Transcript DNMT1 - Ignyta

Regulation of DNA Methylation by Pro-Inflammatory Cytokines
in Rheumatoid Arthritis
Kazuhisa Nakano, David L. Boyle and Gary S. Firestein
Division of Rheumatology, Allergy and Immunology, UCSD School of Medicine, La Jolla, CA
Conclusion: Exposure to pro-inflammatory cytokines in the synovium decreases DNMT
expression and potentially suppresses DNA methylation. The inability of 5-aza-dC to alter
RA FLS global methylation suggests that DNMT3a is primarily responsible for DNA
methylation in RA, while DNMT1 contributes to methylation OA FLS. These data suggest
that the rheumatoid synovium can imprint FLS by altering DNA methylation and potentially
inducing a more aggressive phenotype.
Dose-response
Quantitative real-time polymerase chain reaction (PCR)
qPCR was performed using Assays On Demand (Applied Biosystems) to
determine relative mRNA levels using the GeneAmp 7300 Sequence Detection
System (Applied Biosystems). The data were normalized to GAPDH expression
to obtain relative cell equivalents.
DNMT activity assay
Total DNMT activity was assessed by enzymatic activity assay (DNMT Activity/
Inhibition Assay, Active Motif).
Global Methylation analysis
Global DNA methylation was evaluated with the MDQ1 Imprint methylated DNA
quantification kit (Sigma Aldrich, St. Louis, MO, USA). The methylated fraction
of DNA was identified using 5-methylcytosine mAb and quantified by an ELISAlike reaction. Global DNA methylation is shown as percent methylation of a
control DNA.
1.5
1.2
DNMT1
DNMT3a
1.0
*
0.8
*
0.6
*
*
*
0.4
*
0.2
*
*
*
0.1
1
*
0
0.001 0.01
mRNA expression in unstimulated RA and OA FLS (qPCR)
DNMT1
DNMT3a
2
2.5
0
*
*
*
2
8
24
Time (hrs)
Relative expression
2
1.5
Mean ± SD, n = 4
* p < 0.01 by 2-way ANOVA and contrast testing
DNMT1 and DNMT3a mRNA levels began to decrease in 8 and 2 hours after
exposure to IL-1ß (2 ng/ml ), respectively.
Decreased DNMT function after IL-1 stimulation
Resting FLS
0
OA
RA
RA
OA
RA
Mean ± SD, n=10 each, unpaired t-test
Unstimulated RA and OA FLS expressed similar amounts of DNMT1,
-3a, and -3b mRNA.
Western Blot (Nuclear extracts)
100
80
60
40
20
0
Mean ± SD, n = 6 each, paired t-test
80
FLS were cultured in the presence of a DNMT inhibitor 5-aza-dC (5 uM).
5-aza-dC significantly decreased global DNA methylation
60
40
20
0.6
0.4
0.2
0
RA
Mean ± SD, n=6 each, un-paired t-test
n.s
1.2
Relative expression
Relative expression
Western blot showed abundant DNMT1
and DNMT3a protein. There was no
significant difference between RA and OA
FLS.
5-aza-dC (days)
n.s
DNMT3a
0.8
b-actin
14
0
n.s
DNMT3a
p=0.036
120
0
0.2
0
RA
In resting cultured FLS, there was no difference in global methylation
between RA and OA FLS.
0.4
0.5
OA
40
Mean ± SD, n=10 RA and n=9 OA lines, un-paired t-test
100
0
60
Dose responses with showed significant suppression of DNMT expression
with concentrations of IL-1 as low as 1 pg/ml.
1
0.5
80
OA
1.5
1
100
0
(DNMT activity assay)
0.6
120
20
n.s
0.8
DNMT1
We propose that exposure of pro-inflammatory cytokines can
contribute to DNA hypomethylation through decreased DNMT
expression, thereby contributing to the aggressive phenotype of FLS.
*
0.5
IL-1 (ng/ml)
DNMT3b
DNMT1
PURPOSE AND HYPOTHESIS
*
10
OA
In resting cultured FLS, there was no difference in DNMT function
between RA and OA FLS.
1
0.8
0.6
0.4
0
OA
RA
OA
• Exposure to pro-inflammatory cytokines in the synovium
decreases DNMT expression and potentially suppresses DNA
methylation.
• These data suggest that the rheumatoid synovium can imprint
FLS by altering DNA methylation and potentially inducing a
more aggressive phenotype.
Long-term exposure of IL-1b
0.2
CONCLUSIONS
RA
Mean ± SD, , n=6 each, un-paired t-test
Decreased DNMT gene expression
after cytokine or TLR ligand stimulation
DNMT3a
DNMT1
2.5
DNMT3b
1.6
0.5
*
1.2
1
0.8
0.6
0.4
0.2
0
**
0
*
*
Relative expression
1
*
Relative expression
1.5
p = 0.008
p = 0.026
60
40
Inflammatory cytokines
20
0.6
1.4
2
DNMT activity
(OD/h/mg)
80
Relative expression
When DNA is methylated in the
promoter region of genes, where
transcription is initiated, genes are
silenced and transcription is
prevented.
