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Identification of Compounds Identification of Compounds that Induce the Human that Induce the Human Cathelicidin Gene Through the Cathelicidin Gene Through the Vitamin D and/or Farnesoid X Vitamin D and/or Farnesoid X ReceptorsReceptors
Brenda NiuBrenda Niu
Dr. Adrian F. GombartDr. Adrian F. GombartDept. of Biochemistry and BiophysicsDept. of Biochemistry and BiophysicsLinus Pauling InstituteLinus Pauling InstituteOregon State UniversityOregon State UniversityHHMI 2010 HHMI 2010
VITAMIN DVITAMIN D Obtained through UVB radiation or Obtained through UVB radiation or
foodfood Beneficial for bonesBeneficial for bones Recently linked to innate immune Recently linked to innate immune
systemsystem Regulates cathelicidin Regulates cathelicidin
antimicrobial peptide antimicrobial peptide
(CAMP) gene(CAMP) gene
http://sun-dew.com/wp-content/uploads/2010/02/vitamind-main_Full-300x275.jpg
THE CAMP GENETHE CAMP GENE Regulated by vitamin D only in Regulated by vitamin D only in
humans and primateshumans and primates Promoter contains vitamin D Promoter contains vitamin D
response element (VDRE)response element (VDRE) Produces protein (cathelicidin) Produces protein (cathelicidin)
interferes with cell membrane of interferes with cell membrane of bacteria bacteria pathogen death pathogen death
FXR AND VDRFXR AND VDR
Farnesoid X Receptor (FXR)Farnesoid X Receptor (FXR)
Ligand: primary Ligand: primary bile acids or bile acids or xenobioticsxenobiotics
Vitamin D Receptor (VDR)Vitamin D Receptor (VDR)
Ligand: Ligand: 1,25(OH)1,25(OH)22DD33 or or secondary bile secondary bile acidsacids
http://img.medscape.com/article/712/847/712847-fig4.jpg
(Gombart, 2009)
ACTIVATION OF CAMP
http://www.nature.com/ki/journal/v56/n73s/images/4491279f1.gif
VDRE
or FXR
Activation of CAMP
1,25-Dihydroxy-vitamin D3
1.1. VDR or FXR binds VDR or FXR binds to ligandto ligand
2.2. Forms Forms heterodimers by heterodimers by binding retinoid X binding retinoid X receptors (RXRs)receptors (RXRs)
3.3. Binds to VDREBinds to VDRE
4.4. Activates CAMP Activates CAMP genegene
HYPOTHESISHYPOTHESIS Since the VDR and FXR can bind to Since the VDR and FXR can bind to
other ligands, compounds that other ligands, compounds that resemble either vitamin D or bind to resemble either vitamin D or bind to FXR will activate the VDR or FXR, FXR will activate the VDR or FXR, respectively, and lead to induction of respectively, and lead to induction of the CAMP gene. the CAMP gene.
METHODSMETHODS High throughput screeningHigh throughput screening
Test library of drugs used in NIH Test library of drugs used in NIH clinical trials for CAMP activationclinical trials for CAMP activation
Transfect cells through Transfect cells through electroporationelectroporationTSTA-1, RL, and GFP plasmidsTSTA-1, RL, and GFP plasmids
Dual-glo luciferase assay to test Dual-glo luciferase assay to test for activation of the CAMP genefor activation of the CAMP gene
TSTA-1 CONSTRUCTTSTA-1 CONSTRUCT
TTwo wo SStep tep TTranscriptional ranscriptional AAmplificationmplification hCAMP Promoter
GAL4-VP16 Fusion Protein
5X GAL4 Binding Sites
Firefly Luciferase
Minimal Promoter
DUAL-GLO LUCIFERASE ASSAY
Quantifies gene expression by measuring luminescence from reactions catalyzed by firefly and Renilla luciferase
Luciferase reagent
Stop & glo reagent
Controls: DMSO (-), EtOH (-), 1,25 D3 (+)
Renilla luciferase
Firefly luciferase
RESULTS OF HTSRESULTS OF HTS
CompoundTriplicate
1 Fold Change
Triplicate 2 Fold
Change
Triplicate 3 Fold Change
Average Fold
Change
Cytarabine 4.03 3.74 4.234.00 ±
0.25
Disulfarim 2.74 2.88 3.53.04 ±
0.41Calcipotriol 9.93 9.94 9.89
9.92 ± 0.03
Linezolid 8.25 6.62 7.487.45 ±
0.82Nitazoxanide 4.2 2.44 3.45
3.36 ± 0.88
Pterostilbene 3.28 3.21 3.24
3.24 ± 0.03
Resveratrol 2.84 3.05 2.76
2.88 ± 0.15
CONCLUSIONS
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
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