Class of 2013!!!

This forum made possible through the generous support of SDN members, donors, and sponsors. Thank you.
Members do not see this ad.
riceman04 said:
wait...I thought I heard something about you and Myuu!!!

Don't play innocent...Swarley you better check this guy...he's trying to make moves on Miss Virginia
Click to expand...

😡 What is this. :nono:(Except this is Dikembe Mutombo finger waving)

To quote Alfalfa, "Usually I'm a lover, not a fighter; but in this case I'm willing to make an exception"
 
26. Mackenzie AB, Young MT, Adinolfi E (2005) Pseudoapoptosis
induced by brief activation of ATP-gated P2X7 receptors. J Biol
Chem 280:33968–33976
27. Darville T, Welter-Stahl L, Cruz C (2007) Effect of the purinergic
receptor P2X7 on Chlamydia infection in cervical epithelial cells
and vaginally infected mice. J Immunol 179:3707–30714
28. Li CM, Campbell SJ, Kumararatne DS (2002) Association of a
polymorphism in the P2X7 gene with tuberculosis in a Gambian
population. J Infect Dis 186:1458–1462
29. Barden N, Harvey M, Gagne B (2006) Analysis of single
nucleotide polymorphisms in genes in the chromosome
12Q24.31 region points to P2RX7 as a susceptibility gene to
bipolar affective disorder. Am J Med Genet B Neuropsychiatr
Genet 141:374–382
30. Smart ML, Gu B, Panchal RG (2003) P2X7 receptor cell surface
expression and cytolytic pore formation are regulated by a distal
C-terminal region. J Biol Chem 278:8853–8860
31. Denlinger LC, Fisette PL, Sommer JA (2001) Cutting edge: the
nucleotide receptor P2X7 contains multiple protein- and lipidinteraction
motifs including a potential binding site for bacterial
lipopolysaccharide. J Immunol 167:1871–1876
32. Gu BJ, Zhang W, Worthington RA (2001) A Glu-496 to Ala
polymorphism leads to loss of function of the human P2X7
receptor. J Biol Chem 276:11135–11142
33. Li CM, Campbell SJ, Kumararatne DS (2002) Response
heterogeneity of human macrophages to ATP is associated with
P2X7 receptor expression but not to polymorphisms in the
P2RX7 promoter. FEBS Lett 531:127–131
34. Wiley JS, Dao-Ung LP, Li C (2003) An Ile-568 to Asn
polymorphism prevents normal trafficking and function of the
human P2X7 receptor. J Biol Chem 278:17108–17113
35. Gu BJ, Sluyter R, Skarratt KK (2004) An Arg307 to Gln
polymorphism within the ATP-binding site causes loss of
function of the human P2X7 receptor. J Biol Chem 279:31287–
31295
36. Le Stunff H, Auger R, Kanellopoulos J (2004) The Pro-451 to
Leu polymorphism within the C-terminal tail of P2X7 receptor
impairs cell death but not phospholipase D activation in murine
thymocytes. J Biol Chem 279:16918–16926
37. Cabrini G, Falzoni S, Forchap SL (2005) A His-155 to Tyr
polymorphism confers gain-of-function to the human P2X7
receptor of human leukemic lymphocytes. J Immunol 175:82–89
38. Skarratt KK, Fuller SJ, Sluyter R (2005) A 5′ intronic splice site
polymorphism leads to a null allele of the P2X7 gene in 1–2% of
the Caucasian population. FEBS Lett 579:2675–2678
39. Shemon AN, Sluyter R, Fernando SL (2006) A Thr357 to Ser
polymorphism in homozygous and compound heterozygous
subjects causes absent or reduced P2X7 function and impairs
ATP-induced mycobacterial killing by macrophages. J Biol
Chem 281:2079–2086
40. Denlinger LC, Angelini G, Schell K (2005) Detection of human
P2X7 nucleotide receptor polymorphisms by a novel monocyte
pore assay predictive of alterations in lipopolysaccharideinduced
cytokine production. J Immunol 174:4424–4431
 
41. Denlinger LC, Coursin DB, Schell K (2006) Human P2X7 pore
function predicts allele linkage disequilibrium. Clin Chem
52:995–1004
42. Sjoblom T, Jones S, Wood LD (2006) The consensus coding
sequences of human breast and colorectal cancers. Science
314:268–274
43. Slater M, Danieletto S, Gidley-Baird A (2004) Early prostate
cancer detected using expression of non-functional cytolytic
P2X7 receptors. Histopathology 44:206–215
44. Solini A, Cuccato S, Ferrari D (2008) Increased P2X7 receptor
expression and function in thyroid papillary cancer: a new
potential marker of the disease. Endocrinol 149:389–396
45. Li X, Zhou L, Feng YH (2006) The P2X7 receptor: a novel
biomarker of uterine epithelial cancers. Cancer Epidemiol
Biomarkers Prev 15:1906–1913
46. Zhang XJ, Zheng GG, Ma XT (2004) Expression of P2X7 in
human hematopoietic cell lines and leukemia patients. Leuk Res
28:1313–1322
47. Li X, Qi X, Zhou L (2007) Decreased expression of P2X7 in
endometrial epithelial pre-cancerous and cancer cells. Gynecol
Oncol 106:233–243
Purinergic Signalling
48. Feng YH, Li X, Wang L (2006) A truncated P2X7 receptor
variant (P2X7-j) endogenously expressed in cervical cancer cells
antagonizes the full-length P2X7 receptor through heterooligomerization.
J Biol Chem 281:17228–17237
49. Di Virgilio F, Chiozzi P, Ferrari D (2001) Nucleotide receptors:
an emerging family of regulatory molecules in blood cells. Blood
97:587–600
50. Watters J, Sommer J, Fisette P (2001) P2X7 nucleotide receptor:
modulation of LPS-induced macrophage signaling and mediator
production. Drug Dev Res 53:91–104
51. Lister MF, Sharkey J, Sawatzky DA (2007) The role of the
purinergic P2X7 receptor in inflammation. J Inflamm 4:5
52. Noguchi T, Ishii K, Fukutomi H (2008) Requirement of reactive
oxygen species-dependent activation of ASK1–p38 MAPK
pathway for extracellular ATP-induced apoptosis in macrophage.
J Biol Chem 283:7657–7665
53. Suh BC, Kim JS, Namgung U (2001) P2X7 nucleotide receptor
mediation of membrane pore formation and superoxide generation
in human promyelocytes and neutrophils. J Immunol
166:6754–6763
54. Di Virgilio F, Chiozzi P, Falzoni S (1998) Cytolytic P2X
purinoceptors. Cell Death Differ 5:191–199
55. Stefano L, Rossler OG, Griesemer D (2007) P2X(7) receptor
stimulation upregulates Egr-1 biosynthesis involving a cytosolic
Ca(2+) rise, transactivation of the EGF receptor and phosphorylation
of ERK and Elk-1. J Cell Physiol 213:36–44
 
56. Guerra AN, Gavala ML, Chung HS (2007) Nucleotide receptor
signalling and the generation of reactive oxygen species.
Purinergic Signal 3:39–51
57. Denu JM, Tanner KG (1998) Specific and reversible inactivation
of protein tyrosine phosphatases by hydrogen peroxide: evidence
for a sulfenic acid intermediate and implications for redox
regulation. Biochem 37:5633–5642
58. Guyton KZ, Liu Y, Gorospe M (1996) Activation of mitogenactivated
protein kinase by H2O2. Role in cell survival following
oxidant injury. J Biol Chem 271:4138–4142
59. Iles KE, Dickinson DA, Watanabe N (2002) AP-1 activation
through endogenous H(2)O(2) generation by alveolar macrophages.
Free Radic Biol Med 32:1304–1313
60. Kaul N, Forman HJ (1996) Activation of NF kappa B by the
respiratory burst of macrophages. Free Radic BiolMed 21:401–405
61. Lander HM (1997) An essential role for free radicals and derived
species in signal transduction. FASEB J 11:118–124
62. Lee K, Esselman WJ (2002) Inhibition of PTPs by H(2)O(2)
regulates the activation of distinct MAPK pathways. Free Radic
Biol Med 33:1121–1132
63. Lo YY, Wong JM, Cruz TF (1996) Reactive oxygen species
mediate cytokine activation of c-Jun NH2-terminal kinases. J
Biol Chem 271:15703–15707
64. Curtin JF, Donovan M, Cotter TG (2002) Regulation and
measurement of oxidative stress in apoptosis. J Immunol
Methods 265:49–72
65. Pines A, Perrone L, Bivi N (2005) Activation of APE1/Ref-1 is
dependent on reactive oxygen species generated after purinergic
receptor stimulation by ATP. Nucleic Acids Res 33:4379–4394
66. Adler V, Yin Z, Tew KD (1999) Role of redox potential and
reactive oxygen species in stress signaling. Oncogene 18:6104–
6111
67. Ferrari D, Wesselborg S, Bauer MK (1997) Extracellular ATP
activates transcription factor NF-kappaB through the P2Z
purinoreceptor by selectively targeting NF-kappaB p65. J Cell
Biol 139:1635–1643
68. Aga M, Watters JJ, Pfeiffer ZA (2004) Evidence for nucleotide
receptor modulation of cross talk between MAP kinase and NFkappa
B signaling pathways in murine RAW 264.7 macrophages.
Am J Physiol Cell Physiol 286:C923–C930
69. Aga MJ, Johnson CJ, Hart AP (2002) Modulation of monocytes
signaling and pore formation in response to agonists of the
nucleotide receptor P2X7. J Leukoc Biol 72:222–232
70. Di Virgilio F (1995) The P2Z purinoceptor: an intriguing role in
immunity, inflammation and cell death. Immunol Today 16:524–
528
 
