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

HTS PROGRESSHTS PROGRESS

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

QRT-PCRQRT-PCR

DISULFARIM & DISULFARIM & RESVERATROLRESVERATROL

RESVERATROLRESVERATROL

2’ only, Resv 10-5M

Control

Resveratrol 10-5M

hCAP18 LEVELShCAP18 LEVELS

SYNERGY/SUPPRESSION

1,25 D3

RESVERATROL + 1,25 RESVERATROL + 1,25 D3D3

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

ACKNOWLEDGEMENTS

Dr. Adrian Gombart The Gombart Lab

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