Investigating α7 nicotinic receptor STAT3 signaling and ... 1743/fulltext.pdf Investigating 7 nicotinic receptor STAT3 signaling and cell-dependent expression Thesis Presented By

1

Investigating 7 nicotinic receptor STAT3

signaling and cell-dependent expression

Thesis Presented

By

Thomas M. Koperniak

To

The Bouve Graduate School of Health Sciences

in Partial Fulfillment of the Requirements for the

degree of Doctor of Philosophy in Pharmaceutical Sciences

with specialization in

Pharmacology

NORTHEASTERN UNIVERSITY

BOSTON, MASSACHUSETTS

2012

ii

Northeastern University

Bouve Graduate School of Health Sciences

Thesis title: Investigating 7 nicotinic receptor STAT3 signaling and cell-dependent

expression

Author: Thomas M. Koperniak

Program: Department of Pharmaceutical Sciences

This project satisfies all research requirements for the Doctoral Degree

Thesis Committee (Chairman) ___________________________Date________

___________________________Date________

___________________________Date________

___________________________Date________

___________________________Date________

Director of Graduate School ___________________________Date________

Dean ___________________________Date________

Copy Deposited at Library ___________________________Date________

iii

Northeastern University

Bouve Graduate School of Health Sciences

Thesis title: Investigating 7 nicotinic receptor STAT3 signaling and cell-dependent