1
n.s
n.s
DNA methylation is a major epigenetic determinant that modulates gene
expression, and abnormal methylation is associated with dysregulated cell
function. Our recent studies indicate that RA FLS exhibit a unique DNA
methylation pattern that could contribute to disease pathogenesis (ACR
meeting, 2011)
DNMT1 is primarily a maintenance
methyltransferase that preserves methylation
patterns during cell division.
DNMT3a and DNMT3b can methylate
hemimethylated and unmethylated CpG.
DNMT1
DNMT3a
0
0.0
140
Basal gene expression of DNMTs in FLS
Rheumatoid arthritis fibroblast-like synoviocytes (RA FLS) exhibit a unique
aggressive phenotype that contributes to the cytokine milieu and joint
destruction. While the pathogenesis of partial transformation is not fully
understood, epigenetic mechanisms are one possible explanation.
In mammalian cells, DNMTs are the only
enzymes that have been shown to mediate
the transfer of a methyl group from Sadenosylmethionine (SAM) to cytosine.
There are three enzymatically active
mammalian DNMTs—DNMT1, DNMT3a and
DNMT3b.
n.s
RESULTS
BACKGROUND
DNA methyltransferases (DNMTs) are critical enzymes involved in
establishing proper control of DNA methylation. Global hypomethylation has
been described in RA FLS, but the mechanisms have not been defined.
Time-course
Global DNA methylation
(% of standard)
Nuclear extract (for use in WB and DNMT activity assay) Nuclear extraction
kit (Panomics, Fremont, CA, USA)
Genomic DNA; MagMAX™ DNA Multi-Sample Kit (Applied Biosystems)
Global DNA methylation in cultured FLS
Global DNA methylation (% of standard)
Result: Unstimulated RA and OA FLS expressed similar amounts of DNMT1, -3a, and -3b
mRNA (n=10 each; n.s.). Western blot showed abundant DNMT1 and DNMT3a protein, but
only trace amounts of DNMT3b. DNMT1 and DNMT3a mRNA expression were decreased
when FLS were stimulated with IL-1ß (2 ng/ml) for 24 hr (49±8% and 58±6%
respectively; p<0.01). Dose responses with IL-1 showed significant suppression of DNMT
expression with concentrations as low as 1 pg/ml. DNMT mRNA levels decreased rapidly,
with significant suppression after 2 to 8 hrs of IL-1 stimulation. DNMT functional activity
was also decreased by IL-1 when FLS were cultured continuously for 14 days with IL-1ß
(74.0±6.5% of control, n=11, p=0.0012). 14-days exposure to the DNMT1 inhibitor 5-azadC decreased global DNA methylation in OA FLS (78.7±10% of control, n=6, p<0.05), but
not in RA FLS (105±6.7% of control, n=6, p=0.22).
Fibroblast-like synoviocytes
FLS were obtained from RA and osteoarthritis (OA) synovium at total joint
replacement and studied in the 4th through 7th passage.
Relative expression
Method: FLS were obtained from RA and osteoarthritis (OA) synovium at total joint
replacement and studied in the 4th through 7th passage. Gene expression was determined
by qPCR and protein expression by Western blot analysis. Nuclear extracts and genomic
DNA were purified from control or stimulated FLS. DNMT activity was measured with a
functional assay and global methylation was determined by an immunoassay that detects
methyl-cytosine.
Kinetics of DNMT mRNA regulation by IL-1ß
DNMT activity (OD/h/mg)
Background/Purpose: Rheumatoid arthritis fibroblast-like synoviocytes (RA FLS) exhibit a
unique aggressive phenotype that contributes to the cytokine milieu and joint destruction.
While the pathogenesis of partial transformation is not fully understood, epigenetic
mechanisms are one possible explanation. DNA methylation is a major epigenetic
determinant that modulates gene expression, and abnormal methylation is associated with
dysregulated cell function. DNA methyltransferases (DNMTs) are critical enzymes involved
in establishing proper control of DNA methylation. Global hypomethylation has been
described in RA FLS, but the mechanisms have not been defined. We hypothesized that
persistent exposure of pro-inflammatory cytokines can contribute to DNA hypomethylation
through decreased of DNMT expression, thereby contributing to the aggressive phenotype
of FLS.
METHODS
Relative expression
ABSTRACT
0.5
e.g., IL-1b
0
med
IL-1
med
IL-1
0.4
OA
RA
0.3
0.2
Mean ± SD, n = 6 each, paired t-test
FLS
DNMT1, 3a, 3b
0.1
0.0
When FLS were treated for 14 days with 1 ng/ml of IL-1ß, DNMT functional activity
was significantly decreased.
Mean ± SD, n = 6
**p < 0.05, *p < 0.01 by ANOVA /Dunnett test
DNMT1 and DNMT3a mRNA expression were significantly decreased when FLS were
stimulated with either IL-1ß (2 ng/ml), TNF (50 ng/ml), or LPS (1 ug/ml) for 24 hr.
DNA
hypomethylation
Aggressive phenotype