Nicotine induces EP4 receptor expression in lung carcinoma cells by acting on AP-2α: The intersection between cholinergic and prostanoid signaling

Abstract

It was demonstrated that nicotine increased non-small cell lung cancer cell proliferation through nicotinic acetylcholine receptor -mediated signals. However, the detailed mechanism remains incompletely understood. We evaluated whether nicotine increased EP4 receptor expression in lung carcinoma cells by activating on AP-2α.

Methods: The non-small cell lung cancer cells of A549 and H1838 were cultured and treated with EP4 inhibitor AH23848, also with EP4 and control siRNAs. The extracellular signal-regulated kinases inhibitor PD98059, the p38 mitogen-activated protein kinase inhibitor SB239063, the α7 nicotinic acetylcholine receptor inhibitor α-bungarotoxin, the α4 nicotinic acetylcholine receptor inhibitor dihydro-β-erythroidine, the PI3K inhibitor wortmannin, the PKC inhibitor calphostin C, and the PKA inhibitor H89 have been used to evaluate the effects on proliferations. It indicates that nicotine increases EP4 expression through α7 nicotinic acetylcholine receptor-dependent activations of PI3-K, JNK and PKC pathways that leads to reduction of AP-2α-DNA binding. This, together with the elevated secretion of PGE₂, further enhances the tumor promoting effects of nicotine. These studies suggest a novel molecular mechanism by which nicotine increases non-small cell lung cancer cell proliferation.

Keywords: acetylcholine receptor; cyclooxygenases-2; nicotine; non-small cell lung carcinoma; proliferation.

Figure 1. Nicotine stimulates lung cancer cell growth through induction of EP4

(A) EP4 SiRNA decreased the proliferation of A549 cells induced by nicotine (0.5 μM). (B) AH23848 decreased the proliferation of A549 cells induced by nicotine (0.5 μM). (C) EP4 SiRNA decreased the proliferation of H1838 cells induced by nicotine (0.5 μM). (D) AH23848 decreased the proliferation of H1838 cells induced by nicotine (0.5 μM). (E) Nicotine increased secretion of PGE₂ in dose-dependent manner in A549 cells in ELISA assay. ^*indicates significantly difference from control. ^**indicates significance of combination treatment as compared with nicotine alone (P < 0.05). Con indicates untreated control cells.

Figure 2. The effects of nicotine, acetylcholine, and acetylcholinesterase on EP4 expression in human lung carcinoma cells

(A) Nicotine increased the expression of EP4 in dose-dependent manner in A549 cells. (B) Nicotine increased the expression of EP4 in time-dependent manner in A549 cells. (C) Nicotine increased the expression of EP4 in dose-dependent manner in H1838 cells. (D) Nicotine increased the expression of EP4 in time-dependent manner in H1838 cells. E. Nicotine increased EP4 mRNA expression as determined by real time RT-PCR. GAPDH served as internal control for normalization purposes. ^*indicates significant differences from control (P < 0.05).

Figure 3. Involvements of α7 nAChR, and PI3K, JNK and PKC pathways in the induction of EP4 by nicotine

(A) Acetylcholine increased the expression of EP4 induced by nicotine in dose-dependent in A549 cells. (B) Acetylcholinesterase decreased the expression of EP4 induced by nicotine in dose-dependent in A549 cells. (C) a-bungarotoxin of a a7 nAChR blocker, not dihydro-β-erythroidine of a α4 nAChR inhibitor, decreased the expression of EP4 induced by nicotine in A549 cells. (D) α7 nAChR siRNA (100 nM) decreased the expression of EP4 induced by nicotine in A549 cells. (E) a-bungarotoxin, a a7 nAChR blocker, decreased the proliferation induced by nicotine in A549 cells and H1838 cells. (F) The specific inhibitors of PI3-K (wortmannin, 1 μM), JNK (SP600125, 20 μM) reduced expression of EP4 induced by nicotine in A549 cells. (G) The specific inhibitors of ERK (PD98095, 20 μM), not P38 MAPK (SB239063, 10 μM) had a minor effect on reduction of EP4 induced by nicotine in A549 cells. (H) The specific inhibitors of PKC (calphostin C, 0.5 μM), not PKA (H89, 10 μM) reduced expression of EP4 induced by nicotine in A549 cells. GAPDH served as internal control for normalization purposes.

Figure 4. Nicotine stimulates EP4 promoter activity and affects AP-2α binding activity

(A) The 5′-flanking region of the human EP4 gene wild type and deletion promoter constructs schematics are presented. These regions contain several transcription factor binding sites including AP-2. (B) Nicotine increased EP4 gene promoter activity in A549 cells, transfected with the full-length wild-type EP4 promoter (–1238/+1) luciferase reporter construct and other two EP4 deletion reporter constructs (–238/+1 and –197/+1), not with a shortest EP4 deletion reporter construct (–160/+1). (C) Nicotine decreased the binding ability of AP-2α to the Oligonucleotides which contains the AP-2α site. (D) Nicotine decreased the EP4 promoter DNA quantity binding to AP-2α protein. (E) AP-2α overexpression vector blocked nicotine-induced EP4 protein expression in A549 cells. (F) AP-2α overexpression vector blocked nicotine-induced promoter activity of EP4 in A549cells. (G) Site-directed mutation of AP-2α (–169 bp ) on EP4 promoter blocked nicotine-induced promoter activity of EP4 in A549cells. ^*indicates significance as compared with controls. ^**indicates significance of combination treatment as compared with nicotine alone (P < 0.05). Con, untreated control cells.

Figure 5. The novel mechanism of nicotine increasing EP4 expression and NSCLC proliferation