α4β2 Nicotinic acetylcholine receptors play important roles in the reward pathways for nicotine. We investigated whether receptor up-regulation of α4β2 receptors involves expression changes for non-receptor genes. In a microarray analysis, 10µM nicotine altered expression of 41 genes at 0.25, 1, 8 and 24h in hα4β2 SHEP1 cells. Half of the genes were related to endoplasmic reticulum (ER) chaperones and the remaining genes belonged to inflammation and immune-response pathways.

The first objective was screening genes that alter surface α4β2 expression using correlation analysis and RNA interference. Antagonists potentiate nicotine-induced α4β2 upregulation, and correlation analysis showed that antagonists alone or in combination with nicotine suppressed an ER chaperone CRELD2 message while increasing surface expressing α4β2 receptors. siRNA knockdown of CRELD2 increased basal α4β2 receptor expression, and antagonists alone decreased CRELD2 mRNA in wild type SHEP1 cells lacking α4β2 receptors. These data suggest that ER proteins such as CRELD2 decreases surface α4β2 expression, and may explain antagonist actions in nicotine-induced receptor upregulation.

The second objective was investigating the signaling pathways downstream of α4β2 receptors leading to suppression of immune responses. Nicotine suppresses inflammatory cytokines and chemokines in hα4β2 SHEP1 cells but not in wild type SHEP1 cells. Quantitative RT-PCR (qPCR) corroborated nicotinic suppression of pro-inflammatory cytokines (PICs) IL-1β and IL-6. 10 µM nicotine suppressed basal IL-1β and IL-6 protein expression by blocking NFκB translocation. Nicotine dose-dependently attenuated lipopolysaccharide (LPS)-induced NFκB translocation, IκBα phosphorylation and PIC production. A cell-permeable calcium chelator BAPTA-AM, adenylate cyclase stimulant forskolin and a specific PKA inhibitor PKI 14-22 AMIDE failed to block the effects of nicotine on LPS-induced NFκB translocation and IκBα phosphorylation. The specific JAK2 inhibitor AG-490 and STAT3 inhibitor NSC74859 significantly blocked the anti-inflammatory effects of nicotine. These findings reveal a calcium-and cAMP-PKA independent signaling cascade and suggest a role for JAK2-STAT3 transduction in α4β2-mediated anti-inflammatory actions against endotoxin-induced inflammation.

Nicotine exposure decreased PIC production while upregulating α4β2 receptors. This negative association between nicotine-induced increases in α4β2 receptors and immune suppression may explain the neuroprotective effects observed in chronic smokers against neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease.