P2X7 receptor-deficient mice and uses thereof

Details P2X7 receptor-deficient mice and uses thereof P2X7 receptor-deficient mice and uses thereof

2001-06-06

2004-01-13

Crouch, Deborah (Department: 1632)

Multicellular living organisms and unmodified parts thereof and

Nonhuman animal

Transgenic nonhuman animal

C800S013000, C800S014000, C800S021000, C800S022000, C800S025000, C435S455000, C435S463000, C435S325000, C435S320100

Reexamination Certificate

active

06677501

ABSTRACT:

FIELD OF THE INVENTION
BACKGROUND OF THE INVENTION
Cell surface ATP receptors can be divided into the metabotropic receptor family (P2Y/P2U) and the ionotropic receptor family (or the P
2×
receptor family). Metabotropic receptor family members are G-protein coupled receptors and ionotropic receptor family members are ligand-gated channels. There are eleven metabotropic receptor family members and seven ionotropic receptor family members, P
2×1
R to P
2×7
R.
The P
2×7
receptor (P
2×7
R), like other members of the P2× receptor family, is an ATP-gated ion channel (Surprenant et al. (1996)
Science
272:735-738; Rassendren et al. (1997)
J. Biol. Chem.
272:5482-5486; Michel et al. (1998)
Br. J. Pharmacol.
125:1194-1201). The P
2×7
R, however, demonstrates attributes that clearly distinguish it from other members of the family. For example, the P
2×7
R requires levels of ATP in excess of 1 mM to achieve activation whereas other P
2×
receptors activate at ATP concentrations ≦100 &mgr;M (Greenberg et al. (1988)
J. Biol. Chem.
263:10337-10343; Steinberg et al. (1987)
J. Biol. Chem.
262:8884-8888): the higher concentration requirement reflects, in part, the preference of the P
2×7
R for ATP
4−
as its ligand and the relatively low abundance of this species in media containing physiological concentrations of divalent cations (e.g., Ca
2+
and Mg
2+
). An additional unique feature of the P
2×7
R is found in its conductance properties. All P
2×
receptors demonstrate non-selective channel-like properties following ligation, but the channels formed by the P
2×7
R rapidly transform to “pores” that allow passage of solutes as large as 900 daltons (Steinberg et al. (1987)
J. Biol. Chem.
262:8884-8888; Virgihio et al. (1999)
J. Physiol.
519:335-346). Molecular details of this transformation remain to be described, but domain swapping and deletion experiments have suggested that the carboxy terminal domain of the P
2×7
R participates in pore complex formation; the carboxy terminal domain is significantly longer than the comparable domains in the other P
2×
receptors (North (1996)
Current Opin. Cell Biol.
8:474-483). Possibly as a consequence of this pore-like activity, continuous ligation of the P
2×7
receptor for times greater than 15 minutes can lead to cell death (Di Virgilio (1995)
Immunol. Today
16:524-528; Murgia et al. (1992)
Biochem. J.
288:897-901; Ferrari et al. (1999)
FEBS Let.
447:71-75).
The P
2×7
R displays a restricted cellular distribution, being observed primarily in cells of hematopoietic origin including monocytes and macrophages and some lymphocyte populations (Di Virgilio (1995)
Immunol. Today
16:524-528; Collo et al. (1997)
Neuropharmacol.
36:1277-1283). The receptor also has been reported to exist on microglial cells (Sanz et al. (2000)
J. Immunol.
164:4893-4898), some cancer cells (Wiley et al. (1989)
Blood
73:1316-1323), sperm (Foresta et al. (1996)
Am J. Physiol.
270:C1709-C1714), and dendritic cells (Mutini et al. (1999)
J. Immunol.
163:1958-1965).
The P
2×7
R has been reported to participate in a diverse list of cellular activities including lymphocyte proliferation (Baricordi et al. (1999)
J. Biol. Chem.
274:33206-33208), fertilization (Foresta et al. (1996)
Am J. Physiol.
270:C1709-C1714), giant cell formation (Chiozzi et al. (1997)
J. Cell Biol.
138:697-706), cell death (Murgia et al. (1992)
Biochem. J.
288:897-901; Ferrari et al. (1999)
FEBS Let.
447:71-75), killing of invading mycobacteria (Latumas et al. (1997)
Immunity
7:433-444), and IL-1 posttranslational processing (Hogquist et al. (1991)
Proc. Natl. Acad. Sci.
(
USA
) 88:8485-8489; Perregaux et al. (1994)
J. Biol. Chem.
269:15195-15203). Further, ligation of the P
2×7