71. Ferrari D, Chiozzi P, Falzoni S (1997) ATP-mediated cytotoxicity
in microglial cells. Neuropharmacology 36:1295–1301
72. Grahames CB, Michel AD, Chessell IP (1999) Pharmacological
characterization of ATP- and LPS-induced IL-1beta release in
human monocytes. Br J Pharmacol 127:1915–1921
73. Griffiths RJ, Stam EJ, Downs JT (1995) ATP induces the release
of IL-1 from LPS-primed cells in vivo. J Immunol 154:2821–
2828
74. Solle M, Labasi J, Perregaux DG (2001) Altered cytokine
production in mice lacking P2X(7) receptors. J Biol Chem
276:125–132
75. Tonetti M, Sturla L, Bistolfi T (1994) Extracellular ATP
potentiates nitric oxide synthase expression induced by lipopolysaccharide
in RAW 264.7 murine macrophages. Biochem
Biophys Res Commun 203:430–435
76. Tonetti M, Sturla L, Giovine M (1995) Extracellular ATP
enhances mRNA levels of nitric oxide synthase and TNF-alpha
in lipopolysaccharide-treated RAW 264.7 murine macrophages.
Biochem Biophys Res Commun 214:125–130
77. Cassatella MA, Bazzoni F, Flynn RM (1990) Molecular basis of
interferon-gamma and lipopolysaccharide enhancement of
phagocyte respiratory burst capability. Studies on the gene
expression of several NADPH oxidase components. J Biol Chem
265:20241–20246
78. Quinn MT, Gauss KA (2004) Structure and regulation of the
neutrophil respiratory burst oxidase: comparison with nonphagocyte
oxidases. J Leukoc Biol 76:760–781
79. Sheppard FR, Kelher MR, Moore EE (2005) Structural organization
of the neutrophil NADPH oxidase: phosphorylation and
translocation during priming and activation. J Leukoc Biol
78:1025–1042
80. DeLeo FR, Renee J, McCormick S (1998) Neutrophils exposed
to bacterial lipopolysaccharide upregulate NADPH oxidase
assembly. J Clin Invest 101:455–463
81. Vignais PV (2002) The superoxide-generating NADPH oxidase:
structural aspects and activation mechanism. Cell Mol Life Sci
59:1428–1459
82. Decoursey TE, Ligeti E (2005) Regulation and termination of
NADPH oxidase activity. Cell Mol Life Sci 62:2173–2193
83. Groemping Y, Rittinger K (2005) Activation and assembly of the
NADPH oxidase: a structural perspective. Biochem J 386:401–416
84. Budagian V, Bulanova E, Brovko L (2003) Signaling through
P2X7 receptor in human T cells involves p56lck, MAP kinases,
and transcription factors AP-1 and NF-kappa B. J Biol Chem
278:1549–1560
85. Armstrong S, Korcok J, Sims SM (2007) Activation of
transcription factors by extracellular nucleotides in immune and
related cell types. Purinergic Signal 3:59–69
86. Franco-Martinez S, Nino-Moreno P, Bernal-Silva S (2006)
Expression and function of the purinergic receptor P2X7 in
patients with pulmonary tuberculosis. Clin Exp Immunol
146:253–261
 
