Regulation of the
Human Cathelicidin
Antimicrobial Peptide Gene
by an Alu SINE
by Michael
Power
Dr. Adrian Gombart Laboratory
Linus Pauling Institute/
Department of Biochemistry
and Biophysics
Oregon State University

Vitamin D is important for
human health.
 Bone Health
 Immune Function

70% of Americans are
Vitamin D insufficient.

Vitamin D increases
production of
antimicrobial peptides.
 Defensins
 Cathelicidins
www.healingtherapies.info
Gombart, AF. 2009 Future Microbiol.
Cathelicidin Antimicrobial Peptide
(CAMP)
Part of the innate immune system.
Kills bacteria
Signals other immune cells
Cleans up infection
Effect of CAMP on pathogen
CAMP promoter
CAMP gene
CAMP mRNA
CAMP protein
Izadpanah, A. and Gallo, R. 2005 J Am Acad Dermatol.
Burchi, et al. Arch. Immunol. Ther. Exp. (2010) 58:15–25
Alu Short Interspersed Element
(Alu SINE)
 “Junk DNA”
 300 base pairs
 1 million copies
 10% of genome
Evolution of CAMP promoter
CAMP Promoter
 Insertion in promoter of hCAMP
allows regulatory effect.
 Duplication forms a Vitamin D
Responsive Element (VDRE).
 Mice do not have Alu SINE.
Cordaux R. and Batzer M.A. 2009 Nature Reviews Genetics.
Alu SINE
CAMP Promoter
uVDRE
SINE
Mice
Humans
Gombart AF et al. 2009 BMC Genomics.
A.
Vitamin D induces CAMP
expression in humans, but
not in mice.
LPS and 19kDa induce
CAMP expression in mice,
but not in humans.
hCAP is
human CAMP
LPS and 19kDa are
components of the
bacterial cell
membrane.
Adams, JS et al. 2009 J Immunol.
CRAMP is
mouse CAMP
Human (THP-1)
Question: What causes the
repression of hCAMP when
human cells are treated with
LPS or 19 kDa?
Hypothesis: The Alu SINE causes
the repression of hCAMP in the
presence of LPS and 19 kDa.
Prediction: Deletion of the Alu SINE
will allow induction of hCAMP in the
presence of LPS and FSL-1.
No treatment
LPS (100 ng/ml)
1,25 D₃ (10⁻⁸ M)
FSL-1 (10 ng/ml)
THP-1
Treatment
Gene
Expression
RAW
264.7
Messenger RNA
Real-Time PCR
Complementary
DNA
Average hCAMP Fold Change
1,25 D₃ induces hCAMP.
14
LPS and FSL-1 do not affect hCAMP.
12
10
8
6
4
Average hCAMP Fold Change
2
70
0
untrxt 7/6
1,25 D3 7/6
LPS 7/6
FSL-1 7/6
hCAMP is normalized to 18s.
60
50
40
30
20
hCAMP is normalized to
Beta-Actin.
10
0
untrxt 7/6
-10
1,25 D3 7/6
LPS 7/6
FSL-1 7/6
No mCAMP expression observed.
Sample
Average mCAMP Ct Average 18S Ct
No trxt.
No Detection
12.57
1,25 D3
No Detection
12.18
LPS
No Detection
11.92
FSL-1
No Detection
12.67
29.35
10.0
105 Spleen
Firefly luciferase gene is
inserted under control of
hCAMP promoter.
wildtype
Alu
Promote
SINE
luc
Alu
Promoter
SINE
luc
hCAMP
luc
NotI restriction sites are
engineered on either side of
Alu SINE.
Alu SINE is removed by NotI
restriction enzymes.
NotI
hCAMP
∆ Alu
luc
hCAMP
U937 and
Electroporation
THP-1
No treatment
LPS (100 ng/ml)
1,25 D₃ (10⁻⁷ M)
FSL-1 (10 ng/ml)
Treatment
Gene
Expression
Dual-Luciferase
Assay
Harvest Cells
Expression of hCAMP in U937 cells
0.03
Vitamin D does not affect
expression in cells that
contain NotI plasmid.
0.025
A
v 0.02
g
N
o 0.015
r
m
F 0.01
F
U937 cells grown and
transfected by Chunxiao Guo
respond to 1,25 D3
0.005
0
6.2lucNotI
6.2lucNotI +
1,25 D3
6.2lucNotI +
LPS
6.2luc ∆Alu
6.2luc ∆Alu +
1,25 D3
6.2luc ∆Alu +
LPS
 1,25 D3 induces hCAMP expression in THP-1 cells.
 LPS and FSL-1 do not induce hCAMP expression in THP-1 cells.
 RAW 264.7 cells do not express mCAMP.
Obtain and test mouse cell line J774A.
Expression of mCAMP?
Transfect U937 cells.
Transfect THP-1 cells.
Determine effects of Alu SINE on hCAMP expression.
 Does deletion of the Alu SINE allow induction of hCAMP in the
presence of LPS and FSL-1?
Dr. Gombart, Project Mentor
Dr. Ahern, Program Manager
Mary Fantacone, Lab Manager
Members of the Gombart Laboratory
Linus Pauling Institute
Department of Biochemistry and Biophysics
Howard Hughes Medical Institute
Cripps Scholarship Fund
National Institute of Health