Comparison of Human & Murine Duox2 Activities in vitro

Joanna Wang
1
, R. Steven Esworthy Ph.D
1
, Thomas L. Leto Ph.D
2
, Fong-Fong Chu Ph.D
1

1 Department of cancer Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA, 2 Laboratory of Host Defenses, National Institute
of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, United States of America


control
DUOX2
Inducer
+
Inhibiter
DUOX2
Inducer
+
Inhibiter

DUOX2
Inducer

DUOX2
Inducer

control
Duplicates
H
2
O
2
detection with
plate reader
protein detection with
plate reader
Duox2 transfection
A plasmid containing the Duox2 cDNA from the mice strains C57BL/6 (B6) and
129SI/SvlmJ (129) was transfected into HEK cells expressing DUOXA2 (Duox2
activator). After transfection, cells containing the Duox2 cDNA were selected using a
drug resistant gene . Multiple stable transfectant clones were iosoated and analyzed
individually.
Duox2 activity assay (H2O2 detection assay)
Either monolayer cultures in 12-well plates or trypsinized cells were assayed.
For each transfectant clone, two wells were non-treated and functioned as the
control, two were treated with ionomycin (1 uM), a DUOX2 activator, and two were
treated with ionomycin and DPI ( diphenyleneiodonium sulfate, 10uM ) which acts
as a DUOX2 inhibitor.
Horseradish peroxidase (HRP) and homovanillic acid (HVA) were added for the
Duox2 activity assay, incubated at 37 C for 10 minutes, and were read by a
fluorometric plate reader along with an H
2
O
2
standard.
Each well was rinsed with PBS twice and cells were lysed with .5% TX100 in
PBS
This was followed by BCA protein assay (Pearce), read at A562 with a plate
reader using BSA as a standard.


METHODS
INTRODUCTION
Dual oxidase 2 (DUOX2) in mammals is required for generating H
2
O
2

utilized by thyroid peroxidase (TPO) for the biosynthesis of thyroid
hormones. However, its high level activity can contributes to oxidative
stress especially in mice deficient in glutathione peroxidases (GPx) which
reduces H
2
O
2.

The damage caused by oxidative stress has been implicated in a wide
number of disease processes including cancer, autoimmune disorders,
neuronal degeneration, and colitis.
Ulcerative colitis and closely related Crohn's disease are referred to as
inflammatory bowel disease (IBD).
Approximately 500,000 to 2 million people are affected by the diseases
in the United States. However, not much is known about the disease
mechanism.
Previous studies in Dr. Chus laboratory identify the possible link
between DUOX2 activity and colitis pathogenesis.
Investigating the involvement of DUOX2 activity in colitis disease
process bears significance in disease prevention and treatment values.




ACKNOWLEDGEMENTS
Thanks Dr. Chu and Dr. Esworthy for their guidance and training
in the project.
The summer research fellowship is provided by the Eugene and
Ruth Roberts Summer academy. The funding source is the Rose
Hills Foundation.
Thank you to Dr. Sleeth, Stephanie Patterson, and Dr. Salvaterra
for organizing many wonderful events.
CONCLUSIONS
The enzymatic assay is sensitive and efficient to measure the Duox2
activity in human and mouse alleles.
The clones that expressed high activity will be used to determine
Duox2 protein level in the future.
Hypothesis:
Because colitis was more severe in the 129 strain than B6 strain of GPx1/2
DKO mice, it was expected that there would be stronger Duox2 activity in
the 129 strain.
FUTURE DIRECTIONS
Determine specific activity of Duox2 alleles using Western analysis.
DUOX2 (dual oxidase 2), an enzyme encoded by the DUOX2 gene
catalyzes the reaction to produce H
2
O
2
, which is a member of reactive
oxygen species (ROS) attributed to its high oxidizing capacity. DUOX2
may play a role in the disease process of colitis by affecting microbial
balances in the digestive tract based on the previous studies in Dr. Chus
laboratory. To investigate the involvement of the enzyme in colitis
pathogenesis, DUOX2 activity from human and two mouse strains is
compared in vitro. We observed different levels of DUOX2 activity
between human and mouse alleles. The DUOX2 activity was found to be at
least five-fold higher in human enzyme than mouse enzyme. The enzyme
assay will be used to examine the possible correlation between the DUOX2
activity and the pathological phenotypes two mouse strains with different
colitis severity to study the disease mechanism.
ABSTRACT



Figure 1. This graph represents one of several experiments in which the DUOX2
activity is at least five times higher in human alleles than murine alleles. The data was
normalized against the nmole H
2
O
2
/ mg protein value in HuDuox2 with Ionomycin.
RESULTS
-0.1
0.1
0.3
0.5
0.7
0.9
1.1
hDUOX2 DuoxA2-C 129-c2 129 c-4 B6-p3 B6-c4 R
e
l
a
t
i
v
e

n
m
o
l
e

H
2

O
2


/
m
g

P
r
o
t
e
i
n

Duox2-containing Lysates
Comparison of Duox2 Activity Between Murine and Human
Alleles
Control
Ionomycin
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
129 C2 129 C3 129 C4 129 P4a B6 C3 B6 c4 B6 C6 B6 P2 B6 P3
R
e
l
a
t
i
v
e

n
m
o
l
e

H
2

O
2


/
m
g

P
r
o
t
e
i
n

Duox2-containing Lysates
Comparison of Duox2 Activity Between Alleles From
Different Murine Transfectant Clones
Control
Ionomycin
Ionomycin + DPI
Figure 2. This illustrates the variability in Duox2 activity between alleles from
different murine strains as well as variability among clones of the same strain.
Thomas L. Leto et al., 2009 Antioxidant Redox Signal