Jing ChenProject Advisor: Dr. Adrian F. GombartDepartment of Biochemistry and BiophysicsLinus Pauling InstituteHHMI

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

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

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 continuedVitamin D Signaling Pathway

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 ReceptorActive vitamin D binds to VDR

Goal of Research

Identify molecular mechanisms that

regulate the expression of VDR and

CYP27B1 genes in response to a

pathogen

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.

NFκB

A transcription factor Regulates immune response to infection A target of TLR signaling

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

Using Dominant Negative Factors

Source: Akira, S. J. Biol. Chem. 2003;278:38105-38108

HaCat Cells

An adherent skin cell line Keratinocyte Skin is important in vitamin D synthesis

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

25D310-7 M

25D3 & FSL-1 1:1000

Treat Cells

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

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

Results

*

* = statistically significant

Results continued

** *

* = statistically significant

Results continued

*

**

* = statistically significant

Using Dominant Negative Factors

Source: Akira, S. J. Biol. Chem. 2003;278:38105-38108

Results continuedTransfection of GFP-Ras into HaCat

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

Future Research

Use molecular mechanisms to interfere with TLR pathway components1. Transfection using chemicals

2. Electroporation

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

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-D3-1-α-hydroxylase in human monocytes. Journal of Bone and Mineral Research , 21(1), 37-47.