Download Establishing Mechanisms of Vitamin D Signaling Pathways

ESTABLISHING MECHANISMS
OF VITAMIN D SIGNALING
PATHWAYS
Jing Chen
Project Advisor: Dr. Adrian F. Gombart
Department of Biochemistry and Biophysics
Linus Pauling Institute
HHMI
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
Background continued
Vitamin D Signaling Pathway
Pathogen invades cell
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
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
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
Treat Cells
Untreated
LPS
1 ng/ml
25D3
10-7 M
25D3 &
FSL-1
1:1000
FSL-1
1:1000
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
Methods continued
 Isolate total cellular mRNA from treated
cells
 Make cDNA from mRNA
 Take cDNA samples and prepare a realtime PCR (RT-PCR) plate
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
Induction of CAMP in HaCat
Cells
2.50E-07
Levels of CAMP
*
2.00E-07
1.50E-07
1.00E-07
5.00E-08
0.00E+00
Control
LPS 100 ng/ml
FSL-1 1:1000
Treatments
* = statistically significant
25D3 10-7 M
25D3 + FSL-1
Results continued
Induction of VDR in HaCat Cells
4.50E+00
*
4.00E+00
*
*
*
Fold Change
3.50E+00
3.00E+00
2.50E+00
2.00E+00
1.50E+00
1.00E+00
5.00E-01
0.00E+00
Control
LPS 100 ng/ml
FSL 1:1000
Treatments
* = statistically significant
25D3 10-7 M
25D3 + FSL
Results continued
Induction of CYP27B1 in HaCat
Cells
12
Fold Change
10
*
*
*
8
*
6
4
2
0
Control
LPS 100 ng/ml
FSL 1:1000
Treatments
* = statistically significant
25D3 10-7 M
25D3 + FSL
Using Dominant Negative Factors
Source: Akira, S. J. Biol. Chem. 2003;278:38105-38108
Results continued
Transfection 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 components
1. 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
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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 Ddependent 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.