Ralph H. Loring
Richard C. Deth, Michele Jacob, Akio Ohta, Barbara Waszczak
Date of Award
Doctor of Philosophy
Department or Academic Unit
Bouve College of Health Sciences. Department of Pharmaceutical Sciences
health sciences, alpha4beta2 upregulation, JAK2-STAT3, neurodegeneration, nicotinic acetylcholine receptor, nuclear factor kappa B, pro-inflammatory cytokine
Cytokines, Endoplasmic reticulum, Molecular chaperones, Nicotinic receptors
Alpha4beta2 nicotinic acetylcholine receptors play important roles in the reward pathways for nicotine. We investigated whether receptor upregulation of alpha4beta2 receptors involves expression changes for non-receptor genes. In a microarray analysis, 10 microM nicotine altered expression of 41 genes at 0.25, 1, 8 and 24h in halpha4beta2 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 alpha4beta2 expression using correlation analysis and RNA interference. Antagonists potentiate nicotine-induced alpha4beta2 upregulation, and correlation analysis showed that antagonists alone or in combination with nicotine suppressed an ER chaperone CRELD2 message while increasing surface expressing alpha4beta2 receptors. siRNA knockdown of CRELD2 increased basal alpha4beta2 receptor expression, and antagonists alone decreased CRELD2 mRNA in wild type SHEP1 cells lacking alpha4beta2 receptors. These data suggest that ER proteins such as CRELD2 decreases surface alpha4beta2 expression, and may explain antagonist actions in nicotine-induced receptor upregulation. The second objective was investigating the signaling pathways downstream of alpha4beta2 receptors leading to suppression of immune responses. Nicotine suppresses inflammatory cytokines and chemokines in halpha4beta2 SHEP1 cells but not in wild type SHEP1 cells. Quantitative RT-PCR (qPCR) corroborated nicotinic suppression of pro-inflammatory cytokines (PICs) IL-1beta and IL-6. 10 microM nicotine suppressed basal IL-1beta and IL-6 protein expression by blocking NFkB translocation. Nicotine dose-dependently attenuated lipopolysaccharide (LPS)-induced NFkB translocation, IkB-alpha 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 NFkB translocation and IkB-alpha 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 alpha4beta2-mediated anti-inflammatory actions against endotoxin-induced inflammation. Nicotine exposure decreased PIC production while upregulating alpha4beta2 receptors. This negative association between nicotine-induced increases in alpha4beta2 receptors and immune suppression may explain the neuroprotective effects observed in chronic smokers against neurodegenerative diseases such as Alzheimer's and Parkinson's disease.
Hosur, Vishnu, "Genetic regulation of endoplasmic reticulum chaperones and pro-inflammatory cytokines by neuronal alpha4beta2 nicotinic receptors" (2009). Pharmaceutical Science Dissertations. Paper 10. http://hdl.handle.net/2047/d20000672
Click button above to open, or right-click to save.