Pituitary Adenylate‐Cyclase‐Activating Polypeptide Stimulates Proto‐oncogene Expression and Activates the AP‐1 (c‐Fos/c‐Jun) Transcription Factor in AR4‐2J Pancreatic Carcinoma Cells

Pituitary adenylate‐cyclase‐activating polypeptide (PACAP) has been shown to possess mitogenic activity in various tumor cells. The present study was designed to investigate signal transduction mechanisms and expression of the proto‐oncogenes c‐fos and c‐jun linked to the mitogenic effect of PACAP in the pancreatic carcinoma cell line AR4‐2J. PACAP‐(1–27)‐peptide and PACAP‐(1–38)‐peptide, but not the structurally related vasoactive intestinal polypeptide (VIP), potently stimulated [3H]thymidine incorporation and cell number at doses of 0.1–10 nM. Both molecular forms of PACAP strongly increased formation of cAMP and inositol trisphosphate, elevated cytosolic Ca2+ levels and induced mitogen‐activated protein (MAP) kinase activity. Quantitative reverse‐transcription PCR revealed that PACAP‐(1–27)‐peptide and PACAP‐(1–38)‐peptide elevated c‐fos mRNA levels 50–100‐fold, whereas c‐jun mRNA levels increased only moderately (2–3‐fold). The effect of PACAP on c‐jos and c‐jun expression in AR42J cells was rapid (20 min), transient (1–2 h), dose‐dependent (IC50, 0.5 nM) and was abolished by the specific PACAP receptor antagonist PACAP‐(6–38)‐peptide or inhibitors of protein kinase C or tyrosine kinases. Compared with PACAP, epidermal growth factor and gastrin equipotently stimulated c‐fos transcription whereas VIP, secretin, forskolin or phorbol ester showed only marginal effects. Both PACAP‐(1–27)‐peptide and PACAP‐(1–38)‐peptide strongly increased the DNA binding activity of the c‐fos/c‐jun heterodimer transcription factor AP‐1 at 10 nM and also stimulated AP‐1 transcriptional activity up to 20‐fold in AR4‐2J cells. These findings indicate that the mitogenic effect of PACAP mediated via activation of the GTP‐binding protein coupled PACAP/VIP‐1 (PV1) receptor is linked to the MAP kinase cascade, increased expression of the proto‐oncogenes c‐fos and c‐jun and activation of the heterodimeric transcription factor AP‐1.