UNIVERSITY OF ILLINOIS AT CHICAGO
Department of Psychiatry
Fifth Annual Research Forum – Extravaganza 2014
POSTER TITLE
Ethanol activates Midkine and ALK signaling in neuroblastoma cells and in the
brain
DISEASE/KEY
WORDS:
Alcohol use disorders, signal transduction, amygdala
AUTHORS:
Donghong He, Hu Chen, Hisako Muramatsu , and Amy W. Lasek
MENTEE
CATEGORY:
Research specialist
BACKGROUND:
Midkine (MDK) is a secreted growth factor whose expression is higher in the
prefrontal cortex of chronic alcoholics and in mice genetically predisposed to
consume large amounts of alcohol (ethanol). One of the receptors for MDK is
anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase that regulates
behavioral responses to ethanol in mice. Although MDK and ALK activity appear to
modulate behaviors related to alcohol abuse, it is not known whether ethanol
activates signal transduction through these proteins. We investigated whether
ethanol exposure alters MDK and ALK gene expression and signaling pathways in
neuroblastoma cells in culture. To extend our studies to the brain, we determined if
MDK and ALK are important for the ability of ethanol to increase phosphorylation of
extracellular signal-regulated kinase (ERK), a downstream transducer of MDK and ALK
signaling.
The human neuroblastoma cell lines SH-SY5Y and IMR-32 were treated with various
concentrations of ethanol and tested for gene expression changes using quantitative
real-time PCR (qPCR) and protein expression using western blotting. Activation of
ALK signaling was examined in IMR-32 cells treated with 100 mM ethanol in the
presence and absence of the ALK inhibitor TAE684 or a small-interfering RNA (siRNA)
targeting MDK. Phosphorylation of ALK, ERK, STAT3, and AKT were measured after
ethanol exposure by western blotting. IMR-32 cells were also treated with
recombinant MDK protein and examined for protein phosphorylation. To test if ALK
is important for ERK phosphorylation in response to ethanol in the brain, mice were
treated with 3 g/kg ethanol intraperitoneally after pretreatment with TAE684. Thirty
minutes after ethanol treatment, brains were removed and processed for
immunohistochemistry using antibodies to phosphorylated ERK. ERK phosphorylation
in response to ethanol treatment was also examined in the brains of MDK knockout
mice.
MDK and ALK gene and protein expression increased in response to ethanol
treatment in SH-SY5Y and IMR-32 cells. Ethanol also caused a rapid increase in ALK,
ERK, and STAT3 phosphorylation in IMR-32 cells. Treatment of IMR-32 cells with
recombinant MDK had a similar effect on protein phosphorylation when compared to
ethanol. These effects were blocked by pretreatment with TAE684 or MDK siRNA.
METHODS:
RESULTS:
RESEARCH MENTOR:
Amy Lasek
UNIVERSITY OF ILLINOIS AT CHICAGO
Department of Psychiatry
ERK phosphorylation in the amygdala was increased after ethanol treatment, an
effect that was attenuated by pretreatment with TAE684 and in MDK knockout mice.
CONCLUSIONS:
These results suggest that ethanol activates signaling in the brain through the MDK
and ALK pathways and that the MDK and ALK genes are ethanol-responsive. Since
these genes are important for modulating behavioral responses to ethanol, targeting
ALK and MDK signaling pathways may represent a novel therapeutic strategy for the
treatment of alcohol use disorders.