1 / 20

Brenda Niu Dr. Adrian F. Gombart Dept. of Biochemistry and Biophysics Linus Pauling Institute

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.

arnaud
Download Presentation

Brenda Niu Dr. Adrian F. Gombart Dept. of Biochemistry and Biophysics Linus Pauling Institute

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 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

  2. VITAMIN D • 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

  3. THE CAMP GENE 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

  4. FXR AND VDR Farnesoid X Receptor (FXR) Vitamin D Receptor (VDR) Ligand: 1,25(OH)2D3 or secondary bile acids • Ligand: primary bile acids or xenobiotics (Gombart, 2009) http://img.medscape.com/article/712/847/712847-fig4.jpg

  5. ACTIVATION OF CAMP VDR or FXR binds to ligand Forms heterodimers by binding retinoid X receptors (RXRs) Binds to VDRE Activates CAMP gene or FXR 1,25-Dihydroxy-vitamin D3 VDRE Activation of CAMP http://www.nature.com/ki/journal/v56/n73s/images/4491279f1.gif

  6. HYPOTHESIS 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.   

  7. METHODS • 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

  8. TSTA-1 CONSTRUCT hCAMP Promoter GAL4-VP16 Fusion Protein 5X GAL4 Binding Sites Minimal Promoter Firefly Luciferase Two Step Transcriptional Amplification

  9. 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

  10. HTS PROGRESS

  11. RESULTS OF HTS

  12. QRT-PCR

  13. DISULFARIM & RESVERATROL

  14. RESVERATROL

  15. hCAP18 LEVELS Resveratrol 10-5M Control 2’ only, Resv 10-5M

  16. SYNERGY/SUPPRESSION 1,25 D3

  17. RESVERATROL + 1,25 D3

  18. 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

  19. 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

  20. 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

More Related