1. Jing Chen Project Advisor: Dr. Adrian F. Gombart Department of Biochemistry and Biophysics Linus Pauling Institute HHMI

2. Significance of Findings  Increase our understanding of the innate immune system in humans  Increase our understanding of how the VDR and CYP27B1 genes are involved in innate immunity  May lead to new treatments or medications for human diseases

3. Background  Exposure to sunlight was historically known to cure tuberculosis  Sunlight stimulates the synthesis of vitamin D  Vitamin D stimulates the production of cathelicidin anti-microbial peptide (CAMP) to help fight infections

4. Pathogen invades cell Toll-like receptor signaling activated Increased expression of VDR and CYP27B1 genes Activated vitamin D binds to VDR Production of CAMP increases to fight microbes Vitamin D and VDR go to the nucleus and binds to the vitamin D response element (VDRE) Background continued Vitamin D Signaling Pathway

5. Background continued Adams & Hewison (2008). Nature Clinical Practice Endocrinology & Metabolism, Volume 4, 80-90. TLR = Toll-like receptor allows immune system to recognize microbes by looking at molecular patterns CYP27B1: a gene that encodes an enzyme to convert inactive vitamin D to active vitamin D Active vitamin D VDR = Vitamin D Receptor Active vitamin D binds to VDR

6. Goal of Research Identify molecular mechanisms that regulate the expression of VDR and CYP27B1 genes in response to a pathogen

7. Hypothesis  If toll-like receptor signaling is activated in a cell that encounters a pathogen, then the expression of VDR and CYP27B1 genes are induced by the NFκB transcription factor.

8. NFκB  A transcription factor  Regulates immune response to infection  A target of TLR signaling

9. Methods Overview  Establish a cell line that shows conservation of the vitamin D pathway  Target specific components of the TLR signaling pathway  Determine factors that are necessary for inducing VDR and CYP27B1  Overexpress dominant negative factors to interfere with components of TLR pathway

10. Using Dominant Negative Factors Source: Akira, S. J. Biol. Chem. 2003;278:38105-38108

11. HaCat Cells  An adherent skin cell line  Keratinocyte  Skin is important in vitamin D synthesis

12. Methods LPS: a TLR4 ligand, a component of cell walls in gram-negative bacteria FSL: a TLR2 ligand, a peptide in bacteria 25D3: inactive vitamin D Untreated LPS 1 ng/ml FSL-1 1:1000 25D3 10 -7 M 25D3 & FSL-1 1:1000 Treat Cells

13. Methods continued  Isolate total cellular mRNA from treated cells  Make cDNA from mRNA  Take cDNA samples and prepare a real- time PCR (RT-PCR) plate

14. Methods continued  Amplifies and quantifies DNA samples  Measure the level of CAMP, VDR, and CYP27B1 in each sample  Strong induction of VDR and CYP27B1 genes will make it easier to detect decreases in levels Quantitative Real-time PCR

15. Results * * = statistically significant

16. Results continued * * * * = statistically significant

17. Results continued * * * * = statistically significant

18. Using Dominant Negative Factors Source: Akira, S. J. Biol. Chem. 2003;278:38105-38108

19. Results continued Transfection of GFP-Ras into HaCat

20. Discussion  CAMP, VDR, and CYP27B1 expression in HaCat cells increased after stimulation with vitamin D and a TLR ligand  Established a suitable cell line for transfection of dominant negative factors to interfere with TLR signaling pathway  Vitamin D and TLR signaling are important in a cell’s ability to respond to microbes

21. Future Research  Use molecular mechanisms to interfere with TLR pathway components 1. Transfection using chemicals 2. Electroporation

22. Acknowledgements  HHMI  URISC  NIH Grant 5R01AI065604 – 04 to A.F.G.  OSU Biochemistry and Biophysics Department  Linus Pauling Institute  Gombart Lab -Dr. Adrian F. Gombart -Dr. Tsuyako Saito -Dr. Malcolm Lowry -Mary Fantacone -Chunxiao Guo -Brian Sinnott -Yan Campbell -Jennifer Lam  Dr. Kevin Ahern

23. References Adams, J.S. & Hewison, M. (2008). Unexpected actions of vitamin D: new perspectives on the regulation of innate and adaptive immunity. Nature Clinical Practice Endocrinology & Metabolism, 4, 80-90. Liu, P.T., Schenk, M., Walker, V.P., Dempsey, P.W., Kanchanapoomi, M., Wheelwright, M., et al. (2009). Convergence of IL-1β and VDR activation pathways in human TLR2/1-induced antimicrobial responses. PLoS One 4(6): e5810. doi: 10.1371/journal.pone.0005810. Schauber, J., Dorschner, R.A., Coda, A.B., Buchau, A.S., Liu, P.T., Kiken, D., et al. (2007). Injury enhances TLR2 function and antimicrobial peptide expression through a vitamin D- dependent mechanism. The Journal of Clinical Investigation, 117(3), 803-811. Segaert, S. & Simonart, T. (2008). The epidermal vitamin D system and innate immunity: some more light shed on this unique photoendocrine system? [Editorial]. Dermatology, 217: 7-11. doi: 10.1159/000118506. Stoffels, K., Overbergh, L., Guilietti, A., Verlinden, L., Bouillon, R., & Mathieu, C. (2006). Immune regulation of 25-hydroxyvitamin-D 3 -1-α-hydroxylase in human monocytes. Journal of Bone and Mineral Research, 21(1), 37-47.