However, it is unclear if P2X7 activation can synergize with
other Toll-like receptor (TLR) systems besides TLR4
(which is an LPS receptor). Thus, we have now tested the
capacity of P2X7 ligands to cooperate with other TLR
ligands in the generation of ROS, and as shown in Fig. 2b,
we detect synergistic ROS production after P2X7 agonist
treatment of macrophages primed with other TLR ligands,
namely the lipopeptide Pam3CSK4 (a TLR2/1 ligand) and
zymosan (a TLR2/6 ligand). These data further implicate
P2X7 activation and signaling as a fundamental modulator
of macrophage immune responses in response to multiple
TLR ligands.
Although ROS can be generated by various enzyme
systems, including NADPH oxidase, nitric oxide synthases,
xanthine oxidase, and the mitochondrial respiratory
chain, we first tested the hypothesis that the major
system controlling O2
− production by P2X7 agonists in
macrophages is NADPH oxidase. The NADPH oxidase
complex is comprised of several differentially regulated
subunits that accept an electron from NADPH and donate it
to molecular oxygen (see [78, 79] for comprehensive
reviews). A link between NADPH oxidase activation and
P2X7 signaling in monocytic cells appears plausible in that
Parvathenani et al. demonstrated that P2X7 agonists can
modulate the localization of the NADPH oxidase subunit
p67phox in microglia [16], and Noguchi et al. found that
P2X7 agonist-induced ROS production is sensitive to the
NADPH oxidase inhibitor apocynin and can be attenuated
in RAW 264.7 macrophages by reducing the expression of
the NADPH subunit gp91phox [52]. Similarly, we have
Purinergic Signalling
found that P2X7 agonist-induced JNK phosphorylation in
RAW macrophages is sensitive to the NADPH oxidase
inhibitor diphenyliodonium and that ROS production
stimulated by P2X7 agonists in LPS-primed RAW 264.7
cells is sensitive to the NADPH oxidase inhibitor apocynin
(data not shown).
Many stimuli, including LPS, phorbol 12-myristate 13-
acetate (PMA), and zymosan, increase the expression of
NADPH oxidase subunits in leukocytes and can induce
their spatiotemporal assembly and activation [77, 78, 80].
To further characterize P2X7 modulation of the NADPH
oxidase complex in macrophages, we assessed the effect of
P2X7 agonists on the phosphorylation of a cytosolic subunit
of NADPH oxidase, p40phox. As shown in Fig. 3, treatment
of RAW macrophages with BzATP or high doses of ATP
results in p40phox phosphorylation, and this process is
enhanced after LPS priming. These stimuli had no
detectable effect on total p40phox levels (data not shown).
To test the receptor specificity, we evaluated p40phox
phosphorylation in response to the administration of P2X7
agonists to a RAW 264.7 macrophage cell line expressing a
non-functional P2X7 mutant. We found that although
treatment with PMA was able to induce p40phox phosphorylation
in the P2X7 mutant cell line, P2X7 activity was
critical for BzATP/ATP-induced phosphorylation of p40phox
(Fig. 3). Further support for P2X7 involvement in p40phox
phosphorylation comes from the observation that agonists
(UTP, UDP, ADP, and low ATP doses) selective for other
nucleotide receptors did not stimulate p40phox phosphorylation
(data not shown), illustrating that the pharmacological
profile of p40phox phosphorylation is consistent with
P2X7 stimulation. Together, these data reveal that P2X7
stimulates ROS production in a manner that is concomitant
with the phosphorylation of NADPH oxidase subunits.
Many protein kinases have been implicated in the
stimulus-induced assembly and activation of the NADPH
oxidase complex, including protein kinase C (PKC) isoforms,
MAPKs, and p21-activated kinase [78, 79, 81–83].
Given that P2X7 activation results in the phosphorylation of
NADPH oxidase subunits and subsequent ROS production,
we examined the involvement of the MEK–ERK1/2 MAPK
network in P2X7-stimulated ROS production. To this end,
RAW macrophages were pretreated with increasing concentrations
of U0126, a MEK1/2 inhibitor, followed by
stimulation with BzATP, and then ROS production was
assessed by flow cytometry. We found that U0126
pretreatment is able to decrease P2X7 agonist-stimulated
ROS production and greatly attenuate the enhanced ROS
production found in P2X7 agonist-stimulated LPS-primed
macrophages (Fig. 4). Conversely, general antagonists of
several PKC isoforms (such as Go6976 and rottlerin), as
well as p38 MAPK (i.e., SB203580), had little effect
(0–3%) on P2X7 agonist-stimulated ROS levels in LPSprimed
macrophages. These data support the concept that
the MEK/ERK pathway is critical for P2X7-induced ROS
production (Fig. 4).
P2X7-mediated transcription factor activation
Activation of P2X7 has been linked not only to apoptosis,
but also to lymphocyte cell proliferation [25, 84] and
transcriptional activation in immune cells [85]. As noted
Fig. 2 The P2X7 agonist BzATP stimulates ROS production in
primary human monocytic cells. a RAW 264.7 cells (n=12) were
cultured as described [21] and primary human monocytes (n=5) were
purified and cultured as outlined earlier [105]. The cells were first
treated with HEPES buffer (control) or 1 μg/ml LPS (E. coli, serotype
0111:B4) for 10 h, and then transferred to flow cytometry tubes (5×
105 cells/tube) and loaded with 10 μM 2′,7′-dichloro-dihydrofluorescein
diacetate (DCFDA) for 30 min at 37°C. The cells were then
treated with either HEPES buffer (control) or 250 μM BzATP for
30 min. DCFDA fluorescence was assessed using flow cytometry as
detailed previously [21]. The data are plotted as geometric mean
(±SEM) of DCFDA fluorescence. b RAW 264.7 cells were first
treated with HEPES buffer (control), 1 μg/ml LPS, 1 μg/ml
PamCSK4, or 250 μg/ml zymosan for 10 h, and then transferred to
flow cytometry tubes (5×105 cells/tube) and loaded with 10 μM
DCFDA for 30 min at 37°C. The cells were then treated with either
HEPES (control) or 250 μM BzATP for 30 min. DCFDA fluorescence
was assessed using flow cytometry. The data are plotted as the mean
(±SEM) fold increase over control (n=3). *p<0.05, **p<0.005,
***p<0.001, and &#968;p<0.0001 as compared to control pretreated,
control-induced DCFDA fluorescence
Purinergic Signalling
above, several studies have implicated P2X7 activation in
regulating the processing and release of interleukin family
members and the promotion of iNOS expression. A recent
mRNA microarray analysis comparing P2X7 agonist-treated
peripheral blood mononuclear cells (PBMCs) from
tuberculosis patients with P2X7 agonist-treated cells from
control patients revealed differential gene expression,
suggesting a potentially complex role of P2X7 in gene
regulation [86]. Thus, although P2X7 stimulation appears to
affect gene expression, the mechanisms by which these
events occur are not well-defined. In this regard, a few
studies evaluating the mechanisms of P2X7-induced modulation
of gene expression have suggested a role for several
transcription factors including NF&#954;B, nuclear factor of
activated T cells, signal transducers and activators of
transcription [85], and, as discussed below, CREB and
AP-1.
The transcription factor CREB is a ubiquitously
expressed protein that contributes to many cellular processes
such as glucose homeostasis, growth factor-dependent
cell survival, and inflammatory mediator production [87].
In general, CREB mediates the activation of cAMPresponsive
genes by binding as a dimer to conserved
CREB response elements (CREs) (5&#8242;-TGACGTCA-3&#8242😉 in
the promoter regions of target genes. The activation of
CREB requires the phosphorylation of the protein at Ser-
133 (S133), which promotes the recruitment of the
transcriptional co-activator CREB-binding protein (CBP)
and p300, thereby enabling gene transcription [87, 88].
CREB regulation is recognized to play a role in
leukocyte function [88], yet little is known concerning the
effect of P2X7 stimulation on CREB activation, although
Potucek et al. observed BzATP-induced CREB phosphorylation
in murine microglial cells [89]. In the present report,
we show that stimulation of primary human PBMCs and
RAW 264.7 with P2X7 agonists (BzATP and high doses of
ATP) induces the rapid and transient phosphorylation of
CREB (at Ser-133) and the related factor activating
transcription factor-1 (ATF-1; Fig. 5a, b). As shown in
Fig. 5b, CREB phosphorylation in murine macrophages is
detectable within 5 min of ligand addition, it occurs more
rapidly than in response to LPS, and it is complete within
1 h. In addition, we have observed: (a) a lack of nucleotideinduced
CREB phosphorylation in RAW 264.7 cells
expressing a non-functional P2X7, (b) a gain of nucleotide-
induced CREB phosphorylation in HEK293 cells that
heterologously express human P2X7, and (c) the induction
of CREB/CBP complex formation (which is necessary for
Fig. 3 P2X7 agonists promote p40phox phosphorylation in murine
macrophages. RAW 264.7 cells expressing either wild-type or
defective P2X7 were pretreated with either HEPES buffer (control)
or 1 &#956;g/ml LPS for 10 h. The cells were then treated with either
HEPES (control), 1 &#956;g/ml LPS, 250 &#956;M BzATP, or 3 mM ATP for
30 min or 1 &#956;g/ml PMA for 10 min. Left panel: a phosphorylation of
p40phox in murine macrophages expressing wild-type P2X7 and treated
with either 1 &#956;g/ml PMA (as a positive control) or 250 &#956;M BzATP.
The results of nine independent experiments were combined and are
presented as the mean (±SEM). b Phosphorylation of p40phox in
murine macrophages expressing wild-type P2X7 and treated with
3 mM ATP. The results were obtained from six independent experiments
and plotted as the mean (±SEM). *p<0.05 as compared to
control pretreated, control-induced p40phox phosphorylation. Right
panel: a representative immunoblot displaying p40phox phosphorylation
in response to the indicated stimuli is shown for RAW 264.7 cells
expressing either wild-type (upper blot) or defective P2X7 (lower
blot). Immunoblotting was performed with anti-Grb2 antibody as a
loading control
Purinergic Signalling
CREB transcriptional activation) in macrophages treated
with P2X7 agonists (data not shown, Gavala et al., in
preparation). Altogether these data suggest that P2X7
stimulation modulates monocytic gene expression through
the activation of CREB.
Activating protein-1 is a collective term for dimeric
transcription factors composed of Jun, Fos, or ATF subunits
that bind to a common 12-O-tetradecanoylphorbol-13-
acetate response elements (5&#8242;-TGAGTCA-3&#8242😉 found in the
promoter regions of many inflammatory cytokines and
chemokines [90, 91]. Previous evidence for AP-1 activation
by extracellular ATP comes from studies examining B
lymphocytes. In these experiments, administration of
extracellular ATP to human tonsilar B lymphocytes was
found to increase cFos mRNA levels within 30 min [92].
Furthermore, co-treatment of human fetal astrocytes with
IL-1&#946; and ATP was seen to induce an activation of AP-1
that is more robust than that observed with IL-1&#946; alone
[93], and treatment with P2 receptor antagonists inhibits
cytokine-induced AP-1 activation [93, 94]. It has been
suggested that stimulation of Jurkat T cells induces AP-1
DNA-binding activity that results from increased c-Jun and
cFos expression [84]. Conversely, in the present report, we
show that stimulation of human PBMCs with P2X7
agonists does not appear to enhance cFos expression
(Fig. 6a) but does substantially induce the expression of
the FosB and JunB proteins (Fig. 6b, c). These results are
the first report linking the activation of a nucleotide
receptor to FosB and JunB induction. Interestingly, the
effect of P2X7 activation on FosB levels is detectable
within 1 h of treatment with BzATP alone and suggests that
FosB may be a unique transcriptional regulator of P2X7-
initiated signaling. Electrophoretic mobility shift assays
(EMSAs) in BzATP-treated RAW 264.7 cells reveal an
increase in protein binding to an AP-1 consensus oligonucleotide
compared to vehicle treatment, and this AP-1
complex is supershifted with antibodies directed toward
FosB, JunB, and c-Jun (not shown, Gavala et al., in
preparation). However, stimulation of P2X7 does not induce
AP-1 activation in all phenotypic backgrounds. Stimulation
of N9 and N13 microglial cells with 3 mM ATP inhibits the
basal DNA-binding activity of AP-1, suggesting that P2X7-
Fig. 4 The MEK/ERK cascade appears critical for P2X7-induced
ROS production. RAW 264.7 cells were treated with either HEPES
buffer (control) or 1 &#956;g/ml LPS for 10 h, and then transferred to flow
cytometry tubes (5×105 cells/tube) and loaded with 10 &#956;M DCFDA
for 30 min at 37°C. The cells were then pretreated with DMSO or
increasing concentrations of UO126 (Calbiochem, San Diego, CA,
USA; 130 &#956;M) for 30 min at 37°C. Cells were then treated with either
HEPES (control) or 250 &#956;M BzATP for 30 min. Flow cytometry was
performed by measuring 10,000 cells and excluding propidium iodidestained
(dead) cells from the analysis. The data are plotted as the mean
(±SEM) fold increase over control (n=3). *p<0.01, **p<0.005 as
compared to LPS-pretreated, BzATP-induced DCFDA fluorescence
Fig. 5 P2X7 agonists induce CREB Ser-133 phosphorylation in
monocytic cells. a Isolated peripheral human blood monocytes (1×106
cells/well) were treated with either vehicle (HEPES buffer), 1 mM
ATP, 250 &#956;M BzATP, or 1 &#956;g/ml LPS for 15 min. A representative
immunoblot displaying CREB Ser-133 phosphorylation (pCREB) is
shown (n=4). The pCREB antibody recognizes both pCREB and
phosphorylated ATF-1 (pATF1), thus it is also shown. Immunoblotting
was performed with anti-Grb2 antibody as a loading control. b Murine
RAW 264.7 macrophages (3×105 cells/well) were stimulated with
either vehicle (HEPES), 250 &#956;M BzATP or 1 &#956;g/ml LPS for the
indicated time. A representative immunoblot displaying pCREB/
pATF1 is shown. Immunoblotting was performed with anti-&#946;-tubulin
antibody as a loading control. Results of the three independent
experiments were combined and plotted as the mean (±SEM)
Purinergic Signalling
mediated AP-1 activation is cell type-specific (data not
shown).
The activation of certain protein kinases by P2X7
agonists has been implicated in subsequent AP-1 activation
in several immune cells. Stimulation of Jurkat T cells with
P2X7 agonists induces the activation of the lymphoidspecific
cytoplasmic protein tyrosine kinase p56lck [84].
Although the activation of cFos and c-Jun by P2X7 ligands
occurs in Jurkat T cells, it is absent in a p56lck-deficient
JCaM1T cell line [84], suggesting that this kinase may play
a role in P2X7-induced AP-1 activation in T cells. A role for
MAPKs in P2X7-dependent AP-1 activation in monocytic
cells has also been supported. The transactivation of c-Jun
is augmented by the phosphorylation of Ser-63 and -73 by
JNK1/2. Studies from our lab and others have noted JNK1/
2 activation following stimulation of monocytic cells with
P2X7 agonists [21, 68, 95]. Furthermore, co-stimulation of
RAW 264.7 cells with LPS and BzATP results in enhanced
JNK activation compared to either stimulus alone [68]. Of
note, P2X7 agonist-induced JNK activation in RAW 264.7
cells is attenuated by N-acetylcysteine and ascorbic acid,
implicating a role for ROS in P2X7-dependent AP-1
activation [21]. In addition, we have observed that P2X7
agonist-induced FosB and JunB expression in RAW 264.7
macrophages is abrogated in a dose-dependent fashion by
the MEK1/2 antagonist UO126, suggesting that the MEK/
ERK cascade is upstream of the expression of these AP-1
proteins in macrophages (Gavala et al., in preparation).
Although reporter assays and EMSAs have often been
used to demonstrate activation of transcription factors by
P2X7 agonists, little is known about the target genes of
these transcription factors. To this end, preliminary studies
by our lab have suggested a novel role for CREB in P2X7-
Fig. 6 P2X7 agonists induce AP-1 protein expression in primary
human monocytes. Isolated peripheral human blood monocytes were
stimulated with either vehicle (HEPES), 250 &#956;M BzATP, 1 &#956;g/ml
PMA, or 10 &#956;M ionomycin (c) for 5 min. The media was replaced and
the cells were incubated at 37°C for the indicated times (PMA
treatment was reintroduced to the new media) and immunoblotted for
a cFos, b FosB, or c JunB expression. Immunoblotting was performed
with anti-Grb2 antibody as a loading control. The results are
representative of three independent experiments
Fig. 7 Mutation of two arginine residues in the P2X7 C-terminal
region reduces receptor-mediated pore-forming activity. a The distal
C-terminal sequences of mouse, rat, and human P2X7 were aligned.
The proposed Arg-based ER retention signal is underlined, and
Arg578 and Lys579 are highlighted in red. b COS7 cells were
transfected with pcDNA3, P2X7/pcDNA3, or P2X7 R576A/R578A/
pcDNA3. Sixteen to 24 h later, the cells were treated with 250 &#956;M
BzATP in the presence of 1 &#956;M YO-PRO-1 (Molecular Probes,
Eugene, OR, USA) for 20 min at room temperature. The cells were
fixed with 4% paraformaldehyde, washed, and imaged using a Zeiss
Axioplan 2 microscope. These data represent one of five independent
experiments. P2X7 R576A/R578A/pcDNA3 was generated from
P2X7/pcDNA3 (accession number BC011913) by site-directed mutagenesis.
c COS7 cells were transfected with eYFP-P2X7/pCMV, fixed
the next day with 4% paraformaldehyde, imaged using a Zeiss 200M
Axiovert inverted microscope, and deconvolved. P2X7 was subcloned
into the plasmid eYFP/pCMV, which was a generous gift from Dr.
Melanie Cobb (University of Texas Southwestern Medical Center)
Purinergic Signalling
mediated FosB expression, i.e., transfection of dominantnegative
CREB vectors into HEK293 cells heterologously
expressing P2X7 abrogates P2X7 agonist-induced FosB
expression (Gavala et al., in preparation).
P2X7 contains a trafficking domain in its C-terminus
The intracellular trafficking of plasma membrane receptors is a
critical component of their regulation. P2X7 trafficking has not
been widely explored, and it is unclear how newly synthesized
P2X7 subunits are transported from the endoplasmic
reticulum (ER) to the surface and whether P2X7 internalization
or trafficking to the plasma membrane is controlled by
extracellular signals. It is also not clear whether P2X7 recycles
following internalization or whether the receptor contains
signals within its sequence that regulate its transport through
the cell. Addressing these issues will increase our understanding
of how P2X7 is globally regulated and how cells
mediate their responsiveness to extracellular ATP.
Our lab and those of Wiley, Petrou, and colleagues have
provided evidence supporting the concept that P2X7
contains a trafficking domain in its distal C-terminal region
that is critical for cell surface localization. Point mutations
and deletions within this region reduce P2X7 surface
expression and pore activity [30, 34, 96]. When P2X7 is
truncated at residues 551&#8211;581, it exhibits significantly
reduced activity and plasma membrane localization [30],
and three polymorphisms, I568N, R574H, and R574L, have
been identified in this area of the receptor [34, 42, 97]. T
lymphocytes and natural killer cells from individuals who
are heterozygous for the I568N polymorphism have
reduced ATP-induced pore-forming ability, and exogenously
expressed P2X7 with the I568N mutation is poorly
expressed on the surface of HEK293 cells [34]. We have
previously found that mutation of Arg578 and Lys579,
which are localized in the LPS-binding motif (Fig. 1),
drastically reduces the ability of P2X7 to localize on the
plasma membrane and to promote BzATP-induced cell
death [96]. Although the P2X7 R578E/K579E mutant does
not appear to be stably expressed on the cell surface at 37°C,
it does localize to the plasma membrane at 27°C, suggesting
that the double mutant is internalized more readily than wildtype
receptor. These findings lead us to speculate that P2X7
contains specific signals within its C-terminus that direct its
intracellular trafficking.
One aspect of P2X7 trafficking that is less well
characterized entails the mechanisms by which it is transported
from the ER to the cell surface. Several studies have
shown that newly synthesized subunits of integral membrane
proteins, including N-methyl-D-aspartate (NMDA),
glutamate receptor, and &#947;-aminobutyric acid type B
(GABAB) receptor, are retained in the ER before the
properly assembled receptors and are further transported
through the secretory pathway [98]. The NMDA and
GABAB subunits possess arginine (Arg)-based ER retention/
retrieval signals that prevent improperly assembled
subunits from trafficking to the Golgi apparatus until
assembly is complete and the signal is masked [99, 100].
These ER retention/retrieval sequences were first discovered
in major histocompatibility complex class II proteins
and have also been characterized in inward rectifier
Fig. 8 Proposed model of several
of the key events by which
P2X7 promotes the production
of immune mediators and activates
gene expression. Stimulation
of P2X7 leads to enhanced
ROS formation via the MAPKs
ERK1/2 and the NADPH oxidase
complex and stimulates
gene transcription by activating
CREB and increasing the expression
of the AP-1 proteins
FosB and JunB. In addition,
specific residues in the putative
trafficking domain of P2X7 are
required for proper localization
and full activation of the
receptor
Purinergic Signalling
potassium channel and sulfonylurea receptor subunits [101,
102]. The Arg-based ER retention/retrieval consensus
sequence is &#981;/&#968;/R-R-X-R, where &#981;/&#968; is an aromatic or
bulky hydrophobic residue and X is any amino acid but is
generally not a negatively charged or small, non-polar
residue [98].
In view of the above discussion, we predict that P2X7
contains a RXR retention/retrieval signal at amino acid 576
that lies within the sequence WRIR, which conforms to the
consensus sequence &#981;/&#968;/R-R-X-R (Fig. 7a). To test the
hypothesis that P2X7 contains an RXR ER retention/
retrieval sequence at residue 576, a pore activity assay
was conducted as an indirect readout for cell surface
expression. In this experiment, COS7 cells were transfected
with wild-type or mutant P2X7, stimulated with BzATP, and
imaged for relative dye uptake as a measure of liganddependent
pore formation. As shown in Fig. 7b, mutation
of human R576 and R578 to Ala substantially reduces the
activity of the receptor. Cells take up the dye YO-PRO-1
when wild-type P2X7 is expressed but exhibit drastically
reduced dye uptake in the presence of the RXR mutant or
the vector control. These findings, together with the
information available about Arg-based signals in other
receptors, provide initial support for the idea that P2X7
contains an RXR ER retention/retrieval sequence in its Cterminus
in the area surrounding residue 576.
Although our previous findings revealing that the
P2X7 mutant R578E/K579E does not express on the cell
surface seems counterintuitive to the idea that P2X7
possesses an ER retention/retrieval sequence, we speculate
that cells prematurely traffic the R578E/K579E P2X7
mutant from the ER before it is properly assembled, and
once it reaches the plasma membrane, it becomes quickly
down-regulated because it is non-functional or lacks
appropriate interaction with specific lipids. Alternatively,
the P2X7 R578E/K579E mutation and not the mutation of
an RXR signal prevents P2X7 from localizing to the cell
surface. In this regard, Marshall and colleagues have
shown that a RK/AA mutation in the C-terminus of the
GluR5-2b kainate receptor subunit disrupts ER retention
[103].
Another unresolved issue is whether the trafficking of
P2X7 from intracellular compartments to the plasma
membrane is stimulated by extracellular signals, as it is
for the major insulin-responsive glucose transporter
GLUT4, and has been reported for the NADPH oxidase
complex [79, 80, 104]. We analyzed the localization of
P2X7 fused to enhanced yellow fluorescent protein (eYFP)
and found that it is predominantly localized in intracellular
pools (Fig. 7c). We are now in a position to explore the
critical hypothesis that the trafficking of P2X7 to and from
these intracellular pools to the plasma membrane is
stimulated by extracellular signals.
Summary
The ionotrophic receptor P2X7 is thought to be a major
immune modulator that responds to extracelluar ATP at
sites of inflammation and tissue damage. Although several
human genetic and animal studies have demonstrated the
importance of P2X7 in immune function, many questions
remain concerning P2X7 signaling and the regulation of its
activity via intracellular trafficking. We have investigated
the mechanisms by which P2X7 promotes inflammatory
 