R has been associated with activation of phospholipase D and activation of some forms of NF-&egr;B (Humphreys et al. (1996)
J. Immunol.
157:5627-5637; Ferrari et al. (1997)
J. Cell Biol.
139:1635-1643).
One of the most intriguing activities attributed to the P
2×7
R is its ability to induce posttranslational processing of proIL-1 (Sanz et al. (2000)
J. Immunol.
164:4893-4898; Hogquist et al. (1991)
Proc. Natl. Acad. Sci.
(
USA
) 88:8485-8489; Perregaux et al. (1994)
J. Biol. Chem.
269:15195-15203). Interleukin (IL)-1 is a multipotential inflammatory mediator produced in abundance by activated monocytes and macrophages. The administration of IL-1 to animals has been shown to initiate an inflammatory response, produce fever, and promote tissue degradation. Further, elevated levels of IL-1 have been detected in patients suffering from a number of chronic disorders, including rheumatoid arthritis, Alzheimer's disease, and acute myelocytic leukemia (McNiff et al. (1995)
Cytokine
7:209; Gray et al. (1986)
J. Immunol.
137:3644; Lomedico et al. (1984)
Nature
312:458).
When IL-1 is released from cells, it binds to receptors on target cells and elicits complex signaling cascades leading to the upregulation of gene products that contribute to an inflammatory state including matrix metalloproteinases, cyclooxygenase-2, IL-6, and cellular adhesion molecules (Flannery et al. (1999)
Matrix Biol.
18:225-237; Guzn et al. (1998)
J. Biol. Chem.
273:28670-28676; Allen et al. (2000)
J. Exp. Med.
191:859-869; Bevilacqua et al. (1989)
Science
243:1160-1164). Two distinct gene products, IL-1&agr; and IL-1&bgr;, contribute to IL-1 biological activity. The amino acid sequences of IL-1a and IL-1&bgr; are only 25% identical yet these two polypeptides bind to the same receptors on target cells (Slack et al. (1993)
J. Biol. Chem.
268:2513-2524). Human EL-1&agr; and IL-&bgr; are both initially produced as 31 kDa procytokines containing amino terminal extensions that are subsequently removed by proteolysis. In the case of proIL-1&agr;, the propolypeptide and the 17 kDa cleavage product display equivalent signaling activity indicating that proteolytic cleavage is not necessary to generate a receptor-competent ligand. In contrast, proIL-1&bgr; does not bind to the signaling IL-1 receptor (Mosley et al. (1987)
J. Biol. Chem.
262:2941-2944), and cleavage by caspase-1 is necessary to generate the mature 17 kDa signaling-competent form of this cytokine (Cerretti et al. (1992)
Science
256:97-100; Thornberry et al. (1992)
Nature
356:768-774).
The two forms of IL-1 share another very unusual attribute, both proIL-1&agr; and proIL-1&bgr; are synthesized without a signal sequence (March et al. (1985)
Nature
315:641-647), the peptide epitope required to direct nascent polypeptides to the endoplasmic reticulum (Walter et al. (1994)
Ann. Rev. Cell Biol.
10:87-119). As a result, newly synthesized proIL-1&agr; and proIL-1&bgr; accumulate within the cytoplasmic compartment of producing cells rather than being sequestered to the secretory apparatus. Caspase-1 also is produced as a cytosol-localized proenzyme, the 45 kDa propolypeptide must be proteolytically processed to generate the 20 kDa and 10 kDa subunits which constitute the mature active protease (Thornberry et al. (1992)
Nature
356:768-774; Miller et al. (1993)
J. Biol. Chem.
268:18062-18069; Ayala et al. (1994)
J. Immunol.
153:2592-2599). In activated monocytes and macrophages, therefore, proIL-1&bgr; and procaspase-1 co-exist within the cytoplasm. Mechanisms that control activation of procaspase-1, and in turn cleavage of proIL-1&bgr;, are not well understood. Recent studies, however, have provided evidence that proteolytic processing IL-1&bgr; and release of the mature cytokine product extracellularly do not proceed constitutively. Rather, the post-translational processing of proIL-1 requires that lipopolysaccharide-(LPS)-activated monocytes and/or macrophges encounter an external stimulus that promotes activation of procaspase-1, cleavage of proIL-1&bgr;, and release of the 17 kDa cytokine (Miller et al. (1995)
J. Immunol.
154:1331-1338; Laliberte et al. (1999)
J. Biol. Chem.
274:36944-36951

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