Elena Adinolfi

The P2X7 Receptor: A Key Player in IL-1 Processing and Release

Human IL-1 family proteins are key mediators of the host response to infections, injury, and immunologic challenges. The mechanism by which IL-1 activates proinflammatory responses in target cells, and the plasma membrane receptors involved, is fairly well known. This has led to the development of innovative drugs that block IL-1 downstream to its synthesis and secretion. On the contrary, the mechanism of IL-1 and other IL-1 family members (e.g., IL-18) maturation and release is incompletely understood. Accruing evidence points to a plasma membrane receptor for extracellular ATP, the P2X7 receptor, as a key player in both processes. A deeper understanding of the mechanism by which the P2X7 receptor triggers IL-1 maturation and exteriorization may suggest novel avenues for the treatment of inflammatory diseases and provide a deeper insight in the fundamental mechanism of protease activation and cellular export of proteins lacking a leader sequence.

INCREASED PROLIFERATION RATE OF LYMPHOID CELLS TRANSFECTED WITH THE P2X7 ATP RECEPTOR

Human leukocytes can express the P2X7 purinergic receptor, an ionic channel gated by extracellular ATP, for which the physiological role is only partially understood. Transfection of P2X7 cDNA into lymphoid cells that lack this receptor sustains their proliferation in serum-free medium. Increased proliferation of serum-starved P2X7 transfectants is abolished by the P2X7receptor blocker oxidized ATP or by the ATP hydrolase apyrase. Both wild type and P2X7-transfected lymphoid cells release large amounts of ATP into the culture medium. These data suggest the operation of an ATP-based autocrine/paracrine loop that supports lymphoid cell growth in the absence of serum-derived growth factors.

Expression of P2X7 receptor increases in vivo tumor growth

The P2X7 receptor is an ATP-gated ion channel known for its cytotoxic activity. However, recent evidence suggests a role for P2X7 in cell proliferation. Here, we found that P2X7 exhibits significant growth-promoting effects in vivo. Human embryonic kidney cells expressing P2X7 exhibited a more tumorigenic and anaplastic phenotype than control cells in vivo, and the growth rate and size of these tumors were significantly reduced by intratumoral injection of the P2X7 inhibitor-oxidized ATP. The accelerated growth of P2X7-expressing tumors was characterized by increased proliferation, reduced apoptosis, and a high level of activated transcription factor NFATc1. These tumors also showed a more developed vascular network than control tumors and secreted elevated amounts of VEGF. The growth and neoangiogenesis of P2X7-expressing tumors was blocked by intratumoral injection of the VEGF-blocking antibody Avastin (bevacizumab), pharmacologic P2X7 blockade, or P2X7 silencing in vivo. Immunohistochemistry revealed strong P2X7 positivity in several human cancers. Together, our findings provide direct evidence that P2X7 promotes tumor growth in vivo.

Pseudoapoptosis Induced by Brief Activation of ATP-gated P2X7 Receptors

P2X7 receptors are ATP-gated ion channels primarily expressed on antigen-presenting immune cells where they play a role in the acute inflammatory response. These ion channels couple not only to influx of cations, including calcium, but also to rapid alterations in cell morphology (membrane blebbing, phosphatidylserine exposure, microvesicle shedding). These features resemble the extranuclear events associated with end stages of apoptosis but cell death does not occur if receptor activation is brief. Here we delineate two signaling pathways underlying these apoptotic-like processes. Loss of membrane asymmetry occurs within seconds, which directly triggers cytoskeletal disruption and zeiotic membrane blebbing; this is readily reversible and requires both calcium influx through P2X7 channels and mitochondrial calcium increase but is not associated with cytochrome c release. A slower, calcium-independent, ROCK-1-dependent cascade that does not involve rapid loss of membrane asymmetry but is associated with cytochrome c release is secondarily activated. The ROCK-1 pathway appears largely responsible for cell death, which occurs after prolonged stimulation of P2X7 receptors. We suggest that the former mechanism underlies the reversible pseudoapoptotic events induced by brief activation of P2X7 receptors.