87. Shaywitz AJ, Greenberg ME (1999) CREB: a stimulus-induced
transcription factor activated by a diverse array of extracellular
signals. Annu Rev Biochem 68:821–861
88. Mayr B, Montminy M (2001) Transcriptional regulation by the
phosphorylation-dependent factor CREB. Nat Rev Mol Cell Biol
2:599–609
89. Potucek YD, Crain JM, Watters JJ (2006) Purinergic receptors
modulate MAP kinases and transcription factors that control
Purinergic Signalling
microglial inflammatory gene expression. Neurochem Int
49:204–214
90. Desmet C, Gosset P, Henry E (2005) Treatment of experimental
asthma by decoy-mediated local inhibition of activator protein-1.
Am J Respir Crit Care Med 172:671–678
91. Guo RF, Lentsch AB, Sarma JV (2002) Activator protein-1
activation in acute lung injury. Am J Pathol 161:275–282
92. Padeh S, Cohen A, Roifman CM (1991) ATP-induced activation
of human B-lymphocytes via P2-purinoceptors. J Immunol
146:1626–1632
93. John GR, Simpson JE, Woodroofe MN (2001) Extracellular
nucleotides differentially regulate interleukin-1beta signaling in
primary human astrocytes: implications for inflammatory gene
expression. J Neurosci 21:4134–4142
94. Liu JS, John GR, Sikora A (2000) Modulation of interleukin-
1beta and tumor necrosis factor alpha signaling by P2 purinergic
receptors in human fetal astrocytes. J Neurosci 20:5292–5299
95. Humphreys BD, Rice J, Kertesy SB (2000) Stress-activated
protein kinase/JNK activation and apoptotic induction by the
macrophage P2X7 nucleotide receptor. J Biol Chem 275:26792–
26798
96. Denlinger LC, Sommer JA, Parker K (2003) Mutation of a
dibasic amino acid motif within the C terminus of the P2X7
nucleotide receptor results in trafficking defects and impaired
function. J Immunol 171:1304–1311
97. Fernando SL, Saunders BM, Sluyter R (2005) Gene dosage
determines the negative effects of polymorphic alleles of the
P2X7 receptor on adenosine triphosphate-mediated killing of
mycobacteria by human macrophages. J Infect Dis 192:149–155
98. Michelsen K, Yuan H, Schwappach B (2005) Hide and run.
Arginine-based endoplasmic-reticulum-sorting motifs in the
assembly of heteromultimeric membrane proteins. EMBO Rep
6:717–722
99. Margeta-Mitrovic M, Jan YN, Jan LY (2000) A trafficking
checkpoint controls GABA(B) receptor heterodimerization.
Neuron 27:97–106
 
