Transcript Identification of Compounds that Induce the Human

Identification of Compounds that Induce
the Human Cathelicidin Gene Through
the Vitamin D and/or Farnesoid X
Receptors
Brenda Niu
Dr. Adrian F. Gombart
Dept. of Biochemistry and Biophysics
Linus Pauling Institute
Oregon State University
HHMI 2010
VITAMIN D

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

Obtained through UVB radiation or food
Beneficial for bones
Recently linked to innate immune system
Regulates cathelicidin
antimicrobial peptide
(CAMP) gene
http://sun-dew.com/wp-content/uploads/2010/02/vitamind-main_Full-300x275.jpg
THE CAMP GENE
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Regulated by vitamin D only in humans and
primates
Promoter contains vitamin D response element
(VDRE)
Produces protein (cathelicidin)  interferes
with cell membrane of bacteria  pathogen
death
FXR AND VDR
Farnesoid X Receptor (FXR)

Ligand: primary bile
acids or xenobiotics
Vitamin D Receptor (VDR)
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Ligand: 1,25(OH)2D3 or
secondary bile acids
(Gombart, 2009)
http://img.medscape.com/article/712/847/712847-fig4.jpg
ACTIVATION OF CAMP
1.
2.
3.
4.
VDR or FXR binds to
ligand
Forms heterodimers by
binding retinoid X
receptors (RXRs)
Binds to VDRE
Activates CAMP gene
or FXR
1,25-Dihydroxyvitamin D3
VDRE
Activation of CAMP
http://www.nature.com/ki/journal/v56/n73s/images/4491279f1.gif
HYPOTHESIS
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Since the VDR and FXR can bind to other
ligands, compounds that resemble either vitamin
D or bind to FXR will activate the VDR or
FXR, respectively, and lead to induction of the
CAMP gene.
METHODS
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High throughput screening
 Test library of drugs used in NIH clinical
trials for CAMP activation
 Transfect cells through electroporation
 TSTA-1, RL, and GFP plasmids
 Dual-glo luciferase assay to test for activation
of the CAMP gene
TSTA-1 CONSTRUCT

Two Step Transcriptional Amplification
hCAMP Promoter
GAL4-VP16 Fusion Protein
5X GAL4 Binding Sites
Minimal Promoter
Firefly Luciferase
DUAL-GLO
LUCIFERASE ASSAY

Quantifies gene expression by measuring
luminescence from reactions catalyzed by firefly
and Renilla luciferase
Firefly luciferase
Luciferase reagent 
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Stop & glo reagent 
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Controls: DMSO (-), EtOH (-), 1,25 D3 (+)
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Renilla luciferase
HTS PROGRESS
Duplicate screenings of library
Triplicates if > 2 fold
change
TSTAempty
RESULTS OF HTS
Compound
Cytarabine
Disulfarim
Calcipotriol
Linezolid
Nitazoxanide
Pterostilbene
Resveratrol
Triplicate 1 Triplicate 2 Triplicate 3 Average Fold
Fold Change Fold Change Fold Change Change
4.03
2.74
9.93
8.25
4.2
3.28
2.84
3.74
2.88
9.94
6.62
2.44
3.21
3.05
4.23
3.5
9.89
7.48
3.45
3.24
2.76
4.00 ± 0.25
3.04 ± 0.41
9.92 ± 0.03
7.45 ± 0.82
3.36 ± 0.88
3.24 ± 0.03
2.88 ± 0.15
QRT-PCR
Induction of hCAMP in U937 Cells
120
90.13
Fold Change
100
80
60
40
20
6.79
0
Disulfarim
5.00
0.40
Resveratrol Calcipotriol Linezolid
Compounds
1.03
1.06
EtOH
DMSO
DISULFARIM &
RESVERATROL
Fold Change
Av Fold Change U937 (hCAMP/B actin)
20
18
16
14
12
10
8
6
4
2
0
Compound
RESVERATROL
hCAP18 LEVELS
Resveratrol 10-5M
Control
2’ only, Resv 10-5M
SYNERGY/SUPPRESSION
Fold Change from 1,25 D3 Activation
3.00
Fold Change
2.50
2.00
1.50
1,25 D3
1.00
0.50
0.00
Compound
RESVERATROL + 1,25 D3
Resveratrol and 1,25 D3 hCAMP mRNA Fold Change in U937
cells
600.0
500.0
451.6
Fold Change
400.0
322.9
EtOH
300.0
w/o Res 10-5
200.0
153.6
108.2
100.0
33.4
0.0
1.0
4.4
EtOH
10.3
Vit D10-10
Vit D 10-9
Vit D 10-8
CONCLUSIONS
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20 compounds in the NIH library may suppress
or synergistically activate the CAMP gene with
vitamin D
Resveratrol and pterostilbene activate the
endogenous CAMP gene as well as hCAP18
protein, while also acting synergistically with Vit.
D
FUTURE WORK
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Test resulting 20 compounds with QRT-PCR to
confirm synergy with vitamin D or suppression
Determine the mechanism(s) underlying
activation of the CAMP gene by resveratrol and
pterostilbene
ACKNOWLEDGEMENTS
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Dr. Adrian Gombart
The Gombart Lab
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A special thank you to Brian Sinnott and Dr. Malcolm Lowry
Dr. Kevin Ahern
OSU Biochemistry and Biophysics Department
Linus Pauling Institute
National Institute of Health
Howard Hughes Medical Institute