Extracellular Signal-regulated Kinase 1/2 Activity Is Not ...
Submitted to the Journal of Immunology (2010). ATP as an Extracellular Signal roles as signal...
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Transcript of Submitted to the Journal of Immunology (2010). ATP as an Extracellular Signal roles as signal...
Submitted to the Journal of Immunology (2010)
ATP as an Extracellular Signal
• roles as signal molecule:– DAMP
• inflammatory response1, pain sensation2
– synaptic signaling (neurotransmitter)2,3
– neuron-glia signaling4
– muscle contraction5
adenosine-5'-triphosphate (ATP)
P2RX7
• ligand-gated ion channel
Essential Cell Biology, 2nd Edition (Alberts et al., 2004)
ATP
ATP
in monocytes:
+ P2RX7 inflammatory response
Overall Hypothesis
If P2RX7 is involved in a monocyte’s role of initiating angiogenesis, then P2RX7
activation in monocytes will result in the generation of VEGF.
Results: Figure 1
• Figure 1A and 1B Hypothesis:
If P2RX7 receptors in monocytes are involved in the release of VEGF, then
VEGF concentrations will increase upon stimulation of monocytes with P2RX7
agonists.
Results: Figure 1
• Primary human monocytes
Results: Figure 1
• Primary human monocytes
• BzATP & ATP are P2RX7 agonists
adenosine-5'-triphosphate (ATP)
O
N
NN
N
NH2
OPOPOPO
O O O
O O OOH
O
OO
3 -′ 0-(4-benzoyl) benzoyl ATP (BzATP)
Results: Figure 1
• Primary human monocytes
• BzATP & ATP are P2RX7 agonists
• LPS: known stimulus for VEGF release (positive control)
Modified from www.wikimedia.org
Results: Figure 1 (A)
• Primary human monocytes treated with either:
• VEGF release quantified (Sandwich ELISA) after 24 hours
1. HEPES (control)2. 100 μM BzATP3. 300 μM ATP4. 1 μg/mL LPS (positive control)
Results: Figure 1 (A)
• quantifying VEGF → sandwich ELISA
1 2 3
4 65
Anti-VEGF antibody
VEGF
Enzyme-linked anti-VEGF antibody
Results: Figure 1 (A)
• VEGF release induced by both BzATP “(> 6-fold) and ATP (> 3-fold)
* = p<0.05
# = p<0.005
Results: Figure 1 (B)
• Purpose:– Determine time needed to release significant
VEGF levels following P2RX7 agonist-induced activation
• Primary human monocytes treated with either:
• VEGF release quantified at 1, 2, 4, 8, 16, and 24 hours following treatment
1. 100 μM BzATP2. 1 μg/mL LPS
Results: Figure 1 (B)
• Significant VEGF release occurs after:– BzATP – 1 hour– LPS – 16 hours * = p<0.05
** = p<0.01
# = p<0.005
ψ = p<0.001
Results: Figure 1 (B)
• Problems with Figure 1B:– Why not ATP?
Results: Figure 1 (C)
• Purpose:– Determine dosage BzATP needed for
significant VEGF release after 4 hours
• Primary human monocytes treated with varying amounts of BzATP for 4 hours
• VEGF release quantified
Results: Figure 1 (C)
• Significant VEGF release occurs after 4 hours using 20 μM BzATP
* = p<0.05
** = p<0.01
# = p<0.005
Results: Figure 1 (C)
• Problems:– Std. dev. only visible for 120 μM BzATP– Why not ATP?
Results: Figure 1 (C)
• “Does this give any additional information 1A and B do not give?”
Results: Figure 1 (D)
• Purpose:– Determine if short-term exposure to P2RX7
agonists can induce VEGF release
• Primary human monocytes treated with either:
• Media removed after 5 min. (short- term) or not removed (long-term)
• Cells incubated for 4 hours.• VEGF release quantified
1. 100 μM BzATP2. 300 μM ATP3. 1 μg/mL PMA
Results: Figure 1 (D)
• Both short-term and long-term exposure to BzATP or ATP results in significant VEGF release
* = p<0.05
** = p<0.01
# = p<0.005
• Lingering Question: What if P2RX7 agonists (ATP, BzATP) are stimulating other P2 receptors to stimulate VEGF release, not just P2RX7?
