Related Experiment Videos
A bombesin receptor subtype-3 peptide increases nuclear oncogene expression in a MEK-1 dependent manner in human lung
H C Weber1, J Walters, J Leyton
1Section of Gastroenterology, Boston University School of Medicine, Boston, MA 02118, USA.
Abstract:
A synthetic peptide, (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) was used to investigate the signal transduction mechanisms of bombesin receptor subtype-3. Using NCI-1299#5 human lung cancer cells stably transfected with bombesin receptor subtype-3, 100 nM (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) elevated the cytosolic Ca2+ from 150 to 250 nM within 10 s. Addition of (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) caused phosphorylation of mitogen activated protein kinase in a time- and concentration-dependent manner. The mitogen activated protein kinase phosphorylation caused by (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) was inhibited by 2'-amino-3'-methyoxyflavone (PD98059), a mitogen activated protein kinase kinase (MEK-1) inhibitor. Using a luciferase reporter gene construct, (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) caused Elk-1 activation after 10 min and the increase in Elk-1 activation caused by (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) was inhibited by PD98059 as well as a dominant-negative MEK-1. (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) caused increased c-fos as well as c-jun mRNAs 1 h after addition to NCI-H1299#5 cells. The 47-fold increase in c-fos mRNA caused by 100 nM (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) was inhibited by PD98059, a dominant-negative MEK-1 and a substance P antagonist but not (3-phenylpropanoyl-D-Ala(24), Pro(26), Psi(26,27), Phe(27))GRP-(20-27) (BW2258U89), a GRP receptor antagonist. These results indicate that (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) caused increased nuclear oncogene expression and upstream events include mitogen activated protein kinase phosphorylation and Elk-1 activation.
Insights
A synthetic peptide activates bombesin receptor subtype-3, leading to increased cytosolic calcium and mitogen-activated protein kinase (MAPK) phosphorylation in lung cancer cells. This pathway involves Elk-1 activation and upregulates oncogene expression, suggesting potential therapeutic targets.
Area of Science:
- Molecular Biology
- Cell Signaling
- Oncology
Background:
- Bombesin receptor subtype-3 (BRS-3) plays a role in cell proliferation and survival.
- Understanding BRS-3 signaling is crucial for developing targeted cancer therapies.
- Synthetic bombesin analogs are used to probe receptor function.
Purpose of the Study:
- To investigate the signal transduction mechanisms of bombesin receptor subtype-3 (BRS-3).
- To elucidate the downstream effects of BRS-3 activation by a synthetic peptide.
- To identify key molecular events linking BRS-3 activation to oncogene expression.
Main Methods:
- Utilized NCI-H1299#5 human lung cancer cells stably transfected with BRS-3.
- Measured cytosolic calcium levels and mitogen-activated protein kinase (MAPK) phosphorylation.
- Employed luciferase reporter gene assays for Elk-1 activation and quantified c-fos/c-jun mRNA levels.
Main Results:
- The synthetic peptide (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) rapidly elevated cytosolic Ca2+ and induced time- and concentration-dependent MAPK phosphorylation.
- MAPK phosphorylation and subsequent Elk-1 activation were inhibited by PD98059 (a MAPK kinase inhibitor) and dominant-negative MEK-1.
- The peptide significantly increased c-fos and c-jun mRNA expression, which was attenuated by PD98059, dominant-negative MEK-1, and a substance P antagonist.
Conclusions:
- The synthetic peptide activates BRS-3, initiating a signaling cascade involving MAPK and Elk-1.
- This activation leads to increased expression of oncogenes c-fos and c-jun in lung cancer cells.
- The findings highlight the BRS-3/MAPK/Elk-1 pathway as a critical mediator of bombesin-induced oncogene expression.