100. Standley S, Roche KW, McCallum J (2000) PDZ domain
suppression of an ER retention signal in NMDA receptor NR1
splice variants. Neuron 28:887&#8211;898
101. Schutze MP, Peterson PA, Jackson MR (1994) An N-terminal
double-arginine motif maintains type II membrane proteins in the
endoplasmic reticulum. EMBO J 13:1696&#8211;1705
102. Zerangue N, Schwappach B, Jan YN (1999) A new ER
trafficking signal regulates the subunit stoichiometry of plasma
membrane K(ATP) channels. Neuron 22:537&#8211;548
103. Ren Z, Riley NJ, Needleman LA (2003) Cell surface expression
of GluR5 kainate receptors is regulated by an endoplasmic
reticulum retention signal. J Biol Chem 278:52700&#8211;52709
104. Hou JC, Pessin JE (2007) Ins (endocytosis) and outs (exocytosis)
of GLUT4 trafficking. Curr Opin Cell Biol 19:466&#8211;473
105. Korpi-Steiner NL, Bates ME, Lee WM (2006) Human rhinovirus
induces robust IP-10 release by monocytic cells, which is independent
of viral replication but linked to type I interferon receptor
ligation and STAT1 activation. J Leukoc Biol 80:1364&#8211;1374
Purinergic Signalling
 
here's some more, rice.