expression

Author: Thomas M. Koperniak

Program: Department of Pharmaceutical Sciences

This project satisfies all research requirements for the Doctoral Degree

Thesis Committee (Chairman) ___________________________Date________

___________________________Date________

___________________________Date________

___________________________Date________

___________________________Date________

Director of Graduate School ___________________________Date________

Dean ___________________________Date________

Copy Deposited at Library ___________________________Date________

iv

LIST OF ABBREVIATIONS

5HT Serotonin

AA Arachidonic acid

BGT -Bungarotoxin

AC Adenylate cyclase

AChBP Acetylcholine binding protein

ACh Acetylcholine

AD Alzheimer’s disease

ADP Adenosine diphosphate

ADSS Aged and diluted sidestream smoke

ANOVA Analysis of variation

AS Antisense

ATP Adenosine triphosphate

BAPTA-AM Bis(2-aminophenoxyl)ethane tetraacetic acid

BATH-42 BTB-associated Traf homology 42

BiP Binding immunoglobulin protein

BLAST Basic Local Alignment Search Tool

bp Base pairs

BSA Bovine serum albumin

CA Constitutively active

cAMP Cyclic adenosine monophosphate

CC Coiled-coil domain

cDNA Complementary DNA

CMV Cytomegalovirus

CNS Central nervous system

CREB cAMP response element-binding

CTZ Coelenterazine

DA Dopamine

DHE Dihydro-beta-erythroidine

DHS Donor horse serum

DMEM Dulbecco’s modified Eagle’s medium

DMSO Dimethyl sulfoxide

DN Dominant negative

dsRNA Double stranded RNA

EGFP Enhanced green fluorescent protein

EGFR Epidermal growth factor receptor

EGTA Ethylene glycol tetraacetic acid

ELISA Enzyme-linked immunosorbent assay

ER Endoplasmic reticulum

ERK Extracellular signal-regulated kinase

FBS Fetal bovine serum

FDA Food and Drug Administration

FGF2 Fibroblast growth factor 2

GABA Gamma-aminobutyric acid

GAPDH Glyceraldehyde phosphate dehydrogenase

GFP Green fluorescent protein

v

Glu Glutamate

HBSS Hank’s balanced salt solution

HEK Human embryonic kidney

hRic3 Human Ric3

HRP Horseradish peroxidase

Hsp90 Heat shock protein 90

IBD Inflammatory bowel disease

IDT Integrated DNA Technologies

IL6 Interleukin 6

IP Intraperitoneal

IP3 Inositol phosphate 3

JAK2 Janus kinase 2

KC Keratinocyte

LBD Ligand binding domain

LD-PCR Long distance-polymerase chain reaction

LPS Lipopolysaccharide

MAPK Mitogen-activated protein kinase

MIP2 Macrophage inflammatory protein 2

ML Metridia luciferase

MLA Methyllycaconitine

Mnmc Manumycin A

mRic3 Mouse Ric3

mRNA Messenger RNA

mTOR Mammalian target of rapamycin

NAc Nucleus accumbens

nAChR Nicotinic acetylcholine receptor

NBM Nucleus basilis of Meynert

NFB Nuclear factor kappa B

NMDA N-Methyl-D-aspartate

NMJ Neuromuscular junction

NNK Nicotine-derived nitrosamine ketone

NNN N'-nitrosonornicotine

NNR Neuronal nicotinic receptor

nSCLC Non-small cell lung cancer

p Probability

P9KB pREP9-kanamycin-blasticidin

P9KG pREP9-kanamycin-geneticin

PBS Phosphate buffered saline

PJAK2 Phosphorylated JAK2

PI3K Phosphoinositol-3 kinase

PKA Protein kinase A

PLC Phospholipase C

PM Plasma membrane

PMSF Phenylmethylsulfonyl fluoride

PNS Peripheral nervous system

PPT Pedunculo-pontine nucleus

vi

PSTAT3 Phosphorylated STAT3

RFP Red fluorescent protein

Ric3 Resistance to inhibitors of cholinesterase-3

RIPA Radio-immunoprecipitation assay

RISC RNAi-induced silencing complex

RNAi RNA interference

ROS Reactive oxygen species

RT Reverse transcriptase

S-rRic3 Serine (absent) rat Ric3

S+rRic3 Serine (present) rat Ric3

SCLC Small cell lung cancer

sdrRic3 Splice-deleted rat Ric3

SEAP Secreted alkaline phosphatase

SEM Standard error of the mean

shRNA Short hairpin RNA

siRNA Small interfering RNA

SMART Switching mechanism at 5’ end of RNA template

SN/SNc Substantia nigra

SOCS3 Suppressor of cytokine signaling 3

SS Signal sequence

STAT3 Signal transducer and activator of transcription-3

STR Striatum

TBS Tris buffered saline

TBS-T Tris buffered saline-Triton

THAL Thalamus

TLR Toll-like receptor

TM Transmembrane domain

TNF Tumor necrosis factor

Tyk Tyrosine kinase

UNC-50 Uncoordinated-like protein 50

UT Untransfected

US United States

Vegf Vascular endothelial growth factor

VGCC Voltage-gated calcium channel

VTA Ventral tegmental area

WT Wild-type

XIAP X-linked inhibitor of apoptosis protein

vii

ABSTRACT

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels

consisting of a combination of at least 17 possible subunits. Although most receptor subtypes

are heteromeric, 7 subunits can assemble into homomeric receptors. 7 nicotinic receptors are

the 2 nd

most highly expressed nicotinic receptor in the brain, and their high permeability to

calcium ions allows participation in calcium-dependent processes such as neurotransmitter

release and immune modulation. Although these receptors have been well-studied, 7-mediated

STAT3 signaling and 7 maturation remain unclear. The two major hypotheses we are

investigating stem from these issues. The first hypothesis, regarding 7-mediated STAT3

activation, is that 7 drives STAT3 signaling independently of calcium ion flow. The second

hypothesis, regarding 7 maturation, is that rat pituitary-derived GH4C1 cells possess Resistance

to Inhibitors of Cholinesterase 3 (Ric3) to allow surface 7 expression.

The major advancement of this project is the development of a novel STAT3-Metridia

luciferase (ML) signaling assay using heterologous expression systems. To our knowledge, this

is the only study using heterologous expression systems to investigate such signaling. To study

7-mediated STAT3 signaling, hu