additional unidentified transcription fa ctors bound to the regions from -55/-53 and -37/-34.