SDN2013 said:
However, it is unclear if P2X7 activation can synergize with
other Toll-like receptor (TLR) systems besides TLR4
(which is an LPS receptor). Thus, we have now tested the
capacity of P2X7 ligands to cooperate with other TLR
ligands in the generation of ROS, and as shown in Fig. 2b,
we detect synergistic ROS production after P2X7 agonist
treatment of macrophages primed with other TLR ligands,
namely the lipopeptide Pam3CSK4 (a TLR2/1 ligand) and
zymosan (a TLR2/6 ligand). These data further implicate
P2X7 activation and signaling as a fundamental modulator
of macrophage immune responses in response to multiple
TLR ligands.
Although ROS can be generated by various enzyme
systems, including NADPH oxidase, nitric oxide synthases,
xanthine oxidase, and the mitochondrial respiratory
chain, we first tested the hypothesis that the major
system controlling O2
&#8722; production by P2X7 agonists in
macrophages is NADPH oxidase. The NADPH oxidase
complex is comprised of several differentially regulated
subunits that accept an electron from NADPH and donate it
to molecular oxygen (see [78, 79] for comprehensive
reviews). A link between NADPH oxidase activation and
P2X7 signaling in monocytic cells appears plausible in that
Parvathenani et al. demonstrated that P2X7 agonists can
modulate the localization of the NADPH oxidase subunit
p67phox in microglia [16], and Noguchi et al. found that
P2X7 agonist-induced ROS production is sensitive to the
NADPH oxidase inhibitor apocynin and can be attenuated
in RAW 264.7 macrophages by reducing the expression of
the NADPH subunit gp91phox [52]. Similarly, we have
Purinergic Signalling
found that P2X7 agonist-induced JNK phosphorylation in
RAW macrophages is sensitive to the NADPH oxidase
inhibitor diphenyliodonium and that ROS production
stimulated by P2X7 agonists in LPS-primed RAW 264.7
cells is sensitive to the NADPH oxidase inhibitor apocynin
(data not shown).
Many stimuli, including LPS, phorbol 12-myristate 13-
acetate (PMA), and zymosan, increase the expression of
NADPH oxidase subunits in leukocytes and can induce
their spatiotemporal assembly and activation [77, 78, 80].
To further characterize P2X7 modulation of the NADPH
oxidase complex in macrophages, we assessed the effect of
P2X7 agonists on the phosphorylation of a cytosolic subunit
of NADPH oxidase, p40phox. As shown in Fig. 3, treatment
of RAW macrophages with BzATP or high doses of ATP
results in p40phox phosphorylation, and this process is
enhanced after LPS priming. These stimuli had no
detectable effect on total p40phox levels (data not shown).
To test the receptor specificity, we evaluated p40phox
phosphorylation in response to the administration of P2X7
agonists to a RAW 264.7 macrophage cell line expressing a
non-functional P2X7 mutant. We found that although
treatment with PMA was able to induce p40phox phosphorylation
in the P2X7 mutant cell line, P2X7 activity was
critical for BzATP/ATP-induced phosphorylation of p40phox
(Fig. 3). Further support for P2X7 involvement in p40phox
phosphorylation comes from the observation that agonists
(UTP, UDP, ADP, and low ATP doses) selective for other
nucleotide receptors did not stimulate p40phox phosphorylation
(data not shown), illustrating that the pharmacological
profile of p40phox phosphorylation is consistent with
P2X7 stimulation. Together, these data reveal that P2X7
stimulates ROS production in a manner that is concomitant
with the phosphorylation of NADPH oxidase subunits.
Many protein kinases have been implicated in the
stimulus-induced assembly and activation of the NADPH
oxidase complex, including protein kinase C (PKC) isoforms,
MAPKs, and p21-activated kinase [78, 79, 81&#8211;83].
Given that P2X7 activation results in the phosphorylation of
NADPH oxidase subunits and subsequent ROS production,
we examined the involvement of the MEK&#8211;ERK1/2 MAPK
network in P2X7-stimulated ROS production. To this end,
RAW macrophages were pretreated with increasing concentrations
of U0126, a MEK1/2 inhibitor, followed by
stimulation with BzATP, and then ROS production was
assessed by flow cytometry. We found that U0126
pretreatment is able to decrease P2X7 agonist-stimulated
ROS production and greatly attenuate the enhanced ROS
production found in P2X7 agonist-stimulated LPS-primed
macrophages (Fig. 4). Conversely, general antagonists of
several PKC isoforms (such as Go6976 and rottlerin), as
well as p38 MAPK (i.e., SB203580), had little effect
(0&#8211;3%) on P2X7 agonist-stimulated ROS levels in LPSprimed
macrophages. These data support the concept that
the MEK/ERK pathway is critical for P2X7-induced ROS
production (Fig. 4).
P2X7-mediated transcription factor activation
Activation of P2X7 has been linked not only to apoptosis,
but also to lymphocyte cell proliferation [25, 84] and
transcriptional activation in immune cells [85]. As noted
Fig. 2 The P2X7 agonist BzATP stimulates ROS production in
primary human monocytic cells. a RAW 264.7 cells (n=12) were
cultured as described [21] and primary human monocytes (n=5) were
purified and cultured as outlined earlier [105]. The cells were first
treated with HEPES buffer (control) or 1 &#956;g/ml LPS (E. coli, serotype
0111:B4) for 10 h, and then transferred to flow cytometry tubes (5×
105 cells/tube) and loaded with 10 &#956;M 2&#8242;,7&#8242;-dichloro-dihydrofluorescein
diacetate (DCFDA) for 30 min at 37°C. The cells were then
treated with either HEPES buffer (control) or 250 &#956;M BzATP for
30 min. DCFDA fluorescence was assessed using flow cytometry as
detailed previously [21]. The data are plotted as geometric mean
(±SEM) of DCFDA fluorescence. b RAW 264.7 cells were first
treated with HEPES buffer (control), 1 &#956;g/ml LPS, 1 &#956;g/ml
PamCSK4, or 250 &#956;g/ml zymosan for 10 h, and then transferred to
flow cytometry tubes (5×105 cells/tube) and loaded with 10 &#956;M
DCFDA for 30 min at 37°C. The cells were then treated with either
HEPES (control) or 250 &#956;M BzATP for 30 min. DCFDA fluorescence
was assessed using flow cytometry. The data are plotted as the mean
(±SEM) fold increase over control (n=3). *p<0.05, **p<0.005,
***p<0.001, and &#968;p<0.0001 as compared to control pretreated,
control-induced DCFDA fluorescence
Purinergic Signalling
above, several studies have implicated P2X7 activation in
regulating the processing and release of interleukin family
members and the promotion of iNOS expression. A recent
mRNA microarray analysis comparing P2X7 agonist-treated
peripheral blood mononuclear cells (PBMCs) from
tuberculosis patients with P2X7 agonist-treated cells from
control patients revealed differential gene expression,
suggesting a potentially complex role of P2X7 in gene
regulation [86]. Thus, although P2X7 stimulation appears to
affect gene expression, the mechanisms by which these
events occur are not well-defined. In this regard, a few
studies evaluating the mechanisms of P2X7-induced modulation
of gene expression have suggested a role for several
transcription factors including NF&#954;B, nuclear factor of
activated T cells, signal transducers and activators of
transcription [85], and, as discussed below, CREB and
AP-1.
The transcription factor CREB is a ubiquitously
expressed protein that contributes to many cellular processes
such as glucose homeostasis, growth factor-dependent
cell survival, and inflammatory mediator production [87].
In general, CREB mediates the activation of cAMPresponsive
genes by binding as a dimer to conserved
CREB response elements (CREs) (5&#8242;-TGACGTCA-3&#8242😉 in
the promoter regions of target genes. The activation of
CREB requires the phosphorylation of the protein at Ser-
133 (S133), which promotes the recruitment of the
transcriptional co-activator CREB-binding protein (CBP)
and p300, thereby enabling gene transcription [87, 88].
CREB regulation is recognized to play a role in
leukocyte function [88], yet little is known concerning the
effect of P2X7 stimulation on CREB activation, although
Potucek et al. observed BzATP-induced CREB phosphorylation
in murine microglial cells [89]. In the present report,
we show that stimulation of primary human PBMCs and
RAW 264.7 with P2X7 agonists (BzATP and high doses of
ATP) induces the rapid and transient phosphorylation of
CREB (at Ser-133) and the related factor activating
transcription factor-1 (ATF-1; Fig. 5a, b). As shown in
Fig. 5b, CREB phosphorylation in murine macrophages is
detectable within 5 min of ligand addition, it occurs more
rapidly than in response to LPS, and it is complete within
1 h. In addition, we have observed: (a) a lack of nucleotideinduced
CREB phosphorylation in RAW 264.7 cells
expressing a non-functional P2X7, (b) a gain of nucleotide-
induced CREB phosphorylation in HEK293 cells that
heterologously express human P2X7, and (c) the induction
of CREB/CBP complex formation (which is necessary for
Fig. 3 P2X7 agonists promote p40phox phosphorylation in murine
macrophages. RAW 264.7 cells expressing either wild-type or
defective P2X7 were pretreated with either HEPES buffer (control)
or 1 &#956;g/ml LPS for 10 h. The cells were then treated with either
HEPES (control), 1 &#956;g/ml LPS, 250 &#956;M BzATP, or 3 mM ATP for
30 min or 1 &#956;g/ml PMA for 10 min. Left panel: a phosphorylation of
p40phox in murine macrophages expressing wild-type P2X7 and treated
with either 1 &#956;g/ml PMA (as a positive control) or 250 &#956;M BzATP.
The results of nine independent experiments were combined and are
presented as the mean (±SEM). b Phosphorylation of p40phox in
murine macrophages expressing wild-type P2X7 and treated with
3 mM ATP. The results were obtained from six independent experiments
and plotted as the mean (±SEM). *p<0.05 as compared to
control pretreated, control-induced p40phox phosphorylation. Right
panel: a representative immunoblot displaying p40phox phosphorylation
in response to the indicated stimuli is shown for RAW 264.7 cells
expressing either wild-type (upper blot) or defective P2X7 (lower
blot). Immunoblotting was performed with anti-Grb2 antibody as a
loading control
Purinergic Signalling
CREB transcriptional activation) in macrophages treated
with P2X7 agonists (data not shown, Gavala et al., in
preparation). Altogether these data suggest that P2X7
stimulation modulates monocytic gene expression through
the activation of CREB.
Activating protein-1 is a collective term for dimeric
transcription factors composed of Jun, Fos, or ATF subunits
that bind to a common 12-O-tetradecanoylphorbol-13-
acetate response elements (5&#8242;-TGAGTCA-3&#8242😉 found in the
promoter regions of many inflammatory cytokines and
chemokines [90, 91]. Previous evidence for AP-1 activation
by extracellular ATP comes from studies examining B
lymphocytes. In these experiments, administration of
extracellular ATP to human tonsilar B lymphocytes was
found to increase cFos mRNA levels within 30 min [92].
Furthermore, co-treatment of human fetal astrocytes with
IL-1&#946; and ATP was seen to induce an activation of AP-1
that is more robust than that observed with IL-1&#946; alone
[93], and treatment with P2 receptor antagonists inhibits
cytokine-induced AP-1 activation [93, 94]. It has been
suggested that stimulation of Jurkat T cells induces AP-1
DNA-binding activity that results from increased c-Jun and
cFos expression [84]. Conversely, in the present report, we
show that stimulation of human PBMCs with P2X7
agonists does not appear to enhance cFos expression
(Fig. 6a) but does substantially induce the expression of
the FosB and JunB proteins (Fig. 6b, c). These results are
the first report linking the activation of a nucleotide
receptor to FosB and JunB induction. Interestingly, the
effect of P2X7 activation on FosB levels is detectable
within 1 h of treatment with BzATP alone and suggests that
FosB may be a unique transcriptional regulator of P2X7-
initiated signaling. Electrophoretic mobility shift assays
(EMSAs) in BzATP-treated RAW 264.7 cells reveal an
increase in protein binding to an AP-1 consensus oligonucleotide
compared to vehicle treatment, and this AP-1
complex is supershifted with antibodies directed toward
FosB, JunB, and c-Jun (not shown, Gavala et al., in
preparation). However, stimulation of P2X7 does not induce
AP-1 activation in all phenotypic backgrounds. Stimulation
of N9 and N13 microglial cells with 3 mM ATP inhibits the
basal DNA-binding activity of AP-1, suggesting that P2X7-
