Paulo et al. have performed the first large-scale analysis of the proteomic and phosphoproteomic alterations in pancreatic stellate cells in response to nicotine. Smoking has been correlated with pancreatic cancer risk. Nicotine is a major component of tobacco and is readily absorbed by the body into the bloodstream. Systemic circulation then delivers nicotine to other organs, where it is similarly taken up with ease. Current research suggests that in the case of the pancreas, nicotine may induce fibrosis, which is common to chronic pancreatitis and metastasis, such as that in pancreatic adenocarcinoma. However, the mechanisms are unclear.
What is known is that nicotine binds to nicotinic acetylcholine receptors (nAChR), in particular α7, resulting in intracellular phosphorylation cascades. The neurotoxin α-Bungarotoxin, derived from the venom of the Taiwanese banded krait, also binds to nAChR. It does so irreversibly and competitively, particularly with the α1, α7, and α9 subunits. As pancreatic stellate cells (PaSC) produce acetylcholine, they are vulnerable to nAChR-mediated responses, and therefore to inhibition by α-Bungarotoxin. Paulo et al. have exploited this to quantify the proteins and phosphorylation sites altered by nicotine and α-Bungarotoxin, to unravel the mechanism by which nicotine contributes to pancreatic disease, using PaSCs.
Initial Western blotting comparing PaSC nAChR expression to expression of the same receptors in the neuroblastoma cell line SH-SY5Y showed higher expression of subunits α7 and α9. This correlated well with the subunits that α-Bungarotoxin will bind to (α1, α7 and α9). Paulo et al. then harvested PaSCs after following 1 hour of serum starvation and a further 12 hours of treatment with either nicotine, α-bungarotoxin or the untreated control group. They lysed the cells for protein extraction and tandem mass tag (TMT)-based mass spectrometry analysis using an Orbitrap Fusion mass spectrometer (Thermo Scientific) coupled to an EASY-nLC 1000 liquid chromatography (LC) pump. In order to perform a phosphorylation analysis, they also enriched the phosphopeptides with TiO2.
In total, the investigators quantified over 8,100 proteins and over 16,000 phosphorylation sites across all samples. They found 43 proteins present in greater abundance in nicotine-treated pancreatic cells. Several of these were common to neurons, some were defense proteins, and others such as apolipoprotein B and α-2-macroglobulin are already well defined as biomarkers of chronic pancreatitis. However, proteins were downregulated compared with untreated cells. The downregulated proteins were NEU1 (Sialidase-1), HEXA (β-hexosaminidase subunit alpha) and GLA (α-galactosidase A), all of which function in carbohydrate/lipid catabolism. In the α-bungarotoxin samples, only two proteins were present in greater abundance compared to the untreated cells. These were NRGN and HSBP1, and there were no proteins in lower abundance. Conversely, the researchers found that nicotine altered 132 phosphorylation sites, whereas α-bungarotoxin altered 451.
Paulo et al. suggest that the panel of phosphorylation sites and proteins that their work has generated provides a panel of potential targets that can be further investigated with a more in-depth study of associated pathways.
Paulo, J.A., Gaun, A., and Gygi, S.P. (2015) “Global Analysis of Protein Expression and Phosphorylation Levels in Nicotine-Treated Pancreatic Stellate Cells,” Journal of Proteome Research, 14 (pp. 4246-4256).