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Jing Chen Project Advisor: Dr. Adrian F. Gombart Department of Biochemistry and Biophysics Linus Pauling Institute HHMI . Establishing Mechanisms of Vitamin D Signaling Pathways. Significance of Findings. Increase our understanding of the innate immune system in humans

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Presentation Transcript
slide1
Jing Chen

Project Advisor: Dr. Adrian F. Gombart

Department of Biochemistry and Biophysics

Linus Pauling Institute

HHMI

Establishing Mechanisms of Vitamin D Signaling Pathways

significance of findings
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
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
slide4

Background continued

Pathogen invades cell

Vitamin D Signaling Pathway

Toll-like receptor signaling activated

Increased expression of VDR and CYP27B1 genes

Activated vitamin D binds to VDR

Vitamin D and VDR go to the nucleus and binds to the vitamin D response element (VDRE)

Production of CAMP increases to fight microbes

background continued
Background continued

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

VDR = Vitamin D Receptor

Active vitamin D binds to VDR

Active vitamin D

Adams & Hewison (2008). Nature Clinical Practice Endocrinology & Metabolism, Volume 4, 80-90.

goal of research
Goal of Research

Identify molecular mechanisms that

regulate the expression of VDR and

CYP27B1 genes in response to a

pathogen

hypothesis
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.
slide8
NFκB
  • A transcription factor
  • Regulates immune response to infection
  • A target of TLR signaling
methods overview
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
slide10

Using Dominant Negative Factors

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

hacat cells
HaCat Cells
  • An adherent skin cell line
  • Keratinocyte
  • Skin is important in vitamin D synthesis
methods
Methods

Treat Cells

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

LPS

1 ng/ml

FSL-1

1:1000

Untreated

25D3 & FSL-1 1:1000

25D3

10-7 M

methods continued
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 continued14
Methods continued

Quantitative Real-time PCR

  • 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
results
Results

*

* = statistically significant

results continued
Results continued

*

*

*

* = statistically significant

results continued17
Results continued

*

*

*

* = statistically significant

slide18

Using Dominant Negative Factors

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

results continued19
Results continued

Transfection of GFP-Ras into HaCat

discussion
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
Future Research
  • Use molecular mechanisms to interfere with TLR pathway components
    • Transfection using chemicals
    • Electroporation
acknowledgements
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
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.