– problem: BzATP can stimulate other P2 receptors at high concentrations
• Lingering Question: What if P2RX7 agonists (ATP, BzATP) are stimulating other P2 receptors to stimulate VEGF release, not just P2RX7?
solution
=
use a P2RX7-exclusive antagonist and measure its effect on ATP/BzATP-stimulated VEGF
release
Results: Figure 2
• Figure 2 Hypothesis:
If the BzATP/ATP stimulation of VEGF production is mediated through P2RX7,
then a P2RX7 antagonist will attenuate or decrease VEGF production
Results: Figure 2
• competitive P2RX7-specific antagonist
3-[[5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl]methyl]pyri dine
hydrochloride
tocris.com
Results: Figure 2
• competitive P2RX7-specific antagonist
A438079
tocris.com
Results: Figure 2
• competitive P2RX7-specific antagonist
The Antagonist
tocris.com
Results: Figure 2 (A)
• Methods:– monocytes pretreated w/ vehicle (HEPES) or
antagonist for 30 min at 37°C– monocytes then treated w/ BzATP, ATP or
PMA for 4h– VEGF measured – done in triplicate
Results: Figure 2 (A)
* = p<0.05
** = p<0.01
Results: Figure 2 (A)
• problems with Figure 2A:– why graph percent VEGF release on y-axis
when Figure 1 just used VEGF concentration?• what VEGF level did they use as the 100%
standard for comparison?
– could VEGF attenuation be in response to cytotoxicity of the antagonist?
Results: Figure 2 (A)
• problems with Figure 2A:– why graph percent VEGF release on y-axis
when Figure 1 just used VEGF concentration?• what VEGF level did they use as the 100%
standard for comparison?
– could VEGF attenuation be in response to cytotoxicity of the antagonist?
perform viability assay/control (Fig. 2B)
Results: Figure 2 (B)
• Methods:– pretreated and stimulated similar to
experiment in Fig. 2A– after 4 hours, viability assessed using the
Non-Radioactive Cell Proliferation Assay (NRCPA) from Promega Corp.
– done in triplicate
Results: Figure 2 (B)
• Methods:– NRCPA Overview
• colorimetric method
cell density = 106 cells/mL
Results: Figure 2 (B)
• Methods:– NRCPA Overview
• colorimetric method
substrate of one color
product of another color
wikipedia.org
Results: Figure 2 (B)
• Methods:– NRCPA Overview
Results: Figure 2 (B)
• Methods:– NRCPA Overview
absorbance read @ 490 nm
Results: Figure 2 (B)
Results: Figure 2 (B)
• Figure/Experiment 2 Hypothesis:
If the BzATP/ATP stimulation of VEGF production is mediated through P2RX7, then a P2RX7 antagonist will attenuate or decrease VEGF production
Results and controls → support hypothesis
• P2RX7 = ion channel– significant permeability to Ca2+
Essential Cell Biology, 2nd Edition (Alberts et al., 2004)
ATP
Results: Figure 3
• Figure 3 Hypothesis:
If the P2RX7 stimulation of VEGF production is dependent on the increase in
intracellular Ca2+ mediated through P2RX7, then a cell permeable Ca2+
chelator will attenuate or decrease VEGF production
Results: Figure 3
• BAPTA-AM– cell-permeable Ca2+ chelator
1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
tetrakis(acetoxymethyl ester)
(BAPTA-AM) tocris.com
Results: Figure 3
• BAPTA-AM– cell-permeable Ca2+ chelator
Ca2+
wikipedia.org
Results: Figure 3
• BAPTA-AM– cell-permeable Ca2+ chelator
1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
tetrakis(acetoxymethyl ester)
(BAPTA-AM) tocris.com
Results: Figure 3 (A)
• Methods:– monocytes pretreated w/ vehicle (DMSO) or