Fig. 4 The MEK/ERK cascade appears critical for P2X7-induced
ROS production. RAW 264.7 cells were treated with either HEPES
buffer (control) or 1 &#956;g/ml LPS for 10 h, and then transferred to flow
cytometry tubes (5×105 cells/tube) and loaded with 10 &#956;M DCFDA
for 30 min at 37°C. The cells were then pretreated with DMSO or
increasing concentrations of UO126 (Calbiochem, San Diego, CA,
USA; 130 &#956;M) for 30 min at 37°C. Cells were then treated with either
HEPES (control) or 250 &#956;M BzATP for 30 min. Flow cytometry was
performed by measuring 10,000 cells and excluding propidium iodidestained
(dead) cells from the analysis. The data are plotted as the mean
(±SEM) fold increase over control (n=3). *p<0.01, **p<0.005 as
compared to LPS-pretreated, BzATP-induced DCFDA fluorescence
Fig. 5 P2X7 agonists induce CREB Ser-133 phosphorylation in
monocytic cells. a Isolated peripheral human blood monocytes (1×106
cells/well) were treated with either vehicle (HEPES buffer), 1 mM
ATP, 250 &#956;M BzATP, or 1 &#956;g/ml LPS for 15 min. A representative
immunoblot displaying CREB Ser-133 phosphorylation (pCREB) is
shown (n=4). The pCREB antibody recognizes both pCREB and
phosphorylated ATF-1 (pATF1), thus it is also shown. Immunoblotting
was performed with anti-Grb2 antibody as a loading control. b Murine
RAW 264.7 macrophages (3×105 cells/well) were stimulated with
either vehicle (HEPES), 250 &#956;M BzATP or 1 &#956;g/ml LPS for the
indicated time. A representative immunoblot displaying pCREB/
pATF1 is shown. Immunoblotting was performed with anti-&#946;-tubulin
antibody as a loading control. Results of the three independent
experiments were combined and plotted as the mean (±SEM)
Purinergic Signalling
mediated AP-1 activation is cell type-specific (data not
shown).
The activation of certain protein kinases by P2X7
agonists has been implicated in subsequent AP-1 activation
in several immune cells. Stimulation of Jurkat T cells with
P2X7 agonists induces the activation of the lymphoidspecific
cytoplasmic protein tyrosine kinase p56lck [84].
Although the activation of cFos and c-Jun by P2X7 ligands
occurs in Jurkat T cells, it is absent in a p56lck-deficient
JCaM1T cell line [84], suggesting that this kinase may play
a role in P2X7-induced AP-1 activation in T cells. A role for
MAPKs in P2X7-dependent AP-1 activation in monocytic
cells has also been supported. The transactivation of c-Jun
is augmented by the phosphorylation of Ser-63 and -73 by
JNK1/2. Studies from our lab and others have noted JNK1/
2 activation following stimulation of monocytic cells with
P2X7 agonists [21, 68, 95]. Furthermore, co-stimulation of
RAW 264.7 cells with LPS and BzATP results in enhanced
JNK activation compared to either stimulus alone [68]. Of
note, P2X7 agonist-induced JNK activation in RAW 264.7
cells is attenuated by N-acetylcysteine and ascorbic acid,
implicating a role for ROS in P2X7-dependent AP-1
activation [21]. In addition, we have observed that P2X7
agonist-induced FosB and JunB expression in RAW 264.7
macrophages is abrogated in a dose-dependent fashion by
the MEK1/2 antagonist UO126, suggesting that the MEK/
ERK cascade is upstream of the expression of these AP-1
proteins in macrophages (Gavala et al., in preparation).
Although reporter assays and EMSAs have often been
used to demonstrate activation of transcription factors by
P2X7 agonists, little is known about the target genes of
these transcription factors. To this end, preliminary studies
by our lab have suggested a novel role for CREB in P2X7-
Fig. 6 P2X7 agonists induce AP-1 protein expression in primary
human monocytes. Isolated peripheral human blood monocytes were
stimulated with either vehicle (HEPES), 250 &#956;M BzATP, 1 &#956;g/ml
PMA, or 10 &#956;M ionomycin (c) for 5 min. The media was replaced and
the cells were incubated at 37°C for the indicated times (PMA
treatment was reintroduced to the new media) and immunoblotted for
a cFos, b FosB, or c JunB expression. Immunoblotting was performed
with anti-Grb2 antibody as a loading control. The results are
representative of three independent experiments
Fig. 7 Mutation of two arginine residues in the P2X7 C-terminal
region reduces receptor-mediated pore-forming activity. a The distal
C-terminal sequences of mouse, rat, and human P2X7 were aligned.
The proposed Arg-based ER retention signal is underlined, and
Arg578 and Lys579 are highlighted in red. b COS7 cells were
transfected with pcDNA3, P2X7/pcDNA3, or P2X7 R576A/R578A/
pcDNA3. Sixteen to 24 h later, the cells were treated with 250 &#956;M
BzATP in the presence of 1 &#956;M YO-PRO-1 (Molecular Probes,
Eugene, OR, USA) for 20 min at room temperature. The cells were
fixed with 4% paraformaldehyde, washed, and imaged using a Zeiss
Axioplan 2 microscope. These data represent one of five independent
experiments. P2X7 R576A/R578A/pcDNA3 was generated from
P2X7/pcDNA3 (accession number BC011913) by site-directed mutagenesis.
c COS7 cells were transfected with eYFP-P2X7/pCMV, fixed
the next day with 4% paraformaldehyde, imaged using a Zeiss 200M
Axiovert inverted microscope, and deconvolved. P2X7 was subcloned
into the plasmid eYFP/pCMV, which was a generous gift from Dr.
Melanie Cobb (University of Texas Southwestern Medical Center)
Purinergic Signalling
mediated FosB expression, i.e., transfection of dominantnegative
CREB vectors into HEK293 cells heterologously
expressing P2X7 abrogates P2X7 agonist-induced FosB
expression (Gavala et al., in preparation).
P2X7 contains a trafficking domain in its C-terminus
The intracellular trafficking of plasma membrane receptors is a
critical component of their regulation. P2X7 trafficking has not
been widely explored, and it is unclear how newly synthesized
P2X7 subunits are transported from the endoplasmic
reticulum (ER) to the surface and whether P2X7 internalization
or trafficking to the plasma membrane is controlled by
extracellular signals. It is also not clear whether P2X7 recycles
following internalization or whether the receptor contains
signals within its sequence that regulate its transport through
the cell. Addressing these issues will increase our understanding
of how P2X7 is globally regulated and how cells
mediate their responsiveness to extracellular ATP.
Our lab and those of Wiley, Petrou, and colleagues have
provided evidence supporting the concept that P2X7
contains a trafficking domain in its distal C-terminal region
that is critical for cell surface localization. Point mutations
and deletions within this region reduce P2X7 surface
expression and pore activity [30, 34, 96]. When P2X7 is
truncated at residues 551&#8211;581, it exhibits significantly
reduced activity and plasma membrane localization [30],
and three polymorphisms, I568N, R574H, and R574L, have
been identified in this area of the receptor [34, 42, 97]. T
lymphocytes and natural killer cells from individuals who
are heterozygous for the I568N polymorphism have
reduced ATP-induced pore-forming ability, and exogenously
expressed P2X7 with the I568N mutation is poorly
expressed on the surface of HEK293 cells [34]. We have
previously found that mutation of Arg578 and Lys579,
which are localized in the LPS-binding motif (Fig. 1),
drastically reduces the ability of P2X7 to localize on the
plasma membrane and to promote BzATP-induced cell
death [96]. Although the P2X7 R578E/K579E mutant does
not appear to be stably expressed on the cell surface at 37°C,
it does localize to the plasma membrane at 27°C, suggesting
that the double mutant is internalized more readily than wildtype
receptor. These findings lead us to speculate that P2X7
contains specific signals within its C-terminus that direct its
intracellular trafficking.
One aspect of P2X7 trafficking that is less well
characterized entails the mechanisms by which it is transported
from the ER to the cell surface. Several studies have
shown that newly synthesized subunits of integral membrane
proteins, including N-methyl-D-aspartate (NMDA),
glutamate receptor, and &#947;-aminobutyric acid type B
(GABAB) receptor, are retained in the ER before the
properly assembled receptors and are further transported
through the secretory pathway [98]. The NMDA and
GABAB subunits possess arginine (Arg)-based ER retention/
retrieval signals that prevent improperly assembled
subunits from trafficking to the Golgi apparatus until
assembly is complete and the signal is masked [99, 100].
These ER retention/retrieval sequences were first discovered
in major histocompatibility complex class II proteins
and have also been characterized in inward rectifier
Fig. 8 Proposed model of several
of the key events by which
P2X7 promotes the production
of immune mediators and activates
gene expression. Stimulation
of P2X7 leads to enhanced
ROS formation via the MAPKs
ERK1/2 and the NADPH oxidase
complex and stimulates
gene transcription by activating
CREB and increasing the expression
of the AP-1 proteins
FosB and JunB. In addition,
specific residues in the putative
trafficking domain of P2X7 are
required for proper localization
and full activation of the
receptor
Purinergic Signalling
potassium channel and sulfonylurea receptor subunits [101,
102]. The Arg-based ER retention/retrieval consensus
sequence is &#981;/&#968;/R-R-X-R, where &#981;/&#968; is an aromatic or
bulky hydrophobic residue and X is any amino acid but is
generally not a negatively charged or small, non-polar
residue [98].
In view of the above discussion, we predict that P2X7
contains a RXR retention/retrieval signal at amino acid 576
that lies within the sequence WRIR, which conforms to the
consensus sequence &#981;/&#968;/R-R-X-R (Fig. 7a). To test the
hypothesis that P2X7 contains an RXR ER retention/
retrieval sequence at residue 576, a pore activity assay
was conducted as an indirect readout for cell surface
expression. In this experiment, COS7 cells were transfected
with wild-type or mutant P2X7, stimulated with BzATP, and
imaged for relative dye uptake as a measure of liganddependent
pore formation. As shown in Fig. 7b, mutation
of human R576 and R578 to Ala substantially reduces the
activity of the receptor. Cells take up the dye YO-PRO-1
when wild-type P2X7 is expressed but exhibit drastically
reduced dye uptake in the presence of the RXR mutant or
the vector control. These findings, together with the
information available about Arg-based signals in other
receptors, provide initial support for the idea that P2X7
contains an RXR ER retention/retrieval sequence in its Cterminus
in the area surrounding residue 576.
Although our previous findings revealing that the
P2X7 mutant R578E/K579E does not express on the cell
surface seems counterintuitive to the idea that P2X7
possesses an ER retention/retrieval sequence, we speculate
that cells prematurely traffic the R578E/K579E P2X7
mutant from the ER before it is properly assembled, and
once it reaches the plasma membrane, it becomes quickly
down-regulated because it is non-functional or lacks
appropriate interaction with specific lipids. Alternatively,
the P2X7 R578E/K579E mutation and not the mutation of
an RXR signal prevents P2X7 from localizing to the cell
surface. In this regard, Marshall and colleagues have
shown that a RK/AA mutation in the C-terminus of the
GluR5-2b kainate receptor subunit disrupts ER retention
[103].
Another unresolved issue is whether the trafficking of
P2X7 from intracellular compartments to the plasma
membrane is stimulated by extracellular signals, as it is
for the major insulin-responsive glucose transporter
GLUT4, and has been reported for the NADPH oxidase
complex [79, 80, 104]. We analyzed the localization of
P2X7 fused to enhanced yellow fluorescent protein (eYFP)
and found that it is predominantly localized in intracellular
pools (Fig. 7c). We are now in a position to explore the
critical hypothesis that the trafficking of P2X7 to and from
these intracellular pools to the plasma membrane is
stimulated by extracellular signals.
Summary
The ionotrophic receptor P2X7 is thought to be a major
immune modulator that responds to extracelluar ATP at
sites of inflammation and tissue damage. Although several
human genetic and animal studies have demonstrated the
importance of P2X7 in immune function, many questions
remain concerning P2X7 signaling and the regulation of its
activity via intracellular trafficking. We have investigated
the mechanisms by which P2X7 promotes inflammatory
Click to expand...
 
