CHRNA5 as negative regulator of nicotine signaling in normal and cancer bronchial cells: effects on motility, migration and p63 expression

Abstract

Genome-wide association studies have linked lung cancer risk with a region of chromosome 15q25.1 containing CHRNA3, CHRNA5 and CHRNB4 encoding α3, α5 and β4 subunits of nicotinic acetylcholine receptors (nAChR), respectively. One of the strongest associations was observed for a non-silent single-nucleotide polymorphism at codon 398 in CHRNA5. Here, we have used pharmacological (antagonists) or genetic (RNA interference) interventions to modulate the activity of CHRNA5 in non-transformed bronchial cells and in lung cancer cell lines. In both cell types, silencing CHRNA5 or inhibiting receptors containing nAChR α5 with α-conotoxin MII exerted a nicotine-like effect, with increased motility and invasiveness in vitro and increasing calcium influx. The effects on motility were enhanced by addition of nicotine but blocked by inhibiting CHRNA7, which encodes the homopentameric receptor α7 subunit. Silencing CHRNA5 also decreased the expression of cell adhesion molecules P120 and ZO-1 in lung cancer cells as well as the expression of DeltaNp63α in squamous cell carcinoma cell lines. These results demonstrate a role for CHRNA5 in modulating adhesion and motility in bronchial cells, as well as in regulating p63, a potential oncogene in squamous cell carcinoma.

Fig. 1.

Effects of nicotine, antagonists and CHRNA silencing on the migration capacity of cancer and non-transformed bronchial cells. (A) Non-transformed bronchial cell migration using Boyden Chamber assay. NHBE or 16HBE cells were treated with nicotine (Nic, 1 μM) or α-conotoxin MII (Ctx, 10 nM) or both α-conotoxin MII and α-bungarotoxin (Ctx, 10 nM + Bgtx, 1 μM). Results are expressed as percent of cells detected at the lower side of the Boyden Chamber membrane with respect to untreated cells, counted after 24 h (NHBE) or 26 h (16HBE), as explained in Materials and Methods. Each experiment was repeated three times. *P < 0.005 (Student’s t-test); **α < 0.005 (pairwise non-parametric Wilcoxon test without Bonferroni adjustment). (B) Effects of nicotine, antagonists and of CHRNA silencing on cell migration assay using Boyden Chamber assay, A549 cells (upper panel) and H1299 (lower panel). Cells were transfected with either control siRNA (white bars), CHRNA5 (gray), CHRNA7 (black) or both (hatched) siRNAs. They were then exposed to nicotine (Nic, 1 μM), α-bungarotoxin (Bgtx, 1 μM) or α-conotoxin MII (Ctx, 10 nM). Migration was expressed in percentage of cells at the lower side of the Boyden Chamber membrane with respect to control cells (transfected with control siRNA). At least three independent experiments were performed for each condition. *P < 0.05 (Student’s t-test); **P < 0.05 (pairwise non-parametric Wilcoxon test without Bonferroni adjustment). (C) DNA synthesis as measured by incorporation of BrdU in A549 and H1299 cells after silencing CHRNA5 or/and CHRNA7. Cells were transfected with siRNA 72 h prior to addition of BrdU. BrdU incorporation is expressed in percent with respect to cells transfected with control siRNA. Each experiment was repeated at least three times. *P < 0.05 (Student’s t-test); **α < 0.05 (pairwise non-parametric Wilcoxon test without Bonferroni adjustment).

Fig. 2.

Increased invasion in vitro and decreased expression of adhesion markers after silencing CHRNA5. (A) Three-dimensional cell cultures of A549 or H1299 cells. Upper panels: control cells with control siRNA. Lower panel: cells with siRNA to CHRNA5 (bars: 50 μM). Arrows indicate cells infiltrating the culture substrate. (B) Immunofluorescence for adhesion molecules P120 (left) and ZO-1 (right) in A549 cells after silencing CHRNA5. Upper panels (green fluorescence): P120 and ZO-1. Lower panels: merged pictures of adhesion molecules (green) and nucleus (blue).

Fig. 3a.

Effects of nicotine and antagonists on calcium influx in cells with silenced CHRNA5. Calcium influx in H1299 (A–D), 16HBE (E–F) and NHBE (G–E) was detected using the fluorescent marker Fluo-4. Panels A, C, E and G: microphographs of cells with artificial colors expressing Ca²⁺ levels. Color ranges from blue (low amount of Ca²⁺) to green, yellow and red (high amount of Ca²⁺). Panels B, D, F and G: quantitative analysis of Ca²⁺ influx in H1299 was performed using the Image J analysis program (http://rsbweb.nih.gov/ij/, Jan 2010). Nicotine was added at t = 0 sec (final concentration of nicotine: 1 mM) to cells transfected with siRNA CHRNA5 (A and B) or treated with α-conotoxin MII (Ctx, 10 nM) or α-bungarotoxin (Bgtx, 1 μM) (C–G).

Fig. 4.

Effects of CHRNA silencing on p63 expression. (A) RT-PCR analysis of DeltaNp63 expression in TE1 and TE13 cells after transfection with control siRNA or siRNA CHRNA3, CHRNA5 or CHRNA7. Changes in messenger RNA levels (ΔCt) were calculated with respect to cells treated with control siRNA. *P < 0.05 and ***P < 0.005 (Student’s t-test). (B) Immunofluorescence staining of p63 (Pan-p63 4A4) in TE1 cells (upper panel) and TE13 cells (lower panel) after transfection with control siRNA or siRNA CHRNA3, CHRNA5 or CHRNA7. (C) Western blot analysis of p63 (Pan-p63 4A4) at 48 h after siRNA or scramble RNA transfection. The band at 73 kDa corresponding to DeltaNp63α was identified on the basis of its immunological reactivity and electrophoretic motility as detailed in ref. (30). Ku80 was used as loading control. TE1 cells (upper panel) and TE13 cells (lower panel).

Fig. 5.

Role (α3β2)₂α5 nAChR complexes in regulating nicotine-induced calcium flux: a model. This diagram illustrates the negative role of (α3β2)₂5 nAChR complexes in the regulation of nicotine signaling through homopentameric α7 receptors and its impact on cell adhesion, migration/invasiveness and p63 expression. This model suggests that the susceptibility associated with CHRNA3 variants may be due to differences in their capacity to exert a negative regulation on signaling through α7 receptors. According to this hypothesis, the high-risk α3 protein variant (398N) may exert a less potent negative regulatory effect on nicotine signaling than the low-risk variant (398D) protein variant, thus making bronchial cells more susceptible to the proliferating or motility-promoting effects of nicotine mediated through α7 homopentameric receptor complexes.