BAPTA-AM for 20 min at 37°C– monocytes then treated w/ vehicle (HEPES),
BzATP, or ATP for 4h– VEGF measured– done in triplicate
Results: Figure 3 (A)
* = p<0.05
# = p<0.005
Results: Figure 3 (B)
Results: Figure 3
• problems with Figure 3A and 3B:– better clarity of results if arranged like Fig. 2A
and 2B
Results: Figure 3
• unpublished observation:– “ionomycin treatment alone unable to
stimulate VEGF release”
VEGF
P2RX7
monocyteCa2+ Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Results: Figure 3
• ionomycin = Ca2+ ionophore
tocris.com
Results: Figure 3
• ionomycin = Ca2+ ionophore
wikipedia.org
Results: Figure 3
• Figure 3 Conclusions:– VEGF release attenuated by BAPTA-AM
• though not dose-dependently
– artificial increase in intracellular Ca2+ ≠ VEGF stimulation
Results: Figure 3
Therefore, P2RX7 agonist-induced VEGF release is Ca2+-dependent, but it is not the
exclusive signal for VEGF stimulation
• P2RX7 role in production of ROS
IL-1βnitric oxide synthase
ROS
production of pro-inflammatorymolecules
(Lenertz et al., 2009)
• P2RX7 role in production of ROS
IL-1βnitric oxide synthase
ROS
production of pro-inflammatorymolecules
(Lenertz et al., 2009)
Results: Figure 4
• Figure 4 Hypothesis:
If P2RX7 agonist-induced VEGF release in human monocytes is linked to ROS
production, then P2RX7 agonist-induced VEGF release will be curtailed when
exposed to an antioxidant
Results: Figure 4
• N-AcetylCysteine (NAC)– Antioxidant– Neutralizes reactive oxygen species (ROS)
OHNH3
+
O
SHO
N+
OH
O
SH
H
Cysteine N-AcetylCysteine
Results: Figure 4
• Methods:– Primary human monocytes pretreated with
either HEPES or 10mM NAC for 20 min.– Cells then treated with either 100 μM BzATP
or 300 μM ATP for 4 hours– VEGF release quantified
Results: Figure 4
• NAC pretreatment attenuates P2RX7 agonist-induced VEGF release
Figure 4
• Problems: potentially do a viability for NAC treatment
Results: Figure 4
• Conclusion:– ROS needed for VEGF production
Results: Figure 4
• unpublished observations:– “H2O2 treatment alone, or in combination with
ionomycin treatment, is unable to stimulate VEGF release”
VEGF
P2RX7
ROSROSROS
ROSROS
monocyte
VEGF
P2RX7
monocyteCa2+ Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
ROSROSROS
ROSROS
Results: Figure 4
• Figure 4 Conclusions:– VEGF release attenuated by NAC– artificial increase in ROS, intracellular Ca2+, or
both ≠ VEGF stimulation
Results: Figure 4
Therefore, P2RX7 agonist-induced VEGF release is Ca2+-dependent and ROS-
dependent, but they are not the exclusive signals for P2RX7-mediated VEGF stimulation
Overall Conclusion
VEGF
P2RX7
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
ATP
Ca2+
Ca2+
Ca2+ Ca2+
Ca2+
ROSROSROS
ROSROS
?
monocyte
Implications
• novel action of P2RX7 signaling– aids in monocyte’s role of resolving
inflammation via the production of angiogenic factors
• targeting of P2RX7-dependent VEGF release → tumor treatment (?)
Extra Material
Isolation of Human Blood Monocytes
• Percoll Density Gradient– Separation of cells and
cellular particles – Non-toxic to cells
www.gelifesciences.com
Red blood cells
Granulocytes
Mononuclear cells (Monocytes)
(1aR,1bS,4aR,7aS,7bS,8R,9R,9aS)-9a-(Acetyloxy)-1a,1b,4,
4a,5,7a,7b,8,9,9a-decahydro-4a,7b-dihydroxy-3-(hydroxym ethyl)-1,1,6,8-tetramethyl-5-oxo-1H-
cyclopropa[3,4]benz [1,2-e]azulen-9-yl tetradecanoate
Phorbol 12-myristate 13-acetate (PMA)