Seriously, what is this bulls**t? We're doing ok without these nonsensical posts, guys.
 
I'm hoping we hit it before I have to go to orchestra practice tonight...I'm such a geek.
 
aerobic_cats.jpg
 
Pitt, you effing suck... I got a heart attack reading the email and all it was was a stupid we hope you still wanna be on the waitlist... you should not do this so close to May 15th😡😡😡
 
I prefer factual post padding:

The Baby Ruth candy bar was actually named after Grover Cleveland's daughter Ruth.
 
sarahl86 said:
I'm hoping we hit it before I have to go to orchestra practice tonight...I'm such a geek.
Click to expand...

What instrument do you play? I was a violist for 9 years.

funkydrmonkey said:
Pitt, you effing suck... I got a heart attack reading the email and all it was was a stupid we hope you still wanna be on the waitlist... you should not do this so close to May 15th😡😡😡
Click to expand...

Pitt >> UT-Southwestern for you, funky?

sarahl86 said:
I prefer factual post padding:

The Baby Ruth candy bar was actually named after Grover Cleveland's daughter Ruth.
Click to expand...

I actually knew that one already. Yay!
 
the girl in the middle looks like a guy


Female%20Sprinters.jpg
 
Uma Thurman's father was the first American to be ordained as a Tibetan Buddhist monk.
 
funkydrmonkey said:
Pitt, you effing suck... I got a heart attack reading the email and all it was was a stupid we hope you still wanna be on the waitlist... you should not do this so close to May 15th😡😡😡
Click to expand...

hahahaha...I got the same e-mail...but I kind of like that they do continually maintain some form of correspondence with us
 
we are rapidly approaching 50k...I sense a good deal of post padding on the horizon...
 
Imo this girl is jgberken




Allison_Stokke7.jpg
 
The following are NOT by lear.

There once was an old man of Esser,
Whose knowledge grew lesser and lesser,
It at last grew so small
He knew nothing at all,
And now he's a college professor
 
This one is my favorite limerick.

A girl from the Isle of Wight
Used to travel much faster than light
She set out one day,
In a relative way,
And returned on the previous night.
 
You must log in or register to reply here.

Similar threads

hommefatale
Elective Class Pass/Fail
Replies
3
Views
490
Goro
Goro
L
pass/no pass physical education class
Replies
4
Views
259
LizzyM
LizzyM
H
Not taking planned calculus class anymore
Replies
2
Views
510
holajola
H
aprilandaflower
Course classification of design courses
Replies
1
Views
293
wysdoc
wysdoc
A
Marking an audited class as a repeat?
Replies
1
Views
280
Mr.Smile12
Mr.Smile12
Top