Trophic activity of a naturally occurring truncated isoform of the P2X7 receptor

P2X7 is the largest member of the P2X subfamily of purinergic receptors. A typical feature is the carboxyl tail, which allows formation of a large pore. Recently a naturally occurring truncated P2X7 splice variant, isoform B (P2X7B), has been identified. Here we show that P2X7B expression in HEK293 cells, a cell type lacking endogenous P2X receptors, mediated ATP‐stimulated channel activity but not plasma membrane permeabilization, raised endoplasmic reticulum Ca2+ content, activated the transcription factor NFATc1, increased the cellular ATP content, and stimulated growth. In addition, P2X7B‐transfected HEK293 cells (HEK293‐P2X7B), like most tumor cells, showed strong soft agarinfiltrating ability. When coexpressed with full‐length P2X7 (P2X7A), P2X7B coassembled with P2X7A into a heterotrimer and potentiated all known responses mediated by this latter receptor. P2X7B mRNA was found to be widely distributed in human tissues, especially in the immune and nervous systems, and to a much higher level than P2X7A. Finally, P2X7B expression was increased on mitogenic stimulation of peripheral blood lymphocyte. Altogether, these data show that P2X7B is widely expressed in several human tissues, modulates P2X7A functions, participates in the control of cell growth, and may help understand the role of the P2X7 receptor in the control of normal and cancer cell proliferation.—Adinolfi, E., Cirillo, M., Woltersdorf, R., Falzoni, S., Chiozzi, P., Pellegatti, P., Callegari, M. G., Sandonà, D., Markwardt, F., Schmalzing, G., Di Virgilio, F. Trophic activity of a naturally occurring truncated isoform of the P2X7 receptor. FASEB J. 24, 3393–3404 (2010). www.fasebj.org

P2X(7): a growth-promoting receptor-implications for cancer.

The P2X7 receptor is widely referred to as the paradigmatic cytotoxic nucleotide receptor, and is often taken as an epitome of cytotoxic receptors as a whole. However, cytotoxicity is the result of sustained pharmacological stimulation, which is likely to occur in vivo only under severe pathological conditions. Over the years, we have gathered robust experimental proof that led us to adopt an entirely different view, pointing to P2X7 as a survival/growth-promoting rather than death-inducing receptor. Evidence in favour of this role is manifold: (1) extracellular ATP and benzoyl ATP support cell proliferation in peripheral T lymphocytes via a P2X7-like receptor; (2) P2X7 transfection into several cell lines confers growth advantage; (3) HEK293 cells transfected with P2X7 show enhanced mitochondrial metabolic activity and growth; (4) lipopolysaccharide (LPS)-dependent growth arrest of microglia is mediated via P2X7 down-modulation; (5) several malignant tumours express high P2X7 levels and (6) the ATP concentration in tumour interstitium is several-fold higher than in healthy tissues, to a level in principle sufficient to activate the P2X7 receptor. The molecular basis of P2X7-mediated growth-promoting activity is poorly known, but mitochondria appear to play a central role. A deeper understanding of the role played by P2X7 in cell proliferation might provide an insight into the mechanism of normal and malignant cell growth and suggest novel anti-tumour therapies.

P2X7 receptor: Death or life?

The P2X7 plasma membrane receptor is an intriguing molecule that is endowed with the ability to kill cells, as well as to activate many responses and even stimulate proliferation. Here, the authors give an overview on the multiplicity and complexity of P2X7-mediated responses, discussing recent information on this receptor. Particular attention has been paid to early and late signs of apoptosis and necrosis linked to activation of the receptor and to the emerging field of P2X7 function in carcinogenesis.

Tyrosine phosphorylation of HSP90 within the P2X7 receptor complex negatively regulates P2X7 receptors.

