Elsevier

Regulatory Peptides

Volume 109, Issues 1–3, 15 November 2002, Pages 135-140
Regulatory Peptides

PACAP-27 tyrosine phosphorylates mitogen activated protein kinase and increases VEGF mRNAs in human lung cancer cells

https://doi.org/10.1016/S0167-0115(02)00196-9Get rights and content

Abstract

The effects of pituitary adenylate cyclase activating polypeptide (PACAP) on human lung cancer cell line NCI-1299 mitogen activated protein kinase (MAPK) tyrosine phosphorylation and vascular endothelial cell growth factor (VEGF) expression were investigated. PACAP-27 (100 nM) increased MAPK tyrosine phosphorylation 3-fold, 5 min after addition to NCI-H1299 cells. PACAP caused tyrosine phosphorylation in a concentration-dependent manner being half-maximal at 10 nM PACAP-27. PACAP-27 or PACAP-38 (100 nM) but not PACAP28–38 or VIP caused increased MAPK tyrosine phosphorylation using NCI-H1299 cells. Also, the increase in MAPK tyrosine phosphorylation caused by PACAP-27 was totally inhibited by 10 μM PACAP(6–38), a PAC1 receptor antagonist or 10 μM PD98059, a MAPKK inhibitor. These results suggest that PAC1 receptors regulate tyrosine phosphorylation of MAPK in a MAPKK-dependent manner. PACAP-27 (100 nM) caused increased VEGF mRNA in NCI-H1299 cells after 8 h. The increase in VEGF mRNA caused by PACAP-27 was partially inhibited by PACAP(6–38), PD98059 and H-89. Addition of VIP to NCI-H1299 cells caused increased VEGF mRNA, which was totally inhibited by H89, a PKA inhibitor. These results suggest that PAC1 and VPAC1 receptors regulate VEGF expression in lung cancer cells.

Introduction

Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the vasoactive intestinal peptide (VIP) family of peptides [1]. Two forms of PACAP have been isolated, PACAP-27, which has 70% homology with VIP, and PACAP-38, which contains PACAP-27 plus a C-terminal extension of 11 amino acids [2], [3]. Both PACAP-27 and PACAP-38 bind with high affinity to human lung cancer cells [4] and stimulate their growth. In addition, PACAP stimulates the proliferation of lovocolorectal and rat cerebellar cells [5], [6] and is the most potent stimulant known to cause growth of gastric enterochromaffin cells [7].

PACAP and VIP elevate cAMP [8]. Both VPAC1 and PAC1 receptors interact with a stimulatory guanine nucleotide binding subunit (Gαs) activating adenylate cyclase. In turn, the increase in intracellular cAMP may activate protein kinase (PK) A; H89 is a PKA enzyme inhibitor [9]. In contrast, PAC1 but not VPAC1 receptors interact with a guanine nucleotide binding subunit (Gαq/11) causing phosphatidylinositol (PI) turnover. The inositol-1,4,5-trisphosphate released may cause release of Ca2+ from the endoplasmic reticulum into the cytosol. PACAP-27 but not VIP elevates cytosolic Ca2+ using lung cancer cells [10]. The diacylglycerol released may activate PKC, which in turn phosphorylates substrates such as MAPKK which in turn phosphorylates MAPK; MAPKK is inhibited by PD98059 [11]. Here the effects of PACAP on MAPK tyrosine phosphorylation were investigated using lung cancer cells and NIH/3T3 cells transfected with PAC1 receptors. These results indicate that PAC1 receptors regulate MAPK activity as a result of PI turnover.

Phosphorylated MAPK may enter the nucleus and alter nuclear oncogene expression [12]. PACAP-27 transiently increases c-fos mRNA in lung cancer cells [13]. The effects of PACAP are reversed by the PAC1 receptor antagonist, PACAP(6–38) [14]. Also, PACAP-27 transiently increases c-jun mRNAs in lung cancer cells. After translation, the c-fos and c-jun proteins may form heterodimers and increase expression of growth factor genes. Recently, we showed that VIP and PGE2, which elevate intracellular cAMP in lung cancer cells, increased vascular endothelial cell growth factor (VEGF) mRNAs in lung cancer cell line NCI-H157 [15]. Here the effects of PACAP-27 were investigated on VEGF mRNA using lung cancer cell line NCI-H1299. Our results indicate that PACAP-27 increases MAPK tyrosine phosphorylation in a MAPKK-dependent manner. Also, PACAP-27 increased VEGF mRNAs and the increase in VEGF mRNAs was partially inhibited by PACAP(6–38), H89 and PD98059. In contrast, VIP increased VEGF was inhibited totally by H89 but not PACAP(6–38) or PD98059. These results indicate that both PAC1 and VPAC1 receptors regulate VEGF mRNA in lung cancer cells.

Section snippets

Cell culture

NCI-H1299 and H157 cells were cultured in RPMI-1640 containing 10% heat-inactivated fetal bovine serum (FBS, Life Technologies) [16]. The cells were split weekly with trypsin-EDTA (Life Technologies). The cells were mycoplasma-free and were used when they were in exponential growth phase after incubation at 37 °C in 5% CO2/95% air. NIH/3T3 cells were cultured in DMEM containing 10% calf serum. The cells were transfected with PAC1 receptor splice variants (SVs) using the pCKL-SRα/NEO expression

PACAP-like peptides cause MAPK tyrosine phosphorylation

The ability of PACAP-27 to cause MAPK tyrosine phosphorylation was investigated by Western blot. Fig. 1 shows that 1 nM PACAP-27 weakly, but 10 to 100 nM PACAP-27 strongly caused MAPK tyrosine phosphorylation after addition to NCI-H1299 cells. MAPK tyrosine phosphorylation was maximal (2.8-fold increase) 5 min after addition of PACAP-27 to NCI-H1299 cells (data not shown). These results indicate that PACAP-27 causes MAPK tyrosine phosphorylation in a time- and concentration-dependent manner

Discussion

Previously, we found that PACAP-27 bound with high affinity to lung cancer cells, causing elevated cAMP and phosphatidylinositol turnover [4]. Also, PACAP-27 increased c-fos and c-jun mRNAs [13] and increased the clonal growth of lung cancer cells [4]. In contrast, VIP bound with high affinity causing elevated cAMP but had no effect on PI turnover [8]. Here the mechanisms by which PACAP-27 altered MAPK tyrosine phosphorylation and VEGF mRNAs were investigated.

PACAP-27, but not VIP, caused MAPK

Acknowledgements

The authors thank Drs. D. Brenneman, I. Gozes and S. Wank for helpful discussions.

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