The purinergic P2X7 receptor not only gates the opening of a cationic channel, but also couples to several downstream signaling events such as rapid membrane blebbing, microvesicle shedding, and interleukin-1β release. Protein-protein interactions are likely to be involved in most of these signaling cascades; and recently, a P2X7 receptor-protein complex comprising at least 11 distinct proteins has been identified. We have studied one of these interacting proteins, HSP90, in human embryonic kidney cells expressing either human or rat P2X7 receptors as well as in rat peritoneal macrophages using biochemical (immunoprecipitation and Western blotting) and functional (membrane blebbing and currents) assays. We found that HSP90 was tyrosine-phosphorylated in association with the P2X7 receptor complex, but not in the cytosolic compartment. The HSP90 inhibitor geldanamycin decreased tyrosine phosphorylation of HSP90 and produced a 2-fold increase in the sensitivity of P2X7 receptors to agonist. Protein expression and tyrosine phosphorylation of a mutant P2X7 receptor in which a tyrosine in the C-terminal domain was substituted with phenylalanine (Y550F) were not changed, but tyrosine phosphorylation of HSP90 associated with this mutant P2X7 receptor complex was significantly greater than that associated with the wild-type complex. P2X7-Y550F receptors showed a 15-fold lower sensitivity to agonist, which was reversed by geldanamycin. We conclude that selective tyrosine phosphorylation of P2X7 receptor-associated HSP90 may act as a negative regulator of P2X7 receptor complex formation and function.

Extracellular purines, purinergic receptors and tumor growth.

Virtually, all tumor cells as well as all immune cells express plasma membrane receptors for extracellular nucleosides (adenosine) and nucleotides (ATP, ADP, UTP, UDP and sugar UDP). The tumor microenvironment is characterized by an unusually high concentration of ATP and adenosine. Adenosine is a major determinant of the immunosuppressive tumor milieu. Sequential hydrolysis of extracellular ATP catalyzed by CD39 and CD73 is the main pathway for the generation of adenosine in the tumor interstitium. Extracellular ATP and adenosine mold both host and tumor responses. Depending on the specific receptor activated, extracellular purines mediate immunosuppression or immunostimulation on the host side, and growth stimulation or cytotoxicity on the tumor side. Recent progress in this field is providing the key to decode this complex scenario and to lay the basis to harness the potential benefits for therapy. Preclinical data show that targeting the adenosine-generating pathway (that is, CD73) or adenosinergic receptors (that is, A2A) relieves immunosuppresion and potently inhibits tumor growth. On the other hand, growth of experimental tumors is strongly inhibited by targeting the P2X7 ATP-selective receptor of cancer and immune cells. This review summarizes the recent data on the role played by extracellular purines (purinergic signaling) in host–tumor interaction and highlights novel therapeutic options stemming from recent advances in this field.

Stimulation of P2 (P2X7) receptors in human dendritic cells induces the release of tissue factor-bearing microparticles

Receptors for extracellular nucleotides are the focus of increasing attention for their ability to cause release of plasma membrane vesicles (micropar‐ticles, MPs). Here, we show that monocyte‐derived human dendritic cells (DCs) stimulated with a P2X7 receptor (P2X7R) agonist undergo a large release of MPs endowed with procoagulant activity. Functional and Western blot studies revealed that MPs contain the membrane‐bound form of tissue factor (TF), a glycop‐rotein acting as essential cofactor of activated factor VII and triggering blood coagulation. Quiescent DCs express the membrane‐bound (full length), as well as truncated alternatively spliced TF forms. DC reactivity to anti‐TF Abs disappeared almost completely on stimulation with ATP or benzoyl ATP (BzATP), as shown by immunoblot and confocal microscopy analysis. Concurrently, TF reactivity and activity appeared in the vesicular fraction, indicating that MPs are important carriers for the dissemination of full‐length TF form. Activity of MP‐bound TF, comparable to that of relipidated recombinant TF, was dose dependently inhibited by the addition of a specific anti‐human TF antibody. We infer that a large fraction of this protein, and its procoagulant potential, are “deliverable” after physiological or pathological stimuli. These findings might have implications for triggering and propagating coagulation in healthy and atherosclerotic vessels.—MarcelloBaroni, CinziaPizzirani, MirkoPinotti, DavideFerrari, ElenaAdinolfi, SaraCalzavarini, PierpaoloCaruso, FrancescoBernardi, FrancescoDi Virgilio. Stimulation of P2 (P2X7) receptors in human dendritic cells induces the release of tissue factor‐bearing microparticles. FASEB J. 21, 1926–1933 (2007)